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Sample records for heparan-sulfate proteoglycan glypican

  1. Syndecan heparan sulfate proteoglycans

    DEFF Research Database (Denmark)

    Gomes, Angélica Maciel; Sinkeviciute, Dovile; Multhaupt, Hinke A.B.

    2016-01-01

    Virtually all animal cells express heparan sulfate proteoglycans on the cell surface and in the extracellular matrix. Syndecans are a major group of transmembrane proteoglycans functioning as receptors that mediate signal transmission from the extracellular microenvironment to the cell. Their hep......Virtually all animal cells express heparan sulfate proteoglycans on the cell surface and in the extracellular matrix. Syndecans are a major group of transmembrane proteoglycans functioning as receptors that mediate signal transmission from the extracellular microenvironment to the cell....... Their heparan sulfate chains, due to their vast structural diversity, interact with a wide array of ligands including potent regulators of adhesion, migration, growth and survival. Frequently, ligands interact with cell surface heparan sulfate in conjunction with high affinity receptors. The consequent...... signaling can therefore be complex, but it is now known that syndecans are capable of independent signaling. This review is divided in two sections, and will first discuss how the assembly of heparan sulfate, the anabolic process, encodes information related to ligand binding and signaling. Second, we...

  2. Heparan sulfate proteoglycans in glomerular inflammation.

    NARCIS (Netherlands)

    Rops, A.L.; Vlag, J. van der; Lensen, J.F.M.; Wijnhoven, T.J.M.; Heuvel, L.P.W.J. van den; Kuppevelt, A.H.M.S.M. van; Berden, J.H.M.

    2004-01-01

    Heparan sulfate proteoglycans (HSPGs) are glycoproteins consisting of a core protein to which linear heparan sulfate side chains are covalently attached. These heparan sulfate side chains can be modified at different positions by several enzymes, which include N-deacetylases, N- and

  3. Breast and ovarian cancers: a survey and possible roles for the cell surface heparan sulfate proteoglycans

    DEFF Research Database (Denmark)

    Yoneda, Atsuko; Lendorf, Maria E; Couchman, John R

    2012-01-01

    of breast cancer may also develop ovarian cancer. Here, the authors review the different tumor markers of breast and ovarian carcinoma and discuss the expression, mutations, and possible roles of cell surface heparan sulfate proteoglycans during tumorigenesis of these carcinomas. The focus is on two groups...... of proteoglycans, the transmembrane syndecans and the lipid-anchored glypicans. Both families of proteoglycans have been implicated in cellular responses to growth factors and morphogens, including many now associated with tumor progression....

  4. Heparan sulfate proteoglycans present PCSK9 to the LDL receptor.

    Science.gov (United States)

    Gustafsen, Camilla; Olsen, Ditte; Vilstrup, Joachim; Lund, Signe; Reinhardt, Anika; Wellner, Niels; Larsen, Torben; Andersen, Christian B F; Weyer, Kathrin; Li, Jin-Ping; Seeberger, Peter H; Thirup, Søren; Madsen, Peder; Glerup, Simon

    2017-09-11

    Coronary artery disease is the main cause of death worldwide and accelerated by increased plasma levels of cholesterol-rich low-density lipoprotein particles (LDL). Circulating PCSK9 contributes to coronary artery disease by inducing lysosomal degradation of the LDL receptor (LDLR) in the liver and thereby reducing LDL clearance. Here, we show that liver heparan sulfate proteoglycans are PCSK9 receptors and essential for PCSK9-induced LDLR degradation. The heparan sulfate-binding site is located in the PCSK9 prodomain and formed by surface-exposed basic residues interacting with trisulfated heparan sulfate disaccharide repeats. Accordingly, heparan sulfate mimetics and monoclonal antibodies directed against the heparan sulfate-binding site are potent PCSK9 inhibitors. We propose that heparan sulfate proteoglycans lining the hepatocyte surface capture PCSK9 and facilitates subsequent PCSK9:LDLR complex formation. Our findings provide new insights into LDL biology and show that targeting PCSK9 using heparan sulfate mimetics is a potential therapeutic strategy in coronary artery disease.PCSK9 interacts with LDL receptor, causing its degradation, and consequently reduces the clearance of LDL. Here, Gustafsen et al. show that PCSK9 interacts with heparan sulfate proteoglycans and this binding favors LDLR degradation. Pharmacological inhibition of this binding can be exploited as therapeutic intervention to lower LDL levels.

  5. Changes in expression of proteoglycan core proteins and heparan sulfate enzymes in the developing and adult murine aorta.

    Science.gov (United States)

    Adhikari, Neeta; Carlson, Marjorie; Lerman, Ben; Hall, Jennifer L

    2011-06-01

    Proteoglycan core proteins are linked to four different classes of linear sugar chains referred to as glycosaminoglycans. Heparan sulfate constitutes one of these classes of glycosaminoglycans, and has been shown to be important in developmental processes as well as disease. We designed a low-density gene expression array to identify expression levels of heparan sulfate biosynthetic enzymes and proteoglycan core proteins in the aorta of late stage embryos (E18.5) and adult mice (12 weeks). Significant changes were found in mRNA expression of proteoglycan core proteins syndecan, glypican, decorin, perlecan, and versican from development to adulthood (n = 8, p proteoglycan core proteins and heparan sulfate biosynthetic enzymes in the aorta undergo significant changes in their expression from development to adulthood. These findings may have important biological significance in the specific cell-defined roles of proteoglycan and heparan sulfate related targets in vascular development, maintenance, and response to various perturbations.

  6. The Role(s) of Heparan Sulfate Proteoglycan(s) in the wnt-1 Signaling Pathway

    National Research Council Canada - National Science Library

    Lin, Xinhua

    2000-01-01

    .... I have investigated the role of heparan sulfate proteoglycans (HSPGs) in Wg signaling. HSPGs are cell surface macromolecules that consist of a protein core to which heparan sulfate (HS) glycosaminoglycan (GAG...

  7. Renal localization of heparan sulfate proteoglycan by immunohistochemistry.

    OpenAIRE

    Klein, D. J.; Oegema, T. R.; Eisenstein, R.; Furcht, L.; Michael, A. F.; Brown, D. M.

    1983-01-01

    Glomerular localization of heparan sulfate proteoglycan (HS-proteoglycan) has been studied immunohistochemically with a highly purified antiserum to bovine aorta HS-proteoglycan core protein. The specificity of the antiserum was enhanced by consecutive fibronectin and chondroitin sulfate-dermatan sulfate proteoglycan (CS-DS proteoglycan) affinity chromatography. The affinity-purified HS-proteoglycan antibody lacked cross-reactivity by enzyme-linked immunosorbent assays (ELISA) with CS-DS prot...

  8. Heparan sulfate proteoglycans present PCSK9 to the LDL receptor

    DEFF Research Database (Denmark)

    Gustafsen, Camilla; Olsen, Ditte; Vilstrup, Joachim

    2017-01-01

    and thereby reducing LDL clearance. Here, we show that liver heparan sulfate proteoglycans are PCSK9 receptors and essential for PCSK9-induced LDLR degradation. The heparan sulfate-binding site is located in the PCSK9 prodomain and formed by surface-exposed basic residues interacting with trisulfated heparan...... complex formation. Our findings provide new insights into LDL biology and show that targeting PCSK9 using heparan sulfate mimetics is a potential therapeutic strategy in coronary artery disease.PCSK9 interacts with LDL receptor, causing its degradation, and consequently reduces the clearance of LDL. Here......, Gustafsen et al. show that PCSK9 interacts with heparan sulfate proteoglycans and this binding favors LDLR degradation. Pharmacological inhibition of this binding can be exploited as therapeutic intervention to lower LDL levels....

  9. Heparan sulfate proteoglycans: structure, protein interactions and cell signaling

    Directory of Open Access Journals (Sweden)

    Juliana L. Dreyfuss

    2009-09-01

    Full Text Available Heparan sulfate proteoglycans are ubiquitously found at the cell surface and extracellular matrix in all the animal species. This review will focus on the structural characteristics of the heparan sulfate proteoglycans related to protein interactions leading to cell signaling. The heparan sulfate chains due to their vast structural diversity are able to bind and interact with a wide variety of proteins, such as growth factors, chemokines, morphogens, extracellular matrix components, enzymes, among others. There is a specificity directing the interactions of heparan sulfates and target proteins, regarding both the fine structure of the polysaccharide chain as well precise protein motifs. Heparan sulfates play a role in cellular signaling either as receptor or co-receptor for different ligands, and the activation of downstream pathways is related to phosphorylation of different cytosolic proteins either directly or involving cytoskeleton interactions leading to gene regulation. The role of the heparan sulfate proteoglycans in cellular signaling and endocytic uptake pathways is also discussed.Proteoglicanos de heparam sulfato são encontrados tanto superfície celular quanto na matriz extracelular em todas as espécies animais. Esta revisão tem enfoque nas características estruturais dos proteoglicanos de heparam sulfato e nas interações destes proteoglicanos com proteínas que levam à sinalização celular. As cadeias de heparam sulfato, devido a sua variedade estrutural, são capazes de se ligar e interagir com ampla gama de proteínas, como fatores de crescimento, quimiocinas, morfógenos, componentes da matriz extracelular, enzimas, entreoutros. Existe uma especificidade estrutural que direciona as interações dos heparam sulfatos e proteínas alvo. Esta especificidade está relacionada com a estrutura da cadeia do polissacarídeo e os motivos conservados da cadeia polipeptídica das proteínas envolvidas nesta interação. Os heparam

  10. Heparan sulfate proteoglycans made by different basement-membrane-producing tumors have immunological and structural similarities

    DEFF Research Database (Denmark)

    Wewer, U M; Albrechtsen, R; Hassell, J R

    1985-01-01

    Using immunological assays, we determined the relationship between the heparan sulfate proteoglycans produced by two different murine basement-membrane-producing tumors, i.e., the mouse Engelbreth-Holm-Swarm (EHS) tumor and the L2 rat yolk-sac tumor. Antibodies prepared against the heparan sulfat...... an immunologically and structurally similar type of high-molecular-weight heparan sulfate proteoglycan which subsequently becomes incorporated into basement-membrane-like material....

  11. Nucleolin is a nuclear target of heparan sulfate derived from glypican-1

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Fang [Department of Experimental Medical Science, Division of Neuroscience, Glycobiology Group, Lund University, Biomedical Center A13, SE-221 84 Lund (Sweden); Belting, Mattias [Department of Clinical Sciences, Section of Oncology and Pathology, Lund University, Lund (Sweden); Fransson, Lars-Åke [Department of Experimental Medical Science, Division of Neuroscience, Glycobiology Group, Lund University, Biomedical Center A13, SE-221 84 Lund (Sweden); Mani, Katrin, E-mail: katrin.mani@med.lu.se [Department of Experimental Medical Science, Division of Neuroscience, Glycobiology Group, Lund University, Biomedical Center A13, SE-221 84 Lund (Sweden)

    2017-05-01

    The recycling, S-nitrosylated heparan sulfate (HS) proteoglycan glypican-1 releases anhydromannose (anMan)-containing HS chains by a nitrosothiol-catalyzed cleavage in endosomes that can be constitutive or induced by ascorbate. The HS-anMan chains are then transported to the nucleus. A specific nuclear target for HS-anMan has not been identified. We have monitored endosome-to-nucleus trafficking of HS-anMan by deconvolution and confocal immunofluorescence microscopy using an anMan-specific monoclonal antibody in non-growing, ascorbate-treated, and growing, untreated, wild-type mouse embryonic fibroblasts and hypoxia-exposed Alzheimer mouse Tg2576 fibroblasts and human U87 glioblastoma cells. In all cells, nuclear HS-anMan targeted a limited number of sites of variable size where it colocalized with DNA and nucleolin, an established marker for nucleoli. HS-anMan also colocalized with ethynyl uridine-tagged nascent RNA and two acetylated forms of histone H3. Acute hypoxia increased the formation of HS-anMan in both Tg2576 and U87 cells. A portion of HS-anMan colocalized with nucleolin at small discrete sites, while most of the nucleolin and nascent RNA was dispersed. In U87 cells, HS-anMan, nucleolin and nascent RNA reassembled after prolonged hypoxia. Nucleolar HS may modulate synthesis and/or release of rRNA.

  12. Basement membrane heparan sulfate proteoglycan from the L2 rat yolk sac carcinoma

    DEFF Research Database (Denmark)

    Fenger, M; Wewer, U; Albrechtsen, R

    1984-01-01

    Heparan sulfate proteoglycan from the L2 rat yolk sac carcinoma has been purified and partially characterized. The proteoglycan has an apparent Mr of 750 000, 35% of which represents the core protein. The core protein seems to be homogeneous, whereas the heparan sulfate chains are heterogeneous...... with an Mr of about 50 000-70 000, with 30% of the glucosamine being N-sulfated. Antibodies raised against the core protein of the heparan sulfate proteoglycan reacted with basement membranes of various rat and human tissue....

  13. Heparan sulfate proteoglycan-dependent neutrophil chemotaxis toward PR-39 cathelicidin

    Directory of Open Access Journals (Sweden)

    Patsch Josef R

    2006-11-01

    Full Text Available Abstract Cathelicidins are mammalian proteins containing a C-terminal cationic antimicrobial domain. Porcine PR-39 cathelicidin affects leukocyte biology. Mechanisms of action may involve alteration of heparan sulfate proteoglycan-dependent functions in inflammatory cells. It was tested whether PR-39 affects human neutrophil migration and if such effects involve heparan sulphate proteoglycans. Neutrophils were from forearm venous blood of healthy donors. Migration was tested in modified Boyden chamber assays. Involvement of heparan sulfate proteoglycans was tested by their chemical modification and by the use of specific antibodies. PR-39 induced migration in neutrophils in a concentration dependent manner. Modification of heparan sulfate proteoglycans with sodium chlorate inhibited migration whereas chemotaxis toward the chemoattractant formyl-Met-Leu-Phe was not affected. Removal of heparan sulfates or chondroitin sulfates from the surface of neutrophils by heparinase or chondroitinase inhibited migration toward PR-39. In conclusion, antimicrobial PR-39 stimulates human neutrophil chemotaxis in a heparan sulfate proteoglycan-dependent manner. Involvment of syndecans is likely as both heparinase and chondroitinase were abrogating. Data suggest active participation of heparan sulfate proteoglycans of neutrophils in cathelicidin peptide-mediated regulation of the antimicrobial host defense.

  14. The heparan sulfate-specific epitope 10E4 is NO-sensitive and partly inaccessible in glypican-1.

    NARCIS (Netherlands)

    Mani, K; Cheng, F; Sandgren, S; Born, van den J.; Havsmark, B; Ding, K; Fransson, LA

    2004-01-01

    The monoclonal antibody 10E4, which recognizes an epitope supposed to contain N-unsubstituted glucosamine, is commonly used to trace heparan sulfate proteoglycans. It has not been fully clarified if the N-unsubstituted glucosamine is required for antibody recognition and if all heparan sulfates

  15. Isolation of a Heparan Sulfate-Containing Proteoglycan from Basement Membrane

    Science.gov (United States)

    Hassell, John R.; Gehron Robey, Pamela; Barrach, Hans-Jurgen; Wilczek, Joseph; Rennard, Stephen I.; Martin, George R.

    1980-08-01

    We have isolated a unique, basement membrane proteoglycan from the Engelbreth-Holm-Swarm (EHS) sarcoma. This proteoglycan, estimated to be 0.75× 106 daltons, was found to contain about equal amounts of protein and covalently linked heparan sulfate. Antibody prepared against this proteoglycan reacts with the basement membrane matrix in the tumor and with the basement membranes in skin, kidney, and cornea. These studies indicate that the heparan sulfate proteoglycan is a normal constituent of basement membranes that presumably plays an important role in the organization of basement membrane components and that also may determine the permeability of basement membranes to acidic molecules.

  16. Exploiting Heparan Sulfate Proteoglycans in Human Neurogenesis—Controlling Lineage Specification and Fate

    Directory of Open Access Journals (Sweden)

    Chieh Yu

    2017-10-01

    Full Text Available Unspecialized, self-renewing stem cells have extraordinary application to regenerative medicine due to their multilineage differentiation potential. Stem cell therapies through replenishing damaged or lost cells in the injured area is an attractive treatment of brain trauma and neurodegenerative neurological disorders. Several stem cell types have neurogenic potential including neural stem cells (NSCs, embryonic stem cells (ESCs, induced pluripotent stem cells (iPSCs, and mesenchymal stem cells (MSCs. Currently, effective use of these cells is limited by our lack of understanding and ability to direct lineage commitment and differentiation of neural lineages. Heparan sulfate proteoglycans (HSPGs are ubiquitous proteins within the stem cell microenvironment or niche and are found localized on the cell surface and in the extracellular matrix (ECM, where they interact with numerous signaling molecules. The glycosaminoglycan (GAG chains carried by HSPGs are heterogeneous carbohydrates comprised of repeating disaccharides with specific sulfation patterns that govern ligand interactions to numerous factors including the fibroblast growth factors (FGFs and wingless-type MMTV integration site family (Wnts. As such, HSPGs are plausible targets for guiding and controlling neural stem cell lineage fate. In this review, we provide an overview of HSPG family members syndecans and glypicans, and perlecan and their role in neurogenesis. We summarize the structural changes and subsequent functional implications of heparan sulfate as cells undergo neural lineage differentiation as well as outline the role of HSPG core protein expression throughout mammalian neural development and their function as cell receptors and co-receptors. Finally, we highlight suitable biomimetic approaches for exploiting the role of HSPGs in mammalian neurogenesis to control and tailor cell differentiation into specific lineages. An improved ability to control stem cell specific neural

  17. Heparan sulfate proteoglycans on the cell surface: versatile coordinators of cellular functions

    DEFF Research Database (Denmark)

    Tumova, S; Woods, A; Couchman, J R

    2000-01-01

    Heparan sulfate proteoglycans are complex molecules composed of a core protein with covalently attached glycosaminoglycan chains. While the protein part determines localization of the proteoglycan on the cell surfaces or in the extracellular matrix, the glycosaminoglycan component, heparan sulfate......, mediates interactions with a variety of extracellular ligands such as growth factors and adhesion molecules. Through these interactions, heparan sulfate proteoglycans participate in many events during cell adhesion, migration, proliferation and differentiation. We are determining the multitude...... of proteoglycan functions, as their intricate roles in many pathways are revealed. They act as coreceptors for growth factors, participate in signalling during cell adhesion, modulate the activity of a broad range of molecules, and partake in many developmental and pathological processes, including tumorigenesis...

  18. Renal heparan sulfate proteoglycans modulate fibroblast growth factor 2 signaling in experimental chronic transplant dysfunction.

    Science.gov (United States)

    Katta, Kirankumar; Boersema, Miriam; Adepu, Saritha; Rienstra, Heleen; Celie, Johanna W A M; Mencke, Rik; Molema, Grietje; van Goor, Harry; Berden, Jo H M; Navis, Gerjan; Hillebrands, Jan-Luuk; van den Born, Jacob

    2013-11-01

    Depending on the glycan structure, proteoglycans can act as coreceptors for growth factors. We hypothesized that proteoglycans and their growth factor ligands orchestrate tissue remodeling in chronic transplant dysfunction. We have previously shown perlecan to be selectively up-regulated in the glomeruli and arteries in a rat renal transplantation model. Using the same model, here we present quantitative RT-PCR profiling data on proteoglycans and growth factors from laser-microdissected glomeruli, arterial tunicae mediae, and neointimae at 12 weeks after transplantation. In glomeruli and neointimae of allografts, selective induction of the matrix heparan sulfate proteoglycan perlecan was observed, along with massive accumulation of fibroblast growth factor 2 (FGF2). Profiling the heparan sulfate polysaccharide side chains revealed conversion from a non-FGF2-binding heparan sulfate phenotype in control and isografted kidneys toward a FGF2-binding phenotype in allografts. In vitro experiments with perlecan-positive rat mesangial cells showed that FGF2-induced proliferation is dependent on sulfation and can be inhibited by exogenously added heparan sulfate. These findings indicate that matrix proteoglycans such as perlecan serve as functional docking platforms for FGF2 in chronic transplant dysfunction. We speculate that heparin-like glycomimetics could be a promising intervention to retard development of glomerulosclerosis and neointima formation in chronic transplant dysfunction. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  19. Mammalian tissue distribution of a large heparan sulfate proteoglycan detected by monoclonal antibodies

    DEFF Research Database (Denmark)

    Couchman, J R; Ljubimov, A V

    1989-01-01

    A panel of nine monoclonal antibodies has been characterized, all of which have reactivity with the core protein of a large heparan sulfate proteoglycan derived from the murine EHS tumor matrix. These rat monoclonal antibodies stained mouse basement membranes intensely, including those of all...... muscle, endothelia, peripheral nerve fibers and epithelia so far examined. In addition, two of the monoclonal antibodies show cross-species reactivity, staining bovine and human basement membranes, and immunoprecipitating proteoglycans from human endothelial cell cultures. These antibodies do not......, however, cross-react with avian tissues. These results show the ubiquitous distribution of a heparan sulfate proteoglycan in mammalian tissues, which will be useful in vitro and in vivo for studies on the biology of basement membrane proteoglycans and investigations of possible roles of these molecules...

  20. Heterogeneous distribution of a basement membrane heparan sulfate proteoglycan in rat tissues

    DEFF Research Database (Denmark)

    Couchman, J R

    1987-01-01

    A heparan sulfate proteoglycan (HSPG) synthesized by murine parietal yolk sac (PYS-2) cells has been characterized and purified from culture supernatants. A monospecific polyclonal antiserum was raised against it which showed activity against the HSPG core protein and basement membrane specificity...... HSPG from the murine Engelbreth-Holm swarm tumor. It was, however, confirmed that only a single population of antibodies was present in the serum. Despite the presence of similar epitopes on these two proteoglycans of different hydrodynamic properties, it was apparent that the PYS-2 HSPG represents...... a basement membrane proteoglycan of distinct properties reflected in its restricted distribution in vivo....

  1. Pathogenesis of diabetic vascular disease: evidence for the role of reduced heparan sulfate proteoglycan

    DEFF Research Database (Denmark)

    Jensen, Tonny Joran

    1997-01-01

    Insulin-dependent diabetic patients with increased urinary albumin excretion are characterized by elevated blood pressure and declining kidney function. In addition, such patients have a high risk of atherosclerotic vascular disease, proliferative retinopathy, and cardiomyopathy, suggesting...... that albuminuria is a marker of widespread vascular dysfunction. Increased transport of macromolecules across the vascular wall, elevated plasma levels of von Willebrand factor, and impaired fibrinolytic capacity have been demonstrated in albuminuric patients. The cause of this vascular vulnerability...... in susceptible patients is unknown, but increasing evidence has suggested that loss of the proteoglycan heparan sulfate in the vasculature may explain the widespread nature of the disease. Heparan sulfate is important for the glomerular endothelial cell and basement membrane charge densities, the anticoagulant...

  2. Syndecan 4 heparan sulfate proteoglycan is a selectively enriched and widespread focal adhesion component

    DEFF Research Database (Denmark)

    Woods, A; Couchman, J R

    1994-01-01

    Focal adhesion formation in fibroblasts results from complex transmembrane signaling processes initiated by extracellular matrix molecules. Although a role for integrins with attendant tyrosine kinases has been established, there is evidence that cell surface heparan sulfate proteoglycans (HSPGs......) are also involved with an associated role of protein kinase C. The identity of the proteoglycan has remained elusive, but we now report that syndecan 4 (ryudocan/amphiglycan) is present in focal adhesions of a number of cell types. Affinity-purified antibodies raised against a unique portion...

  3. Adhesion defective BHK cell mutant has cell surface heparan sulfate proteoglycan of altered properties

    DEFF Research Database (Denmark)

    Couchman, J R; Austria, R; Woods, A

    1988-01-01

    In the light of accumulating data that implicate cell surface heparan sulfate proteoglycans (HSPGs) with a role in cell interactions with extracellular matrix molecules such as fibronectin, we have compared the properties of these molecules in wild-type BHK cells and an adhesion-defective ricin......-resistant mutant (RicR14). Our results showed that the mutant, unlike BHK cells, cannot form focal adhesions when adherent to planar substrates in the presence of serum. Furthermore, while both cell lines possess similar amounts of cell surface HSPG with hydrophobic properties, that of RicR14 cells had decreased...... sulfation, reduced affinity for fibronectin and decreased half-life on the cell surface when compared to the normal counterpart. Our conclusions based on this data are that these altered properties may, in part, account for the adhesion defect in the ricin-resistant mutant. Whether this results from...

  4. Immunohistochemical localization of chondroitin sulfate, chondroitin sulfate proteoglycan, heparan sulfate proteoglycan, entactin, and laminin in basement membranes of postnatal developing and adult rat lungs

    DEFF Research Database (Denmark)

    Sannes, P L; Burch, K K; Khosla, J

    1993-01-01

    Histologic preparations of lungs from 1-, 5-, 10-, 18-, and 25-day-old postnatal and adult rats were examined immunohistochemically with antibodies specific against chondroitin sulfate (CS), basement membrane chondroitin sulfate proteoglycan (BM-CSPG), heparan sulfate proteoglycan (HSPG), entactin...

  5. Changes in cardiac heparan sulfate proteoglycan expression and streptozotocin-induced diastolic dysfunction in rats

    Directory of Open Access Journals (Sweden)

    Cestari Ismar N

    2011-04-01

    Full Text Available Abstract Background Changes in the proteoglycans glypican and syndecan-4 have been reported in several pathological conditions, but little is known about their expression in the heart during diabetes. The aim of this study was to investigate in vivo heart function changes and alterations in mRNA expression and protein levels of glypican-1 and syndecan-4 in cardiac and skeletal muscles during streptozotocin (STZ-induced diabetes. Methods Diabetes was induced in male Wistar rats by STZ administration. The rats were assigned to one of the following groups: control (sham injection, after 24 hours, 10 days, or 30 days of STZ administration. Echocardiography was performed in the control and STZ 10-day groups. Western and Northern blots were used to quantify protein and mRNA levels in all groups. Immunohistochemistry was performed in the control and 30-day groups to correlate the observed mRNA changes to the protein expression. Results In vivo cardiac functional analysis performed using echocardiography in the 10-day group showed diastolic dysfunction with alterations in the peak velocity of early (E diastolic filling and isovolumic relaxation time (IVRT indices. These functional alterations observed in the STZ 10-day group correlated with the concomitant increase in syndecan-4 and glypican-1 protein expression. Cardiac glypican-1 mRNA and skeletal syndecan-4 mRNA and protein levels increased in the STZ 30-day group. On the other hand, the amount of glypican in skeletal muscle was lower than that in the control group. The same results were obtained from immunohistochemistry analysis. Conclusion Our data suggest that membrane proteoglycans participate in the sequence of events triggered by diabetes and inflicted on cardiac and skeletal muscles.

  6. ScFv Anti-Heparan Sulfate Antibodies Unexpectedly Activate Endothelial and Cancer Cells through p38 MAPK: Implications for Antibody-Based Targeting of Heparan Sulfate Proteoglycans in Cancer

    NARCIS (Netherlands)

    Christianson, H.C.; Kuppevelt, A.H. van; Belting, M.

    2012-01-01

    Tumor development requires angiogenesis and anti-angiogenic therapies have been introduced in the treatment of cancer. In this context, heparan sulfate proteoglycans (HSPGs) emerge as interesting targets, owing to their function as co-receptors of major, pro-angiogenic factors. Accordingly, previous

  7. Nerve injury induces the expression of syndecan-1 heparan sulfate proteoglycan in primary sensory neurons.

    Science.gov (United States)

    Murakami, K; Tanaka, T; Bando, Y; Yoshida, S

    2015-08-06

    Heparan sulfate proteoglycans (HSPGs) have important functions in development of the central nervous system; however, their functions in nerve injury are not yet fully understood. We previously reported the expression of syndecan-1, a type of HSPG, in cranial motor neurons after nerve injury, suggesting the importance of syndecan-1 in the pathology of motor nerve injury. In this study, we examined the expression of syndecan-1, a type of HSPG, in primary sensory neurons after nerve injury in mice. Sciatic nerve axotomy strongly induced the expression of syndecan-1 in a subpopulation of injured dorsal root ganglion (DRG) neurons, which were small in size and had CGRP- or isolectin B4-positive fibers. Syndecan-1 was also distributed in the dorsal horn of the spinal cord ipsilateral to the axotomy, and located on the membrane of axons in lamina II of the dorsal horn. Not only sciatic nerve axotomy, infraorbital nerve axotomy also induced the expression of syndecan-1 in trigeminal ganglion neurons. Moreover, syndecan-1 knockdown in cultured DRG neurons induced a shorter neurite extension. These results suggest that syndecan-1 expression in injured primary sensory neurons may have functional roles in nerve regeneration and synaptic plasticity, resulting in the development of neuropathic pain. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

  8. Heparanase facilitates cell adhesion and spreading by clustering of cell surface heparan sulfate proteoglycans.

    Directory of Open Access Journals (Sweden)

    Flonia Levy-Adam

    2008-06-01

    Full Text Available Heparanase is a heparan sulfate (HS degrading endoglycosidase participating in extracellular matrix degradation and remodeling. Apart of its well characterized enzymatic activity, heparanase was noted to exert also enzymatic-independent functions. Non-enzymatic activities of heparanase include enhanced adhesion of tumor-derived cells and primary T-cells. Attempting to identify functional domains of heparanase that would serve as targets for drug development, we have identified heparin binding domains of heparanase. A corresponding peptide (residues Lys(158-Asp(171, termed KKDC was demonstrated to physically associate with heparin and HS, and to inhibit heparanase enzymatic activity. We hypothesized that the pro-adhesive properties of heparanase are mediated by its interaction with cell surface HS proteoglycans, and utilized the KKDC peptide to examine this possibility. We provide evidence that the KKDC peptide interacts with cell membrane HS, resulting in clustering of syndecan-1 and syndecan-4. We applied classical analysis of cell morphology, fluorescent and time-lapse microscopy and demonstrated that the KKDC peptide efficiently stimulates the adhesion and spreading of various cell types, mediated by PKC, Src, and the small GTPase Rac1. These results support, and further substantiate the notion that heparanase function is not limited to its enzymatic activity.

  9. Inhibition of SARS Pseudovirus Cell Entry by Lactoferrin Binding to Heparan Sulfate Proteoglycans

    Science.gov (United States)

    Lang, Jianshe; Yang, Ning; Deng, Jiejie; Liu, Kangtai; Yang, Peng; Zhang, Guigen; Jiang, Chengyu

    2011-01-01

    It has been reported that lactoferrin (LF) participates in the host immune response against Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) invasion by enhancing NK cell activity and stimulating neutrophil aggregation and adhesion. We further investigated the role of LF in the entry of SARS pseudovirus into HEK293E/ACE2-Myc cells. Our results reveal that LF inhibits SARS pseudovirus infection in a dose-dependent manner. Further analysis suggested that LF was able to block the binding of spike protein to host cells at 4°C, indicating that LF exerted its inhibitory function at the viral attachment stage. However, LF did not disrupt the interaction of spike protein with angiotensin-converting enzyme 2 (ACE2), the functional receptor of SARS-CoV. Previous studies have shown that LF colocalizes with the widely distributed cell-surface heparan sulfate proteoglycans (HSPGs). Our experiments have also confirmed this conclusion. Treatment of the cells with heparinase or exogenous heparin prevented binding of spike protein to host cells and inhibited SARS pseudovirus infection, demonstrating that HSPGs provide the binding sites for SARS-CoV invasion at the early attachment phase. Taken together, our results suggest that, in addition to ACE2, HSPGs are essential cell-surface molecules involved in SARS-CoV cell entry. LF may play a protective role in host defense against SARS-CoV infection through binding to HSPGs and blocking the preliminary interaction between SARS-CoV and host cells. Our findings may provide further understanding of SARS-CoV pathogenesis and aid in treatment of this deadly disease. PMID:21887302

  10. Role of cellular heparan sulfate proteoglycans in infection of human adenovirus serotype 3 and 35.

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    Sebastian Tuve

    2008-10-01

    Full Text Available Species B human adenoviruses (Ads are increasingly associated with outbreaks of acute respiratory disease in U.S. military personnel and civil population. The initial interaction of Ads with cellular attachment receptors on host cells is via Ad fiber knob protein. Our previous studies showed that one species B Ad receptor is the complement receptor CD46 that is used by serotypes 11, 16, 21, 35, and 50 but not by serotypes 3, 7, and 14. In this study, we attempted to identify yet-unknown species B cellular receptors. For this purpose we used recombinant Ad3 and Ad35 fiber knobs in high-throughput receptor screening methods including mass spectrometry analysis and glycan arrays. Surprisingly, we found that the main interacting surface molecules of Ad3 fiber knob are cellular heparan sulfate proteoglycans (HSPGs. We subsequently found that HSPGs acted as low-affinity co-receptors for Ad3 but did not represent the main receptor of this serotype. Our study also revealed a new CD46-independent infection pathway of Ad35. This Ad35 infection mechanism is mediated by cellular HSPGs. The interaction of Ad35 with HSPGs is not via fiber knob, whereas Ad3 interacts with HSPGs via fiber knob. Both Ad3 and Ad35 interacted specifically with the sulfated regions within HSPGs that have also been implicated in binding physiologic ligands. In conclusion, our findings show that Ad3 and Ad35 directly utilize HSPGs as co-receptors for infection. Our data suggest that adenoviruses evolved to simulate the presence of physiologic HSPG ligands in order to increase infection.

  11. Heparan sulfate proteoglycans of rat embryo fibroblasts. A hydrophobic form may link cytoskeleton and matrix components

    DEFF Research Database (Denmark)

    Woods, A; Couchman, J R; Höök, M

    1985-01-01

    properties in that it showed no affinity for octyl-Sepharose and could not be inserted into liposomes. The other HSPG type had an estimated Mr of 3-5 X 10(5), was retained on octyl-Sepharose, and could be inserted into liposomes. In addition, the cells contained low molecular weight heparan sulfate...

  12. Border patrol: insights into the unique role of perlecan/heparan sulfate proteoglycan 2 at cell and tissue borders.

    Science.gov (United States)

    Farach-Carson, Mary C; Warren, Curtis R; Harrington, Daniel A; Carson, Daniel D

    2014-02-01

    The extracellular matrix proteoglycan (ECM) perlecan, also known as heparan sulfate proteoglycan 2 or HSPG2, is one of the largest (>200 nm) and oldest (>550 M years) extracellular matrix molecules. In vertebrates, perlecan's five-domain structure contains numerous independently folding modules with sequence similarities to other ECM proteins, all connected like cars into one long, diverse complex train following a unique N-terminal domain I decorated with three long glycosaminoglycan chains, and an additional glycosaminoglycan attachment site in the C-terminal domain V. In lower invertebrates, perlecan is not typically a proteoglycan, possessing the majority of the core protein modules, but lacking domain I where the attachment sites for glycosaminoglycan chains are located. This suggests that uniting the heparan sulfate binding growth factor functions of domain I and the core protein functions of the rest of the molecule in domains II-V occurred later in evolution for a new functional purpose. In this review, we surveyed several decades of pertinent literature to ask a fundamental question: Why did nature design this protein uniquely as an extraordinarily long multifunctional proteoglycan with a single promoter regulating expression, rather than separating these functions into individual proteins that could be independently regulated? We arrived at the conclusion that the concentration of perlecan at functional borders separating tissues and tissue layers is an ancient key function of the core protein. The addition of the heparan sulfate chains in domain I likely occurred as an additional means of binding the core protein to other ECM proteins in territorial matrices and basement membranes, and as a means to reserve growth factors in an on-site depot to assist with rapid repair of those borders when compromised, such as would occur during wounding. We propose a function for perlecan that extends its role from that of an extracellular scaffold, as we previously

  13. Intraepithelial expression of perlecan, a basement membrane-type heparan sulfate proteoglycan reflects dysplastic changes of the oral mucosal epithelium.

    Science.gov (United States)

    Ikarashi, Terué; Ida-Yonemochi, Hiroko; Ohshiro, Kazufumi; Cheng, Jun; Saku, Takashi

    2004-02-01

    Intercellular deposition of perlecan, a major heparan sulfate proteoglycan (HSPG) of the basement membrane, is known to result in characteristic stellate reticulum-like structures in ameloblastomas or tooth germs. Although enlargement of the intercellular space is one of the histological characteristics of epithelial dysplasia of oral mucosa, the mode of expression of perlecan is poorly understood in these epithelial lesions. Eighty-two biopsy specimens consisting of normal and hyperplastic epithelium, epithelial dysplasia, and squamous cell carcinomas were examined for both perlecan core protein and heparan sulfate (HS) chains by immunohistochemistry and in situ hybridization. In normal and hyperplastic epithelium, perlecan core protein and HS chains were localized in the cell border of parabasal cells and lower prickle cells, and HS chains were also found in basal cells. With an increase in the severity of epithelial dysplasia, the core protein was heavily and extensively deposited in the interepithelial space as well as in the cytoplasm of epithelial cells from the basal to the surface layers. Its gene expression was confirmed in the cells around the protein deposits. On the other hand, HS chains were enhanced in mild dysplasia, but decreased in moderate and severe dysplasias. In squamous cell carcinomas, either the core protein or HS chains were found scarcely in tumor cells but abundantly in the stromal space. The findings indicate that perlecan is localized in the intercellular space of the oral epithelia, and that it is over-expressed in dysplastic epithelial cells and is deposited in their interepithelial space, which results in the histology of reduction of cellular cohesion.

  14. In vivo turnover of the basement membrane and other heparan sulfate proteoglycans of rat glomerulus

    DEFF Research Database (Denmark)

    Beavan, L A; Davies, M; Couchman, J R

    1989-01-01

    The metabolic turnover of rat glomerular proteoglycans in vivo was investigated. Newly synthesized proteoglycans were labeled during a 7-h period after injecting sodium [35S]sulfate intraperitoneally. At the end of the labeling period a chase dose of sodium sulfate was given. Subsequently at defi...

  15. A targeted glycan-related gene screen reveals heparan sulfate proteoglycan sulfation regulates WNT and BMP trans-synaptic signaling.

    Directory of Open Access Journals (Sweden)

    Neil Dani

    Full Text Available A Drosophila transgenic RNAi screen targeting the glycan genome, including all N/O/GAG-glycan biosynthesis/modification enzymes and glycan-binding lectins, was conducted to discover novel glycan functions in synaptogenesis. As proof-of-product, we characterized functionally paired heparan sulfate (HS 6-O-sulfotransferase (hs6st and sulfatase (sulf1, which bidirectionally control HS proteoglycan (HSPG sulfation. RNAi knockdown of hs6st and sulf1 causes opposite effects on functional synapse development, with decreased (hs6st and increased (sulf1 neurotransmission strength confirmed in null mutants. HSPG co-receptors for WNT and BMP intercellular signaling, Dally-like Protein and Syndecan, are differentially misregulated in the synaptomatrix of these mutants. Consistently, hs6st and sulf1 nulls differentially elevate both WNT (Wingless; Wg and BMP (Glass Bottom Boat; Gbb ligand abundance in the synaptomatrix. Anterograde Wg signaling via Wg receptor dFrizzled2 C-terminus nuclear import and retrograde Gbb signaling via synaptic MAD phosphorylation and nuclear import are differentially activated in hs6st and sulf1 mutants. Consequently, transcriptional control of presynaptic glutamate release machinery and postsynaptic glutamate receptors is bidirectionally altered in hs6st and sulf1 mutants, explaining the bidirectional change in synaptic functional strength. Genetic correction of the altered WNT/BMP signaling restores normal synaptic development in both mutant conditions, proving that altered trans-synaptic signaling causes functional differentiation defects.

  16. A Haploid Genetic Screen Identifies Heparan Sulfate Proteoglycans Supporting Rift Valley Fever Virus Infection.

    Science.gov (United States)

    Riblett, Amber M; Blomen, Vincent A; Jae, Lucas T; Altamura, Louis A; Doms, Robert W; Brummelkamp, Thijn R; Wojcechowskyj, Jason A

    2015-11-18

    Rift Valley fever virus (RVFV) causes recurrent insect-borne epizootics throughout the African continent, and infection of humans can lead to a lethal hemorrhagic fever syndrome. Deep mutagenesis of haploid human cells was used to identify host factors required for RVFV infection. This screen identified a suite of enzymes involved in glycosaminoglycan (GAG) biogenesis and transport, including several components of the cis-oligomeric Golgi (COG) complex, one of the central components of Golgi complex trafficking. In addition, disruption of PTAR1 led to RVFV resistance as well as reduced heparan sulfate surface levels, consistent with recent observations that PTAR1-deficient cells exhibit altered Golgi complex morphology and glycosylation defects. A variety of biochemical and genetic approaches were utilized to show that both pathogenic and attenuated RVFV strains require GAGs for efficient infection on some, but not all, cell types, with the block to infection being at the level of virion attachment. Examination of other members of the Bunyaviridae family for GAG-dependent infection suggested that the interaction with GAGs is not universal among bunyaviruses, indicating that these viruses, as well as RVFV on certain cell types, employ additional unidentified virion attachment factors and/or receptors. Rift Valley fever virus (RVFV) is an emerging pathogen that can cause severe disease in humans and animals. Epizootics among livestock populations lead to high mortality rates and can be economically devastating. Human epidemics of Rift Valley fever, often initiated by contact with infected animals, are characterized by a febrile disease that sometimes leads to encephalitis or hemorrhagic fever. The global burden of the pathogen is increasing because it has recently disseminated beyond Africa, which is of particular concern because the virus can be transmitted by widely distributed mosquito species. There are no FDA-licensed vaccines or antiviral agents with activity

  17. Cell surface heparan sulfate proteoglycans control adhesion and invasion of breast carcinoma cells

    DEFF Research Database (Denmark)

    Lim, Hooi Ching; Multhaupt, Hinke A. B.; Couchman, John R.

    2015-01-01

    phenotype of mammary carcinoma cells. Finally, both syndecan-2 and caveolin-2 were upregulated in tissue arrays from breast cancer patients compared to normal mammary tissue. Moreover their expression levels were correlated in triple negative breast cancers. Conclusion: Cell surface proteoglycans, notably...

  18. Binding of beta-VLDL to heparan sulfate proteoglycans requires lipoprotein lipase, whereas ApoE only modulates binding affinity.

    Science.gov (United States)

    de Beer, F; Hendriks, W L; van Vark, L C; Kamerling, S W; van Dijk, K W; Hofker, M H; Smelt, A H; Havekes, L M

    1999-03-01

    The binding of beta-VLDL to heparan sulfate proteoglycans (HSPG) has been reported to be stimulated by both apoE and lipoprotein lipase (LPL). In the present study we investigated the effect of the isoform and the amount of apoE per particle, as well as the role of LPL on the binding of beta-VLDL to HSPG. Therefore, we isolated beta-VLDL from transgenic mice, expressing either APOE*2(Arg158-->Cys) or APOE*3-Leiden (E2-VLDL and E3Leiden-VLDL, respectively), as well as from apoE-deficient mice containing no apoE at all (Enull-VLDL). In the absence of LPL, the binding affinity and maximal binding capacity of all beta-VLDL samples for HSPG-coated microtiter plates was very low. Addition of LPL to this cell-free system resulted in a 12- to 55-fold increase in the binding affinity and a 7- to 15-fold increase in the maximal binding capacity (Bmax). In the presence of LPL, the association constant (Ka) tended to increase in the order Enull-VLDL

  19. An affinity adsorption media that mimics heparan sulfate proteoglycans for the treatment of drug-resistant bacteremia

    Science.gov (United States)

    McCrea, Keith R.; Ward, Robert S.

    2016-06-01

    Removal of several drug-resistant bacteria from blood by affinity adsorption onto a heparin-functional media is reported. Heparin is a chemical analogue of heparan sulfate (HS) proteoglycans, found on transmembrane proteins of endothelial cells. Many blood-borne human pathogens, including bacteria, viruses, parasites, and fungi have been reported to target HS as an initial step in their pathogenesis. Here, we demonstrate the binding and removal of Methicillin-resistant Staphylococcus aureus (MRSA), Extended-Spectrum Betalactamase Klebsiella pneumoniae (ESBL), and two Carbapenem-resistant Enterobacteriaceae (both CRE Escherichia coli and CRE K. pneumoniae) using 300 μm polyethylene beads surface modified with end-point-attached heparin. Depending on the specific bacteria, the amount removed ranged between 39% (ESBL) and 99.9% (CRE). The total amount of bacteria adsorbed ranged between 2.8 × 105 and 8.6 × 105 colony forming units (CFU) per gram of adsorption media. Based on a polymicrobial challenge which showed no competitive binding, MRSA and CRE apparently utilize different binding sequences on the immobilized heparin ligand. Since the total circulating bacterial load during bacteremia seldom exceeds 5 × 105 CFUs, it appears possible to significantly reduce bacterial concentration in infected patients by multi-pass recirculation of their blood through a small extracorporeal affinity filter containing the heparin-functional adsorption media. This 'dialysis-like therapy' is expected to improve patient outcomes and reduce the cost of care, particularly when there are no anti-infective drugs available to treat the infection.

  20. ScFv anti-heparan sulfate antibodies unexpectedly activate endothelial and cancer cells through p38 MAPK: implications for antibody-based targeting of heparan sulfate proteoglycans in cancer.

    Directory of Open Access Journals (Sweden)

    Helena C Christianson

    Full Text Available Tumor development requires angiogenesis and anti-angiogenic therapies have been introduced in the treatment of cancer. In this context, heparan sulfate proteoglycans (HSPGs emerge as interesting targets, owing to their function as co-receptors of major, pro-angiogenic factors. Accordingly, previous studies have suggested anti-tumor effects of heparin, i.e. over-sulfated HS, and various heparin mimetics; however, a significant drawback is their unspecific mechanism of action and potentially serious side-effects related to their anticoagulant properties. Here, we have explored the use of human ScFv anti-HS antibodies (αHS as a more rational approach to target HSPG function in endothelial cells (ECs. αHS were initially selected for their recognition of HS epitopes localized preferentially to the vasculature of patient glioblastoma tumors, i.e. highly angiogenic brain tumors. Unexpectedly, we found that these αHS exhibited potent pro-angiogenic effects in primary human ECs. αHS were shown to stimulate EC differentiation, which was associated with increased EC tube formation and proliferation. Moreover, αHS supported EC survival under hypoxia and starvation, i.e. conditions typical of the tumor microenvironment. Importantly, αHS-mediated proliferation was efficiently counter-acted by heparin and was absent in HSPG-deficient mutant cells, confirming HS-specific effects. On a mechanistic level, binding of αHS to HSPGs of ECs as well as glioblastoma cells was found to trigger p38 MAPK-dependent signaling resulting in increased proliferation. We conclude that several αHS that recognize HS epitopes abundant in the tumor vasculature may elicit a pro-angiogenic response, which has implications for the development of antibody-based targeting of HSPGs in cancer.

  1. Adeno-Associated Virus Type 12 (AAV12): a Novel AAV Serotype with Sialic Acid- and Heparan Sulfate Proteoglycan-Independent Transduction Activity▿

    Science.gov (United States)

    Schmidt, Michael; Voutetakis, Antonis; Afione, Sandra; Zheng, Changyu; Mandikian, Danielle; Chiorini, John A.

    2008-01-01

    Recombinant adeno-associated virus (rAAV) is a promising vector for gene therapy. Recent isolations of novel AAV serotypes have led to significant advances by broadening the tropism and increasing the efficiency of gene transfer to the desired target cell. However, a major concern that remains is the strong preexisting immune responses to several vectors. In this paper, we describe the isolation and characterization of AAV12, an AAV serotype with unique biological and immunological properties. In contrast to those of all other reported AAVs, AAV12 cell attachment and transduction do not require cell surface sialic acids or heparan sulfate proteoglycans. Furthermore, rAAV12 is resistant to neutralization by circulating antibodies from human serum. The feasibility of rAAV12 as a vector was demonstrated in a mouse model in which muscle and salivary glands were transduced. These characteristics make rAAV12 an interesting candidate for gene transfer applications. PMID:18045941

  2. Heparan sulfate biosynthesis

    DEFF Research Database (Denmark)

    Multhaupt, Hinke A B; Couchman, John R

    2012-01-01

    Heparan sulfate is perhaps the most complex polysaccharide known from animals. The basic repeating disaccharide is extensively modified by sulfation and uronic acid epimerization. Despite this, the fine structure of heparan sulfate is remarkably consistent with a particular cell type. This suggests...... that the synthesis of heparan sulfate is tightly controlled. Although genomics has identified the enzymes involved in glycosaminoglycan synthesis in a number of vertebrates and invertebrates, the regulation of the process is not understood. Moreover, the localization of the various enzymes in the Golgi apparatus has......-quality resolution of the distribution of enzymes. The EXT2 protein, which when combined as heterodimers with EXT1 comprises the major polymerase in heparan sulfate synthesis, has been studied in depth. All the data are consistent with a cis-Golgi distribution and provide a starting point to establish whether all...

  3. Heparan sulfate 6-O-sulfotransferase is essential for muscle development in zebrafish

    OpenAIRE

    Bink, R.J.; Habuchi, H; Lele, Z.; Dolk, E.; Joore, J.; Rauch, G.; Geisler, R.; Wilson, S.W.; Hertog, J. den; Kimata, K.; Zivkovic, D.

    2003-01-01

    Heparan sulfate proteoglycans function in development and disease. They consist of a core protein with attached heparan sulfate chains that are altered by a series of carbohydrate-modifying enzymes and sulfotransferases. Here, we report on the identification and characterization of a gene encoding zebrafish heparan sulfate 6-O-sulfotransferase (hs6st) that shows high homology to other heparan sulfate 6-O-sulfotransferases. When expressed as a fusion protein in cultured cells, the protein show...

  4. Heparan sulfate proteoglycans mediate interstitial flow mechanotransduction regulating MMP-13 expression and cell motility via FAK-ERK in 3D collagen.

    Directory of Open Access Journals (Sweden)

    Zhong-Dong Shi

    2011-01-01

    Full Text Available Interstitial flow directly affects cells that reside in tissues and regulates tissue physiology and pathology by modulating important cellular processes including proliferation, differentiation, and migration. However, the structures that cells utilize to sense interstitial flow in a 3-dimensional (3D environment have not yet been elucidated. Previously, we have shown that interstitial flow upregulates matrix metalloproteinase (MMP expression in rat vascular smooth muscle cells (SMCs and fibroblasts/myofibroblasts via activation of an ERK1/2-c-Jun pathway, which in turn promotes cell migration in collagen. Herein, we focused on uncovering the flow-induced mechanotransduction mechanism in 3D.Cleavage of rat vascular SMC surface glycocalyx heparan sulfate (HS chains from proteoglycan (PG core proteins by heparinase or disruption of HS biosynthesis by silencing N-deacetylase/N-sulfotransferase 1 (NDST1 suppressed interstitial flow-induced ERK1/2 activation, interstitial collagenase (MMP-13 expression, and SMC motility in 3D collagen. Inhibition or knockdown of focal adhesion kinase (FAK also attenuated or blocked flow-induced ERK1/2 activation, MMP-13 expression, and cell motility. Interstitial flow induced FAK phosphorylation at Tyr925, and this activation was blocked when heparan sulfate proteoglycans (HSPGs were disrupted. These data suggest that HSPGs mediate interstitial flow-induced mechanotransduction through FAK-ERK. In addition, we show that integrins are crucial for mechanotransduction through HSPGs as they mediate cell spreading and maintain cytoskeletal rigidity.We propose a conceptual mechanotransduction model wherein cell surface glycocalyx HSPGs, in the presence of integrin-mediated cell-matrix adhesions and cytoskeleton organization, sense interstitial flow and activate the FAK-ERK signaling axis, leading to upregulation of MMP expression and cell motility in 3D. This is the first study to describe a flow-induced mechanotransduction

  5. Single Particle Tracking Confirms That Multivalent Tat Protein Transduction Domain-induced Heparan Sulfate Proteoglycan Cross-linkage Activates Rac1 for Internalization*

    Science.gov (United States)

    Imamura, Junji; Suzuki, Yasuhiro; Gonda, Kohsuke; Roy, Chandra Nath; Gatanaga, Hiroyuki; Ohuchi, Noriaki; Higuchi, Hideo

    2011-01-01

    The mechanism by which HIV-1-Tat protein transduction domain (TatP) enters the cell remains unclear because of an insufficient understanding of the initial kinetics of peptide entry. Here, we report the successful visualization and tracking of TatP molecular kinetics on the cell surface with 7-nm spatial precision using quantum dots. Strong cell binding was only observed with a TatP valence of ≥8, whereas monovalent TatP binding was negligible. The requirement of the cell-surface heparan sulfate (HS) chains of HS proteoglycans (HSPGs) for TatP binding and intracellular transport was demonstrated by the enzymatic removal of HS and simultaneous observation of two individual particles. Multivalent TatP induces HSPG cross-linking, recruiting activated Rac1 to adjacent lipid rafts and thereby enhancing the recruitment of TatP/HSPG to actin-associated microdomains and its internalization by macropinocytosis. These findings clarify the initial binding mechanism of TatP to the cell surface and demonstrate the importance of TatP valence for strong surface binding and signal transduction. Our data also shed light on the ability of TatP to exploit the machinery of living cells, using HSPG signaling to activate Rac1 and alter TatP mobility and internalization. This work should guide the future design of TatP-based peptides as therapeutic nanocarriers with efficient transduction. PMID:21199870

  6. Single particle tracking confirms that multivalent Tat protein transduction domain-induced heparan sulfate proteoglycan cross-linkage activates Rac1 for internalization.

    Science.gov (United States)

    Imamura, Junji; Suzuki, Yasuhiro; Gonda, Kohsuke; Roy, Chandra Nath; Gatanaga, Hiroyuki; Ohuchi, Noriaki; Higuchi, Hideo

    2011-03-25

    The mechanism by which HIV-1-Tat protein transduction domain (TatP) enters the cell remains unclear because of an insufficient understanding of the initial kinetics of peptide entry. Here, we report the successful visualization and tracking of TatP molecular kinetics on the cell surface with 7-nm spatial precision using quantum dots. Strong cell binding was only observed with a TatP valence of ≥8, whereas monovalent TatP binding was negligible. The requirement of the cell-surface heparan sulfate (HS) chains of HS proteoglycans (HSPGs) for TatP binding and intracellular transport was demonstrated by the enzymatic removal of HS and simultaneous observation of two individual particles. Multivalent TatP induces HSPG cross-linking, recruiting activated Rac1 to adjacent lipid rafts and thereby enhancing the recruitment of TatP/HSPG to actin-associated microdomains and its internalization by macropinocytosis. These findings clarify the initial binding mechanism of TatP to the cell surface and demonstrate the importance of TatP valence for strong surface binding and signal transduction. Our data also shed light on the ability of TatP to exploit the machinery of living cells, using HSPG signaling to activate Rac1 and alter TatP mobility and internalization. This work should guide the future design of TatP-based peptides as therapeutic nanocarriers with efficient transduction.

  7. Shear stress modulation of smooth muscle cell marker genes in 2-D and 3-D depends on mechanotransduction by heparan sulfate proteoglycans and ERK1/2.

    Directory of Open Access Journals (Sweden)

    Zhong-Dong Shi

    2010-08-01

    Full Text Available During vascular injury, vascular smooth muscle cells (SMCs and fibroblasts/myofibroblasts (FBs/MFBs are exposed to altered luminal blood flow or transmural interstitial flow. We investigate the effects of these two types of fluid flows on the phenotypes of SMCs and MFBs and the underlying mechanotransduction mechanisms.Exposure to 8 dyn/cm(2 laminar flow shear stress (2-dimensional, 2-D for 15 h significantly reduced expression of alpha-smooth muscle actin (alpha-SMA, smooth muscle protein 22 (SM22, SM myosin heavy chain (SM-MHC, smoothelin, and calponin. Cells suspended in collagen gels were exposed to interstitial flow (1 cmH(2O, approximately 0.05 dyn/cm(2, 3-D, and after 6 h of exposure, expression of SM-MHC, smoothelin, and calponin were significantly reduced, while expression of alpha-SMA and SM22 were markedly enhanced. PD98059 (an ERK1/2 inhibitor and heparinase III (an enzyme to cleave heparan sulfate significantly blocked the effects of laminar flow on gene expression, and also reversed the effects of interstitial flow on SM-MHC, smoothelin, and calponin, but enhanced interstitial flow-induced expression of alpha-SMA and SM22. SMCs and MFBs have similar responses to fluid flow. Silencing ERK1/2 completely blocked the effects of both laminar flow and interstitial flow on SMC marker gene expression. Western blotting showed that both types of flows induced ERK1/2 activation that was inhibited by disruption of heparan sulfate proteoglycans (HSPGs.The results suggest that HSPG-mediated ERK1/2 activation is an important mechanotransduction pathway modulating SMC marker gene expression when SMCs and MFBs are exposed to flow. Fluid flow may be involved in vascular remodeling and lesion formation by affecting phenotypes of vascular wall cells. This study has implications in understanding the flow-related mechanobiology in vascular lesion formation, tumor cell invasion, and stem cell differentiation.

  8. Microglial Heparan Sulfate Proteoglycans Facilitate the Cluster-of-Differentiation 14 (CD14)/Toll-like Receptor 4 (TLR4)-Dependent Inflammatory Response*

    Science.gov (United States)

    O'Callaghan, Paul; Li, Jin-Ping; Lannfelt, Lars; Lindahl, Ulf; Zhang, Xiao

    2015-01-01

    Microglia rapidly mount an inflammatory response to pathogens in the central nervous system (CNS). Heparan sulfate proteoglycans (HSPGs) have been attributed various roles in inflammation. To elucidate the relevance of microglial HSPGs in a pro-inflammatory response we isolated microglia from mice overexpressing heparanase (Hpa-tg), the HS-degrading endoglucuronidase, and challenged them with lipopolysaccharide (LPS), a bacterial endotoxin. Prior to LPS-stimulation, the LPS-receptor cluster-of-differentiation 14 (CD14) and Toll-like receptor 4 (TLR4; essential for the LPS response) were similarly expressed in Ctrl and Hpa-tg microglia. However, compared with Ctrl microglia, Hpa-tg cells released significantly less tumor necrosis factor-α (TNFα), essentially failed to up-regulate interleukin-1β (IL1β) and did not initiate synthesis of proCD14. Isolated primary astroyctes expressed TLR4, but notably lacked CD14 and in contrast to microglia, LPS challenge induced a similar TNFα response in Ctrl and Hpa-tg astrocytes, while neither released IL1β. The astrocyte TNFα-induction was thus attributed to CD14-independent TLR4 activation and was unaffected by the cells HS status. Equally, the suppressed LPS-response in Hpa-tg microglia indicated a loss of CD14-dependent TLR4 activation, suggesting that microglial HSPGs facilitate this process. Indeed, confocal microscopy confirmed interactions between microglial HS and CD14 in LPS-stimulated microglia and a potential HS-binding motif in CD14 was identified. We conclude that microglial HSPGs facilitate CD14-dependent TLR4 activation and that heparanase can modulate this mechanism. PMID:25869127

  9. Real time monitoring of the effects of Heparan Sulfate Proteoglycan (HSPG) and surface charge on the cell adhesion process using thickness shear mode (TSM) sensor.

    Science.gov (United States)

    Ergezen, E; Hong, S; Barbee, K A; Lec, R

    2007-04-15

    The effects of Heparan Sulfate Proteoglycan (HSPG) and surface charge on the cellular interactions of the cell membrane with different substrates to determine the kinetics of cell adhesion was studied using thickness shear mode (TSM) sensor. The TSM sensor was operated at its first, third, fifth and seventh harmonics. Since the penetration depth of the shear wave decreases with increases in frequency, the multi-resonance operation of the TSM sensor was used to monitor the changes in the kinetics of the cell-substrate interaction at different distances from the sensor surface. During the sedimentation and the initial attachment of the cells on the sensor surface, the changes in the sensor resonant frequency and the magnitude response were monitored. First, HSPGs were partially digested with the enzyme Heparinase III to evaluate the effect of HSPG on the cell adhesion process. The results indicated that HSPG did not have any effect on the kinetics of the initial attachment, but it did reduce the strength of steady-state cell adhesion. Next, we investigated the effect of the electrostatic interactions of the cell membrane with the substrate on the cell adhesion. In this case, the sensor surface was coated with positively charged Poly-D-Lysine (PDL). It was observed that electrostatic interaction of the negatively charged cell membrane with the PDL surface promoted the initial cell adhesion but did not support long-term cell adhesion. The multi-resonant TSM technique was shown to be a very promising method for monitoring specific interfacial effects involving in cell adhesion process in real-time.

  10. Modulators of axonal growth and guidance at the brain midline with special reference to glial heparan sulfate proteoglycans

    Directory of Open Access Journals (Sweden)

    CAVALCANTE LENY A.

    2002-01-01

    Full Text Available Bilaterally symmetric organisms need to exchange information between the left and right sides of their bodies to integrate sensory input and to coordinate motor control. Thus, an important choice point for developing axons is the Central Nervous System (CNS midline. Crossing of this choice point is influenced by highly conserved, soluble or membrane-bound molecules such as the L1 subfamily, laminin, netrins, slits, semaphorins, Eph-receptors and ephrins, etc. Furthermore, there is much circumstantial evidence for a role of proteoglycans (PGs or their glycosaminoglycan (GAG moieties on axonal growth and guidance, most of which was derived from simplified models. A model of intermediate complexity is that of cocultures of young neurons and astroglial carpets (confluent cultures obtained from medial and lateral sectors of the embryonic rodent midbrain soon after formation of its commissures. Neurite production in these cocultures reveals that, irrespective of the previous location of neurons in the midbrain, medial astrocytes exerted an inhibitory or non-permissive effect on neuritic growth that was correlated to a higher content of both heparan and chondroitin sulfates (HS and CS. Treatment with GAG lyases shows minor effects of CS and discloses a major inhibitory or non-permissive role for HS. The results are discussed in terms of available knowledge on the binding of HSPGs to interative proteins and underscore the importance of understanding glial polysaccharide arrays in addition to its protein complement for a better understanding of neuron-glial interactions.

  11. Heparan sulfate 6-O-Sulfotransferase is essential for muscle development in zebrafish

    NARCIS (Netherlands)

    Bink, R.J.; Habuchi, H.; Lele, Z.; Dolk, E.; Joore, J.; Rauch, G.; Geisler, R.; Wilson, S.W.; Hertog, J. den; Kimata, K.; Zivkovic, D.

    2003-01-01

    Heparan sulfate proteoglycans function in development and disease. They consist of a core protein with attached heparan sulfate chains that are altered by a series of carbohydrate-modifying enzymes and sulfotransferases. Here, we report on the identification and characterization of a gene

  12. Glypican Is a Modulator of Netrin-Mediated Axon Guidance.

    Directory of Open Access Journals (Sweden)

    Cassandra R Blanchette

    2015-07-01

    Full Text Available Netrin is a key axon guidance cue that orients axon growth during neural circuit formation. However, the mechanisms regulating netrin and its receptors in the extracellular milieu are largely unknown. Here we demonstrate that in Caenorhabditis elegans, LON-2/glypican, a heparan sulfate proteoglycan, modulates UNC-6/netrin signaling and may do this through interactions with the UNC-40/DCC receptor. We show that developing axons misorient in the absence of LON-2/glypican when the SLT-1/slit guidance pathway is compromised and that LON-2/glypican functions in both the attractive and repulsive UNC-6/netrin pathways. We find that the core LON-2/glypican protein, lacking its heparan sulfate chains, and secreted forms of LON-2/glypican are functional in axon guidance. We also find that LON-2/glypican functions from the epidermal substrate cells to guide axons, and we provide evidence that LON-2/glypican associates with UNC-40/DCC receptor-expressing cells. We propose that LON-2/glypican acts as a modulator of UNC-40/DCC-mediated guidance to fine-tune axonal responses to UNC-6/netrin signals during migration.

  13. Glypican Is a Modulator of Netrin-Mediated Axon Guidance.

    Science.gov (United States)

    Blanchette, Cassandra R; Perrat, Paola N; Thackeray, Andrea; Bénard, Claire Y

    2015-07-01

    Netrin is a key axon guidance cue that orients axon growth during neural circuit formation. However, the mechanisms regulating netrin and its receptors in the extracellular milieu are largely unknown. Here we demonstrate that in Caenorhabditis elegans, LON-2/glypican, a heparan sulfate proteoglycan, modulates UNC-6/netrin signaling and may do this through interactions with the UNC-40/DCC receptor. We show that developing axons misorient in the absence of LON-2/glypican when the SLT-1/slit guidance pathway is compromised and that LON-2/glypican functions in both the attractive and repulsive UNC-6/netrin pathways. We find that the core LON-2/glypican protein, lacking its heparan sulfate chains, and secreted forms of LON-2/glypican are functional in axon guidance. We also find that LON-2/glypican functions from the epidermal substrate cells to guide axons, and we provide evidence that LON-2/glypican associates with UNC-40/DCC receptor-expressing cells. We propose that LON-2/glypican acts as a modulator of UNC-40/DCC-mediated guidance to fine-tune axonal responses to UNC-6/netrin signals during migration.

  14. Chondroitin 6-sulfate proteoglycan but not heparan sulfate proteoglycan is abnormally expressed in skin basement membrane from patients with dominant and recessive dystrophic epidermolysis bullosa

    DEFF Research Database (Denmark)

    Fine, J D; Couchman, J R

    1989-01-01

    or more forms of epidermolysis bullosa (EB), a disease known to have specific alterations in skin BM, we have examined by indirect immunofluorescence 31 specimens of clinically normal skin from 28 EB patients (simplex, 5; junctional, 8; dominant dystrophic [DDEB], 9; recessive dystrophic [RDEB], 9...... in human skin, and that its absent or reduced binding in dystrophic EB skin BM may reflect either absence of associated core protein or posttranslational alterations in the proteoglycan side chains....

  15. Heparan sulfate and cell division

    Directory of Open Access Journals (Sweden)

    Porcionatto M.A.

    1999-01-01

    Full Text Available Heparan sulfate is a component of vertebrate and invertebrate tissues which appears during the cytodifferentiation stage of embryonic development. Its structure varies according to the tissue and species of origin and is modified during neoplastic transformation. Several lines of experimental evidence suggest that heparan sulfate plays a role in cellular recognition, cellular adhesion and growth control. Heparan sulfate can participate in the process of cell division in two distinct ways, either as a positive or negative modulator of cellular proliferation, or as a response to a mitogenic stimulus.

  16. On the roles and regulation of chondroitin sulfate and heparan sulfate in zebrafish pharyngeal cartilage morphogenesis

    DEFF Research Database (Denmark)

    Holmborn, Katarina; Habicher, Judith; Kasza, Zsolt

    2012-01-01

    The present study addresses the roles of heparan sulfate (HS) proteoglycans and chondroitin sulfate (CS) proteoglycans in the development of zebrafish pharyngeal cartilage structures. uxs1 and b3gat3 mutants, predicted to have impaired biosynthesis of both HS and CS because of defective formation...

  17. Recombinant heparan sulfate for use in tissue engineering applications

    DEFF Research Database (Denmark)

    Whitelock, J.; Ma, J.L.; Davies, N.

    2008-01-01

    Background: Heparan sulfate (HS) is an important component of many extracellular matrices that interacts with mitogens and morphogens to guide and control tissue and organ development. These interactions are controlled by its structure, which varies when produced by different cell types and diffe......Background: Heparan sulfate (HS) is an important component of many extracellular matrices that interacts with mitogens and morphogens to guide and control tissue and organ development. These interactions are controlled by its structure, which varies when produced by different cell types...... in the presence of Medium 199. It was purified as a proteoglycan with a molecular weight between 75 and 150 kDa, which was decorated with HS, chondroitin sulfate (CS) and keratan sulfate (KS) in a similar way to the full-length perlecan from the same cells. Compositional analysis of the glycosaminoglycan (GAG...

  18. Heparan sulfate mediates trastuzumab effect in breast cancer cells

    Science.gov (United States)

    2013-01-01

    Background Trastuzumab is an antibody widely used in the treatment of breast cancer cases that test positive for the human epidermal growth factor receptor 2 (HER2). Many patients, however, become resistant to this antibody, whose resistance has become a major focus in breast cancer research. But despite this interest, there are still no reliable markers that can be used to identify resistant patients. A possible role of several extracellular matrix (ECM) components—heparan sulfate (HS), Syn-1(Syndecan-1) and heparanase (HPSE1)—in light of the influence of ECM alterations on the action of several compounds on the cells and cancer development, was therefore investigated in breast cancer cell resistance to trastuzumab. Methods The cDNA of the enzyme responsible for cleaving HS chains from proteoglycans, HPSE1, was cloned in the pEGFP-N1 plasmid and transfected into a breast cancer cell lineage. We evaluated cell viability after trastuzumab treatment using different breast cancer cell lines. Trastuzumab and HS interaction was investigated by confocal microscopy and Fluorescence Resonance Energy Transfer (FRET). The profile of sulfated glycosaminoglycans was also investigated by [35S]-sulfate incorporation. Quantitative RT-PCR and immunofluorescence were used to evaluate HPSE1, HER2 and Syn-1 mRNA expression. HPSE1 enzymatic activity was performed using biotinylated heparan sulfate. Results Breast cancer cell lines responsive to trastuzumab present higher amounts of HER2, Syn-1 and HS on the cell surface, but lower levels of secreted HS. Trastuzumab and HS interaction was proven by FRET analysis. The addition of anti-HS to the cells or heparin to the culture medium induced resistance to trastuzumab in breast cancer cells previously sensitive to this monoclonal antibody. Breast cancer cells transfected with HPSE1 became resistant to trastuzumab, showing lower levels of HER2, Syn-1 and HS on the cell surface. In addition, HS shedding was increased significantly in

  19. Two distinct sites in sonic Hedgehog combine for heparan sulfate interactions and cell signaling functions

    DEFF Research Database (Denmark)

    Chang, Shu-Chun; Mulloy, Barbara; Magee, Anthony I

    2011-01-01

    Hedgehog (Hh) proteins are morphogens that mediate many developmental processes. Hh signaling is significant for many aspects of embryonic development, whereas dysregulation of this pathway is associated with several types of cancer. Hh proteins require heparan sulfate proteoglycans (HSPGs) for t...

  20. Proteoglycans in liver cancer.

    Science.gov (United States)

    Baghy, Kornélia; Tátrai, Péter; Regős, Eszter; Kovalszky, Ilona

    2016-01-07

    Proteoglycans are a group of molecules that contain at least one glycosaminoglycan chain, such as a heparan, dermatan, chondroitin, or keratan sulfate, covalently attached to the protein core. These molecules are categorized based on their structure, localization, and function, and can be found in the extracellular matrix, on the cell surface, and in the cytoplasm. Cell-surface heparan sulfate proteoglycans, such as syndecans, are the primary type present in healthy liver tissue. However, deterioration of the liver results in overproduction of other proteoglycan types. The purpose of this article is to provide a current summary of the most relevant data implicating proteoglycans in the development and progression of human and experimental liver cancer. A review of our work and other studies in the literature indicate that deterioration of liver function is accompanied by an increase in the amount of chondroitin sulfate proteoglycans. The alteration of proteoglycan composition interferes with the physiologic function of the liver on several levels. This article details and discusses the roles of syndecan-1, glypicans, agrin, perlecan, collagen XVIII/endostatin, endocan, serglycin, decorin, biglycan, asporin, fibromodulin, lumican, and versican in liver function. Specifically, glypicans, agrin, and versican play significant roles in the development of liver cancer. Conversely, the presence of decorin could potentially provide protective effects.

  1. Role of Heparan Sulfate in Cellular Infection of Integrin-Binding Coxsackievirus A9 and Human Parechovirus 1 Isolates

    NARCIS (Netherlands)

    Merilahti, Pirjo; Karelehto, Eveliina; Susi, Petri

    2016-01-01

    Heparan sulfate/heparin class of proteoglycans (HSPG) have been shown to function in cellular attachment and infection of numerous viruses including picornaviruses. Coxsackievirus A9 (CV-A9) and human parechovirus 1 (HPeV-1) are integrin-binding members in the family Picornaviridae. CV-A9 Griggs and

  2. Structural Aspects of N-Glycosylations and the C-terminal Region in Human Glypican-1.

    Science.gov (United States)

    Awad, Wael; Adamczyk, Barbara; Örnros, Jessica; Karlsson, Niclas G; Mani, Katrin; Logan, Derek T

    2015-09-18

    Glypicans are multifunctional cell surface proteoglycans involved in several important cellular signaling pathways. Glypican-1 (Gpc1) is the predominant heparan sulfate proteoglycan in the developing and adult human brain. The two N-linked glycans and the C-terminal domain that attach the core protein to the cell membrane are not resolved in the Gpc1 crystal structure. Therefore, we have studied Gpc1 using crystallography, small angle x-ray scattering, and chromatographic approaches to elucidate the composition, structure, and function of the N-glycans and the C terminus and also the topology of Gpc1 with respect to the membrane. The C terminus is shown to be highly flexible in solution, but it orients the core protein transverse to the membrane, directing a surface evolutionarily conserved in Gpc1 orthologs toward the membrane, where it may interact with signaling molecules and/or membrane receptors on the cell surface, or even the enzymes involved in heparan sulfate substitution in the Golgi apparatus. Furthermore, the N-glycans are shown to extend the protein stability and lifetime by protection against proteolysis and aggregation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Functional Requirements for Heparan Sulfate Biosynthesis in Morphogenesis and Nervous System Development in C. elegans.

    Science.gov (United States)

    Blanchette, Cassandra R; Thackeray, Andrea; Perrat, Paola N; Hekimi, Siegfried; Bénard, Claire Y

    2017-01-01

    The regulation of cell migration is essential to animal development and physiology. Heparan sulfate proteoglycans shape the interactions of morphogens and guidance cues with their respective receptors to elicit appropriate cellular responses. Heparan sulfate proteoglycans consist of a protein core with attached heparan sulfate glycosaminoglycan chains, which are synthesized by glycosyltransferases of the exostosin (EXT) family. Abnormal HS chain synthesis results in pleiotropic consequences, including abnormal development and tumor formation. In humans, mutations in either of the exostosin genes EXT1 and EXT2 lead to osteosarcomas or multiple exostoses. Complete loss of any of the exostosin glycosyltransferases in mouse, fish, flies and worms leads to drastic morphogenetic defects and embryonic lethality. Here we identify and study previously unavailable viable hypomorphic mutations in the two C. elegans exostosin glycosyltransferases genes, rib-1 and rib-2. These partial loss-of-function mutations lead to a severe reduction of HS levels and result in profound but specific developmental defects, including abnormal cell and axonal migrations. We find that the expression pattern of the HS copolymerase is dynamic during embryonic and larval morphogenesis, and is sustained throughout life in specific cell types, consistent with HSPGs playing both developmental and post-developmental roles. Cell-type specific expression of the HS copolymerase shows that HS elongation is required in both the migrating neuron and neighboring cells to coordinate migration guidance. Our findings provide insights into general principles underlying HSPG function in development.

  4. Functional Requirements for Heparan Sulfate Biosynthesis in Morphogenesis and Nervous System Development in C. elegans.

    Directory of Open Access Journals (Sweden)

    Cassandra R Blanchette

    2017-01-01

    Full Text Available The regulation of cell migration is essential to animal development and physiology. Heparan sulfate proteoglycans shape the interactions of morphogens and guidance cues with their respective receptors to elicit appropriate cellular responses. Heparan sulfate proteoglycans consist of a protein core with attached heparan sulfate glycosaminoglycan chains, which are synthesized by glycosyltransferases of the exostosin (EXT family. Abnormal HS chain synthesis results in pleiotropic consequences, including abnormal development and tumor formation. In humans, mutations in either of the exostosin genes EXT1 and EXT2 lead to osteosarcomas or multiple exostoses. Complete loss of any of the exostosin glycosyltransferases in mouse, fish, flies and worms leads to drastic morphogenetic defects and embryonic lethality. Here we identify and study previously unavailable viable hypomorphic mutations in the two C. elegans exostosin glycosyltransferases genes, rib-1 and rib-2. These partial loss-of-function mutations lead to a severe reduction of HS levels and result in profound but specific developmental defects, including abnormal cell and axonal migrations. We find that the expression pattern of the HS copolymerase is dynamic during embryonic and larval morphogenesis, and is sustained throughout life in specific cell types, consistent with HSPGs playing both developmental and post-developmental roles. Cell-type specific expression of the HS copolymerase shows that HS elongation is required in both the migrating neuron and neighboring cells to coordinate migration guidance. Our findings provide insights into general principles underlying HSPG function in development.

  5. Applications of heparin and heparan sulfate microarrays.

    Science.gov (United States)

    Yin, Jian; Seeberger, Peter H

    2010-01-01

    Carbohydrate microarrays have become crucial tools for revealing the biological interactions and functions of glycans, primarily because the microarray format enables the investigation of large numbers of carbohydrates at a time. Heparan sulfate (HS) and heparin are the most structurally complex glycosaminoglycans (GAGs). In this chapter, we describe the preparation of a small library of HS/heparin oligosaccharides, and the fabrication of HS/heparin microarrays that have been used to establish HS/heparin-binding profiles. Fibroblast growth factors (FGFs), natural cytotoxicity receptors (NCRs), and chemokines were screened to illuminate the very important biological functions of these glycans. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  6. The proteoglycan repertoire of lymphoid cells.

    Science.gov (United States)

    Fadnes, Bodil; Husebekk, Anne; Svineng, Gunbjørg; Rekdal, Øystein; Yanagishita, Masaki; Kolset, Svein O; Uhlin-Hansen, Lars

    2012-10-01

    Proteoglycans have been studied to a limited extent in lymphoid cells. In this study we have investigated the expression of proteoglycans in B-cells, CD4+ T-cells, CD8+ T-cells, natural killer cells, as well as in nine different cell lines established from patients with lymphoid malignancies. Serglycin was the major proteoglycan expressed at mRNA level by the primary lymphocytes. None of the syndecans or glycpicans was detected at mRNA level in the primary lymphocytes, except for syndecan-4 in CD4+ T-cells and CD8+ T-cells. All lymphoid cell lines expressed serglycin mRNA, as well as one or several members of the syndecan and glypican families. Further, increased synthesis of proteoglycans was found in the cell lines compared to the primary lymphocytes, as well as the presence of heparan sulfate on the cell surface of five of the cells lines. Western blot analysis showed a close correlation between serglycin mRNA level and expression of serglycin core protein. Our results show that serglycin is a major proteoglycan in all the normal lymphoid cells and that these cells carry little, or none, proteoglycans on the cell surface. Serglycin was also a major proteoglycan in the malignant lymphoid cells, but these also expressed one or more types of cell surface proteoglycans. Thus, malignant transformation of lymphoid cells may be followed by increased synthesis of proteoglycans and expression of cell surface proteoglycans.

  7. Glypican4 modulates lateral line collective cell migration non cell-autonomously.

    Science.gov (United States)

    Venero Galanternik, Marina; Lush, Mark E; Piotrowski, Tatjana

    2016-11-15

    Collective cell migration is an essential process during embryonic development and diseases such as cancer, and still much remains to be learned about how cell intrinsic and environmental cues are coordinated to guide cells to their targets. The migration-dependent development of the zebrafish sensory lateral line proves to be an excellent model to study how proteoglycans control collective cell migration in a vertebrate. Proteoglycans are extracellular matrix glycoproteins essential for the control of several signaling pathways including Wnt/β-catenin, Fgf, BMP and Hh. In the lateral line primordium the modified sugar chains on proteoglycans are important regulators of cell polarity, ligand distribution and Fgf signaling. At least five proteoglycans show distinct expression patterns in the primordium; however, their individual functions have not been studied. Here, we describe the function of glypican4 during zebrafish lateral line development. glypican4 is expressed in neuromasts, interneuromast cells and muscle cells underlying the lateral line. knypek fr6 /glypican4 mutants show severe primordium migration defects and the primordium often U-turns and migrates back toward the head. Our analysis shows that Glypican4 regulates the feedback loop between Wnt/β-catenin/Fgf signaling in the primordium redundantly with other Heparan Sulfate Proteoglycans. In addition, the primordium migration defect is caused non-cell autonomously by the loss of cxcl12a-expressing muscle precursors along the myoseptum via downregulation of Hh. Our results show that glypican4 has distinct functions in primordium cells and cells in the environment and that both of these functions are essential for collective cell migration. Copyright © 2016. Published by Elsevier Inc.

  8. The function of heparan sulfate during branching morphogenesis

    Science.gov (United States)

    Patel, Vaishali N.; Pineda, Dallas L.; Hoffman, Matthew P.

    2016-01-01

    Branching morphogenesis is a fundamental process in the development of diverse epithelial organs such as the lung, kidney, liver, pancreas, prostate, salivary, lacrimal and mammary glands. A unifying theme during organogenesis is the importance of epithelial cell interactions with the extracellular matrix (ECM) and growth factors (GFs). The diverse developmental mechanisms giving rise to these epithelial organs involve many organ-specific GFs, but a unifying paradigm during organogenesis is the regulation of GF activity by heparan sulfates (HS) on the cell surface and in the ECM. This primarily involves the interactions of GFs with the sulfated side-chains of HS proteoglycans. HS is one of the most diverse biopolymers and modulates GF binding and signaling at the cell surface and in the ECM of all tissues. Here, we review what is known about how HS regulates branching morphogenesis of epithelial organs with emphasis on the developing salivary gland, which is a classic model to investigate epithelial-ECM interactions. We also address the structure, biosynthesis, turnover and function of HS during organogenesis. Understanding the regulatory mechanisms that control HS dynamics may aid in the development of therapeutic interventions for diseases and novel strategies for tissue engineering and regenerative medicine. PMID:27609403

  9. Novel heparan sulfate-binding peptides for blocking herpesvirus entry.

    Directory of Open Access Journals (Sweden)

    Pranay Dogra

    Full Text Available Human cytomegalovirus (HCMV infection can lead to congenital hearing loss and mental retardation. Upon immune suppression, reactivation of latent HCMV or primary infection increases morbidity in cancer, transplantation, and late stage AIDS patients. Current treatments include nucleoside analogues, which have significant toxicities limiting their usefulness. In this study we screened a panel of synthetic heparin-binding peptides for their ability to prevent CMV infection in vitro. A peptide designated, p5+14 exhibited ~ 90% reduction in murine CMV (MCMV infection. Because negatively charged, cell-surface heparan sulfate proteoglycans (HSPGs, serve as the attachment receptor during the adsorption phase of the CMV infection cycle, we hypothesized that p5+14 effectively competes for CMV adsorption to the cell surface resulting in the reduction in infection. Positively charged Lys residues were required for peptide binding to cell-surface HSPGs and reducing viral infection. We show that this inhibition was not due to a direct neutralizing effect on the virus itself and that the peptide blocked adsorption of the virus. The peptide also inhibited infection of other herpesviruses: HCMV and herpes simplex virus 1 and 2 in vitro, demonstrating it has broad-spectrum antiviral activity. Therefore, this peptide may offer an adjunct therapy for the treatment of herpes viral infections and other viruses that use HSPGs for entry.

  10. Toxoplasma gondii RON4 binds to heparan sulfate on the host cell surface.

    Science.gov (United States)

    Takemae, Hitoshi; Kobayashi, Kyousuke; Sugi, Tatsuki; Han, Yongmei; Gong, Haiyan; Ishiwa, Akiko; Recuenco, Frances C; Murakoshi, Fumi; Takano, Ryo; Murata, Yuho; Nagamune, Kisaburo; Horimoto, Taisuke; Akashi, Hiroomi; Kato, Kentaro

    2018-04-01

    Toxoplasma gondii rhoptry neck protein 4 (TgRON4) is a component of the moving junction, a key structure for host cell invasion. We previously showed that host cellular β-tubulin is a binding partner of TgRON4 in the invasion process. Here, to identify other binding partners of TgRON4 in the host cell, we examined the binding of TgRON4 to components of the host cell surface. TgRON4 binds to various mammalian cells, but this binding disappeared in glycosaminoglycan- and heparan sulfate-deficient CHO cells and after heparitinase treatment of mammalian cells. The C-terminal half of TgRON4 showed relatively strong binding to cells and heparin agarose. A glycoarray assay indicated that TgRON4 binds to heparin and modified heparin derivatives. Immunoprecipitation of T. gondii-infected CHO cell lysates showed that TgRON4 interacts with glypican 1 during Toxoplasma invasion. This interaction suggests a role for heparan sulfate in parasite invasion. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Differential expression of heparan sulfate domains in rat spleen.

    NARCIS (Netherlands)

    Dam, G.B. ten; Hafmans, T.G.M.; Veerkamp, J.H.; Kuppevelt, A.H.M.S.M. van

    2003-01-01

    The microarchitecture of the spleen is composed of a meshwork of reticulum cells and their matrix. Heparan sulfates (HS) are important components of this meshwork and are involved in processes such as cell adhesion, cell migration, and cytokine/growth factor binding. The expression of HS epitopes

  12. Heparan sulfate alterations in extracellular matrix structures and fibroblast growth factor-2 signaling impairment in the aged neurogenic niche.

    Science.gov (United States)

    Yamada, Taihei; Kerever, Aurelien; Yoshimura, Yusuke; Suzuki, Yuji; Nonaka, Risa; Higashi, Kyohei; Toida, Toshihiko; Mercier, Frederic; Arikawa-Hirasawa, Eri

    2017-08-01

    Adult neurogenesis in the subventricular zone of the lateral ventricle decreases with age. In the subventricular zone, the specialized extracellular matrix structures, known as fractones, contact neural stem cells and regulate neurogenesis. Fractones are composed of extracellular matrix components, such as heparan sulfate proteoglycans. We previously found that fractones capture and store fibroblast growth factor 2 (FGF-2) via heparan sulfate binding, and may deliver FGF-2 to neural stem cells in a timely manner. The heparan sulfate (HS) chains in the fractones of the aged subventricular zone are modified based on immunohistochemistry. However, how aging affects fractone composition and subsequent FGF-2 signaling and neurogenesis remains unknown. The formation of the FGF-fibroblast growth factor receptor-HS complex is necessary to activate FGF-2 signaling and induce the phosphorylation of extracellular signal-regulated kinase (Erk1/2). In this study, we observed a reduction in HS 6-O-sulfation, which is critical for FGF-2 signal transduction, and failure of the FGF-2-induced phosphorylation of Erk1/2 in the aged subventricular zone. In addition, we observed increased HS 6-O-endo-sulfatase, an enzyme that may be responsible for the HS modifications in aged fractones. In conclusion, the data revealed that heparan sulfate 6-O-sulfation is reduced and FGF-2-dependent Erk1/2 signaling is impaired in the aged subventricular zone. HS modifications in fractones might play a role in the reduced neurogenic activity in aging brains. © 2017 International Society for Neurochemistry.

  13. A role for heparan sulfate in viral surfing

    Energy Technology Data Exchange (ETDEWEB)

    Oh, Myung-Jin; Akhtar, Jihan [Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL 60612 (United States); Desai, Prashant [Viral Oncology Program, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, 1650 Orleans Street, Baltimore, MD 21231 (United States); Shukla, Deepak, E-mail: dshukla@uic.edu [Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL 60612 (United States); Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612 (United States)

    2010-01-01

    Heparan sulfate (HS) moieties on cell surfaces are known to provide attachment sites for many viruses including herpes simplex virus type-1 (HSV-1). Here, we demonstrate that cells respond to HSV-1 infection by enhancing filopodia formation. Filopodia express HS and are subsequently utilized for the transport of HSV-1 virions to cell bodies in a surfing-like phenomenon, which is facilitated by the underlying actin cytoskeleton and is regulated by transient activation of a small Rho GTPase, Cdc42. We also demonstrate that interaction between a highly conserved herpesvirus envelope glycoprotein B (gB) and HS is required for surfing. A HSV-1 mutant that lacks gB fails to surf and quantum dots conjugated with gB demonstrate surfing-like movements. Our data demonstrates a novel use of a common receptor, HS, which could also be exploited by multiple viruses and quite possibly, many additional ligands for transport along the plasma membrane.

  14. A Heparan Sulfate-Binding Cell Penetrating Peptide for Tumor Targeting and Migration Inhibition

    Directory of Open Access Journals (Sweden)

    Chien-Jung Chen

    2015-01-01

    Full Text Available As heparan sulfate proteoglycans (HSPGs are known as co-receptors to interact with numerous growth factors and then modulate downstream biological activities, overexpression of HS/HSPG on cell surface acts as an increasingly reliable prognostic factor in tumor progression. Cell penetrating peptides (CPPs are short-chain peptides developed as functionalized vectors for delivery approaches of impermeable agents. On cell surface negatively charged HS provides the initial attachment of basic CPPs by electrostatic interaction, leading to multiple cellular effects. Here a functional peptide (CPPecp has been identified from critical HS binding region in hRNase3, a unique RNase family member with in vitro antitumor activity. In this study we analyze a set of HS-binding CPPs derived from natural proteins including CPPecp. In addition to cellular binding and internalization, CPPecp demonstrated multiple functions including strong binding activity to tumor cell surface with higher HS expression, significant inhibitory effects on cancer cell migration, and suppression of angiogenesis in vitro and in vivo. Moreover, different from conventional highly basic CPPs, CPPecp facilitated magnetic nanoparticle to selectively target tumor site in vivo. Therefore, CPPecp could engage its capacity to be developed as biomaterials for diagnostic imaging agent, therapeutic supplement, or functionalized vector for drug delivery.

  15. Heparan sulfate dependent signaling of fibroblast growth factor (FGF) 18 by chondrocyte-derived perlecan

    Science.gov (United States)

    Chuang, Christine Y.; Lord, Megan S.; Melrose, James; Rees, Martin D.; Knox, Sarah M.; Freeman, Craig; Iozzo, Renato V.; Whitelock, John M.

    2010-01-01

    Perlecan is a large multi-domain proteoglycan which is essential for normal cartilage development. In this study perlecan was localized in the pericellular matrix of hypertrophic chondrocytes in developing human cartilage rudiments. Perlecan immunopurified from medium conditioned by cultured human fetal chondrocytes was found to be substituted with heparan sulfate (HS), chondroitin sulfate (CS) and keratan sulfate (KS). Ligand and carbohydrate engagement (LACE) assays demonstrated that immunopurified chondrocyte-derived perlecan formed HS dependent ternary complexes with fibroblast growth factors (FGF) 2 and either FGFR receptors (FGFRs) 1 or 3, however these complexes were not biologically active in the BaF32 cell system. Chondrocyte-derived perlecan also formed HS dependent ternary complexes with FGF18 and FGFR3. The proliferation of BaF32 cells expressing FGFR3 was promoted by chondrocyte-derived perlecan in the presence of FGF18 and this activity was reduced by digesting the HS with either heparinase III or mammalian heparanase. These data suggest that FGF2 and 18 bind to discrete structures on the HS chains attached to chondrocyte-derived perlecan which modulate the growth factor activities. The presence and activity of mammalian heparanase may be important in the turnover of HS and subsequent signaling required for the establishment and maintenance of functional osteo-chondral junctions in long bone growth. PMID:20507176

  16. Heparan sulfate regulates fibrillin-1 N- and C-terminal interactions

    DEFF Research Database (Denmark)

    Cain, Stuart A; Baldwin, Andrew K; Mahalingam, Yashithra

    2008-01-01

    in response to soluble PF1. Within domains encoded by exons 59-62 near the fibrillin-1 C terminus are novel conformation-dependent high affinity heparin and tropoelastin binding sites. Heparin disrupted tropoelastin binding but did not disrupt N- and C-terminal fibrillin-1 interactions. Thus, fibrillin-1 N......-terminal interactions with heparin/heparan sulfate directly influence cell behavior, whereas C-terminal interactions with heparin/heparan sulfate regulate elastin deposition. These data highlight how heparin/heparan sulfate controls fibrillin-1 interactions....

  17. Structure-Activity Relationships of Bioengineered Heparin/Heparan Sulfates Produced in Different Bioreactors

    Directory of Open Access Journals (Sweden)

    Ha Na Kim

    2017-05-01

    Full Text Available Heparin and heparan sulfate are structurally-related carbohydrates with therapeutic applications in anticoagulation, drug delivery, and regenerative medicine. This study explored the effect of different bioreactor conditions on the production of heparin/heparan sulfate chains via the recombinant expression of serglycin in mammalian cells. Tissue culture flasks and continuously-stirred tank reactors promoted the production of serglycin decorated with heparin/heparan sulfate, as well as chondroitin sulfate, while the serglycin secreted by cells in the tissue culture flasks produced more highly-sulfated heparin/heparan sulfate chains. The serglycin produced in tissue culture flasks was effective in binding and signaling fibroblast growth factor 2, indicating the utility of this molecule in drug delivery and regenerative medicine applications in addition to its well-known anticoagulant activity.

  18. Tetrasulfated Disaccharide Unit in Heparan Sulfate: ENZYMATIC FORMATION AND TISSUE DISTRIBUTION*

    OpenAIRE

    Mochizuki, Hideo; Yoshida, Keiichi; Shibata, Yuniko; Kimata, Koji

    2008-01-01

    We previously reported that the heparan sulfate 3-O-sulfotransferase (3OST)-5 produces a novel component of heparan sulfate, i.e. the tetrasulfated disaccharide (Di-tetraS) unit (Mochizuki, H., Yoshida, K., Gotoh, M., Sugioka, S., Kikuchi, N., Kwon, Y.-D., Tawada, A., Maeyama, K., Inaba, N., Hiruma, T., Kimata, K., and Narimatsu, H. (2003) J. Biol. Chem.278 ,26780 -2678712740361). In the present study, we investigated the potential of other 3OST isoforms to produce Di-...

  19. Distribution of Heparan Sulfate Oligosaccharides in Murine Mucopolysaccharidosis Type IIIA

    Directory of Open Access Journals (Sweden)

    Kerryn Mason

    2014-12-01

    Full Text Available Heparan sulfate (HS catabolism begins with endo-degradation of the polysaccharide to smaller HS oligosaccharides, followed by the sequential action of exo-enzymes to reduce these oligosaccharides to monosaccharides and inorganic sulfate. In mucopolysaccharidosis type IIIA (MPS IIIA the exo-enzyme, N-sulfoglucosamine sulfohydrolase, is deficient resulting in an inability to hydrolyze non-reducing end glucosamine N-sulfate esters. Consequently, partially degraded HS oligosaccharides with non-reducing end glucosamine sulfate esters accumulate. We investigated the distribution of these HS oligosaccharides in tissues of a mouse model of MPS IIIA using high performance liquid chromatography electrospray ionization-tandem mass spectrometry. Oligosaccharide levels were compared to total uronic acid (UA, which was used as a measure of total glycosaminoglycan. Ten oligosaccharides, ranging in size from di- to hexasaccharides, were present in all the tissues examined including brain, spleen, lung, heart, liver, kidney and urine. However, the relative levels varied up to 10-fold, suggesting different levels of HS turnover and storage. The relationship between the di- and tetrasaccharides and total UA was tissue specific with spleen and kidney showing a different disaccharide:total UA ratio than the other tissues. The hexasaccharides showed a stronger correlation with total UA in all tissue types suggesting that hexasaccharides may more accurately reflect the storage burden in these tissues.

  20. The Synthetic Antimicrobial Peptide 19-2.5 Interacts with Heparanase and Heparan Sulfate in Murine and Human Sepsis.

    Directory of Open Access Journals (Sweden)

    Lukas Martin

    Full Text Available Heparanase is an endo-β-glucuronidase that cleaves heparan sulfate side chains from their proteoglycans. Thereby, heparanase liberates highly potent circulating heparan sulfate-fragments (HS-fragments and triggers the fatal and excessive inflammatory response in sepsis. As a potential anti-inflammatory agent for sepsis therapy, peptide 19-2.5 belongs to the class of synthetic anti-lipopolysaccharide peptides; however, its activity is not restricted to Gram-negative bacterial infection. We hypothesized that peptide 19-2.5 interacts with heparanase and/or HS, thereby reducing the levels of circulating HS-fragments in murine and human sepsis. Our data indicate that the treatment of septic mice with peptide 19-2.5 compared to untreated control animals lowers levels of plasma heparanase and circulating HS-fragments and reduces heparanase activity. Additionally, mRNA levels of heparanase in heart, liver, lung, kidney and spleen are downregulated in septic mice treated with peptide 19-2.5 compared to untreated control animals. In humans, plasma heparanase level and activity are elevated in septic shock. The ex vivo addition of peptide 19-2.5 to plasma of septic shock patients decreases heparanase activity but not heparanase level. Isothermal titration calorimetry revealed a strong exothermic reaction between peptide 19-2.5 and heparanase and HS-fragments. However, a saturation character has been identified only in the peptide 19-2.5 and HS interaction. In conclusion, the findings of our current study indicate that peptide 19-2.5 interacts with heparanase, which is elevated in murine and human sepsis and consecutively attenuates the generation of circulating HS-fragments in systemic inflammation. Thus, peptide 19-2.5 seems to be a potential anti-inflammatory agent in sepsis.

  1. Autism-like socio-communicative deficits and stereotypies in mice lacking heparan sulfate

    Science.gov (United States)

    Irie, Fumitoshi; Badie-Mahdavi, Hedieh; Yamaguchi, Yu

    2012-01-01

    Heparan sulfate regulates diverse cell-surface signaling events, and its roles in the development of the nervous system recently have been increasingly uncovered by studies using genetic models carrying mutations of genes encoding enzymes for its synthesis. On the other hand, the role of heparan sulfate in the physiological function of the adult brain has been poorly characterized, despite several pieces of evidence suggesting its role in the regulation of synaptic function. To address this issue, we eliminated heparan sulfate from postnatal neurons by conditionally inactivating Ext1, the gene encoding an enzyme essential for heparan sulfate synthesis. Resultant conditional mutant mice show no detectable morphological defects in the cytoarchitecture of the brain. Remarkably, these mutant mice recapitulate almost the full range of autistic symptoms, including impairments in social interaction, expression of stereotyped, repetitive behavior, and impairments in ultrasonic vocalization, as well as some associated features. Mapping of neuronal activation by c-Fos immunohistochemistry demonstrates that neuronal activation in response to social stimulation is attenuated in the amygdala in these mice. Electrophysiology in amygdala pyramidal neurons shows an attenuation of excitatory synaptic transmission, presumably because of the reduction in the level of synaptically localized AMPA-type glutamate receptors. Our results demonstrate that heparan sulfate is critical for normal functioning of glutamatergic synapses and that its deficiency mediates socio-communicative deficits and stereotypies characteristic for autism. PMID:22411800

  2. Sugar Antennae for Guidance Signals: Syndecans and Glypicans Integrate Directional Cues for Navigating Neurons

    Directory of Open Access Journals (Sweden)

    Christa Rhiner

    2006-01-01

    Full Text Available Attractive and repulsive signals guide migrating nerve cells in all directions when the nervous system starts to form. The neurons extend thin processes, axons, that connect over wide distances with other brain cells to form a complicated neuronal network. One of the most fascinating questions in neuroscience is how the correct wiring of billions of nerve cells in our brain is controlled. Several protein families are known to serve as guidance cues for navigating neurons and axons. Nevertheless, the combinatorial potential of these proteins seems to be insufficient to sculpt the entire neuronal network and the appropriate formation of connections. Recently, heparan sulfate proteoglycans (HSPGs, which are present on the cell surface of neurons and in the extracellular matrix through which neurons and axons migrate, have been found to play a role in regulating cell migration and axon guidance. Intriguingly, the large number of distinct modifications that can be put onto the sugar side chains of these PGs would in principle allow for an enormous diversity of HSPGs, which could help in regulating the vast number of guidance choices taken by individual neurons. In this review, we will focus on the role of the cell surface HSPGs syndecan and glypican and specific HS modifications in promoting neuronal migration, axon guidance, and synapse formation.

  3. Humanization of high-affinity antibodies targeting glypican-3 in hepatocellular carcinoma.

    Science.gov (United States)

    Zhang, Yi-Fan; Ho, Mitchell

    2016-09-26

    Glypican-3 (GPC3) is a cell-surface heparan sulfate proteoglycan highly expressed in hepatocellular carcinoma (HCC). We have generated a group of high-affinity mouse monoclonal antibodies targeting GPC3. Here, we report the humanization and testing of these antibodies for clinical development. We compared the affinity and cytotoxicity of recombinant immunotoxins containing mouse single-chain variable regions fused with a Pseudomonas toxin. To humanize the mouse Fvs, we grafted the combined KABAT/IMGT complementarity determining regions (CDR) into a human IgG germline framework. Interestingly, we found that the proline at position 41, a non-CDR residue in heavy chain variable regions (VH), is important for humanization of mouse antibodies. We also showed that two humanized anti-GPC3 antibodies (hYP7 and hYP9.1b) in the IgG format induced antibody-dependent cell-mediated cytotoxicity and complement-dependent-cytotoxicity in GPC3-positive cancer cells. The hYP7 antibody was tested and showed inhibition of HCC xenograft tumor growth in nude mice. This study successfully humanizes and validates high affinity anti-GPC3 antibodies and sets a foundation for future development of these antibodies in various clinical formats in the treatment of liver cancer.

  4. The BDLF3 gene product of Epstein-Barr virus, gp150, mediates non-productive binding to heparan sulfate on epithelial cells and only the binding domain of CD21 is required for infection.

    Science.gov (United States)

    Chesnokova, Liudmila S; Valencia, Sarah M; Hutt-Fletcher, Lindsey M

    2016-07-01

    The cell surface molecules used by Epstein-Barr virus (EBV) to attach to epithelial cells are not well-defined, although when CD21, the B cell receptor for EBV is expressed epithelial cell infection increases disproportionately to the increase in virus bound. Many herpesviruses use low affinity charge interactions with molecules such as heparan sulfate to attach to cells. We report here that the EBV glycoprotein gp150 binds to heparan sulfate proteoglycans, but that attachment via this glycoprotein is not productive of infection. We also report that only the aminoterminal two short consensus repeats of CD21 are required for efficient infection, This supports the hypothesis that, when expressed on an epithelial cell CD21 serves primarily to cluster the major attachment protein gp350 in the virus membrane and enhance access of other important glycoproteins to the epithelial cell surface. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Enterovirus 71 uses cell surface heparan sulfate glycosaminoglycan as an attachment receptor.

    Science.gov (United States)

    Tan, Chee Wah; Poh, Chit Laa; Sam, I-Ching; Chan, Yoke Fun

    2013-01-01

    Enterovirus 71 (EV-71) infections are usually associated with mild hand, foot, and mouth disease in young children but have been reported to cause severe neurological complications with high mortality rates. To date, four EV-71 receptors have been identified, but inhibition of these receptors by antagonists did not completely abolish EV-71 infection, implying that there is an as yet undiscovered receptor(s). Since EV-71 has a wide range of tissue tropisms, we hypothesize that EV-71 infections may be facilitated by using receptors that are widely expressed in all cell types, such as heparan sulfate. In this study, heparin, polysulfated dextran sulfate, and suramin were found to significantly prevent EV-71 infection. Heparin inhibited infection by all the EV-71 strains tested, including those with a single-passage history. Neutralization of the cell surface anionic charge by polycationic poly-d-lysine and blockage of heparan sulfate by an anti-heparan sulfate peptide also inhibited EV-71 infection. Interference with heparan sulfate biosynthesis either by sodium chlorate treatment or through transient knockdown of N-deacetylase/N-sulfotransferase-1 and exostosin-1 expression reduced EV-71 infection in RD cells. Enzymatic removal of cell surface heparan sulfate by heparinase I/II/III inhibited EV-71 infection. Furthermore, the level of EV-71 attachment to CHO cell lines that are variably deficient in cell surface glycosaminoglycans was significantly lower than that to wild-type CHO cells. Direct binding of EV-71 particles to heparin-Sepharose columns under physiological salt conditions was demonstrated. We conclude that EV-71 infection requires initial binding to heparan sulfate as an attachment receptor.

  6. The role of syndecan-1 in cellular signaling and its effects on heparan sulfate biosynthesis in mesenchymal tumors

    Directory of Open Access Journals (Sweden)

    Tünde eSzatmári

    2013-12-01

    Full Text Available Proteoglycans and in particular the syndecans are involved in the differentiation process across the epithelial-mesenchymal axis, principally through their ability to bind growth factors and modulate their downstream signalling. Malignant tumors have individual proteoglycan profiles, which are closely associated with their differentiation and biological behavior, mesenchymal tumors showing a different profile from that of epithelial tumors. Syndecan-1 is the main syndecan of epithelial malignancies, whereas in sarcomas its expression level is generally low, in accordance with their mesenchymal phenotype and highly malignant behaviour. This proteoglycan is often overexpressed in adenocarcinoma cells, whereas mesothelioma and fibrosarcoma cells express syndecan-2 and syndecan-4 more abundantly. Increased expression of syndecan-1 in mesenchymal tumors changes the tumor cell morphology to an epithelioid direction whereas downregulation results in a change in shape from polygonal to spindle-like morphology. Although syndecan-1 plays major roles on the cell surface, there are also intracellular functions, which are not very well studied. On the functional level, syndecan-1 affects mesenchymal tumor cell proliferation, adhesion, migration and motility, and the effect varies with the different domains of the core protein. Syndecan-1 may exert stimulatory or inhibitory effects, depending on the concentration of various mitogens, enzymes and signalling molecules, the ratio between the shed and membrane-associated syndecan-1 and histological grade of the tumour. Growth factor signaling seems to be delicately controlled by regulatory loops involving the syndecan expression levels and their sulfation patterns. Overexpression of syndecan-1 modulates the biosynthesis and sulfation of heparan sulfate and it also affects the expression of other proteoglycans. On transcriptomic level, syndecan-1 modulation results in profound effects on genes involved in

  7. Basement membrane proteoglycans are of epithelial origin in rodent skin

    DEFF Research Database (Denmark)

    Yamane, Y; Yaoita, H; Couchman, J R

    1996-01-01

    Basement membrane proteoglycans in mammalian skin comprise at least one chondroitin sulfate proteoglycan and heparan sulfate proteoglycans, including perlecan. In this study, the origins of basement membrane chondroitin sulfate proteoglycan and perlecan were investigated both in vivo and in vitro...... proteoglycan and rat and mouse perlecan. While the isolated rat epidermis was shown to completely lack rat basement membrane chondroitin sulfate proteoglycan and rat basement membrane heparan sulfate proteoglycans, including perlecan, immunofluorescence staining of tissue sections from the grafted sites...... on mice demonstrated the presence of rat basement membrane chondroitin sulfate proteoglycan and rat perlecan on interfollicular and follicular basement membranes including that separating dermal papillae from adjacent hair follicle epithelium. In contrast, the basement membranes of all dermal capillaries...

  8. Effect of heparan sulfate and gold nanoparticles on muscle development during embryogenesis

    DEFF Research Database (Denmark)

    Zielinska, Marlena; Sawosz, Ewa; Grodzik, Marta

    2011-01-01

    Purpose: It was hypothesized that heparan sulfate (HS) as an essential compound for myogenesis and nanoparticles of gold (nano-Au) ashighly reactive compounds can affect muscle development as a consequence of molecular regulation of muscle cell formation, and that these effects may be enhanced by...

  9. RB4CD12 epitope expression and heparan sulfate disaccharide composition in brain vasculature

    NARCIS (Netherlands)

    Hosono-Fukao, T.; Ohtake-Niimi, S.; Nishitsuji, K.; Hossain, M.M.; Kuppevelt, A.H.M.S.M. van; Michikawa, M.; Uchimura, K.

    2011-01-01

    RB4CD12 is a phage display antibody that recognizes a heparan sulfate (HS) glycosaminoglycan epitope. The epitope structure is proposed to contain a trisulfated disaccharide, [-IdoA(2-OSO(3))-GlcNSO(3) (6-OSO(3))-], which supports HS binding to various macromolecules such as growth factors and

  10. The Effect of a Synthetic Heparan Sulfate on the Healing of Colonic Anastomoses

    DEFF Research Database (Denmark)

    Nerstrøm, Malene; Krarup, Peter-Martin; Jorgensen, Lars Nannestad

    2017-01-01

    BACKGROUND: The mimetic compound OTR4120 may replace endogenous-degraded heparan sulfates that normally maintain the bioactivity of growth factors that are important for tissue repair. Herein, we investigated the effect of OTR4120 on the healing of normal colonic anastomoses. METHODS: We evaluated...

  11. A NEW ELISA FOR THE DETECTION OF ANTI-HEPARAN SULFATE REACTIVITY, USING PHOTOBIOTINYLATED ANTIGEN

    NARCIS (Netherlands)

    HYLKEMA, MN; KRAMERS, C; VANDERWAL, TJ; VANBRUGGEN, MCJ; SWAAK, AJG; BERDEN, JHM; SMEENK, RJT; Hylkema, Machteld

    1994-01-01

    Autoantibodies reacting with a great variety of autoantigens are characteristic for the autoimmune disease systemic lupus erythematosus (SLE). Although reactivity with heparan sulfate (HS) in sera of patients with SLE is found in association with the occurrence of nephritis, the aetiological

  12. The agouti-related peptide binds heparan sulfate through segments critical for its orexigenic effects.

    Science.gov (United States)

    Palomino, Rafael; Lee, Hsiau-Wei; Millhauser, Glenn L

    2017-05-05

    Syndecans potently modulate agouti-related peptide (AgRP) signaling in the central melanocortin system. Through heparan sulfate moieties, syndecans are thought to anchor AgRP near its receptor, enhancing its orexigenic effects. Original work proposed that the N-terminal domain of AgRP facilitates this interaction. However, this is not compatible with evidence that this domain is posttranslationally cleaved. Addressing this long-standing incongruity, we used calorimetry and magnetic resonance to probe interactions of AgRP peptides with glycosaminoglycans, including heparan sulfate. We show that mature, cleaved, C-terminal AgRP, not the N-terminal domain, binds heparan sulfate. NMR shows that the binding site consists of regions distinct from the melanocortin receptor-binding site. Using a library of designed AgRP variants, we find that the strength of the syndecan interaction perfectly tracks orexigenic action. Our data provide compelling evidence that AgRP is a heparan sulfate-binding protein and localizes critical regions in the AgRP structure required for this interaction. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Antiviral activity of human lactoferrin : Inhibition of alphavirus interaction with heparan sulfate

    NARCIS (Netherlands)

    Waarts, Barry-Lee; Aneke, Onwuchekwa J.C.; Smit, Jolanda; Kimata, Koji; Bittman, Robert; Meijer, Dirk K.F.; Wilschut, Jan

    2005-01-01

    Human lactoferrin is a component of the non-specific immune system with distinct antiviral properties. We used alphaviruses, adapted to interaction with heparan sulfate (HS), as a tool to investigate the mechanism of lactoferrin's antiviral activity. Lactoferrin inhibited infection of BHK-21 cells

  14. Perlecan and basement membrane-chondroitin sulfate proteoglycan (bamacan) are two basement membrane chondroitin/dermatan sulfate proteoglycans in the Engelbreth-Holm-Swarm tumor matrix

    DEFF Research Database (Denmark)

    Couchman, J R; Kapoor, R; Sthanam, M

    1996-01-01

    The presence of proteoglycans bearing galactosaminoglycan chains has been reported, but none has been identified previously in the matrix of the Engelbreth-Holm-Swarm tumor, which is a source of several basement membrane components. This tumor matrix contains perlecan, a large, low buoyant density...... heparan sulfate proteoglycan, widespread in many basement membranes and connective tissues. We now identify two distinct proteoglycan species from this tumor source, which are substituted with galactosaminoglycans and which show basement membrane localization by immunohistochemistry. One species...... is perlecan but, in addition to being present as a heparan sulfate proteoglycan, it is also present as a hybrid molecule, with dermatan sulfate chains. A minor population of perlecan apparently lacks heparan sulfate chains totally, and some of this is substituted with chondroitin sulfate. The second species...

  15. Impact of Heparan Sulfate Binding on Transduction of Retina by Recombinant Adeno-Associated Virus Vectors

    Science.gov (United States)

    Boye, Sanford L.; Bennett, Antonette; Scalabrino, Miranda L.; McCullough, K. Tyler; Van Vliet, Kim; Choudhury, Shreyasi; Ruan, Qing; Peterson, James

    2016-01-01

    ABSTRACT Adeno-associated viruses (AAVs) currently are being developed to efficiently transduce the retina following noninvasive, intravitreal (Ivt) injection. However, a major barrier encountered by intravitreally delivered AAVs is the inner limiting membrane (ILM), a basement membrane rich in heparan sulfate (HS) proteoglycan. The goal of this study was to determine the impact of HS binding on retinal transduction by Ivt-delivered AAVs. The heparin affinities of AAV2-based tyrosine-to-phenylalanine (Y-F) and threonine-to-valine (T-V) capsid mutants, designed to avoid proteasomal degradation during cellular trafficking, were established. In addition, the impact of grafting HS binding residues onto AAV1, AAV5, and AAV8(Y733F) as well as ablation of HS binding by AAV2-based vectors on retinal transduction was investigated. Finally, the potential relationship between thermal stability of AAV2-based capsids and Ivt-mediated transduction was explored. The results show that the Y-F and T-V AAV2 capsid mutants bind heparin but with slightly reduced affinity relative to that of AAV2. The grafting of HS binding increased Ivt transduction by AAV1 but not by AAV5 or AAV8(Y733F). The substitution of any canonical HS binding residues ablated Ivt-mediated transduction by AAV2-based vectors. However, these same HS variant vectors displayed efficient retinal transduction when delivered subretinally. Notably, a variant devoid of canonical HS binding residues, AAV2(4pMut)ΔHS, was remarkably efficient at transducing photoreceptors. The disparate AAV phenotypes indicate that HS binding, while critical for AAV2-based vectors, is not the sole determinant for transduction via the Ivt route. Finally, Y-F and T-V mutations alter capsid stability, with a potential relationship existing between stability and improvements in retinal transduction by Ivt injection. IMPORTANCE AAV has emerged as the vector of choice for gene delivery to the retina, with attention focused on developing vectors

  16. Role of glycanation and convertase maturation of the soluble Glypican-3 in inhibiting proliferation of hepatocellular carcinoma cells.

    Science.gov (United States)

    Saad, Ahmad; Liet, Benjamin; Joucla, Gilles; Santarelli, Xavier; Charpentier, Justine; Claverol, Stéphane; Grosset, Christophe F; Trézéguet, Véronique

    2018-01-18

    Glypican 3 (GPC3) is a complex heparan sulfate proteoglycan associated with the outer surface of the plasma membrane by a glycosyl-phosphatidylinositol anchor (GPI). It is also N-glycosylated and processed by a furine-like convertase. GPC3 has numerous biological functions. While undetectable in normal liver tissue, it is abnormally and highly overexpressed in hepatocellular carcinoma (HCC). Interestingly, proliferation of HCC cells such as HepG2 and HuH7 is inhibited when they express a soluble form of GPC3 after lentiviral transduction. To get more insight into the role of some of its post-translational modifications, we have designed a mutant GPC3, sGPC3m, without its GPI anchor, convertase cleavage site and glycosaminoglycan chains. The highly pure sGPC3m protein strongly inhibited HuH7 and HepG2 cell proliferation in vitro, and induced a significant increase in their cell doubling time. It changed the HuH7 cell morphology but not that of HepG2. It induced HuH7 cell nuclear area oversize and adherent cell junction restructuration. Unexpectedly, for both cell types the apoptosis, cell division and the β-catenin levels were not altered though the growth inhibition was very efficient. Overall, our data show that glycanation and convertase maturation are not required for sGPC3m to inhibit HCC cell proliferation.

  17. Functional Importance of a Proteoglycan Coreceptor in Pathologic Lymphangiogenesis.

    Science.gov (United States)

    Johns, Scott C; Yin, Xin; Jeltsch, Michael; Bishop, Joseph R; Schuksz, Manuela; El Ghazal, Roland; Wilcox-Adelman, Sarah A; Alitalo, Kari; Fuster, Mark M

    2016-07-08

    Lymphatic vessel growth is mediated by major prolymphangiogenic factors, such as vascular endothelial growth factor (VEGF-C) and VEGF-D, among other endothelial effectors. Heparan sulfate is a linear polysaccharide expressed on proteoglycan core proteins on cell membranes and matrix, playing roles in angiogenesis, although little is known about any function(s) in lymphatic remodeling in vivo. To explore the genetic basis and mechanisms, whereby heparan sulfate proteoglycans mediate pathological lymphatic remodeling. Lymphatic endothelial deficiency in the major heparan sulfate biosynthetic enzyme N-deacetylase/N-sulfotransferase-1 (Ndst1; involved in glycan-chain sulfation) was associated with reduced lymphangiogenesis in pathological models, including spontaneous neoplasia. Mouse mutants demonstrated tumor-associated lymphatic vessels with apoptotic nuclei. Mutant lymphatic endothelia demonstrated impaired mitogen (Erk) and survival (Akt) pathway signaling and reduced VEGF-C-mediated protection from starvation-induced apoptosis. Lymphatic endothelial-specific Ndst1 deficiency (in Ndst1(f/f)Prox1(+/CreERT2) mice) was sufficient to inhibit VEGF-C-dependent lymphangiogenesis. Lymphatic heparan sulfate deficiency reduced phosphorylation of the major lymphatic growth receptor VEGF receptor-3 in response to multiple VEGF-C species. Syndecan-4 was the dominantly expressed heparan sulfate proteoglycan in mouse lymphatic endothelia, and pathological lymphangiogenesis was impaired in Sdc4((-/-)) mice. On the lymphatic cell surface, VEGF-C induced robust association between syndecan-4 and VEGF receptor-3, which was sensitive to glycan disruption. Moreover, VEGF receptor-3 mitogen and survival signaling was reduced in the setting of Ndst1 or Sdc4 deficiency. These findings demonstrate the genetic importance of heparan sulfate and the major lymphatic proteoglycan syndecan-4 in pathological lymphatic remodeling. This may introduce novel future strategies to alter pathological

  18. Heparan sulfate chain valency controls syndecan-4 function in cell adhesion

    DEFF Research Database (Denmark)

    Gopal, Sandeep; Bober, Adam; Whiteford, James R

    2010-01-01

    , clustering of one-chain syndecan-4 forms with antibodies overcame the block, indicating that valency of interactions with ligands is a key component of syndecan-4 function. Measurements of focal contact/adhesion size and focal adhesion kinase phosphorylation correlated with syndecan-4 status and alpha...... of the core protein cytoplasmic domain, though not interactions with PDZ proteins. A second key requirement is multiple heparan sulfate chains. Mutant syndecan-4 with no chains, or only one chain, failed to restore the wild type phenotype, while those expressing two or three were competent. However......-smooth muscle actin organization, being reduced where syndecan-4 function was compromised by a lack of multiple heparan sulfate chains....

  19. Transmembrane proteoglycans control stretch-activated channels to set cytosolic calcium levels

    DEFF Research Database (Denmark)

    Gopal, Sandeep; Søgaard, Pernille; Multhaupt, Hinke A B

    2015-01-01

    Transmembrane heparan sulfate proteoglycans regulate multiple aspects of cell behavior, but the molecular basis of their signaling is unresolved. The major family of transmembrane proteoglycans is the syndecans, present in virtually all nucleated cells, but with mostly unknown functions. Here, we...

  20. Regulation of cytoskeletal organization by syndecan transmembrane proteoglycans

    DEFF Research Database (Denmark)

    Yoneda, Atsuko; Couchman, John R

    2003-01-01

    Syndecans, a family of transmembrane proteoglycans, interact with numerous extracellular ligands through specific sequences in their heparan sulfate chains and have been considered to be co-receptors for matrix molecules and growth factors. In addition to their roles as co-receptors, many studies...

  1. Host cell heparan sulfate glycosaminoglycans are ligands for OspF-related proteins of the Lyme disease spirochete.

    Science.gov (United States)

    Lin, Yi-Pin; Bhowmick, Rudra; Coburn, Jenifer; Leong, John M

    2015-10-01

    Borrelia burgdorferi, the agent of Lyme disease, spreads from the site of the tick bite to tissues such as heart, joints and the nervous tissues. Host glycosaminoglycans, highly modified repeating disaccharides that are present on cell surfaces and in extracellular matrix, are common targets of microbial pathogens during tissue colonization. While several dermatan sulfate-binding B. burgdorferi adhesins have been identified, B. burgdorferi adhesins documented to promote spirochetal binding to heparan sulfate have not yet been identified. OspEF-related proteins (Erps), a large family of plasmid-encoded surface lipoproteins that are produced in the mammalian host, can be divided into the OspF-related, OspEF-leader peptide (Elp) and OspE-related subfamilies. We show here that a member of the OspF-related subfamily, ErpG, binds to heparan sulfate and when produced on the surface of an otherwise non-adherent B. burgdorferi strain, ErpG promotes heparan sulfate-mediated bacterial attachment to the glial but not the endothelial, synovial or respiratory epithelial cells. Six other OspF-related proteins were capable of binding heparan sulfate, whereas representative OspE-related and Elp proteins lacked this activity. These results indicate that OspF-related proteins are heparan sulfate-binding adhesins, at least one of which promotes bacterial attachment to glial cells. © 2015 John Wiley & Sons Ltd.

  2. Basement membrane proteoglycans and development

    DEFF Research Database (Denmark)

    Couchman, J R; Abrahamson, D R; McCarthy, K J

    1993-01-01

    Basement membranes contain distinct collagen, glycoprotein and proteoglycan species, and these exhibit considerable heterogeneity in isoform or type when different tissue types are compared. Additionally, many components are differentially expressed in organogenesis. We have considered the distri......Basement membranes contain distinct collagen, glycoprotein and proteoglycan species, and these exhibit considerable heterogeneity in isoform or type when different tissue types are compared. Additionally, many components are differentially expressed in organogenesis. We have considered...... the distributions in glomerulogenesis of two distinct basement membrane proteoglycans, a small heparan sulfate proteoglycan and a chondroitin sulfate proteoglycan (BM-CSPG). While the former was present in all kidney basement membranes through development, the latter was apparently regulated in distribution. BM...

  3. The proteoglycan (heparan sulfate proteoglycan) binding domain of APRIL serves as a platform for ligand multimerization and cross-linking

    NARCIS (Netherlands)

    Kimberley, Fiona C.; van Bostelen, Liesbeth; Cameron, Katherine; Hardenberg, Gijs; Marquart, J. Arnoud; Hahne, Michael; Medema, Jan Paul

    2009-01-01

    A proliferation-inducing ligand (APRIL) (also known as TALL-2 and TRDL-1) is a member of the tumor necrosis factor (TNF) superfamily that has tumorigenic properties but is also important for the induction of humoral immune responses. APRIL binds two TNF receptors: transmembrane activator and calcium

  4. HSulf sulfatases catalyze processive and oriented 6-O-desulfation of heparan sulfate that differentially regulates fibroblast growth factor activity

    NARCIS (Netherlands)

    Seffouh, A.; Milz, F.; Przybylski, C.; Laguri, C.; Oosterhof, A.; Bourcier, S.; Sadir, R.; Dutkowski, E.; Daniel, R.; Kuppevelt, A.H.M.S.M. van; Dierks, T.; Lortat-Jacob, H.; Vives, R.R.

    2013-01-01

    Sulfs are extracellular sulfatases that have emerged recently as critical regulators of heparan sulfate (HS) activities through their ability to catalyze specific 6-O-desulfation of the polysaccharide. Consequently, Sulfs have been involved in many physiological and pathological processes, and

  5. Adaptation of alphaviruses to heparan sulfate : Interaction of Sindbis and Semliki Forest viruses with liposomes containing lipid-conjugated heparin

    NARCIS (Netherlands)

    Smit, JM; Waarts, BL; Kimata, K; Klimstra, WB; Bittman, R; Wilschut, J

    2002-01-01

    Passage of Sindbis virus (SIN) in BHK-21 cells has been shown to select for virus mutants with high affinity for the glycosaminoglycan heparan sulfate (HS). Three loci in the viral spike protein E2 (E2:1, E2:70, and E2:114) have been identified that mutate during adaptation and independently confer

  6. Important role of heparan sulfate in postnatal islet growth and insulin secretion

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, Iwao; Noguchi, Naoya [Department of Advanced Biological Sciences for Regeneration (Kotobiken Medical Laboratories), Tohoku University Graduate School of Medicine, Sendai 980-8575 (Japan); Nata, Koji [Department of Medical Biochemistry, Iwate Medical University School of Pharmacy, Yahaba-cho 028-3603 (Japan); Yamada, Shuhei; Kaneiwa, Tomoyuki; Mizumoto, Shuji [Laboratory of Proteoglycan Signaling and Therapeutics, Hokkaido University Graduate School of Life Science, Sapporo 001-0021 (Japan); Ikeda, Takayuki [Department of Advanced Biological Sciences for Regeneration (Kotobiken Medical Laboratories), Tohoku University Graduate School of Medicine, Sendai 980-8575 (Japan); Sugihara, Kazushi; Asano, Masahide [Division of Transgenic Animal Science, Advanced Science Research Center, Kanazawa University, Kanazawa 920-8640 (Japan); Yoshikawa, Takeo [Department of Advanced Biological Sciences for Regeneration (Kotobiken Medical Laboratories), Tohoku University Graduate School of Medicine, Sendai 980-8575 (Japan); Yamauchi, Akiyo [Department of Biochemistry, Nara Medical University, Kashihara 634-8521 (Japan); Shervani, Nausheen Jamal; Uruno, Akira [Department of Advanced Biological Sciences for Regeneration (Kotobiken Medical Laboratories), Tohoku University Graduate School of Medicine, Sendai 980-8575 (Japan); Kato, Ichiro [Department of Biochemistry, University of Toyama Graduate School of Medicine and Pharmaceutical Sciences, Toyama 930-0194 (Japan); Unno, Michiaki [Department of Surgery, Tohoku University Graduate School of Medicine, Sendai 980-8574 (Japan); Sugahara, Kazuyuki [Laboratory of Proteoglycan Signaling and Therapeutics, Hokkaido University Graduate School of Life Science, Sapporo 001-0021 (Japan); Takasawa, Shin [Department of Biochemistry, Nara Medical University, Kashihara 634-8521 (Japan); and others

    2009-05-22

    Heparan sulfate (HS) binds with several signaling molecules and regulates ligand-receptor interactions, playing an essential role in embryonic development. Here we showed that HS was intensively expressed in pancreatic islet {beta}-cells after 1 week of age in mice. The enzymatic removal of HS in isolated islets resulted in attenuated glucose-induced insulin secretion with a concomitant reduction in gene expression of several key components in the insulin secretion machinery. We further depleted islet HS by inactivating the exostosin tumor-like 3 gene specifically in {beta}-cells. These mice exhibited abnormal islet morphology with reduced {beta}-cell proliferation after 1 week of age and glucose intolerance due to defective insulin secretion. These results demonstrate that islet HS is involved in the regulation of postnatal islet maturation and required to ensure normal insulin secretion.

  7. Role of heparan sulfates and glycosphingolipids in the pore formation of basic polypeptides of cobra cardiotoxin.

    Science.gov (United States)

    Wu, Wen-Guey; Tjong, Siu-Cin; Wu, Po-Long; Kuo, Je-Hung; Wu, Karen

    2010-01-01

    Cobra venom contains cardiotoxins (CTXs) that induce tissue necrosis and systolic heart arrest in bitten victims. CTX-induced membrane pore formation is one of the major mechanisms responsible for the venom's designated cytotoxicity. This chapter examines how glycoconjugates such as heparan sulfates (HS) and glycosphingolipids, located respectively in the extracellular matrix and lipid bilayers of the cell membranes, facilitate CTX pore formation. Evidences for HS-facilitated cell surface retention and glycosphingolipid-facilitated membrane bilayer insertion of CTX are reviewed. We suggest that similar physical steps could play a role in the mediation of other pore forming toxins (PFT). The membrane pores formed by PFT are expected to have limited lifetime on biological cell surface as a result of membrane dynamics during endocytosis and/or rearrangement of lipid rafts.

  8. Uncovering Biphasic Catalytic Mode of C5-epimerase in Heparan Sulfate Biosynthesis*

    Science.gov (United States)

    Sheng, Juzheng; Xu, Yongmei; Dulaney, Steven B.; Huang, Xuefei; Liu, Jian

    2012-01-01

    Heparan sulfate (HS), a highly sulfated polysaccharide, is biosynthesized through a pathway involving several enzymes. C5-epimerase (C5-epi) is a key enzyme in this pathway. C5-epi is known for being a two-way catalytic enzyme, displaying a “reversible” catalytic mode by converting a glucuronic acid to an iduronic acid residue, and vice versa. Here, we discovered that C5-epi can also serve as a one-way catalyst to convert a glucuronic acid to an iduronic acid residue, displaying an “irreversible” catalytic mode. Our data indicated that the reversible or irreversible catalytic mode strictly depends on the saccharide substrate structures. The biphasic mode of C5-epi offers a novel mechanism to regulate the biosynthesis of HS with the desired biological functions. PMID:22528493

  9. Role of Heparan Sulfate in Cellular Infection of Integrin-Binding Coxsackievirus A9 and Human Parechovirus 1 Isolates.

    Science.gov (United States)

    Merilahti, Pirjo; Karelehto, Eveliina; Susi, Petri

    2016-01-01

    Heparan sulfate/heparin class of proteoglycans (HSPG) have been shown to function in cellular attachment and infection of numerous viruses including picornaviruses. Coxsackievirus A9 (CV-A9) and human parechovirus 1 (HPeV-1) are integrin-binding members in the family Picornaviridae. CV-A9 Griggs and HPeV-1 Harris (prototype) strains have been reported not to bind to heparin, but it was recently shown that some CV-A9 isolates interact with heparin in vitro via VP1 protein with a specific T132R/K mutation. We found that the infectivity of both CV-A9 Griggs and HPeV-1 Harris was reduced by sodium chlorate and heparinase suggestive of HSPG interactions. We analyzed the T132 site in fifty-four (54) CV-A9 clinical isolates and found that only one of them possessed T132/R mutation while the other nine (9) had T132K. We then treated CV-A9 Griggs and HPeV-1 Harris and eight CV-A9 and six HPeV-1 clinical isolates with heparin and protamine. Although infectivity of Griggs strain was slightly reduced (by 25%), heparin treatment did not affect the infectivity of the CV-A9 isolates that do not possess the T132R/K mutation, which is in line with the previous findings. Some of the HPeV-1 isolates were also affected by heparin treatment, which suggested that there may be a specific heparin binding site in HPeV-1. In contrast, protamine (a specific inhibitor of heparin) completely inhibited the infection of both prototypes and clinical CV-A9 and HPeV-1 isolates. We conclude that T132R/K mutation has a role in heparin binding of CV-A9, but we also show data, which suggest that there are other HSPG binding sites in CV-A9. In all, we suggest that HSPGs play a general role in both CV-A9 and HPeV-1 infections.

  10. Role of Heparan Sulfate in Cellular Infection of Integrin-Binding Coxsackievirus A9 and Human Parechovirus 1 Isolates.

    Directory of Open Access Journals (Sweden)

    Pirjo Merilahti

    Full Text Available Heparan sulfate/heparin class of proteoglycans (HSPG have been shown to function in cellular attachment and infection of numerous viruses including picornaviruses. Coxsackievirus A9 (CV-A9 and human parechovirus 1 (HPeV-1 are integrin-binding members in the family Picornaviridae. CV-A9 Griggs and HPeV-1 Harris (prototype strains have been reported not to bind to heparin, but it was recently shown that some CV-A9 isolates interact with heparin in vitro via VP1 protein with a specific T132R/K mutation. We found that the infectivity of both CV-A9 Griggs and HPeV-1 Harris was reduced by sodium chlorate and heparinase suggestive of HSPG interactions. We analyzed the T132 site in fifty-four (54 CV-A9 clinical isolates and found that only one of them possessed T132/R mutation while the other nine (9 had T132K. We then treated CV-A9 Griggs and HPeV-1 Harris and eight CV-A9 and six HPeV-1 clinical isolates with heparin and protamine. Although infectivity of Griggs strain was slightly reduced (by 25%, heparin treatment did not affect the infectivity of the CV-A9 isolates that do not possess the T132R/K mutation, which is in line with the previous findings. Some of the HPeV-1 isolates were also affected by heparin treatment, which suggested that there may be a specific heparin binding site in HPeV-1. In contrast, protamine (a specific inhibitor of heparin completely inhibited the infection of both prototypes and clinical CV-A9 and HPeV-1 isolates. We conclude that T132R/K mutation has a role in heparin binding of CV-A9, but we also show data, which suggest that there are other HSPG binding sites in CV-A9. In all, we suggest that HSPGs play a general role in both CV-A9 and HPeV-1 infections.

  11. Basement membrane chondroitin sulfate proteoglycans: localization in adult rat tissues

    DEFF Research Database (Denmark)

    McCarthy, K J; Couchman, J R

    1990-01-01

    Heparan sulfate proteoglycans have been described as the major proteoglycan component of basement membranes. However, previous investigators have also provided evidence for the presence of chondroitin sulfate glycosaminoglycan in these structures. Recently we described the production...... and characterization of core protein-specific monoclonal antibodies (MAb) against a chondroitin sulfate proteoglycan (CSPG) present in Reichert's membrane, a transient extra-embryonic structure of rodents. This CSPG was also demonstrated to be present in adult rat kidney. We report here the tissue distribution...... sulfate proteoglycans previously described....

  12. Lung heparan sulfates modulate Kfc during increased vascular pressure: evidence for glycocalyx-mediated mechanotransduction

    Science.gov (United States)

    Cluff, Mark; Kingston, Joseph; Hill, Denzil; Chen, Haiyan; Hoehne, Soeren; Malleske, Daniel T.; Kaur, Rajwinederjit

    2012-01-01

    Lung endothelial cells respond to changes in vascular pressure through mechanotransduction pathways that alter barrier function via non-Starling mechanism(s). Components of the endothelial glycocalyx have been shown to participate in mechanotransduction in vitro and in systemic vessels, but the glycocalyx's role in mechanosensing and pulmonary barrier function has not been characterized. Mechanotransduction pathways may represent novel targets for therapeutic intervention during states of elevated pulmonary pressure such as acute heart failure, fluid overload, and mechanical ventilation. Our objective was to assess the effects of increasing vascular pressure on whole lung filtration coefficient (Kfc) and characterize the role of endothelial heparan sulfates in mediating mechanotransduction and associated increases in Kfc. Isolated perfused rat lung preparation was used to measure Kfc in response to changes in vascular pressure in combination with superimposed changes in airway pressure. The roles of heparan sulfates, nitric oxide, and reactive oxygen species were investigated. Increases in capillary pressure altered Kfc in a nonlinear relationship, suggesting non-Starling mechanism(s). nitro-l-arginine methyl ester and heparanase III attenuated the effects of increased capillary pressure on Kfc, demonstrating active mechanotransduction leading to barrier dysfunction. The nitric oxide (NO) donor S-nitrosoglutathione exacerbated pressure-mediated increase in Kfc. Ventilation strategies altered lung NO concentration and the Kfc response to increases in vascular pressure. This is the first study to demonstrate a role for the glycocalyx in whole lung mechanotransduction and has important implications in understanding the regulation of vascular permeability in the context of vascular pressure, fluid status, and ventilation strategies. PMID:22160307

  13. An integrated approach using orthogonal analytical techniques to characterize heparan sulfate structure.

    Science.gov (United States)

    Beccati, Daniela; Lech, Miroslaw; Ozug, Jennifer; Gunay, Nur Sibel; Wang, Jing; Sun, Elaine Y; Pradines, Joël R; Farutin, Victor; Shriver, Zachary; Kaundinya, Ganesh V; Capila, Ishan

    2017-02-01

    Heparan sulfate (HS), a glycosaminoglycan present on the surface of cells, has been postulated to have important roles in driving both normal and pathological physiologies. The chemical structure and sulfation pattern (domain structure) of HS is believed to determine its biological function, to vary across tissue types, and to be modified in the context of disease. Characterization of HS requires isolation and purification of cell surface HS as a complex mixture. This process may introduce additional chemical modification of the native residues. In this study, we describe an approach towards thorough characterization of bovine kidney heparan sulfate (BKHS) that utilizes a variety of orthogonal analytical techniques (e.g. NMR, IP-RPHPLC, LC-MS). These techniques are applied to characterize this mixture at various levels including composition, fragment level, and overall chain properties. The combination of these techniques in many instances provides orthogonal views into the fine structure of HS, and in other instances provides overlapping / confirmatory information from different perspectives. Specifically, this approach enables quantitative determination of natural and modified saccharide residues in the HS chains, and identifies unusual structures. Analysis of partially digested HS chains allows for a better understanding of the domain structures within this mixture, and yields specific insights into the non-reducing end and reducing end structures of the chains. This approach outlines a useful framework that can be applied to elucidate HS structure and thereby provides means to advance understanding of its biological role and potential involvement in disease progression. In addition, the techniques described here can be applied to characterization of heparin from different sources.

  14. Therapeutically targeting glypican-2 via single-domain antibody-based chimeric antigen receptors and immunotoxins in neuroblastoma.

    Science.gov (United States)

    Li, Nan; Fu, Haiying; Hewitt, Stephen M; Dimitrov, Dimiter S; Ho, Mitchell

    2017-08-08

    Neuroblastoma is a childhood cancer that is fatal in almost half of patients despite intense multimodality treatment. This cancer is derived from neuroendocrine tissue located in the sympathetic nervous system. Glypican-2 (GPC2) is a cell surface heparan sulfate proteoglycan that is important for neuronal cell adhesion and neurite outgrowth. In this study, we find that GPC2 protein is highly expressed in about half of neuroblastoma cases and that high GPC2 expression correlates with poor overall survival compared with patients with low GPC2 expression. We demonstrate that silencing of GPC2 by CRISPR-Cas9 or siRNA results in the inhibition of neuroblastoma tumor cell growth. GPC2 silencing inactivates Wnt/β-catenin signaling and reduces the expression of the target gene N-Myc, an oncogenic driver of neuroblastoma tumorigenesis. We have isolated human single-domain antibodies specific for GPC2 by phage display technology and found that the single-domain antibodies can inhibit active β-catenin signaling by disrupting the interaction of GPC2 and Wnt3a. To explore GPC2 as a potential target in neuroblastoma, we have developed two forms of antibody therapeutics, immunotoxins and chimeric antigen receptor (CAR) T cells. Immunotoxin treatment was demonstrated to inhibit neuroblastoma growth in mice. CAR T cells targeting GPC2 eliminated tumors in a disseminated neuroblastoma mouse model where tumor metastasis had spread to multiple clinically relevant sites, including spine, skull, legs, and pelvis. This study suggests GPC2 as a promising therapeutic target in neuroblastoma.

  15. Internalization and trafficking of cell surface proteoglycans and proteoglycan-binding ligands.

    Science.gov (United States)

    Payne, Christine K; Jones, Sara A; Chen, Chen; Zhuang, Xiaowei

    2007-04-01

    Using multicolor live cell imaging in combination with biochemical assays, we have investigated an endocytic pathway mediated by cell surface proteoglycans, primary receptors for many cationic ligands. We have characterized this pathway for a variety of proteoglycan-binding ligands including cationic polymers, lipids and polypeptides. Following clathrin- and caveolin-independent, but flotillin- and dynamin-dependent internalization, proteoglycan-bound ligands associate with flotillin-1-positive vesicles and are efficiently trafficked to late endosomes. The route to late endosomes differs considerably from that following clathrin-mediated endocytosis. The proteoglycan-dependent pathway to late endosomes does not require microtubule-dependent transport or phosphatidyl-inositol-3-OH kinase-dependent sorting from early endosomes. The pathway taken by these ligands is identical to that taken by an antibody against heparan sulfate proteoglycans, suggesting that this mechanism may be used generally by cell surface proteoglycans and proteoglycan-binding ligands that lack secondary receptors.

  16. Proteoglycans and Diabetes.

    Science.gov (United States)

    Hiebert, Linda M

    2017-01-01

    Most proteoglycans are heterogeneous molecules composed of a protein core with glycosaminoglycans (GAGs) attached. GAGs are highly negatively charged molecules that readily bind to enzymes, growth factors, cytokines etc. and as such have many functions. The role played by proteoglycans in diabetes has only recently been investigated. The importance of proteoglycans and the effects of diabetes on proteoglycans are discussed. Possible strategies for reducing diabetic complications associated with preventing proteoglycan destruction are examined. Proteoglycans are altered in the endothelium, vascular wall, kidney, retina, heart, gut epithelial cells, bone and cartilage with diabetes. A decrease in proteoglycans, associated with hyperglycemic conditions, is reported to be due to a decrease in proteoglycan synthesis or an increase in destruction. Destruction may be a result of an upregulation of enzymes that degrade GAGs or destruction by reactive oxygen species. Several studies suggest that upregulation of heparanase and its destruction of heparan sulfate proteoglycans may be responsible for many of the complications associated with diabetes particularly in the kidney and blood vessels leading to chronic kidney disease, atherosclerosis and acute coronary syndrome. Preliminary studies suggest that administration of GAGs may be beneficial in reducing or delaying the harmful consequences of diabetes in the kidney and retina. Changes in proteoglycans are partially responsible for diabetic complications. Recent studies demonstrate that administration of GAGs may reduce or delay diabetic complications. Further studies are required to understand the alterations in proteoglycans associated with diabetes, and the protective potential of administered GAGs. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  17. THYROID HORMONE TREATED ASTROCYTES INDUCE MATURATION OF CEREBRAL CORTICAL NEURONS THROUGH MODULATION OF PROTEOGLYCAN LEVELS

    Directory of Open Access Journals (Sweden)

    Romulo Sperduto Dezonne

    2013-08-01

    Full Text Available Proper brain neuronal circuitry formation and synapse development is dependent on specific cues, either genetic or epigenetic, provided by the surrounding neural environment. Within these signals, thyroid hormones (T3 and T4 play crucial role in several steps of brain morphogenesis including proliferation of progenitor cells, neuronal differentiation, maturation, migration, and synapse formation. The lack of thyroid hormones during childhood is associated with several impair neuronal connections, cognitive deficits, and mental disorders. Many of the thyroid hormones effects are mediated by astrocytes, although the mechanisms underlying these events are still unknown. In this work, we investigated the effect of 3, 5, 3’-triiodothyronine-treated (T3-treated astrocytes on cerebral cortex neuronal differentiation. Culture of neural progenitors from embryonic cerebral cortex mice onto T3-treated astrocyte monolayers yielded an increment in neuronal population, followed by enhancement of neuronal maturation, arborization and neurite outgrowth. In addition, real time PCR assays revealed an increase in the levels of the heparan sulfate proteoglycans, Glypican 1 (GPC-1 and Syndecans 3 e 4 (SDC-3 e SDC-4, followed by a decrease in the levels of the chondroitin sulfate proteoglycan, Versican. Disruption of glycosaminoglycan chains by chondroitinase AC or heparanase III completely abolished the effects of T3-treated astrocytes on neuronal morphogenesis. Our work provides evidence that astrocytes are key mediators of T3 actions on cerebral cortex neuronal development and identified potential molecules and pathways involved in neurite extension; which might eventually contribute to a better understanding of axonal regeneration, synapse formation and neuronal circuitry recover.

  18. Synthesis of 3-O-sulfonated heparan sulfate octasaccharides that inhibit the herpes simplex virus type 1 host-cell interaction

    Science.gov (United States)

    Hu, Yu-Peng; Lin, Shu-Yi; Huang, Cheng-Yen; Zulueta, Medel Manuel L.; Liu, Jing-Yuan; Chang, Wen; Hung, Shang-Cheng

    2011-07-01

    Cell surface carbohydrates play significant roles in a number of biologically important processes. Heparan sulfate, for instance, is a ubiquitously distributed polysulfated polysaccharide that is involved, among other things, in the initial step of herpes simplex virus type 1 (HSV-1) infection. The virus interacts with cell-surface heparan sulfate to facilitate host-cell attachment and entry. 3-O-Sulfonated heparan sulfate has been found to function as an HSV-1 entry receptor. Achieving a complete understanding of these interactions requires the chemical synthesis of such oligosaccharides, but this remains challenging. Here, we present a convenient approach for the synthesis of two irregular 3-O-sulfonated heparan sulfate octasaccharides, making use of a key disaccharide intermediate to acquire different building blocks for the oligosaccharide chain assembly. Despite substantial structural differences, the prepared 3-O-sulfonated sugars blocked viral infection in a dosage-dependent manner with remarkable similarity to one another.

  19. Distribution of two basement membrane proteoglycans through hair follicle development and the hair growth cycle in the rat

    DEFF Research Database (Denmark)

    Couchman, J R; King, J L; McCarthy, K J

    1990-01-01

    The distribution of two distinct populations of basement membrane proteoglycans has been monitored through hair growth development in the rat embryo and subsequent hair growth cycle. An antiserum against a small heparan sulfate proteoglycan uniformly stained the dermal-epidermal junction...... of embryonic rats throughout the period of hair follicle formation. On the other hand, monoclonal antibodies recognizing a basement membrane-specific chondroitin sulfate proteoglycan only weakly stained 16-d embryo dermal-epidermal junction, but strong staining was associated with hair follicle buds...... as they developed. Through the hair growth cycle, it was found that the heparan sulfate proteoglycan persisted around the follicles, while the chondroitin sulfate proteoglycan decreased in amount through catagen until it was undetectable at the base and dermal papilla of the telogen follicle. As anagen commenced...

  20. Syndecan-4 proteoglycan cytoplasmic domain and phosphatidylinositol 4,5-bisphosphate coordinately regulate protein kinase C activity

    DEFF Research Database (Denmark)

    Oh, E S; Woods, A; Lim, S T

    1998-01-01

    Phosphatidylinositol 4,5-bisphosphate (PIP2) is involved in the organization of the actin cytoskeleton by regulating actin-associated proteins. The transmembrane heparan sulfate proteoglycan syndecan-4 also plays a critical role in protein kinase C (PKC) signaling in the formation of focal...

  1. Combining measurements to estimate properties and characterization extent of complex biochemical mixtures; applications to Heparan Sulfate

    Science.gov (United States)

    Pradines, Joël R.; Beccati, Daniela; Lech, Miroslaw; Ozug, Jennifer; Farutin, Victor; Huang, Yongqing; Gunay, Nur Sibel; Capila, Ishan

    2016-04-01

    Complex mixtures of molecular species, such as glycoproteins and glycosaminoglycans, have important biological and therapeutic functions. Characterization of these mixtures with analytical chemistry measurements is an important step when developing generic drugs such as biosimilars. Recent developments have focused on analytical methods and statistical approaches to test similarity between mixtures. The question of how much uncertainty on mixture composition is reduced by combining several measurements still remains mostly unexplored. Mathematical frameworks to combine measurements, estimate mixture properties, and quantify remaining uncertainty, i.e. a characterization extent, are introduced here. Constrained optimization and mathematical modeling are applied to a set of twenty-three experimental measurements on heparan sulfate, a mixture of linear chains of disaccharides having different levels of sulfation. While this mixture has potentially over two million molecular species, mathematical modeling and the small set of measurements establish the existence of nonhomogeneity of sulfate level along chains and the presence of abundant sulfate repeats. Constrained optimization yields not only estimations of sulfate repeats and sulfate level at each position in the chains but also bounds on these levels, thereby estimating the extent of characterization of the sulfation pattern which is achieved by the set of measurements.

  2. Heparan sulfate glycosaminoglycans modulate migration and survival in zebrafish primordial germ cells.

    Science.gov (United States)

    Wei, Ke-Hsuan; Liu, I-Hsuan

    2014-06-01

    Early in embryonic development, primordial germ cells (PGCs) are specified and migrate from the site of their origin to where the gonad develops, following a specific route. Heparan sulfate glycosaminoglycans (HS-GAGs) are ubiquitous in extracellular matrix and the cell surface and have long been speculated to play a role during the migration of PGCs. In line with this speculation, whole-mount immunohistochemistry revealed the existence of HS-GAGs in the vicinity of migrating PGCs in early zebrafish embryos. To examine the roles of HS-GAGs during PGC migration, zebrafish heparanase 1 (hpse1), which degrades HS-GAGs, was cloned and overexpressed specifically in PGCs. The guidance signal for the migration of PGCs was disrupted with the overexpression of hpse1, as cluster formation and marginal localization at the blastoderm were significantly perturbed at 6 hours postfertilization. Furthermore, the number of PGCs was significantly decreased with the lack of vicinal HS-GAGs, as observed in the whole-mount in situ hybridization and quantitative PCR of the PGC marker gene vasa. Terminal deoxynucleotidyl transferase dUTP nick-end labeling indicated significantly increased apoptosis in PGCs overexpressing hpse1, suggesting that HS-GAGs contribute to the maintenance of PGC survival. In conclusion, HS-GAGs play multifaceted roles in PGCs during migration and are required both for guidance signals and multiplication of PGCs. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Targeting phosphatase-dependent proteoglycan switch for rheumatoid arthritis therapy.

    Science.gov (United States)

    Doody, Karen M; Stanford, Stephanie M; Sacchetti, Cristiano; Svensson, Mattias N D; Coles, Charlotte H; Mitakidis, Nikolaos; Kiosses, William B; Bartok, Beatrix; Fos, Camille; Cory, Esther; Sah, Robert L; Liu-Bryan, Ru; Boyle, David L; Arnett, Heather A; Mustelin, Tomas; Corr, Maripat; Esko, Jeffrey D; Tremblay, Michel L; Firestein, Gary S; Aricescu, A Radu; Bottini, Nunzio

    2015-05-20

    Despite the availability of several therapies for rheumatoid arthritis (RA) that target the immune system, a large number of RA patients fail to achieve remission. Joint-lining cells, called fibroblast-like synoviocytes (FLS), become activated during RA and mediate joint inflammation and destruction of cartilage and bone. We identify RPTPσ, a transmembrane tyrosine phosphatase, as a therapeutic target for FLS-directed therapy. RPTPσ is reciprocally regulated by interactions with chondroitin sulfate or heparan sulfate containing extracellular proteoglycans in a mechanism called the proteoglycan switch. We show that the proteoglycan switch regulates FLS function. Incubation of FLS with a proteoglycan-binding RPTPσ decoy protein inhibited cell invasiveness and attachment to cartilage by disrupting a constitutive interaction between RPTPσ and the heparan sulfate proteoglycan syndecan-4. RPTPσ mediated the effect of proteoglycans on FLS signaling by regulating the phosphorylation and cytoskeletal localization of ezrin. Furthermore, administration of the RPTPσ decoy protein ameliorated in vivo human FLS invasiveness and arthritis severity in the K/BxN serum transfer model of RA. Our data demonstrate that FLS are regulated by an RPTPσ-dependent proteoglycan switch in vivo, which can be targeted for RA therapy. We envision that therapies targeting the proteoglycan switch or its intracellular pathway in FLS could be effective as a monotherapy or in combination with currently available immune-targeted agents to improve control of disease activity in RA patients. Copyright © 2015, American Association for the Advancement of Science.

  4. Retention of the Structure and Function of Heparan Sulfate Biomaterials After Gamma Irradiation.

    Science.gov (United States)

    Smith, Raymond A A; Chua, R J E; Carnachan, Susan M; Tan, Clarissa L L; Sims, Ian M; Hinkley, Simon F R; Nurcombe, Victor; Cool, Simon M

    2017-11-30

    Heparan sulfate (HS) is a highly heterogeneous polysaccharide implicated in many important biological processes. Our previous work has demonstrated that a particular affinity-selected HS (referred to henceforth as "HS3") is capable of enhancing the osteogenic effects of bone morphogenetic protein 2 (BMP2). Here, we gamma-irradiated HS with 26 kGy of ionizing radiation to determine how this affected the structure, composition, and function. Initial structural studies were performed on a commercial preparation of HS as a proof-of-concept. Gamma irradiation of this HS preparation did not significantly alter its structure or composition compared to nonirradiated material, as demonstrated by proton nuclear magnetic resonance spectroscopy, molecular weight analysis using size exclusion chromatography, and disaccharide compositional analysis. When HS3 was gamma irradiated, no significant effect on binding affinity toward BMP2 was observed, based on competitive surface plasmon resonance and differential scanning fluorimetry assays. Furthermore, irradiation did not significantly affect HS3's ability to synergistically enhance the osteogenic effects of BMP2 in vitro; as measured by the relative abundance of osteogenic transcripts in transdifferentiating C2C12 murine myoblasts. Additionally, no significant differences were observed in the levels of alkaline phosphatase (ALP) or calcium deposition in C2C12s treated with BMP2, together with the irradiated, or nonirradiated HS3. Irradiation of HS3 incorporated into collagen type I sponges did not affect its ability to enhance BMP2-mediated ALP expression in C2C12 cells. Our data confirm that gamma irradiation is a cost-effective and viable solution for the sterilization of HS species that allows the retention of its structure and biological function. The work suggests an effective way to incorporate clinically compatible HS species into orthotic implants, scaffolds, and other medical devices for use in the treatment of a range

  5. Synthetic heparan sulfate oligosaccharides inhibit endothelial cell functions essential for angiogenesis.

    Directory of Open Access Journals (Sweden)

    Claire L Cole

    2010-07-01

    Full Text Available Heparan sulfate (HS is an important regulator of the assembly and activity of various angiogenic signalling complexes. However, the significance of precisely defined HS structures in regulating cytokine-dependent angiogenic cellular functions and signalling through receptors regulating angiogenic responses remains unclear. Understanding such structure-activity relationships is important for the rational design of HS fragments that inhibit HS-dependent angiogenic signalling complexes.We synthesized a series of HS oligosaccharides ranging from 7 to 12 saccharide residues that contained a repeating disaccharide unit consisting of iduronate 2-O-sulfate linked to glucosamine with or without N-sulfate. The ability of oligosaccharides to compete with HS for FGF2 and VEGF165 binding significantly increased with oligosaccharide length and sulfation. Correspondingly, the inhibitory potential of oligosaccharides against FGF2- and VEGF165-induced endothelial cell responses was greater in longer oligosaccharide species that were comprised of disaccharides bearing both 2-O- and N-sulfation (2SNS. FGF2- and VEGF165-induced endothelial cell migration were inhibited by longer 2SNS oligosaccharide species with 2SNS dodecasaccharide activity being comparable to that of receptor tyrosine kinase inhibitors targeting FGFR or VEGFR-2. Moreover, the 2SNS dodecasaccharide ablated FGF2- or VEGF165-induced phosphorylation of FAK and assembly of F-actin in peripheral lamellipodia-like structures. In contrast, FGF2-induced endothelial cell proliferation was only moderately inhibited by longer 2SNS oligosaccharides. Inhibition of FGF2- and VEGF165-dependent endothelial tube formation strongly correlated with oligosaccharide length and sulfation with 10-mer and 12-mer 2SNS oligosaccharides being the most potent species. FGF2- and VEGF165-induced activation of MAPK pathway was inhibited by biologically active oligosaccharides correlating with the specific phosphorylation

  6. Heparan Sulfate Regulates the Structure and Function of Osteoprotegerin in Osteoclastogenesis.

    Science.gov (United States)

    Li, Miaomiao; Yang, Shuying; Xu, Ding

    2016-11-11

    Osteoprotegerin (OPG), a decoy receptor secreted by osteoblasts, is a major negative regulator of bone resorption. It functions by neutralizing the receptor activator of nuclear factor κB ligand (RANKL), which plays a central role in promoting osteoclastogenesis. OPG is known to be a high-affinity heparan sulfate (HS)-binding protein. Presumably, HS could regulate the function of OPG and affect how it inhibits RANKL. However, the molecular detail of HS-OPG interaction remains poorly understood, which hinders our understanding of how HS functions in osteoclastogenesis. Here we report mapping of the HS-binding site of OPG. The HS-binding site, identified by mutagenesis study, consists of eight basic residues that are located mostly at the junction of the second death domain and the C-terminal domain. We further show that heparin-derived dodecasaccharide is able to induce dimerization of OPG monomers with a stoichiometry of 1:1. Small-angle X-ray scattering analysis revealed that upon binding of HS, OPG undergoes a dramatic conformational change, resulting in a more compact and less flexible structure. Importantly, we present here three lines of evidence that HS, OPG, and RANKL form a stable ternary complex. Using a HS binding-deficient OPG mutant, we further show that in an osteoblast/bone marrow macrophage co-culture system, immobilization of OPG by HS at the osteoblast cell surface substantially lowers the inhibitory threshold of OPG toward RANKL. These discoveries strongly suggest that HS plays an active role in regulating OPG-RANKL interaction and osteoclastogenesis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Increased deposition of glycosaminoglycans and altered structure of heparan sulfate in idiopathic pulmonary fibrosis.

    Science.gov (United States)

    Westergren-Thorsson, Gunilla; Hedström, Ulf; Nybom, Annika; Tykesson, Emil; Åhrman, Emma; Hornfelt, Marie; Maccarana, Marco; van Kuppevelt, Toin H; Dellgren, Göran; Wildt, Marie; Zhou, Xiao-Hong; Eriksson, Leif; Bjermer, Leif; Hallgren, Oskar

    2017-02-01

    Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant deposition of extracellular matrix (ECM) constituents, including glycosaminoglycans (GAGs), that may play a role in remodelling processes by influencing critical mediators such as growth factors. We hypothesize that GAGs may be altered in IPF and that this contribute to create a pro-fibrotic environment. The aim of this study was therefore to examine the fine structure of heparan sulfate (HS), chondroitin/dermatan sulfate (CS/DS) and hyaluronan (HA) in lung samples from IPF patients and from control subjects. GAGs in lung samples from severe IPF patients and donor lungs were analyzed with HPLC. HS was assessed by immunohistochemistry and collagen was quantified as hydroxyproline content. The total amount of HS, CS/DS and HA was increased in IPF lungs but there was no significant difference in the total collagen content. We found a relative increase in total sulfation of HS due to increment of 2-O, 6-O and N-sulfation and a higher proportion of sulfation in CS/DS. Highly sulfated HS was located in the border zone between denser areas and more normal looking alveolar parenchyma in basement membranes of blood vessels and airways, that were immuno-positive for perlecan, as well as on the cell surface of spindle-shaped cells in the alveolar interstitium. These findings show for the first time that both the amount and structure of glycosaminoglycans are altered in IPF. These changes may contribute to the tissue remodelling in IPF by altering growth factor retention and activity, creating a pro-fibrotic ECM landscape. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  8. Gas-Phase Analysis of the Complex of Fibroblast GrowthFactor 1 with Heparan Sulfate: A Traveling Wave Ion Mobility Spectrometry (TWIMS) and Molecular Modeling Study

    Science.gov (United States)

    Zhao, Yuejie; Singh, Arunima; Xu, Yongmei; Zong, Chengli; Zhang, Fuming; Boons, Geert-Jan; Liu, Jian; Linhardt, Robert J.; Woods, Robert J.; Amster, I. Jonathan

    2017-01-01

    Fibroblast growth factors (FGFs) regulate several cellular developmental processes by interacting with cell surface heparan proteoglycans and transmembrane cell surface receptors (FGFR). The interaction of FGF with heparan sulfate (HS) is known to induce protein oligomerization, increase the affinity of FGF towards its receptor FGFR, promoting the formation of the HS-FGF-FGFR signaling complex. Although the role of HS in the signaling pathways is well recognized, the details of FGF oligomerization and formation of the ternary signaling complex are still not clear, with several conflicting models proposed in literature. Here, we examine the effect of size and sulfation pattern of HS upon FGF1 oligomerization, binding stoichiometry and conformational stability, through a combination of ion mobility (IM) and theoretical modeling approaches. Ion mobility-mass spectrometry (IMMS) of FGF1 in the presence of several HS fragments ranging from tetrasaccharide (dp4) to dodecasaccharide (dp12) in length was performed. A comparison of the binding stoichiometry of variably sulfated dp4 HS to FGF1 confirmed the significance of the previously known high-affinity binding motif in FGF1 dimerization, and demonstrated that certain tetrasaccharide-length fragments are also capable of inducing dimerization of FGF1. The degree of oligomerization was found to increase in the presence of dp12 HS, and a general lack of specificity for longer HS was observed. Additionally, collision cross-sections (CCSs) of several FGF1-HS complexes were calculated, and were found to be in close agreement with experimental results. Based on the (CCSs) a number of plausible binding modes of 2:1 and 3:1 FGF1-HS are proposed.

  9. LARGE2-dependent glycosylation confers laminin-binding ability on proteoglycans.

    Science.gov (United States)

    Inamori, Kei-Ichiro; Beedle, Aaron M; de Bernabé, Daniel Beltrán-Valero; Wright, Michael E; Campbell, Kevin P

    2016-12-01

    Both LARGE1 (formerly LARGE) and its paralog LARGE2 are bifunctional glycosyltransferases with xylosy- and glucuronyltransferase activities, and are capable of synthesizing polymers composed of a repeating disaccharide [-3Xylα1,3GlcAβ1-]. Post-translational modification of the O-mannosyl glycan of α-dystroglycan (α-DG) with the polysaccharide is essential for it to act as a receptor for ligands in the extracellular matrix (ECM), and both LARGE paralogs contribute to the modification in vivo. LARGE1 and LARGE2 have different tissue distribution profiles and enzymatic properties; however, the functional difference of the homologs remains to be determined, and α-DG is the only known substrate for the modification by LARGE1 or LARGE2. Here we show that LARGE2 can modify proteoglycans (PGs) with the laminin-binding glycan. We found that overexpression of LARGE2, but not LARGE1, mediates the functional modification on the surface of DG-/-, Pomt1-/- and Fktn-/- embryonic stem cells. We identified a heparan sulfate-PG glypican-4 as a substrate for the LARGE2-dependent modification by affinity purification and subsequent mass spectrometric analysis. Furthermore, we showed that LARGE2 could modify several additional PGs with the laminin-binding glycan, most likely within the glycosaminoglycan (GAG)-protein linkage region. Our results indicate that LARGE2 can modify PGs with the GAG-like polysaccharide composed of xylose and glucuronic acid to confer laminin binding. Thus, LARGE2 may play a differential role in stabilizing the basement membrane and modifying its functions by augmenting the interactions between laminin globular domain-containing ECM proteins and PGs. © The Author 2016. Published by Oxford University Press.

  10. Pectin of Prunus domestica L. alters sulfated structure of cell-surface heparan sulfate in differentiated Caco-2 cells through stimulation of heparan sulfate 6-O-endosulfatase-2.

    Science.gov (United States)

    Nishida, Mitsutaka; Murata, Kazuma; Kanamaru, Yoshihiro; Yabe, Tomio

    2014-01-01

    Although previous reports have suggested that pectin induces morphological changes of the small intestine in vivo, the molecular mechanisms have not been elucidated. As heparan sulfate plays important roles in development of the small intestine, to verify the involvement of heparan sulfate (HS) in the pectin-induced morphological changes of the small intestine, the effects of pectin from Prunus domestica L. on cell-surface HS were investigated using differentiated Caco-2 cells. Disaccharide compositional analysis revealed that sulfated structures of HS were markedly changed by pectin administration. Real-time RT-PCR showed that pectin upregulated human HS 6-O-endosulfatase-2 (HSulf-2) expression and markedly inhibited HSulf-1 expression. Furthermore, inhibition analysis suggested that pretreatment with fibronectin III1C fragment, RGD peptide, and ERK1/2 inhibitor suppressed pectin-induced HSulf-2 expression. These observations indicate that pectin induced the expression of HSulf-2 through the interaction with fibronectin, α5β1 integrin, and ERK1/2, thereby regulating the sulfated structure of HS on differentiated Caco-2 cells.

  11. Genomic organization of the human heparan sulfate-N-deacetylase/N-sulfotransferase gene: Exclusion from a causative role in the pathogenesis of Treacher Collins syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Gladwin, A.J.; Dixon, J.; Loftus, S.K.; Wasmuth, J.J.; Dixon, M.J. [Univ. of Manchester (United Kingdom)]|[Univ. of California, Irvine, CA (United States)

    1996-03-05

    Heparan sulfate-N-deacetylase/N-sulfotransferase (HSST) catalyzes both the N-deacetylation and the N-sulfation of heparan sulfate. Previous studies have resulted in the isolation of the human HSST gene from within the Treacher Collins syndrome locus (TCOF1) critical region on 5q. In the present study, the genomic organization of the HSST gene has been elucidated, and the 14 exons identified have been tested for TCOF1-specific mutations. As a result of these studies, mutations within the coding sequence and adjacent splice junctions of HSST can be excluded from a causative role in the pathogenesis of Treacher Collins syndrome. 13 refs., 1 fig., 2 tabs.

  12. HABA-based ionic liquid matrices for UV-MALDI-MS analysis of heparin and heparan sulfate oligosaccharides.

    Science.gov (United States)

    Przybylski, Cedric; Gonnet, Florence; Bonnaffé, David; Hersant, Yael; Lortat-Jacob, Hugues; Daniel, Regis

    2010-02-01

    Polysulfated carbohydrates such as heparin (HP) and heparan sulfate (HS) are not easily amenable to usual ultraviolet matrix-assisted laser desorption/ionization-mass spectrometry (UV-MALDI)-MS analysis due to the thermal lability of their O- and N-SO(3) moieties, and their poor ionization efficiency with common crystalline matrices. Recently, ionic liquid matrices showed considerable advantages over conventional matrices for MALDI-MS of acidic compounds. Two new ionic liquid matrices (ILMs) based on the combination of 2-(4-hydroxyphenylazo)benzoic acid (HABA) with 1,1,3,3-tetramethylguanidine and spermine were evaluated in the study herein. Both ILMs were successfully applied to the analysis of synthetic heparin oligosaccharides of well-characterized structures as well as to heparan sulfate-derived oligosaccharides from enzymatic depolymerization. HABA-based ILMs showed improved signal-to-noise ratio as well as a decrease of fragmentation/desulfation processes and cation exchange. Sulfated oligosaccharides were detected with higher sensitivity than usual crystalline matrices, and their intact fully O- and N-sulfated species [M-Na](-) were easily observed on mass spectra. MALDI-MS characterization of challenging analytes such as heparin octasaccharide carrying 8-O and 4 N-sulfo groups, and heparin octadecasulfated dodecasaccharide was successfully achieved.

  13. cDNA cloning of the basement membrane chondroitin sulfate proteoglycan core protein, bamacan: a five domain structure including coiled-coil motifs

    DEFF Research Database (Denmark)

    Wu, R R; Couchman, J R

    1997-01-01

    Basement membranes contain several proteoglycans, and those bearing heparan sulfate glycosaminoglycans such as perlecan and agrin usually predominate. Most mammalian basement membranes also contain chondroitin sulfate, and a core protein, bamacan, has been partially characterized. We have now....../translation product from a full-length bamacan cDNA. The unusual structure of this proteoglycan is indicative of specific functional roles in basement membrane physiology, commensurate with its distinct expression in development and changes in disease models....

  14. Positional information in axolotl and mouse limb extracellular matrix is mediated via heparan sulfate and fibroblast growth factor during limb regeneration in the axolotl (Ambystoma mexicanum).

    Science.gov (United States)

    Phan, Anne Q; Lee, Jangwoo; Oei, Michelle; Flath, Craig; Hwe, Caitlyn; Mariano, Rachele; Vu, Tiffany; Shu, Cynthia; Dinh, Andrew; Simkin, Jennifer; Muneoka, Ken; Bryant, Susan V; Gardiner, David M

    2015-08-01

    Urodele amphibians are unique among adult vertebrates in their ability to regenerate complex body structures after traumatic injury. In salamander regeneration, the cells maintain a memory of their original position and use this positional information to recreate the missing pattern. We used an in vivo gain-of-function assay to determine whether components of the extracellular matrix (ECM) have positional information required to induce formation of new limb pattern during regeneration. We discovered that salamander limb ECM has a position-specific ability to either inhibit regeneration or induce de novo limb structure, and that this difference is dependent on heparan sulfates that are associated with differential expression of heparan sulfate sulfotransferases. We also discovered that an artificial ECM containing only heparan sulfate was sufficient to induce de novo limb pattern in salamander limb regeneration. Finally, ECM from mouse limbs is capable of inducing limb pattern in axolotl blastemas in a position-specific, developmental-stage-specific, and heparan sulfate-dependent manner. This study demonstrates a mechanism for positional information in regeneration and establishes a crucial functional link between salamander regeneration and mammals.

  15. Mutations in Biosynthetic Enzymes for the Protein Linker Region of Chondroitin/Dermatan/Heparan Sulfate Cause Skeletal and Skin Dysplasias

    Directory of Open Access Journals (Sweden)

    Shuji Mizumoto

    2015-01-01

    Full Text Available Glycosaminoglycans, including chondroitin, dermatan, and heparan sulfate, have various roles in a wide range of biological events such as cell signaling, cell proliferation, tissue morphogenesis, and interactions with various growth factors. Their polysaccharides covalently attach to the serine residues on specific core proteins through the common linker region tetrasaccharide, -xylose-galactose-galactose-glucuronic acid, which is produced through the stepwise addition of respective monosaccharides by four distinct glycosyltransferases. Mutations in the human genes encoding the glycosyltransferases responsible for the biosynthesis of the linker region tetrasaccharide cause a number of genetic disorders, called glycosaminoglycan linkeropathies, including Desbuquois dysplasia type 2, spondyloepimetaphyseal dysplasia, Ehlers-Danlos syndrome, and Larsen syndrome. This review focused on recent studies on genetic diseases caused by defects in the biosynthesis of the common linker region tetrasaccharide.

  16. An unusual dependence of human herpesvirus-8 glycoproteins-induced cell-to-cell fusion on heparan sulfate

    Energy Technology Data Exchange (ETDEWEB)

    Tiwari, Vaibhav [Department of Ophthalmology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Department of Basic Medical Sciences, College of Osteopathic Medicine of the Pacific and College of Optometry, Western University of Health Sciences, Pomona, CA 91766 (United States); Darmani, Nissar A.; Thrush, Gerald R. [Department of Basic Medical Sciences, College of Osteopathic Medicine of the Pacific and College of Optometry, Western University of Health Sciences, Pomona, CA 91766 (United States); Shukla, Deepak, E-mail: dshukla@uic.edu [Department of Ophthalmology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612 (United States)

    2009-12-18

    Human herpesvirus-8 (HHV-8) is known to interact with cell surface heparan sulfate (HS) for entry into a target cell. Here we investigated the role of HS during HHV-8 glycoproteins-induced cell fusion. Interestingly, the observed fusion demonstrated an unusual dependence on HS as evident from following lines of evidence: (1) a significant reduction in cell-to-cell fusion occurred when target cells were treated with heparinase; (2) in a competition assay, when the effector cells expressing HHV-8 glycoproteins were challenged with soluble HS, cell-to-cell fusion was reduced; and, (3) co-expression of HHV-8 glycoproteins gH-gL on target cells resulted in inhibition of cell surface HS expression. Taken together, our results indicate that cell surface HS can play an additional role during HHV-8 pathogenesis.

  17. Cell entry and trafficking of human adenovirus bound to blood factor X is determined by the fiber serotype and not hexon:heparan sulfate interaction.

    Directory of Open Access Journals (Sweden)

    Stéphanie Corjon

    Full Text Available Human adenovirus serotype 5 (HAdV5-based vectors administered intravenously accumulate in the liver as the result of their direct binding to blood coagulation factor X (FX and subsequent interaction of the FX-HAdV5 complex with heparan sulfate proteoglycan (HSPG at the surface of liver cells. Intriguingly, the serotype 35 fiber-pseudotyped vector HAdV5F35 has liver transduction efficiencies 4-logs lower than HAdV5, even though both vectors carry the same hexon capsomeres. In order to reconcile this apparent paradox, we investigated the possible role of other viral capsid proteins on the FX/HSPG-mediated cellular uptake of HAdV5-based vectors. Using CAR- and CD46-negative CHO cells varying in HSPG expression, we confirmed that FX bound to serotype 5 hexon protein and to HAdV5 and HAdV5F35 virions via its Gla-domain, and enhanced the binding of both vectors to surface-immobilized hypersulfated heparin and cellular HSPG. Using penton mutants, we found that the positive effect of FX on HAdV5 binding to HSPG and cell transduction did not depend on the penton base RGD and fiber shaft KKTK motifs. However, we found that FX had no enhancing effect on the HAdV5F35-mediated cell transduction, but a negative effect which did not involve the cell attachment or endocytic step, but the intracellular trafficking and nuclear import of the FX-HAdV5F35 complex. By cellular imaging, HAdV5F35 particles were observed to accumulate in the late endosomal compartment, and were released in significant amounts into the extracellular medium via exocytosis. We showed that the stability of serotype 5 hexon:FX interaction was higher at low pH compared to neutral pH, which could account for the retention of FX-HAdV5F35 complexes in the late endosomes. Our results suggested that, despite the high affinity interaction of hexon capsomeres to FX and cell surface HSPG, the adenoviral fiber acted as the dominant determinant of the internalization and trafficking pathway of HAdV5

  18. Lung heparan sulfates modulate K(fc) during increased vascular pressure: evidence for glycocalyx-mediated mechanotransduction.

    Science.gov (United States)

    Dull, Randal O; Cluff, Mark; Kingston, Joseph; Hill, Denzil; Chen, Haiyan; Hoehne, Soeren; Malleske, Daniel T; Kaur, Rajwinederjit

    2012-05-01

    Lung endothelial cells respond to changes in vascular pressure through mechanotransduction pathways that alter barrier function via non-Starling mechanism(s). Components of the endothelial glycocalyx have been shown to participate in mechanotransduction in vitro and in systemic vessels, but the glycocalyx's role in mechanosensing and pulmonary barrier function has not been characterized. Mechanotransduction pathways may represent novel targets for therapeutic intervention during states of elevated pulmonary pressure such as acute heart failure, fluid overload, and mechanical ventilation. Our objective was to assess the effects of increasing vascular pressure on whole lung filtration coefficient (K(fc)) and characterize the role of endothelial heparan sulfates in mediating mechanotransduction and associated increases in K(fc). Isolated perfused rat lung preparation was used to measure K(fc) in response to changes in vascular pressure in combination with superimposed changes in airway pressure. The roles of heparan sulfates, nitric oxide, and reactive oxygen species were investigated. Increases in capillary pressure altered K(fc) in a nonlinear relationship, suggesting non-Starling mechanism(s). nitro-l-arginine methyl ester and heparanase III attenuated the effects of increased capillary pressure on K(fc), demonstrating active mechanotransduction leading to barrier dysfunction. The nitric oxide (NO) donor S-nitrosoglutathione exacerbated pressure-mediated increase in K(fc). Ventilation strategies altered lung NO concentration and the K(fc) response to increases in vascular pressure. This is the first study to demonstrate a role for the glycocalyx in whole lung mechanotransduction and has important implications in understanding the regulation of vascular permeability in the context of vascular pressure, fluid status, and ventilation strategies.

  19. Differential expression of proteoglycans in tissue remodeling and lymphangiogenesis after experimental renal transplantation in rats.

    Science.gov (United States)

    Rienstra, Heleen; Katta, Kirankumar; Celie, Johanna W A M; van Goor, Harry; Navis, Gerjan; van den Born, Jacob; Hillebrands, Jan-Luuk

    2010-02-05

    Chronic transplant dysfunction explains the majority of late renal allograft loss and is accompanied by extensive tissue remodeling leading to transplant vasculopathy, glomerulosclerosis and interstitial fibrosis. Matrix proteoglycans mediate cell-cell and cell-matrix interactions and play key roles in tissue remodeling. The aim of this study was to characterize differential heparan sulfate proteoglycan and chondroitin sulfate proteoglycan expression in transplant vasculopathy, glomerulosclerosis and interstitial fibrosis in renal allografts with chronic transplant dysfunction. Renal allografts were transplanted in the Dark Agouti-to-Wistar Furth rat strain combination. Dark Agouti-to-Dark Agouti isografts and non-transplanted Dark Agouti kidneys served as controls. Allograft and isograft recipients were sacrificed 66 and 81 days (mean) after transplantation, respectively. Heparan sulfate proteoglycan (collXVIII, perlecan and agrin) and chondroitin sulfate proteoglycan (versican) expression, as well as CD31 and LYVE-1 (vascular and lymphatic endothelium, respectively) expression were (semi-) quantitatively analyzed using immunofluorescence. Arteries with transplant vasculopathy and sclerotic glomeruli in allografts displayed pronounced neo-expression of collXVIII and perlecan. In contrast, in interstitial fibrosis expression of the chondroitin sulfate proteoglycan versican dominated. In the cortical tubular basement membranes in both iso- and allografts, induction of collXVIII was detected. Allografts presented extensive lymphangiogenesis (pproteoglycans being expressed are tightly associated with tissue remodeling after renal transplantation. Therefore, proteoglycans might be potential targets for clinical intervention in renal chronic transplant dysfunction.

  20. Proteoglycans and their roles in brain cancer.

    Science.gov (United States)

    Wade, Anna; Robinson, Aaron E; Engler, Jane R; Petritsch, Claudia; James, C David; Phillips, Joanna J

    2013-05-01

    Glioblastoma, a malignant brain cancer, is characterized by abnormal activation of receptor tyrosine kinase signalling pathways and a poor prognosis. Extracellular proteoglycans, including heparan sulfate and chondroitin sulfate, play critical roles in the regulation of cell signalling and migration via interactions with extracellular ligands, growth factor receptors and extracellular matrix components, as well as intracellular enzymes and structural proteins. In cancer, proteoglycans help drive multiple oncogenic pathways in tumour cells and promote critical tumour-microenvironment interactions. In the present review, we summarize the evidence for proteoglycan function in gliomagenesis and examine the expression of proteoglycans and their modifying enzymes in human glioblastoma using data obtained from The Cancer Genome Atlas (http://cancergenome.nih.gov/). Furthermore, we demonstrate an association between specific proteoglycan alterations and changes in receptor tyrosine kinases. Based on these data, we propose a model in which proteoglycans and their modifying enzymes promote receptor tyrosine kinase signalling and progression in glioblastoma, and we suggest that cancer-associated proteoglycans are promising biomarkers for disease and therapeutic targets. © 2013 The Authors Journal compilation © 2013 FEBS.

  1. The effect of heparan sulfate application on bone formation during distraction osteogenesis.

    Directory of Open Access Journals (Sweden)

    Marie Gdalevitch

    Full Text Available Bone morphogenetic proteins (BMPs are recognized for their ability to induce bone formation in vivo and in vitro. Their osteogenic and osteoinductive properties are tightly regulated by the secretion of specific BMP antagonists, which have been shown to physically bind and sometimes be blocked by the extracellular proteoglycan heparan sulphate side chains (from hereon referred to as HS. The purpose of this study was to investigate if local application of 5 µg of HS proteoglycan to a bone regenerate site in a mouse model of distraction osteogenesis (DO can accelerate bone healing and affect the expression of key members of the BMP signaling pathway. DO was performed on the right tibia of 115 adult male wild-type mice. At mid-distraction (day 11, half the group was injected locally with 5 µg of HS, while the other half was injected with saline. The mice were sacrificed at 2 time-points: mid-consolidation (34 days and full consolidation (51 days. The distracted tibial zone was then collected for analysis by μCT, radiology, biomechanical testing, immunohistochemistry, and histology. While μCT data showed no statistically significant difference in bone formation, the results of biomechanical testing in stiffness and ultimate force were significantly lower in the HS-injected bones at 51 days, compared to controls. Immunohistochemistry results also suggested a decrease in expression of several key members of the BMP signaling pathway at 34 days. Furthermore, wound dehiscence and infection rates were significantly elevated in the HS group compared to the controls, which resulted in a higher rate of euthanasia in the treatment group. Our findings demonstrate that exogenous application of 5 µg of HS in the distracted gap of a murine model had a negative impact on bone and wound healing.

  2. Interaction of the amyloid precursor protein-like protein 1 (APLP1) E2 domain with heparan sulfate involves two distinct binding modes

    Energy Technology Data Exchange (ETDEWEB)

    Dahms, Sven O., E-mail: sdahms@fli-leibniz.de [Leibniz Institute for Age Research (FLI), Beutenbergstrasse 11, 07745 Jena (Germany); Mayer, Magnus C. [Freie Universität Berlin, Thielallee 63, 14195 Berlin (Germany); Miltenyi Biotec GmbH, Robert-Koch-Strasse 1, 17166 Teterow (Germany); Roeser, Dirk [Leibniz Institute for Age Research (FLI), Beutenbergstrasse 11, 07745 Jena (Germany); Multhaup, Gerd [McGill University Montreal, Montreal, Quebec H3G 1Y6 (Canada); Than, Manuel E., E-mail: sdahms@fli-leibniz.de [Leibniz Institute for Age Research (FLI), Beutenbergstrasse 11, 07745 Jena (Germany)

    2015-03-01

    Two X-ray structures of APLP1 E2 with and without a heparin dodecasaccharide are presented, revealing two distinct binding modes of the protein to heparan sulfate. The data provide a mechanistic explanation of how APP-like proteins bind to heparan sulfates and how they specifically recognize nonreducing structures of heparan sulfates. Beyond the pathology of Alzheimer’s disease, the members of the amyloid precursor protein (APP) family are essential for neuronal development and cell homeostasis in mammals. APP and its paralogues APP-like protein 1 (APLP1) and APP-like protein 2 (APLP2) contain the highly conserved heparan sulfate (HS) binding domain E2, which effects various (patho)physiological functions. Here, two crystal structures of the E2 domain of APLP1 are presented in the apo form and in complex with a heparin dodecasaccharide at 2.5 Å resolution. The apo structure of APLP1 E2 revealed an unfolded and hence flexible N-terminal helix αA. The (APLP1 E2){sub 2}–(heparin){sub 2} complex structure revealed two distinct binding modes, with APLP1 E2 explicitly recognizing the heparin terminus but also interacting with a continuous heparin chain. The latter only requires a certain register of the sugar moieties that fits to a positively charged surface patch and contributes to the general heparin-binding capability of APP-family proteins. Terminal binding of APLP1 E2 to heparin specifically involves a structure of the nonreducing end that is very similar to heparanase-processed HS chains. These data reveal a conserved mechanism for the binding of APP-family proteins to HS and imply a specific regulatory role of HS modifications in the biology of APP and APP-like proteins.

  3. A single amino acid substitution in the cytoplasmic tail of the glycoprotein B of herpes simplex virus 1 affects both syncytium formation and binding to intracellular heparan sulfate.

    Science.gov (United States)

    Diakidi-Kosta, A; Michailidou, G; Kontogounis, G; Sivropoulou, A; Arsenakis, M

    2003-05-01

    Herpes simplex virus 1 (HSV-1) (S) is a spontaneous syncytial mutant derived from the prototype HSV-1(F) after extensive plaque purification, and produces large syncytial plaques on Vero cells. Marker transfer experiments and DNA sequence analysis mapped the syncytial phenotype to a T-C base substitution at codon 787 of the cytoplasmic domain of mature gB, that results in Leu to Pro substitution and consequently belongs to the syn 3 locus. Both the cytoplasmic and the extracellular domains of gB are active in the fusion event since the addition of anti-gB monoclonal antibodies that recognize the extracellular domain of gB prevent HSV-1(S) induced cell fusion. Similarly, gD also participates in cell fusion since addition of anti-gD monoclonal antibodies also prevent HSV-1(S) induced cell fusion. Furthermore the glycoproteins B and D formed complexes in cells infected with mutant or wild type viruses. The amount of gB bound to total heparan sulfate is lower in the mutant than in the wild type strain. This difference becomes particularly profound when gB is associated with a portion of heparan sulfate intercalated to the membranes. The discrepancy in the binding of the mutant and wild type gB to heparan sulfate may be related to the mechanism of cell fusion induced by HSV-1(S).

  4. Basement membrane chondroitin sulfate proteoglycan alterations in a rat model of polycystic kidney disease

    DEFF Research Database (Denmark)

    Ehara, T; Carone, F A; McCarthy, K J

    1994-01-01

    Alterations in basement membrane components, notably proteoglycans, in a rat model of polycystic kidney disease have been investigated. Rats were fed phenol II (2-amino-4-hydroxyphenyl-5-phenyl thiazole) for 4 days and then changed to normal diet for a 7-day recovery period. Marked dilation...... membrane heparan sulfate proteoglycan core protein related to perlecan did not diminish but rather stained affected tubules intensely, whereas laminin, on the other hand, was apparently diminished in the basement membranes of the cystic tubules. Type IV collagen staining did not change through disease...... in this disease....

  5. Improved liquid chromatography-MS/MS of heparan sulfate oligosaccharides via chip-based pulsed makeup flow.

    Science.gov (United States)

    Huang, Yu; Shi, Xiaofeng; Yu, Xiang; Leymarie, Nancy; Staples, Gregory O; Yin, Hongfeng; Killeen, Kevin; Zaia, Joseph

    2011-11-01

    Microfluidic chip-based hydrophilic interaction chromatography (HILIC) is a useful separation system for liquid chromatography-mass spectrometry (LC-MS) in compositional profiling of heparan sulfate (HS) oligosaccharides; however, ions observed using HILIC LC-MS are low in charge. Tandem MS of HS oligosaccharide ions with low charge results in undesirable losses of SO(3) from precursor ions during collision induced dissociation. One solution is to add metal cations to stabilize sulfate groups. Another is to add a nonvolatile, polar compound such as sulfolane, a molecule known to supercharge proteins, to produce a similar effect for oligosaccharides. We demonstrate use of a novel pulsed makeup flow (MUF) HPLC-chip. The chip enables controlled application of additives during specified chromatographic time windows and thus minimizes the extent to which nonvolatile additives build up in the ion source. The pulsed MUF system was applied to LC-MS/MS of HS oligosaccharides. Metal cations and sulfolane were tested as additives. The most promising results were obtained for sulfolane, for which supercharging of the oligosaccharide ions increased their signal strengths relative to controls. Tandem MS of these supercharged precursor ions showed decreased abundances of product ions from sulfate losses yet more abundant product ions from backbone cleavages.

  6. Bovine Lactoferrin Inhibits Dengue Virus Infectivity by Interacting with Heparan Sulfate, Low-Density Lipoprotein Receptor, and DC-SIGN

    Directory of Open Access Journals (Sweden)

    Jo-Mei Chen

    2017-09-01

    Full Text Available Bovine lactoferrin (bLF presents in milk and has been shown to inhibit several viral infections. Effective drugs are unavailable for the treatment of dengue virus (DENV infection. In this study, we evaluated the antiviral effect of bLF against DENV infection in vivo and in vitro. Bovine LF significantly inhibited the infection of the four serotypes of DENV in Vero cells. In the time-of-drug addition test, DENV-2 infection was remarkably inhibited when bLF was added during or prior to the occurrence of virus attachment. We also revealed that bovine LF blocks binding between DENV-2 and the cellular membrane by interacting with heparan sulfate (HS, dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN, and low-density lipoprotein receptors (LDLR. In addition, bLF inhibits DENV-2 infection and decreases morbidity in a suckling mouse challenge model. This study supports the finding that bLF may inhibit DENV infection by binding to the potential DENV receptors.

  7. Collagens and proteoglycans of the corneal extracellular matrix

    Directory of Open Access Journals (Sweden)

    Y.M. Michelacci

    2003-08-01

    Full Text Available The cornea is a curved and transparent structure that provides the initial focusing of a light image into the eye. It consists of a central stroma that constitutes 90% of the corneal depth, covered anteriorly with epithelium and posteriorly with endothelium. Its transparency is the result of the regular spacing of collagen fibers with remarkably uniform diameter and interfibrillar space. Corneal collagen is composed of heterotypic fibrils consisting of type I and type V collagen molecules. The cornea also contains unusually high amounts of type VI collagen, which form microfibrillar structures, FACIT collagens (XII and XIV, and other nonfibrillar collagens (XIII and XVIII. FACIT collagens and other molecules, such as leucine-rich repeat proteoglycans, play important roles in modifying the structure and function of collagen fibrils.Proteoglycans are macromolecules composed of a protein core with covalently linked glycosaminoglycan side chains. Four leucine-rich repeat proteoglycans are present in the extracellular matrix of corneal stroma: decorin, lumican, mimecan and keratocan. The first is a dermatan sulfate proteoglycan, and the other three are keratan sulfate proteoglycans. Experimental evidence indicates that the keratan sulfate proteoglycans are involved in the regulation of collagen fibril diameter, and dermatan sulfate proteoglycan participates in the control of interfibrillar spacing and in the lamellar adhesion properties of corneal collagens. Heparan sulfate proteoglycans are minor components of the cornea, and are synthesized mainly by epithelial cells. The effect of injuries on proteoglycan synthesis is discussed.

  8. The anticancer activity of lytic peptides is inhibited by heparan sulfate on the surface of the tumor cells.

    Science.gov (United States)

    Fadnes, Bodil; Rekdal, Oystein; Uhlin-Hansen, Lars

    2009-06-15

    Cationic antimicrobial peptides (CAPs) with antitumor activity constitute a promising group of novel anticancer agents. These peptides induce lysis of cancer cells through interactions with the plasma membrane. It is not known which cancer cell membrane components influence their susceptibility to CAPs. We have previously shown that CAPs interact with the two glycosaminoglycans (GAGs), heparan sulfate (HS) and chondroitin sulfate (CS), which are present on the surface of most cells. The purpose of this study was to investigate the role of the two GAGs in the cytotoxic activity of CAPs. Various cell lines, expressing different levels of cell surface GAGs, were exposed to bovine lactoferricin (LfcinB) and the designer peptide, KW5. The cytotoxic effect of the peptides was investigated by use of the colorimetric MTT viability assay. The cytotoxic effect on wild type CHO cells, expressing normal amounts of GAGs on the cell surface, and the mutant pgsA-745, that has no expression of GAGs on the cell surface, was also investigated. We show that cells not expressing HS were more susceptible to CAPs than cells expressing HS at the cell surface. Further, exogenously added heparin inhibited the cytotoxic effect of the peptides. Chondroitin sulfate had no effect on the cytotoxic activity of KW5 and only minor effects on LfcinB cytotoxicity. Our results show for the first time that negatively charged molecules at the surface of cancer cells inhibit the cytotoxic activity of CAPs. Our results indicate that HS at the surface of cancer cells sequesters CAPs away from the phospholipid bilayer and thereby impede their ability to induce cytolysis.

  9. The anticancer activity of lytic peptides is inhibited by heparan sulfate on the surface of the tumor cells

    Directory of Open Access Journals (Sweden)

    Rekdal Øystein

    2009-06-01

    Full Text Available Abstract Background Cationic antimicrobial peptides (CAPs with antitumor activity constitute a promising group of novel anticancer agents. These peptides induce lysis of cancer cells through interactions with the plasma membrane. It is not known which cancer cell membrane components influence their susceptibility to CAPs. We have previously shown that CAPs interact with the two glycosaminoglycans (GAGs, heparan sulfate (HS and chondroitin sulfate (CS, which are present on the surface of most cells. The purpose of this study was to investigate the role of the two GAGs in the cytotoxic activity of CAPs. Methods Various cell lines, expressing different levels of cell surface GAGs, were exposed to bovine lactoferricin (LfcinB and the designer peptide, KW5. The cytotoxic effect of the peptides was investigated by use of the colorimetric MTT viability assay. The cytotoxic effect on wild type CHO cells, expressing normal amounts of GAGs on the cell surface, and the mutant pgsA-745, that has no expression of GAGs on the cell surface, was also investigated. Results We show that cells not expressing HS were more susceptible to CAPs than cells expressing HS at the cell surface. Further, exogenously added heparin inhibited the cytotoxic effect of the peptides. Chondroitin sulfate had no effect on the cytotoxic activity of KW5 and only minor effects on LfcinB cytotoxicity. Conclusion Our results show for the first time that negatively charged molecules at the surface of cancer cells inhibit the cytotoxic activity of CAPs. Our results indicate that HS at the surface of cancer cells sequesters CAPs away from the phospholipid bilayer and thereby impede their ability to induce cytolysis.

  10. Deliberate attenuation of chikungunya virus by adaptation to heparan sulfate-dependent infectivity: a model for rational arboviral vaccine design.

    Directory of Open Access Journals (Sweden)

    Christina L Gardner

    2014-02-01

    Full Text Available Mosquito-borne chikungunya virus (CHIKV is a positive-sense, single-stranded RNA virus from the genus Alphavirus, family Togaviridae, which causes fever, rash and severe persistent polyarthralgia in humans. Since there are currently no FDA licensed vaccines or antiviral therapies for CHIKV, the development of vaccine candidates is of critical importance. Historically, live-attenuated vaccines (LAVs for protection against arthropod-borne viruses have been created by blind cell culture passage leading to attenuation of disease, while maintaining immunogenicity. Attenuation may occur via multiple mechanisms. However, all examined arbovirus LAVs have in common the acquisition of positively charged amino acid substitutions in cell-surface attachment proteins that render virus infection partially dependent upon heparan sulfate (HS, a ubiquitously expressed sulfated polysaccharide, and appear to attenuate by retarding dissemination of virus particles in vivo. We previously reported that, like other wild-type Old World alphaviruses, CHIKV strain, La Réunion, (CHIKV-LR, does not depend upon HS for infectivity. To deliberately identify CHIKV attachment protein mutations that could be combined with other attenuating processes in a LAV candidate, we passaged CHIKV-LR on evolutionarily divergent cell-types. A panel of single amino acid substitutions was identified in the E2 glycoprotein of passaged virus populations that were predicted to increase electrostatic potential. Each of these substitutions was made in the CHIKV-LR cDNA clone and comparisons of the mutant viruses revealed surface exposure of the mutated residue on the spike and sensitivity to competition with the HS analog, heparin, to be primary correlates of attenuation in vivo. Furthermore, we have identified a mutation at E2 position 79 as a promising candidate for inclusion in a CHIKV LAV.

  11. Heparan sulfate, heparin, and heparinase activity detection on polyacrylamide gel electrophoresis using the fluorochrome tris(2,2'-bipyridine) ruthenium (II).

    Science.gov (United States)

    Rozenberg, G I; Espada, J; de Cidre, L L; Eiján, A M; Calvo, J C; Bertolesi, G E

    2001-01-01

    The paper shows the ability of the fluorochrome tris(2,2'-bipyridine) ruthenium (II) (Rubipy) to detect heparan sulfate, heparin, and heparinase activity of M3 murine mammary adenocarcinoma cells as well as bacterial heparinases I, II, and III in native polyacrylamide gel electrophoresis (PAGE). The technique is based on the electrophoretic mobility of high molecular weight heparins and subsequent staining with Rubipy (50 micrograms/mL). The minimum content of heparin detected by fluorescence in a UV transilluminator was 25-50 ng. The number of Rubipy molecules bound to heparin, determined in relationship to the number of disaccharide units (DU), showed that two to six heparin disaccharide units are bound by each fluorochrome molecule. Scatchard plot analysis showed one Rubipy-binding site (Kd = (8.56 +/- 2.97) x 10(-5) M). Heparinase activity was determined by densitometric analysis of the fluorescence intensity of the heparin-containing band of the gel. While heparinase I (EC 4.2.2.7.) degraded heparin and, to a lower degree, partially N-desulfated N-acetylated heparin (N-des N-Ac), heparinase II (no EC number) could efficiently degrade heparan sulfate (HS) and partially N-des N-Ac heparin. Finally, heparinase III (EC 4.2.2.8.) degraded HS almost exclusively. Only heparin and N-des N-Ac heparin were substrates for M3 tumor cell heparinases. We describe a qualitative, sensitive and simple method to detect heparinase activity and determine its substrate specificity using Rubipy fluorescence with heparin and heparan sulfate in multiple biological samples tested in parallel.

  12. Ultrastructural localization of the core protein of a basement membrane-specific chondroitin sulfate proteoglycan in adult rat skin

    DEFF Research Database (Denmark)

    McCarthy, K J; Horiguchi, Y; Couchman, J R

    1990-01-01

    , fibronectin, and entactin/nidogen. IN this paper we show, using core protein-specific antibodies, the presence of a newly described basement membrane-specific chondroitin sulfate proteoglycan at the epithelial/mesenchymal interface of adult rat skin. Ultrastructurally, this antigen was proven to reside......Basement membranes are complex extracellular matrices present at epithelial/mesenchymal interfaces of tissues. The dermal-epidermal junction has been shown to contain numerous components, some of the most well known being laminin, types IV and VII collagens, heparan sulfate proteoglycan...

  13. Heparin/heparan sulfates bind to and modulate neuronal L-type (Cav1.2) voltage-dependent Ca2+ channels

    DEFF Research Database (Denmark)

    Garau, Gianpiero; Magotti, Paola; Heine, Martin

    2015-01-01

    Our previous studies revealed that L-type voltage-dependent Ca2+ channels (Cav1.2 L-VDCCs) are modulated by the neural extracellular matrix backbone, polyanionic glycan hyaluronic acid. Here we used isothermal titration calorimetry and screened a set of peptides derived from the extracellular...... domains of Cav1.2α1 to identify putative binding sites between the channel and hyaluronic acid or another class of polyanionic glycans, such as heparin/heparan sulfates. None of the tested peptides showed detectable interaction with hyaluronic acid, but two peptides derived from the first pore...

  14. Decoding the Matrix: Instructive Roles of Proteoglycan Receptors.

    Science.gov (United States)

    Neill, Thomas; Schaefer, Liliana; Iozzo, Renato V

    2015-08-04

    The extracellular matrix is a dynamic repository harboring instructive cues that embody substantial regulatory dominance over many evolutionarily conserved intracellular activities, including proliferation, apoptosis, migration, motility, and autophagy. The matrix also coordinates and parses hierarchical information, such as angiogenesis, tumorigenesis, and immunological responses, typically providing the critical determinants driving each outcome. We provide the first comprehensive review focused on proteoglycan receptors, that is, signaling transmembrane proteins that use secreted proteoglycans as ligands, in addition to their natural ligands. The majority of these receptors belong to an exclusive subset of receptor tyrosine kinases and assorted cell surface receptors that specifically bind, transduce, and modulate fundamental cellular processes following interactions with proteoglycans. The class of small leucine-rich proteoglycans is the most studied so far and constitutes the best understood example of proteoglycan-receptor interactions. Decorin and biglycan evoke autophagy and immunological responses that deter, suppress, or exacerbate pathological conditions such as tumorigenesis, angiogenesis, and chronic inflammatory disease. Basement membrane-associated heparan sulfate proteoglycans (perlecan, agrin, and collagen XVIII) represent a unique cohort and provide proteolytically cleaved bioactive fragments for modulating cellular behavior. The receptors that bind the genuinely multifactorial and multivalent proteoglycans represent a nexus in understanding basic biological pathways and open new avenues for therapeutic and pharmacological intervention.

  15. The heparan sulfate motif (GlcNS6S-IdoA2S)3, common in heparin, has a strict topography and is involved in cell behavior and disease.

    NARCIS (Netherlands)

    Smits, N.C.; Kurup, S.; Rops, A.L.; Dam, G.B. ten; Massuger, L.F.A.G.; Hafmans, T.G.M.; Turnbull, J.E.; Spillmann, D.; Li, J.P.; Kennel, S.J.; Wall, J.S.; Shworak, N.W.; Dekhuijzen, P.N.R.; Vlag, J. van der; Kuppevelt, A.H.M.S.M. van

    2010-01-01

    Heparan sulfate (HS) is a structurally complex polysaccharide that interacts with a broad spectrum of extracellular effector ligands and thereby is thought to regulate a diverse array of biologic processes. The specificity of HS-ligand interactions is determined by the arrangement of sulfate groups

  16. Cloning of the human heparan sulfate-N-deacetylase/N-sulfotransferase gene from the Treacher Collins syndrome candidate region at 5q32-q33.1

    Energy Technology Data Exchange (ETDEWEB)

    Dixon, J.; Loftus, S.K.; Gladwin, A.J. [Univ. of Manchester (United Kingdom)] [and others

    1995-03-20

    Treacher Collins syndrome is an autosomal dominant disorder of craniofacial development, the features of which include conductive hearing loss and cleft palate. Previous studies have shown that the Treacher Collins syndrome locus is flanked by D5S519 and SPARC, and a yeast artificial chromosome contig encompassing this {open_quotes}critical region{close_quotes} has been completed. In the current investigation a cosmid containing D5S519 has been used to screen a human placental cDNA library. This has resulted in the cloning of the human heparan sulfate-N-deacetylase/N-sulfotransferase gene. Two different mRNA species that have identical protein coding sequences but that differ in the size and sequence of the 3{prime} untranslated regions (3{prime}UTR) have been identified. The smaller species has a 3{prime}UTR of 1035 bp, whereas that of the larger is 4878 bp. 24 refs., 3 figs.

  17. Mutation of the dengue virus type 2 envelope protein heparan sulfate binding sites or the domain III lateral ridge blocks replication in Vero cells prior to membrane fusion

    Energy Technology Data Exchange (ETDEWEB)

    Roehrig, John T., E-mail: jtr1@cdc.gov [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Butrapet, Siritorn; Liss, Nathan M. [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Bennett, Susan L. [Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523 (United States); Luy, Betty E.; Childers, Thomas; Boroughs, Karen L.; Stovall, Janae L.; Calvert, Amanda E. [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Blair, Carol D. [Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523 (United States); Huang, Claire Y.-H. [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States)

    2013-07-05

    Using an infectious cDNA clone we engineered seven mutations in the putative heparan sulfate- and receptor-binding motifs of the envelope protein of dengue virus serotype 2, strain 16681. Four mutant viruses, KK122/123EE, E202K, G304K, and KKK305/307/310EEE, were recovered following transfection of C6/36 cells. A fifth mutant, KK291/295EE, was recovered from C6/36 cells with a compensatory E295V mutation. All mutants grew in and mediated fusion of virus-infected C6/36 cells, but three of the mutants, KK122/123EE, E202K, G304K, did not grow in Vero cells without further modification. Two Vero cell lethal mutants, KK291/295EV and KKK307/307/310EEE, failed to replicate in DC-SIGN-transformed Raji cells and did not react with monoclonal antibodies known to block DENV attachment to Vero cells. Additionally, both mutants were unable to initiate negative-strand vRNA synthesis in Vero cells by 72 h post-infection, suggesting that the replication block occurred prior to virus-mediated membrane fusion. - Highlights: • Heparan sulfate- and receptor-binding motifs of DENV2 envelope protein were mutated. • Four mutant viruses were isolated—all could fuse C6/36 cells. • Two of these mutants were lethal in Vero cells without further modification. • Lethal mutations were KK291/295EV and KKK305/307/310EEE. • Cell attachment was implicated as the replication block for both mutants.

  18. Natural variation in the heparan sulfate binding domain of the eastern equine encephalitis virus E2 glycoprotein alters interactions with cell surfaces and virulence in mice.

    Science.gov (United States)

    Gardner, Christina L; Choi-Nurvitadhi, Jo; Sun, Chengqun; Bayer, Avraham; Hritz, Jozef; Ryman, Kate D; Klimstra, William B

    2013-08-01

    Recently, we compared amino acid sequences of the E2 glycoprotein of natural North American eastern equine encephalitis virus (NA-EEEV) isolates and demonstrated that naturally circulating viruses interact with heparan sulfate (HS) and that this interaction contributes to the extreme neurovirulence of EEEV (C. L. Gardner, G. D. Ebel, K. D. Ryman, and W. B. Klimstra, Proc. Natl. Acad. Sci. U. S. A., 108:16026-16031, 2011). In the current study, we have examined the contribution to HS binding of each of three lysine residues in the E2 71-to-77 region that comprise the primary HS binding site of wild-type (WT) NA-EEEV viruses. We also report that the original sequence comparison identified five virus isolates, each with one of three amino acid differences in the E2 71-to-77 region, including mutations in residues critical for HS binding by the WT virus. The natural variant viruses, which possessed either a mutation from lysine to glutamine at E2 71, a mutation from lysine to threonine at E2 71, or a mutation from threonine to lysine at E2 72, exhibited altered interactions with heparan sulfate and cell surfaces and altered virulence in a mouse model of EEEV disease. An electrostatic map of the EEEV E1/E2 heterotrimer based upon the recent Chikungunya virus crystal structure (J. E. Voss, M. C. Vaney, S. Duquerroy, C. Vonrhein, C. Girard-Blanc, E. Crublet, A. Thompson, G. Bricogne, and F. A. Rey, Nature, 468:709-712, 2010) showed the HS binding site to be at the apical surface of E2, with variants affecting the electrochemical nature of the binding site. Together, these results suggest that natural variation in the EEEV HS binding domain may arise during EEEV sylvatic cycles and that this variation may influence receptor interaction and the severity of EEEV disease.

  19. Dengue virus induces expression of CXC chemokine ligand 10/IFN-gamma-inducible protein 10, which competitively inhibits viral binding to cell surface heparan sulfate.

    Science.gov (United States)

    Chen, Jia-Perng; Lu, Hsin-Lin; Lai, Szu-Liang; Campanella, Gabriele S; Sung, Jui-Ming; Lu, Mei-Yi; Wu-Hsieh, Betty A; Lin, Yi-Ling; Lane, Thomas E; Luster, Andrew D; Liao, Fang

    2006-09-01

    Dengue virus is an arthropod-borne flavivirus that causes a mild febrile illness, dengue fever, or a potentially fatal syndrome, dengue hemorrhagic fever/dengue shock syndrome. Chemokines primarily orchestrate leukocyte recruitment to the areas of viral infection, which makes them critical mediators of immune and inflammatory responses. In the present study, we investigated the induction and function of chemokines in mice early after infection with dengue virus in vivo. We found that CXCL10/IFN-gamma-inducible protein 10 (IP-10) expression was rapidly and transiently induced in liver following infection. The expressed CXCL10/IP-10 likely mediates the recruitment of activated NK cells, given that anti-CXCL10/IP-10-treated mice showed diminished NK cell infiltration and reduced hepatic expression of effector molecules in activated NK cells after dengue virus infection. Of particular interest, we found that CXCL10/IP-10 also was able to inhibit viral binding to target cells in vitro. Further investigation revealed that various CXCL10/IP-10 mutants, in which the residues that mediate the interaction between the chemokine and heparan sulfate were substituted, failed to exert the inhibitory effect on dengue binding, which suggests that CXCL10/IP-10 competes with dengue virus for binding to heparan sulfate on the cell surface. Moreover, subsequent plaque assays showed that this inhibition of dengue binding blocked viral uptake and replication. The inhibitory effect of CXCL10/IP-10 on the binding of dengue virus to cells may represent a novel contribution of this chemokine to the host defense against viral infection.

  20. SPECT imaging of peripheral amyloid in mice by targeting hyper-sulfated heparan sulfate proteoglycans with specific scFv antibodies.

    NARCIS (Netherlands)

    Wall, J.S.; Richey, T.; Stuckey, A.; Donnell, R.; Oosterhof, A.; Kuppevelt, T. van; Smits, N.C.; Kennel, S.J.

    2012-01-01

    INTRODUCTION: Amyloid deposits are associated with a broad spectrum of disorders including monoclonal gammopathies, chronic inflammation, and Alzheimer's disease. In all cases, the amyloid pathology contains, in addition to protein fibrils, a plethora of associated molecules, including high

  1. Distribution, ultrastructural localization, and ontogeny of the core protein of a heparan sulfate proteoglycan in human skin and other basement membranes

    DEFF Research Database (Denmark)

    Horiguchi, Y; Couchman, J R; Ljubimov, A V

    1989-01-01

    components, such as laminin and type IV collagen. Immunoelectron microscopy on adult skin and neonatal foreskin showed staining primarily within the lamina densa (LD) and sub-lamina densa regions of the dermoepidermal junction (DEJ) and vascular BM. In neonatal foreskin, additional staining was noted...

  2. Low density lipoprotein receptor internalizes low density and very low density lipoproteins that are bound to heparan sulfate proteoglycans via lipoprotein lipase

    NARCIS (Netherlands)

    Mulder, M.; Lombardi, P.; Jansen, H.; Berkel, T.J.C. van; Frants, R.R.; Havekes, L.M.

    1993-01-01

    It has previously been shown that lipoprotein lipase (LPL) enhances the binding of low density lipoproteins (LDL) and very low density lipoproteins (VLDL) to HepG2 cells and fibroblasts, up to 80-fold. This increase in binding is LDL receptor-independent and is due to a bridging of LPL between

  3. Single Particle Tracking Confirms That Multivalent Tat Protein Transduction Domain-induced Heparan Sulfate Proteoglycan Cross-linkage Activates Rac1 for Internalization*

    OpenAIRE

    Imamura, Junji; Suzuki, Yasuhiro; Gonda, Kohsuke; Roy, Chandra Nath; Gatanaga, Hiroyuki; Ohuchi, Noriaki; Higuchi, Hideo

    2011-01-01

    The mechanism by which HIV-1-Tat protein transduction domain (TatP) enters the cell remains unclear because of an insufficient understanding of the initial kinetics of peptide entry. Here, we report the successful visualization and tracking of TatP molecular kinetics on the cell surface with 7-nm spatial precision using quantum dots. Strong cell binding was only observed with a TatP valence of ≥8, whereas monovalent TatP binding was negligible. The requirement of the cell-surface heparan sulf...

  4. Embryonic lung morphogenesis in organ culture: experimental evidence for a proteoglycan function in the extracellular matrix

    Science.gov (United States)

    Spooner, B. S.; Bassett, K. E.; Spooner, B. S. Jr

    1993-01-01

    The lung rudiment, isolated from mid-gestation (11 day) mouse embryos, can undergo morphogenesis in organ culture. Observation of living rudiments, in culture, reveals both growth and ongoing bronchiolar branching activity. To detect proteoglycan (PG) biosynthesis, and deposition in the extracellular matrix, rudiments were metabolically labeled with radioactive sulfate, then fixed, embedded, sectioned and processed for autoradiography. The sulfated glycosaminoglycan (GAG) types, composing the carbohydrate component of the proteoglycans, were evaluated by selective GAG degradative approaches that showed chondroitin sulfate PG principally associated with the interstitial matrix, and heparan sulfate PG principally associated with the basement membrane. Experiments using the proteoglycan biosynthesis disrupter, beta-xyloside, suggest that when chondroitin sulfate PG deposition into the ECM is perturbed, branching morphogenesis is compromised.

  5. Faulty Initiation of Proteoglycan Synthesis Causes Cardiac and Joint Defects

    Science.gov (United States)

    Baasanjav, Sevjidmaa; Al-Gazali, Lihadh; Hashiguchi, Taishi; Mizumoto, Shuji; Fischer, Bjoern; Horn, Denise; Seelow, Dominik; Ali, Bassam R.; Aziz, Samir A.A.; Langer, Ruth; Saleh, Ahmed A.H.; Becker, Christian; Nürnberg, Gudrun; Cantagrel, Vincent; Gleeson, Joseph G.; Gomez, Delphine; Michel, Jean-Baptiste; Stricker, Sigmar; Lindner, Tom H.; Nürnberg, Peter; Sugahara, Kazuyuki; Mundlos, Stefan; Hoffmann, Katrin

    2011-01-01

    Proteoglycans are a major component of extracellular matrix and contribute to normal embryonic and postnatal development by ensuring tissue stability and signaling functions. We studied five patients with recessive joint dislocations and congenital heart defects, including bicuspid aortic valve (BAV) and aortic root dilatation. We identified linkage to chromosome 11 and detected a mutation (c.830G>A, p.Arg277Gln) in B3GAT3, the gene coding for glucuronosyltransferase-I (GlcAT-I). The enzyme catalyzes an initial step in the synthesis of glycosaminoglycan side chains of proteoglycans. Patients' cells as well as recombinant mutant protein showed reduced glucuronyltransferase activity. Patient fibroblasts demonstrated decreased levels of dermatan sulfate, chondroitin sulfate, and heparan sulfate proteoglycans, indicating that the defect in linker synthesis affected all three lines of O-glycanated proteoglycans. Further studies demonstrated that GlcAT-I resides in the cis and cis-medial Golgi apparatus and is expressed in the affected tissues, i.e., heart, aorta, and bone. The study shows that reduced GlcAT-I activity impairs skeletal as well as heart development and results in variable combinations of heart malformations, including mitral valve prolapse, ventricular septal defect, and bicuspid aortic valve. The described family constitutes a syndrome characterized by heart defects and joint dislocations resulting from altered initiation of proteoglycan synthesis (Larsen-like syndrome, B3GAT3 type). PMID:21763480

  6. 159 MOLECULAR EVALUATION OF GLYPICAN 3 GENE ...

    African Journals Online (AJOL)

    1111

    Department, National Liver Institute, Menoufiya University , Shebin. El-Kom, 3 Microbiology ... Glypican-3 (GPC3) is a member of the glypican family of heparin- sulphate ... (Roche Molecular Biochemicals, Mannheium, Germany), 0.2 mM.

  7. Proteoglycans in cancer biology, tumour microenvironment and angiogenesis

    Science.gov (United States)

    Iozzo, Renato V; Sanderson, Ralph D

    2011-01-01

    Proteoglycans, key molecular effectors of cell surface and pericellular microenvironments, perform multiple functions in cancer and angiogenesis by virtue of their polyhedric nature and their ability to interact with both ligands and receptors that regulate neoplastic growth and neovascularization. Some proteoglycans such as perlecan, have pro- and anti-angiogenic activities, whereas other proteoglycans, such as syndecans and glypicans, can also directly affect cancer growth by modulating key signalling pathways. The bioactivity of these proteoglycans is further modulated by several classes of enzymes within the tumour microenvironment: (i) sheddases that cleave transmembrane or cell-associated syndecans and glypicans, (ii) various proteinases that cleave the protein core of pericellular proteoglycans and (iii) heparanases and endosulfatases which modify the structure and bioactivity of various heparan sulphate proteoglycans and their bound growth factors. In contrast, some of the small leucine-rich proteoglycans, such as decorin and lumican, act as tumour repressors by physically antagonizing receptor tyrosine kinases including the epidermal growth factor and the Met receptors or integrin receptors thereby evoking anti-survival and pro-apoptotic pathways. In this review we will critically assess the expanding repertoire of molecular interactions attributed to various proteoglycans and will discuss novel proteoglycan functions modulating cancer progression, invasion and metastasis and how these factors regulate the tumour microenvironment. PMID:21155971

  8. Control of extracellular matrix assembly by syndecan-2 proteoglycan

    DEFF Research Database (Denmark)

    Klass, C M; Couchman, J R; Woods, A

    2000-01-01

    Extracellular matrix (ECM) deposition and organization is maintained by transmembrane signaling and integrins play major roles. We now show that a second transmembrane component, syndecan-2 heparan sulfate proteoglycan, is pivotal in matrix assembly. Chinese Hamster Ovary (CHO) cells were stably...... transfected with full length (S2) or truncated syndecan-2 lacking the C-terminal 14 amino acids of the cytoplasmic domain (S2deltaS). No differences in the amount of matrix assembly were noted with S2 cells, but those expressing S2deltaS could not assemble laminin or fibronectin into a fibrillar matrix....... The loss of matrix formation was not caused by a failure to synthesize or externalize ECM components as determined by metabolic labeling or due to differences in surface expression of alpha5 or beta1 integrin. The matrix assembly defect was at the cell surface, since S2deltaS cells also lost the ability...

  9. Glypican-3 distinguishes aggressive from non-aggressive odontogenic tumors: a preliminary study.

    Science.gov (United States)

    Mendes, Ramon Barreto; Dias, Rosane Borges; Figueiredo, Andreia Leal; Gurgel, Clarissa Araújo; Santana Filho, Manoel; Melo, Leonardo Araújo; Trierveiler, Marília; Cury, Patrícia Ramos; Leonardi, Rosalia; Dos Santos, Jean Nunes

    2017-04-01

    Glypican-3 is a cell surface proteoglycan that is found in embrionary tissues, and there are no studies investigating this protein in odontogenic tumor. Thus, the aim of this study was to investigate glypican-3 in a series of aggressive and non-aggressive odontogenic tumors. Fifty-nine cases of tumors were divided into aggressive odontogenic tumors (20 solid ameloblastomas, four unicystic ameloblastoma, 28 KOTs including five associated with Gorlin-Goltz syndrome) and non-aggressive odontogenic tumors (five adenomatoid odontogenic tumors and two calcifying cystic odontogenic tumors) and analyzed for glypican-3 using immunohistochemistry. Glypican-3 was observed in seven solid ameloblastoma and eighteen keratocystic odontogenic tumors including three of the five syndromic cases, but there was no significant difference between syndromic and sporadic cases (P > 0.05; Fisher's exact Test). All cases of unicystic ameloblastoma (n = 4), adenomatoid odontogenic tumor (n = 5), and calcifying cystic odontogenic tumor (n = 2) were negative. This provided insights into the presence of glypican-3 in odontogenic tumors. This protein distinguished aggressive from non-aggressive odontogenic tumors. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  10. Fibroblast invasive migration into fibronectin/fibrin gels requires a previously uncharacterized dermatan sulfate-CD44 proteoglycan

    DEFF Research Database (Denmark)

    Clark, Richard A F; Lin, Fubao; Greiling, Doris

    2004-01-01

    of fibronectin. Several integrins-alpha 4 beta 1, alpha 5 beta 1, and alpha v beta 3-with known fibronectin binding affinity were necessary for this invasive migration. Here we examined another family of cell surface receptors: the proteoglycans. We found that dermatan sulfate was required for fibroblast...... migration into a fibronectin/fibrin gel. This conclusion was based on beta-xyloside inhibition of glycanation and specific glycosaminoglycan degradation. CD44, a cell surface receptor known to bind hyaluronan, not infrequently exists as a proteoglycan, decorated with various glycosaminoglycan chains...... with chondroitin sulfate and dermatan sulfate, but not heparan sulfate, after a 24 h incubation with platelet-derived growth factor, the stimulus used in the migration assay. These results demonstrate that dermatan sulfate-CD44H proteoglycan is essential for fibroblast migration into fibrin clots and that platelet...

  11. Mutation of the dengue virus type 2 envelope protein heparan sulfate binding sites or the domain III lateral ridge blocks replication in Vero cells prior to membrane fusion.

    Science.gov (United States)

    Roehrig, John T; Butrapet, Siritorn; Liss, Nathan M; Bennett, Susan L; Luy, Betty E; Childers, Thomas; Boroughs, Karen L; Stovall, Janae L; Calvert, Amanda E; Blair, Carol D; Huang, Claire Y-H

    2013-07-05

    Using an infectious cDNA clone we engineered seven mutations in the putative heparan sulfate- and receptor-binding motifs of the envelope protein of dengue virus serotype 2, strain 16681. Four mutant viruses, KK122/123EE, E202K, G304K, and KKK305/307/310EEE, were recovered following transfection of C6/36 cells. A fifth mutant, KK291/295EE, was recovered from C6/36 cells with a compensatory E295V mutation. All mutants grew in and mediated fusion of virus-infected C6/36 cells, but three of the mutants, KK122/123EE, E202K, G304K, did not grow in Vero cells without further modification. Two Vero cell lethal mutants, KK291/295EV and KKK307/307/310EEE, failed to replicate in DC-SIGN-transformed Raji cells and did not react with monoclonal antibodies known to block DENV attachment to Vero cells. Additionally, both mutants were unable to initiate negative-strand vRNA synthesis in Vero cells by 72h post-infection, suggesting that the replication block occurred prior to virus-mediated membrane fusion. Published by Elsevier Inc.

  12. Profiling sulfation/epimerization pattern of full-length heparan sulfate by NMR following cell culture 13C-glucose metabolic labeling.

    Science.gov (United States)

    Pegeot, Mathieu; Sadir, Rabia; Eriksson, Inger; Kjellen, Lena; Simorre, Jean-Pierre; Gans, Pierre; Lortat-Jacob, Hugues

    2015-02-01

    Through its ability to interact with proteins, heparan sulfate (HS) fulfills a large variety of functions. Protein binding depends on the level of HS sulfation and epimerization which are cell specific and dynamically regulated. Characterization of this molecule, however, has been restricted to oligosaccharide fragments available in large amount for structural investigation or to sulfate distribution through compositional analysis. Here we developed a (1)H-(13)C 2D NMR-based approach, directly performed on HS isolated from (13)C-labeled cells. By integrating the peak volumes measured at different chemical shifts, this non-destructive analysis allows us to determine both the sulfation and the iduronic/glucuronic profiles of the polysaccharide. Applied to wild-type and N-deacetylase/N-sulfotransferase-deficient fibroblasts as well as to epithelial cells differentiation, it also gives insights into the functional relationships existing between HS biosynthetic enzymes. This approach should be of significant interest to better understand HS changes that occur through physiologic regulations or during pathological development. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  13. Secreted NS1 of dengue virus attaches to the surface of cells via interactions with heparan sulfate and chondroitin sulfate E.

    Directory of Open Access Journals (Sweden)

    Panisadee Avirutnan

    2007-11-01

    Full Text Available Dengue virus (DENV nonstructural protein-1 (NS1 is a secreted glycoprotein that is absent from viral particles but accumulates in the supernatant and on the plasma membrane of cells during infection. Immune recognition of cell surface NS1 on endothelial cells has been hypothesized as a mechanism for the vascular leakage that occurs during severe DENV infection. However, it has remained unclear how NS1 becomes associated with the plasma membrane, as it contains no membrane-spanning sequence motif. Using flow cytometric and ELISA-based binding assays and mutant cell lines lacking selective glycosaminoglycans, we show that soluble NS1 binds back to the surface of uninfected cells primarily via interactions with heparan sulfate and chondroitin sulfate E. DENV NS1 binds directly to the surface of many types of epithelial and mesenchymal cells yet attaches poorly to most peripheral blood cells. Moreover, DENV NS1 preferentially binds to cultured human microvascular compared to aortic or umbilical cord vein endothelial cells. This binding specificity was confirmed in situ as DENV NS1 bound to lung and liver but not intestine or brain endothelium of mouse tissues. Differential binding of soluble NS1 by tissue endothelium and subsequent recognition by anti-NS1 antibodies could contribute to the selective vascular leakage syndrome that occurs during severe secondary DENV infection.

  14. Binding of the chemokine CXCL12α to its natural extracellular matrix ligand heparan sulfate enables myoblast adhesion and facilitates cell motility.

    Science.gov (United States)

    Thakar, Dhruv; Dalonneau, Fabien; Migliorini, Elisa; Lortat-Jacob, Hugues; Boturyn, Didier; Albiges-Rizo, Corinne; Coche-Guerente, Liliane; Picart, Catherine; Richter, Ralf P

    2017-04-01

    The chemokine CXCL12α is a potent chemoattractant that guides the migration of muscle precursor cells (myoblasts) during myogenesis and muscle regeneration. To study how the molecular presentation of chemokines influences myoblast adhesion and motility, we designed multifunctional biomimetic surfaces as a tuneable signalling platform that enabled the response of myoblasts to selected extracellular cues to be studied in a well-defined environment. Using this platform, we demonstrate that CXCL12α, when presented by its natural extracellular matrix ligand heparan sulfate (HS), enables the adhesion and spreading of myoblasts and facilitates their active migration. In contrast, myoblasts also adhered and spread on CXCL12α that was quasi-irreversibly surface-bound in the absence of HS, but were essentially immotile. Moreover, co-presentation of the cyclic RGD peptide as integrin ligand along with HS-bound CXCL12α led to enhanced spreading and motility, in a way that indicates cooperation between CXCR4 (the CXCL12α receptor) and integrins (the RGD receptors). Our findings reveal the critical role of HS in CXCL12α induced myoblast adhesion and migration. The biomimetic surfaces developed here hold promise for mechanistic studies of cellular responses to different presentations of biomolecules. They may be broadly applicable for dissecting the signalling pathways underlying receptor cross-talks, and thus may guide the development of novel biomaterials that promote highly specific cellular responses. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Proteoglycans and axon guidance: a new relationship between old partners.

    Science.gov (United States)

    Masu, Masayuki

    2016-10-01

    Neural circuits are formed with great precision during development. Accumulated evidence over the past three decades has demonstrated that growing axons are navigated toward their targets by the combined actions of attractants and repellents together with their receptors. It has long been known that proteoglycans, glycosylated proteins possessing covalently attached glycosaminoglycans, play a critical role in axon guidance; however, the molecular mechanisms by which proteoglycans regulate axon behaviors remain largely unknown. Glycosaminoglycans such as heparan sulfate and chondroitin sulfate are large linear polysaccharides composed of repeating disaccharide units that are highly modified by specific sulfation and epimerization. Recent biochemical and molecular biological studies have identified the enzymes that are involved in the biosynthesis of glycosaminoglycans. Interestingly, many mutants lacking glycosaminoglycan-synthesizing enzymes or proteoglycans in several model organisms show defects in specific nerve tract formation. In parallel, detailed biochemical studies have identified the molecular interactions between axon guidance molecules and glycosaminoglycans that have specific modification in their sugar chains. This review summarizes the structure and function of axon guidance molecules and glycosaminoglycans, and then tries to combine the knowledge from these studies to understand the role of proteoglycans from a new vantage point. Deciphering the sugar code is important for understanding the complicated nature of proteoglycans in axon guidance. Neural circuits are formed by the combined actions of axon guidance molecules. Proteoglycans play critical roles in regulating axon guidance through the interaction between signaling molecules and glycosaminoglycan chains attached to the core protein. This paper summarizes the structure and functions of axon guidance molecules and glycosaminoglycans and reviews the molecular mechanisms by which proteoglycans

  16. The role of proteoglycans in pulmonary edema development.

    Science.gov (United States)

    Negrini, Daniela; Passi, Alberto; Moriondo, Andrea

    2008-04-01

    Pulmonary gas exchange critically depends upon the hydration state and the thinness of the interstitial tissue layer within the alveolo-capillary membrane. In the interstitium, fluid freely moving within the fibrous extracellular matrix (ECM) equilibrates with water chemically bound to hyaluronic acid and proteoglycans (PGs). The dynamic equilibrium between these two phases is set and maintained by the transendothelial fluid and solutes exchanges, by the convective outflows into the lymphatic system, and by the mechanical and hydrophilic properties of the solid elements of the ECM. The fibrous ECM components, in particular the chondroitin sulfate proteoglycan (CS-PG) and the heparan-sulfate proteoglycan (HS-PG) families, play a major role in the maintenance of tissue fluid homeostasis. In fact, they provide: (a) a perivascular and interstitial highly restrictive sieve with respect to plasma proteins, thus modulating both interstitial protein concentration and transendothelial fluid filtration; (b) a mechanical support to lymphatic vessels sustaining and modulating their draining function, and (c) a rigid three-dimensional low-compliant scaffold opposing fluid accumulation into the interstitial space. Fragmentation of PG induced by increased plasma volume, by degradation through proteolytic or inflammatory agents, by exposure to inspiratory gas mixture with modified oxygen fraction, or by increased tissue strain/stress invariably results in the progressive loosening of PG intermolecular bonds with other ECM components. The loss of the PGs regulatory functions compromises the protective role of the tissue solid matrix progressively leading to interstitial and eventually severe lung edema.

  17. Improvements in the order, isotropy and electron density of glypican-1 crystals by controlled dehydration

    Energy Technology Data Exchange (ETDEWEB)

    Awad, Wael [Lund University, Box 124, 221 00 Lund (Sweden); Cairo University, Cairo (Egypt); Svensson Birkedal, Gabriel [Lund University, Biomedical Center A13, 221 84 Lund (Sweden); Thunnissen, Marjolein M. G. M. [Lund University, Box 124, 221 00 Lund (Sweden); Lund University, Box 188, 221 00 Lund (Sweden); Mani, Katrin [Lund University, Biomedical Center A13, 221 84 Lund (Sweden); Logan, Derek T., E-mail: derek.logan@biochemistry.lu.se [Lund University, Box 124, 221 00 Lund (Sweden)

    2013-12-01

    The anisotropy of crystals of glypican-1 was significantly reduced by controlled dehydration using the HC1 device, allowing the building of previously disordered parts of the structure. The use of controlled dehydration for improvement of protein crystal diffraction quality is increasing in popularity, although there are still relatively few documented examples of success. A study has been carried out to establish whether controlled dehydration could be used to improve the anisotropy of crystals of the core protein of the human proteoglycan glypican-1. Crystals were subjected to controlled dehydration using the HC1 device. The optimal protocol for dehydration was developed by careful investigation of the following parameters: dehydration rate, final relative humidity and total incubation time T{sub inc}. Of these, the most important was shown to be T{sub inc}. After dehydration using the optimal protocol the crystals showed significantly reduced anisotropy and improved electron density, allowing the building of previously disordered parts of the structure.

  18. Mutant Fibulin-3 Causes Proteoglycan Accumulation and Impaired Diffusion Across Bruch's Membrane.

    Science.gov (United States)

    Zayas-Santiago, Astrid; Cross, Samuel D; Stanton, James B; Marmorstein, Alan D; Marmorstein, Lihua Y

    2017-06-01

    The mutation R345W in EFEMP1 (fibulin-3) causes macular degeneration. This study sought to determine whether proteoglycan content and diffusion across Bruch's membrane are altered in Efemp1ki/ki mice carrying this mutation or in Efemp1-/- mice. Proteoglycans in mouse Bruch's membranes were stained with Cupromeronic Blue (CB). Heparan sulfated proteoglycan (HSPG) and chondroitin/dermatan sulfate proteoglycan (C/DSPG) distributions were visualized following treatments with chondroitinase ABC (C-ABC) or nitrous acid. Total sulfated glycosaminoglycans (sGAGs) in Bruch's membrane/choroid (BrM/Ch) were measured with dimethylmethylene blue (DMMB). Matrix metalloprotease (MMP)-2, MMP-9, and tissue inhibitor of metalloproteinase (TIMP)-3 were examined by immunofluorescence and quantified using Image J. Molecules with different Stokes radius (Rs) were allowed simultaneously to diffuse through mouse BrM/Ch mounted in a modified Ussing chamber. Samples were quantified using gel exclusion chromatography. HSPGs and C/DSPGs were markedly increased in Efemp1ki/ki Bruch's membrane, and MMP-2 and MMP-9 were decreased, but TIMP-3 was increased. Diffusion across Efemp1ki/ki Bruch's membrane was impaired. In contrast, the proteoglycan amount in Efemp1-/- Bruch's membrane was not significantly different, but the size of proteoglycans was much larger. MMP-2, MMP-3, and TIMP-3 levels were similar to that of Efemp1+/+ mice, but they were localized diffusely in retinal pigment epithelium (RPE) cells instead of Bruch's membrane. Diffusion across Efemp1-/- Bruch's membrane was enhanced. Mutant fibulin-3 causes proteoglycan accumulation, reduction of MMP-2 and MMP-9, but increase of TIMP-3, and impairs diffusion across Bruch's membrane. Fibulin-3 ablation results in altered sizes of proteoglycans, altered distributions of MMP-2, MMP-9, and TIMP-3, and enhances diffusion across Bruch's membrane.

  19. Discovery of a Novel Molecule that Regulates Tumor Growth and Metastasis

    Directory of Open Access Journals (Sweden)

    Chery A. Whipple

    2008-01-01

    Full Text Available The heparan sulfate proteoglycan, Glypican-1 (GPC1, significantly impacts the growth of pancreatic cancer cells in vivo and markedly attenuates tumor angiogenesis and metastasis in athymic mice. Interestingly, both cancer cell–derived and host-derived GPC1 play an important role in tumor development and spread. These data suggest that GPC1 may be a valid therapeutic target for pancreatic cancer.

  20. An epidermal microRNA regulates neuronal migration through control of the cellular glycosylation state

    DEFF Research Database (Denmark)

    Pedersen, Mikael Egebjerg; Snieckute, Goda; Kagias, Konstantinos

    2013-01-01

    biosynthetic pathway: a chondroitin synthase (SQV-5; squashed vulva-5) and a uridine 5'-diphosphate-sugar transporter (SQV-7). Loss of mir-79 causes neurodevelopmental defects through SQV-5 and SQV-7 dysregulation in the epidermis. This results in a partial shutdown of heparan sulfate biosynthesis...... that impinges on a LON-2/glypican pathway and disrupts neuronal migration. Our results identify a regulatory axis controlled by a conserved microRNA that maintains proteoglycan homeostasis in cells....

  1. Recombinant Domain V of Human Perlecan Is a Bioactive Vascular Proteoglycan.

    Science.gov (United States)

    Rnjak-Kovacina, Jelena; Tang, Fengying; Lin, Xiaoting; Whitelock, John M; Lord, Megan S

    2017-12-01

    The C-terminal domain V of the extracellular matrix proteoglycan perlecan plays unique and often divergent roles in a number of biological processes, including angiogenesis, vascular cell interactions, wound healing, and autophagy. Recombinant forms of domain V have been proposed as therapeutic agents for the treatment of cancer, stroke, and the development of cardiovascular devices and bioartificial tissues. However, the effect of domain V appears to be related to the differences in domain V structure and function observed in different expression systems and environments and exactly how this occurs is not well understood. In this study, the sequence from amino acid 3626 to 4391 of the perlecan protein core, which includes domain V, is expressed in HEK-293 cells and purified as a secreted product from conditioned media. This recombinant domain V (rDV) is expressed as a proteoglycan decorated with heparan sulfate and chondroitin sulfate chains and supports endothelial cell interactions to the same extent as full-length perlecan. This expression system serves as an important model of recombinant proteoglycan expression, as well as a source of biologically active rDV for therapeutic applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. The P2X7 receptor regulates proteoglycan expression in the corneal stroma

    Science.gov (United States)

    Mankus, Courtney; Chi, Cheryl; Rich, Celeste; Ren, Ruiyi

    2012-01-01

    Purpose Previously, the authors demonstrated that the lack of the P2X7 receptor impairs epithelial wound healing and stromal collagen organization in the cornea. The goal here is to characterize specific effects of the P2X7 receptor on components of the corneal stroma extracellular matrix. Methods Unwounded corneas from P2X7 knockout mice (P2X7−/−) and C57BL/6J wild type mice (WT) were fixed and prepared for quantitative and qualitative analysis of protein expression and localization using Real Time PCR and immunohistochemistry. Corneas were stained also with Cuprolinic blue for electron microscopy to quantify proteoglycan sulfation in the stroma. Results P2X7−/− mice showed decreased mRNA expression in the major components of the corneal stroma: collagen types I and V and small leucine-rich proteoglycans decorin, keratocan, and lumican. In contrast P2X7−/− mice showed increased mRNA expression in lysyl oxidase and biglycan. Additionally, we observed increases in syndecan 1, perlecan, and type III collagen. There was a loss of perlecan along the basement membrane and enhanced expression throughout the stroma, in contrast with the decreased localization of other proteoglycans throughout the stroma. In the absence of lyase digestion there was a significantly smaller number of proteoglycan units per 100 nm of collagen fibrils in the P2X7−/− compared to WT mice. While digestion was more pronounced in the WT group, double digestion with Keratanase I and Chondroitinase ABC removed 88% of the GAG filaments in the WT, compared to 72% of those in the P2X7−/− mice, indicating that there are more heparan sulfate proteoglycans in the latter. Conclusions Our results indicate that loss of P2X7 alters both the expression of proteins and the sulfation of proteoglycans in the corneal stroma. PMID:22275804

  3. The P2X(7) receptor regulates proteoglycan expression in the corneal stroma.

    Science.gov (United States)

    Mankus, Courtney; Chi, Cheryl; Rich, Celeste; Ren, Ruiyi; Trinkaus-Randall, Vickery

    2012-01-01

    Previously, the authors demonstrated that the lack of the P2X(7) receptor impairs epithelial wound healing and stromal collagen organization in the cornea. The goal here is to characterize specific effects of the P2X(7) receptor on components of the corneal stroma extracellular matrix. Unwounded corneas from P2X(7) knockout mice (P2X(7) (-/-)) and C57BL/6J wild type mice (WT) were fixed and prepared for quantitative and qualitative analysis of protein expression and localization using Real Time PCR and immunohistochemistry. Corneas were stained also with Cuprolinic blue for electron microscopy to quantify proteoglycan sulfation in the stroma. P2X(7) (-/-) mice showed decreased mRNA expression in the major components of the corneal stroma: collagen types I and V and small leucine-rich proteoglycans decorin, keratocan, and lumican. In contrast P2X(7) (-/-) mice showed increased mRNA expression in lysyl oxidase and biglycan. Additionally, we observed increases in syndecan 1, perlecan, and type III collagen. There was a loss of perlecan along the basement membrane and enhanced expression throughout the stroma, in contrast with the decreased localization of other proteoglycans throughout the stroma. In the absence of lyase digestion there was a significantly smaller number of proteoglycan units per 100 nm of collagen fibrils in the P2X(7) (-/-) compared to WT mice. While digestion was more pronounced in the WT group, double digestion with Keratanase I and Chondroitinase ABC removed 88% of the GAG filaments in the WT, compared to 72% of those in the P2X(7) (-/-) mice, indicating that there are more heparan sulfate proteoglycans in the latter. Our results indicate that loss of P2X(7) alters both the expression of proteins and the sulfation of proteoglycans in the corneal stroma.

  4. Development and Growth of the Avian Pectoralis Major (Breast Muscle: Function of Syndecan-4 and Glypican-1 in Adult Myoblast Proliferation and Differentiation

    Directory of Open Access Journals (Sweden)

    Sandra G. Velleman

    2017-08-01

    Full Text Available Muscle fiber number is determined around the time hatch with continued posthatch muscle growth being mediated by the adult myoblast, satellite cell, population of cells. Satellite cells are dynamic in their expression of proteins including the cell membrane associated proteoglycans, syndecan-4 and glypican-1. These proteoglycans play roles in organizing the extracellular environment in the satellite cell niche, cytoskeletal structure, cell-to-cell adhesion, satellite cell migration, and signal transduction. This review article focuses on syndecan-4 and glypican-1 as both are capable of regulating satellite cell responsiveness to fibroblast growth factor 2. Fibroblast growth factor 2 is a potent stimulator of muscle cell proliferation and a strong inhibitor of differentiation. Proteoglycans are composed of a central core protein defined functional domains, and covalently attached glycosaminoglycans and N-glycosylation chains. The functional association of these components with satellite cell function is discussed as well as an emerging role for microRNA regulation of syndecan-4 and glypican-1.

  5. Glypican-3 Targeting of Liver Cancer Cells Using Multifunctional Nanoparticles

    Directory of Open Access Journals (Sweden)

    James O. Park

    2011-01-01

    Full Text Available Imaging is essential in accurately detecting, staging, and treating primary liver cancer (hepatocellular carcinoma [HCC], one of the most prevalent and lethal malignancies. We developed a novel multifunctional nanoparticle (NP specifically targeting glypican-3 (GPC3, a proteoglycan implicated in promotion of cell growth that is overexpressed in most HCCs. Quantitative real-time polymerase chain reaction was performed to confirm the differential GPC3 expression in two human HCC cells, Hep G2 (high and HLF (negligible. These cells were treated with biotin-conjugated GPC3 monoclonal antibody (αGPC3 and subsequently targeted using superparamagnetic iron oxide NPs conjugated to streptavidin and Alexa Fluor 647. Flow cytometry demonstrated that only GPC3-expressing Hep G2 cells were specifically targeted using this αGPC3-NP conjugate (fourfold mean fluorescence over nontargeted NP, and magnetic resonance imaging (MRI experiments showed similar findings (threefold R2 relaxivity. Confocal fluorescence microscopy localized the αGPC3 NPs only to the cell surface of GPC3-expressing Hep G2 cells. Further characterization of this construct demonstrated a negatively charged, monodisperse, 50 nm NP, ideally suited for tumor targeting. This GPC3-specific NP system, with dual-modality imaging capability, may enhance pretreatment MRI, enable refined intraoperative HCC visualization by near-infrared fluorescence, and be potentially used as a carrier for delivery of tumor-targeted therapies, improving patient outcomes.

  6. Function of Membrane-Associated Proteoglycans in the Regulation of Satellite Cell Growth.

    Science.gov (United States)

    Song, Yan

    2016-01-01

    Muscle growth can be divided into embryonic and postnatal periods. During the embryonic period, mesenchymal stem cells proliferate and differentiate to form muscle fibers. Postnatal muscle growth (hypertrophy) is characterized by the enlargement of existing muscle fiber size. Satellite cells (also known as adult myoblasts) are responsible for hypertrophy. The activity of satellite cells can be regulated by their extracellular matrix (ECM). The ECM is composed of collagens, proteoglycans, non-collagenous glycoproteins, cytokines and growth factors. Proteoglycans contain a central core protein with covalently attached glycosaminoglycans (GAGs: chondroitin sulfate, keratan sulfate, dermatan sulfate, and heparan sulfate) and N- or O-linked glycosylation chains. Membrane-associated proteoglycans attach to the cell membrane either through a glycosylphosphatidylinositol anchor or transmembrane domain. The GAGs can bind proteins including cytokines and growth factors. Both cytokines and growth factors play important roles in regulating satellite cell growth and development. Cytokines are generally associated with immune cells. However, cytokines can also affect muscle cell development. For instance, interleukin-6, tumor necrosis factor-α, and leukemia inhibitory factor have been reported to affect the proliferation and differentiation of satellite cells and myoblasts. Growth factors are potent stimulators or inhibitors of satellite cell proliferation and differentiation. The proper function of some cytokines and growth factors requires an interaction with the cell membrane-associated proteoglycans to enhance the affinity to bind to their primary receptors to initiate downstream signal transduction. This chapter is focused on the interaction of membrane-associated proteoglycans with cytokines and growth factors, and their role in satellite cell growth and development.

  7. NCBI nr-aa BLAST: CBRC-TGUT-37-0500 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TGUT-37-0500 ref|NP_005520.4| heparan sulfate proteoglycan 2 [Homo sapiens] em...b|CAH71870.1| heparan sulfate proteoglycan 2 [Homo sapiens] emb|CAI12125.1| heparan sulfate proteoglycan 2 [Homo sapiens] NP_005520.4 4e-24 49% ...

  8. α3 Chains of type V collagen regulate breast tumour growth via glypican-1

    Science.gov (United States)

    Huang, Guorui; Ge, Gaoxiang; Izzi, Valerio; Greenspan, Daniel S.

    2017-01-01

    Pericellular α3(V) collagen can affect the functioning of cells, such as adipocytes and pancreatic β cells. Here we show that α3(V) chains are an abundant product of normal mammary gland basal cells, and that α3(V) ablation in a mouse mammary tumour model inhibits mammary tumour progression by reducing the proliferative potential of tumour cells. These effects are shown to be primarily cell autonomous, from loss of α3(V) chains normally produced by tumour cells, in which they affect growth by enhancing the ability of cell surface proteoglycan glypican-1 to act as a co-receptor for FGF2. Thus, a mechanism is presented for microenvironmental influence on tumour growth. α3(V) chains are produced in both basal-like and luminal human breast tumours, and its expression levels are tightly coupled with those of glypican-1 across breast cancer types. Evidence indicates α3(V) chains as potential targets for inhibiting tumour growth and as markers of oncogenic transformation. PMID:28102194

  9. Pectin from Prunus domestica L. induces proliferation of IEC-6 cells through the alteration of cell-surface heparan sulfate on differentiated Caco-2 cells in co-culture.

    Science.gov (United States)

    Nishida, Mitsutaka; Murata, Kazuma; Oshima, Kazuya; Itoh, Chihiro; Kitaguchi, Kohji; Kanamaru, Yoshihiro; Yabe, Tomio

    2015-05-01

    Dietary fiber intake provides various physiological and metabolic effects for human health. Pectin, a water-soluble dietary fiber, induces morphological changes of the small intestine in vivo. However, the molecular mechanisms underlying pectin-derived morphological alterations have not been elucidated. Previously, we found that pectin purified from Prunus domestica L. altered the sulfated structure of cell-surface heparan sulfate (HS) on differentiated Caco-2 cells via fibronectin and α5β1 integrin. In this study, we investigated the biological significance of the effect of pectin on HS in differentiated Caco-2 cells. An in vitro intestinal epithelium model was constructed by co-culture of differentiated Caco-2 cells and rat IEC-6 cells, which were used as models of intestinal epithelium and intestinal crypt cells, respectively. We found that pectin-treated differentiated Caco-2 cells promoted growth of IEC-6 cells. Real-time RT-PCR analysis and western blotting showed that relative mRNA and protein expression levels of Wnt3a were upregulated by pectin treatment in differentiated Caco-2 cells. Analysis by surface plasmon resonance spectroscopy demonstrated that pectin-induced structural alteration of HS markedly decreased the interaction with Wnt3a. However, depression in the secretion of Wnt3a from Caco-2 cells by anti-Wnt3a antibody did not affect the proliferation of IEC-6 cells in co-culture system. These observations indicated that pectin altered the sulfated structure of cell-surface HS to promote secretion of Wnt3a from differentiated Caco-2 cells and Wnt3a indirectly stimulated the proliferation of IEC-6 cells.

  10. Hair follicle proteoglycans

    DEFF Research Database (Denmark)

    Couchman, J R

    1993-01-01

    Proteoglycans are polymorphic macromolecules present in all mammalian tissues, including the skin and its appendages. They consist of a core protein to which one or more glycosaminoglycan chains are covalently attached. Broadly, they can be divided into classes based on location and core protein...... structure. These classes include cell surface proteoglycans, basement membrane proteoglycans, small leucine-rich proteoglycans, large proteoglycans aggregating with hyaluronan, and intracellular granule proteoglycans. They have a wide range of functions, but little is known of the proteoglycans...... that are present in the epithelial and stromal compartments of hair follicles. However, the transmembrane proteoglycan syndecan may be important in follicle morphogenesis, both with respect to the epithelium and dermal papilla cells. Syndecan may possess both heparan and chondroitin sulfate chains, interacts...

  11. The transition of mouse pluripotent stem cells from the naïve to the primed state requires Fas signaling through 3-O sulfated heparan sulfate structures recognized by the HS4C3 antibody

    Energy Technology Data Exchange (ETDEWEB)

    Hirano, Kazumi [Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577 (Japan); Van Kuppevelt, Toin H. [Department of Biochemistry, Nijmegen Center for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, 280 P.O. Box 9101, 6500 HB Nijmegen (Netherlands); Nishihara, Shoko, E-mail: shoko@soka.ac.jp [Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577 (Japan)

    2013-01-18

    Highlights: ► Fas transcript increases during the transition from the naïve to the primed state. ► 3OST-5 transcript, the HS4C3 epitope synthesis gene, increases during the transition. ► Fas signaling regulates the transition from the naïve to the primed state. ► HS4C3-binding epitope regulates the transition from the naïve to the primed state. ► Fas signaling is regulated by the HS4C3 epitope during the transition. -- Abstract: The characteristics of pluripotent embryonic stem cells of human and mouse are different. The properties of human embryonic stem cells (hESCs) are similar to those of mouse epiblast stem cells (mEpiSCs), which are in a later developmental pluripotency state, the so-called “primed state” compared to mouse embryonic stem cells (mESCs) which are in a naïve state. As a result of the properties of the primed state, hESCs proliferate slowly, cannot survive as single cells, and can only be transfected with genes at low efficiency. Generating hESCs in the naïve state is necessary to overcome these problems and allow their application in regenerative medicine. Therefore, clarifying the mechanism of the transition between the naïve and primed states in pluripotent stem cells is important for the establishment of stable methods of generating naïve state hESCs. However, the signaling pathways which contribute to the transition between the naïve and primed states are still unclear. In this study, we carried out induction from mESCs to mEpiSC-like cells (mEpiSCLCs), and observed an increase in the activation of Fas signaling during the induction. The expression of Fgf5, an epiblast marker, was diminished by inhibition of Fas signaling using the caspase-8 and -3 blocking peptides, IETD and DEVD, respectively. Furthermore, during the induction, we observed increased expression of 3-O sulfated heparan sulfate (HS) structures synthesized by HS 3-O-sulfotransferase (3OST), which are recognized by the HS4C3 antibody (HS4C3-binding epitope

  12. Copy number variation analysis implicates the cell polarity gene glypican 5 as a human spina bifida candidate gene

    Science.gov (United States)

    Bassuk, Alexander G.; Muthuswamy, Lakshmi B.; Boland, Riley; Smith, Tiffany L.; Hulstrand, Alissa M.; Northrup, Hope; Hakeman, Matthew; Dierdorff, Jason M.; Yung, Christina K.; Long, Abby; Brouillette, Rachel B.; Au, Kit Sing; Gurnett, Christina; Houston, Douglas W.; Cornell, Robert A.; Manak, J. Robert

    2013-01-01

    Neural tube defects (NTDs) are common birth defects of complex etiology. Family and population-based studies have confirmed a genetic component to NTDs. However, despite more than three decades of research, the genes involved in human NTDs remain largely unknown. We tested the hypothesis that rare copy number variants (CNVs), especially de novo germline CNVs, are a significant risk factor for NTDs. We used array-based comparative genomic hybridization (aCGH) to identify rare CNVs in 128 Caucasian and 61 Hispanic patients with non-syndromic lumbar-sacral myelomeningocele. We also performed aCGH analysis on the parents of affected individuals with rare CNVs where parental DNA was available (42 sets). Among the eight de novo CNVs that we identified, three generated copy number changes of entire genes. One large heterozygous deletion removed 27 genes, including PAX3, a known spina bifida-associated gene. A second CNV altered genes (PGPD8, ZC3H6) for which little is known regarding function or expression. A third heterozygous deletion removed GPC5 and part of GPC6, genes encoding glypicans. Glypicans are proteoglycans that modulate the activity of morphogens such as Sonic Hedgehog (SHH) and bone morphogenetic proteins (BMPs), both of which have been implicated in NTDs. Additionally, glypicans function in the planar cell polarity (PCP) pathway, and several PCP genes have been associated with NTDs. Here, we show that GPC5 orthologs are expressed in the neural tube, and that inhibiting their expression in frog and fish embryos results in NTDs. These results implicate GPC5 as a gene required for normal neural tube development. PMID:23223018

  13. Proteoglycans in prostate cancer.

    Science.gov (United States)

    Edwards, Iris J

    2012-02-21

    The complexity and diversity of proteoglycan structure means that they have a range of functions that regulate cell behavior. Through multiple interactions of their core proteins and glycosaminoglycans with extracellular matrix proteins, growth factors and chemokines, proteoglycans affect cell signaling, motility, adhesion, growth and apoptosis. Progressive changes in proteoglycans occur in the tumor microenvironment, but neither the source nor consequences of those changes are well understood. Proteoglycans studied in prostate cancer include versican--a hyalectan regulator of cell adhesion and migration-and the small leucine-rich proteoglycans decorin, biglycan and lumican, which have roles in cell signaling and tissue organization. Studies support an inhibitory role in prostate cancer for decorin and lumican. Conversely, the basement membrane proteoglycan perlecan might be a tumor promoter through upregulation of sonic hedgehog signaling. Loss of the growth-inhibitory cell-surface proteoglycans syndecan-1 and betaglycan in early prostate cancer might facilitate progression, but syndecan-1 effects are pleiotropic and its renewed expression in advanced tumors might adversely affect outcome. Importantly, cellular changes and enzymatic activity in the developing tumor can alter proteoglycan composition and structure to modify their function. Emerging studies suggest that cancers, including those of the prostate, use these changes to promote their own survival, growth, and spread.

  14. Proteoglycan isolation and analysis

    DEFF Research Database (Denmark)

    Woods, A; Couchman, J R

    2001-01-01

    Proteoglycans can be difficult molecules to isolate and analyze due to large mass, charge, and tendency to aggregate or form macromolecular complexes. This unit describes detailed methods for purification of matrix, cell surface, and cytoskeleton-linked proteoglycans. Methods for analysis...

  15. Effects of two amino acid substitutions in the capsid proteins on the interaction of two cell-adapted PanAsia-1 strains of foot-and-mouth disease virus serotype O with heparan sulfate receptor

    Science.gov (United States)

    2014-01-01

    Background Some cell-adapted strains of foot-and-mouth disease virus (FMDV) can utilize heparan sulfate (HS) as a receptor to facilitate viral infection in cultured cells. A number of independent sites on the capsid that might be involved in FMDV-HS interaction have been studied. However, the previously reported residues do not adequately explain HS-dependent infection of two cell-adapted PanAsia-1 strains (O/Tibet/CHA/6/99tc and O/Fujian/CHA/9/99tc) of FMDV serotype O. To identify the molecular determinant(s) for the interaction of O/Tibet/CHA/6/99tc and O/Fujian/CHA/9/99tc with HS receptor, several chimeric viruses and site-directed mutants were generated by using an infectious cDNA of a non-HS-utilizing rescued virus (Cathay topotype) as the genomic backbone. Phenotypic properties of these viruses were determined by plaque assays and virus adsorption and penetration assays in cultured cells. Results Only two of the rescued viruses encoding VP0 of O/Tibet/CHA/6/99tc or VP1 of O/Fujian/CHA/9/99tc formed plaques on wild-type Chinese hamster ovary (WT-CHO; HS+) cells, but not on HS-negative pgsD-677 cells. The formation of plaques by these two chimeric viruses on WT-CHO cells could be abolished by the introduction of single amino acid mutations Gln-2080 → Leu in VP2 of O/Tibet/CHA/6/99tc and Lys-1083 → Glu in VP1 of O/Fujian/CHA/9/99tc, respectively. Nonetheless, the introduced mutation Leu-2080 → Gln in VP2 of O/Fujian/CHA/9/99tc for the construction of expectant recombinant plasmid led to non-infectious progeny virus in baby hamster kidney 21 (BHK-21) cells, and the site-directed mutant encoding Glu-1083 → Lys in VP1 of O/Tibet/CHA/6/99tc did not acquire the ability to produce plaques on WT-CHO cells. Significant differences in the inhibition of the infectivity of four HS-utilizing viruses by heparin and RGD-containing peptide were observed in BHK-21 cells. Interestingly, the chimeric virus encoding VP0 of O/Fujian/CHA/9/99tc, and the site

  16. Changes in heparan sulfate are associated with delayed wound repair, altered cell migration, adhesion and contractility in the galactosyltransferase I (beta4GalT-7) deficient form of Ehlers-Danlos syndrome.

    NARCIS (Netherlands)

    Gotte, M.; Spillmann, D.; Yip, G.W.; Versteeg, E.M.M.; Echtermeyer, F.G.; Kuppevelt, A.H.M.S.M. van; Kiesel, L.

    2008-01-01

    Reduced activity of beta4-galactosyltransferase 7 (beta4GalT-7), an enzyme involved in synthesizing the glycosaminoglycan linkage region of proteoglycans, is associated with the progeroid form of Ehlers-Danlos syndrome (EDS). In the invertebrates Drosophila melanogaster and Caenorhabditis elegans,

  17. Transmembrane Signaling Proteoglycans

    DEFF Research Database (Denmark)

    Couchman, John R

    2010-01-01

    Virtually all metazoan cells contain at least one and usually several types of transmembrane proteoglycans. These are varied in protein structure and type of polysaccharide, but the total number of vertebrate genes encoding transmembrane proteoglycan core proteins is less than 10. Some core...... proteins, including those of the syndecans, always possess covalently coupled glycosaminoglycans; others do not. Syndecan has a long evolutionary history, as it is present in invertebrates, but many other transmembrane proteoglycans are vertebrate inventions. The variety of proteins......, and linkage to PDZ protein networks. Many transmembrane proteoglycans associate on the cell surface with metzincin proteases and can be shed by them. Work with model systems in vivo and in vitro reveal roles in growth, adhesion, migration, and metabolism. Furthermore, a wide range of phenotypes for the core...

  18. In vivo imaging of hepatocellular carcinoma using a glypican-3-binding peptide based probe

    Science.gov (United States)

    Zhang, Qi; Han, Zhihao; Zhang, Wancun; Qian, Zhiyu; Gu, Yueqing

    2017-02-01

    Hepatocellular carcinoma (HCC) has been the third most common cause of cancer-related death worldwide. Glypican-3 (GPC3) is a heparin sulfate proteoglycan linked to the cell membrane by a glycosyl-phosphatidylinositol anchor (GPI) and is expressed by 75% of all hepatocellular carcinomas but undetectable in healthy liver tissue or liver with focal lesions. What's more, GPC3 has been gradually applied in clinical applications as a specific indicator for the early detection and prognosis of HCC. As GPC3 can also regulate many pathways in HCC pathogenesis including Wnt, Hh and Yap signaling, it has been shown that GPC3 knockdown can inhibit HCC growth, reinforcing the important roles of GPC3 in HCC development. For HCC early detection, we designed a peptide targeting GPC3 that allows to establish a fluorescent dyes-labeled probe. Firstly, according to the structure of the GPC3 antibody GC33 and the positive peptide reported in the literature, we generated a peptide consisting of twelve amino acids named 12P that may bind to GPC3 with tight binding ability and specificity. In vitro testing, we utilized FCM and laser confocal microscopy to verify its specificity of targeting to the high expression cells of GPC3. What's more, we linked 12P with a near infrared dye to verify its in vivo targeting ability. All results indicated that 12P possessed potent binding capacity which could be used as a targeting module in GPC3 detection probe.

  19. Molecular evaluation of Glypican 3 gene expression in Egyptian ...

    African Journals Online (AJOL)

    of hepatocellular carcinoma (HCC) has been increasing in Egypt with a doubling in the incidence rate in the past 10 years. . The genetic alterations that take place in the transformation of normal cells to neoplastic cells give an insight into the molecular mechanism of the disease. Glypican 3 (GPC3) is protein that plays a ...

  20. Growth plate regulation and osteochondroma formation: insights from tracing proteoglycans in zebrafish models and human cartilage.

    Science.gov (United States)

    de Andrea, Carlos E; Prins, Frans A; Wiweger, Malgorzata I; Hogendoorn, Pancras C W

    2011-06-01

    Proteoglycans are secreted into the extracellular matrix of virtually all cell types and function in several cellular processes. They consist of a core protein onto which glycosaminoglycans (e.g., heparan or chondroitin sulphates), are attached. Proteoglycans are important modulators of gradient formation and signal transduction. Impaired biosynthesis of heparan sulphate glycosaminoglycans causes osteochondroma, the most common bone tumour to occur during adolescence. Cytochemical staining with positively charged dyes (e.g., polyethyleneimine-PEI) allows, visualisation of proteoglycans and provides a detailed description of how proteoglycans are distributed throughout the cartilage matrix. PEI staining was studied by electron and reflection contrast microscopy in human growth plates, osteochondromas and five different proteoglycan-deficient zebrafish mutants displaying one of the following skeletal phenotypes: dackel (dak/ext2), lacking heparan sulphate and identified as a model for human multiple osteochondromas; hi307 (β3gat3), deficient for most glycosaminoglycans; pinscher (pic/slc35b2), presenting with defective sulphation of glycosaminoglycans; hi954 (uxs1), lacking most glycosaminoglycans; and knypek (kny/gpc4), missing the protein core of the glypican-4 proteoglycan. The panel of genetically well-characterized proteoglycan-deficient zebrafish mutants serves as a convincing and comprehensive study model to investigate proteoglycan distribution and the relation of this distribution to the model mutation status. They also provide insight into the distributions and gradients that can be expected in the human homologue. Human growth plate, wild-type zebrafish and fish mutants with mild proteoglycan defects (hi307 and kny) displayed proteoglycans distributed in a gradient throughout the matrix. Although the mutants pic and hi954, which had severely impaired proteoglycan biosynthesis, showed no PEI staining, dak mutants demonstrated reduced PEI staining and no

  1. Glypican-3 antibodies: a new therapeutic target for liver cancer

    OpenAIRE

    Ho, Mingqian Feng, Mitchell

    2013-01-01

    Glypican-3 (GPC3) is an emerging therapeutic target in hepatocellular carcinoma (HCC), even though the biological function of GPC3 remains elusive. Currently human (MDX-1414 and HN3) and humanized mouse (GC33 and YP7) antibodies that target GPC3 for HCC treatment are under different stages of preclinical or clinical development. Humanized mouse antibody GC33 is being evaluated in a phase II clinical trial. Human antibodies MDX-1414 and HN3 are under different stages of preclinical evaluation....

  2. Pentagone internalises glypicans to fine-tune multiple signalling pathways

    Science.gov (United States)

    Norman, Mark; Vuilleumier, Robin; Springhorn, Alexander; Gawlik, Jennifer; Pyrowolakis, George

    2016-01-01

    Tight regulation of signalling activity is crucial for proper tissue patterning and growth. Here we investigate the function of Pentagone (Pent), a secreted protein that acts in a regulatory feedback during establishment and maintenance of BMP/Dpp morphogen signalling during Drosophila wing development. We show that Pent internalises the Dpp co-receptors, the glypicans Dally and Dally-like protein (Dlp), and propose that this internalisation is important in the establishment of a long range Dpp gradient. Pent-induced endocytosis and degradation of glypicans requires dynamin- and Rab5, but not clathrin or active BMP signalling. Thus, Pent modifies the ability of cells to trap and transduce BMP by fine-tuning the levels of the BMP reception system at the plasma membrane. In addition, and in accordance with the role of glypicans in multiple signalling pathways, we establish a requirement of Pent for Wg signalling. Our data propose a novel mechanism by which morphogen signalling is regulated. DOI: http://dx.doi.org/10.7554/eLife.13301.001 PMID:27269283

  3. Heparanase activates the syndecan-syntenin-ALIX exosome pathway.

    Science.gov (United States)

    Roucourt, Bart; Meeussen, Sofie; Bao, Jie; Zimmermann, Pascale; David, Guido

    2015-04-01

    Exosomes are secreted vesicles of endosomal origin involved in signaling processes. We recently showed that the syndecan heparan sulfate proteoglycans control the biogenesis of exosomes through their interaction with syntenin-1 and the endosomal-sorting complex required for transport accessory component ALIX. Here we investigated the role of heparanase, the only mammalian enzyme able to cleave heparan sulfate internally, in the syndecan-syntenin-ALIX exosome biogenesis pathway. We show that heparanase stimulates the exosomal secretion of syntenin-1, syndecan and certain other exosomal cargo, such as CD63, in a concentration-dependent manner. In contrast, exosomal CD9, CD81 and flotillin-1 are not affected. Conversely, reduction of endogenous heparanase reduces the secretion of syntenin-1-containing exosomes. The ability of heparanase to stimulate exosome production depends on syntenin-1 and ALIX. Syndecans, but not glypicans, support exosome biogenesis in heparanase-exposed cells. Finally, heparanase stimulates intraluminal budding of syndecan and syntenin-1 in endosomes, depending on the syntenin-ALIX interaction. Taken together, our findings identify heparanase as a modulator of the syndecan-syntenin-ALIX pathway, fostering endosomal membrane budding and the biogenesis of exosomes by trimming the heparan sulfate chains on syndecans. In addition, our data suggest that this mechanism controls the selection of specific cargo to exosomes.

  4. High Expression of Glypican-1 Predicts Dissemination and Poor Prognosis in Glioblastomas.

    Science.gov (United States)

    Saito, Taiichi; Sugiyama, Kazuhiko; Hama, Seiji; Yamasaki, Fumiyuki; Takayasu, Takeshi; Nosaka, Ryo; Onishi, Shumpei; Muragaki, Yoshihiro; Kawamata, Takakazu; Kurisu, Kaoru

    2017-09-01

    Glioblastoma (GBM) relapses locally or in a disseminated pattern and is highly resistant to chemoradiotherapy. Although dissemination is associated with poor prognosis for patients with GBM, the clinicopathologic factors that promote dissemination have not been elucidated. Glypican-1 (GPC-1) is a heparin sulfate proteoglycan that is attached to the extracytoplasmic surface of the cell membrane and regulates cell motility. The aim of this study was to determine whether GPC-1 expression correlated with GBM dissemination and patient prognosis. GPC-1 expression was examined by immunohistochemistry in 53 patients with GBM who received radiotherapy and temozolomide treatment. We assessed the relationship between dissemination and clinicopathologic factors, including GPC-1 expression. We also evaluated the relationship between GPC-1 expression and overall survival (OS) by uni- and multivariate analyses of a range of clinicopathologic factors, including age, Karnofsky Performance Status, extent of resection, and O6-methylguanine-DNA methyltransferase (MGMT) status. Logistic regression analysis revealed that GPC-1 expression correlated with dissemination (P = 0.0116). Log-rank tests revealed that age, Karnofsky Performance Status, extent of resection, MGMT status, dissemination (P = 0.0008) and GPC-1 expression (P = 0.0011) were significantly correlated with OS. Multivariate analysis indicated that age, MGMT status, and GPC-1 expression were significantly correlated with OS. GPC-1 expression had the highest hazard ratio (2.392) among all regressors. GPC-1 expression significantly correlated with OS in patients with GBM who received radiotherapy and temozolomide treatment. GPC-1 expression can help predict the occurrence of dissemination and shorter OS in patients with GBM. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Perlecan domain 1 recombinant proteoglycan augments BMP-2 activity and osteogenesis.

    Science.gov (United States)

    Decarlo, Arthur A; Belousova, Maria; Ellis, April L; Petersen, Donald; Grenett, Hernan; Hardigan, Patrick; O'Grady, Robert; Lord, Megan; Whitelock, John M

    2012-09-11

    Many growth factors, such as bone morphogenetic protein (BMP)-2, have been shown to interact with polymers of sulfated disacharrides known as heparan sulfate (HS) glycosaminoglycans (GAGs), which are found on matrix and cell-surface proteoglycans throughout the body. HS GAGs, and some more highly sulfated forms of chondroitin sulfate (CS), regulate cell function by serving as co-factors, or co-receptors, in GF interactions with their receptors, and HS or CS GAGs have been shown to be necessary for inducing signaling and GF activity, even in the osteogenic lineage. Unlike recombinant proteins, however, HS and CS GAGs are quite heterogenous due, in large part, to post-translational addition, then removal, of sulfate groups to various positions along the GAG polymer. We have, therefore, investigated whether it would be feasible to deliver a DNA pro-drug to generate a soluble HS/CS proteoglycan in situ that would augment the activity of growth-factors, including BMP-2, in vivo. Utilizing a purified recombinant human perlecan domain 1 (rhPln.D1) expressed from HEK 293 cells with HS and CS GAGs, tight binding and dose-enhancement of rhBMP-2 activity was demonstrated in vitro. In vitro, the expressed rhPln.D1 was characterized by modification with sulfated HS and CS GAGs. Dose-enhancement of rhBMP-2 by a pln.D1 expression plasmid delivered together as a lyophilized single-phase on a particulate tricalcium phosphate scaffold for 6 or more weeks generated up to 9 fold more bone volume de novo on the maxillary ridge in a rat model than in control sites without the pln.D1 plasmid. Using a significantly lower BMP-2 dose, this combination provided more than 5 times as much maxillary ridge augmentation and greater density than rhBMP-2 delivered on a collagen sponge (InFuse™). A recombinant HS/CS PG interacted strongly and functionally with BMP-2 in binding and cell-based assays, and, in vivo, the pln.247 expression plasmid significantly improved the dose-effectiveness of BMP-2

  6. Perlecan domain 1 recombinant proteoglycan augments BMP-2 activity and osteogenesis

    Directory of Open Access Journals (Sweden)

    DeCarlo Arthur A

    2012-09-01

    Full Text Available Abstract Background Many growth factors, such as bone morphogenetic protein (BMP-2, have been shown to interact with polymers of sulfated disacharrides known as heparan sulfate (HS glycosaminoglycans (GAGs, which are found on matrix and cell-surface proteoglycans throughout the body. HS GAGs, and some more highly sulfated forms of chondroitin sulfate (CS, regulate cell function by serving as co-factors, or co-receptors, in GF interactions with their receptors, and HS or CS GAGs have been shown to be necessary for inducing signaling and GF activity, even in the osteogenic lineage. Unlike recombinant proteins, however, HS and CS GAGs are quite heterogenous due, in large part, to post-translational addition, then removal, of sulfate groups to various positions along the GAG polymer. We have, therefore, investigated whether it would be feasible to deliver a DNA pro-drug to generate a soluble HS/CS proteoglycan in situ that would augment the activity of growth-factors, including BMP-2, in vivo. Results Utilizing a purified recombinant human perlecan domain 1 (rhPln.D1 expressed from HEK 293 cells with HS and CS GAGs, tight binding and dose-enhancement of rhBMP-2 activity was demonstrated in vitro. In vitro, the expressed rhPln.D1 was characterized by modification with sulfated HS and CS GAGs. Dose-enhancement of rhBMP-2 by a pln.D1 expression plasmid delivered together as a lyophilized single-phase on a particulate tricalcium phosphate scaffold for 6 or more weeks generated up to 9 fold more bone volume de novo on the maxillary ridge in a rat model than in control sites without the pln.D1 plasmid. Using a significantly lower BMP-2 dose, this combination provided more than 5 times as much maxillary ridge augmentation and greater density than rhBMP-2 delivered on a collagen sponge (InFuse™. Conclusions A recombinant HS/CS PG interacted strongly and functionally with BMP-2 in binding and cell-based assays, and, in vivo, the pln.247 expression plasmid

  7. Proteoglycan form and function: A comprehensive nomenclature of proteoglycans.

    Science.gov (United States)

    Iozzo, Renato V; Schaefer, Liliana

    2015-03-01

    We provide a comprehensive classification of the proteoglycan gene families and respective protein cores. This updated nomenclature is based on three criteria: Cellular and subcellular location, overall gene/protein homology, and the utilization of specific protein modules within their respective protein cores. These three signatures were utilized to design four major classes of proteoglycans with distinct forms and functions: the intracellular, cell-surface, pericellular and extracellular proteoglycans. The proposed nomenclature encompasses forty-three distinct proteoglycan-encoding genes and many alternatively-spliced variants. The biological functions of these four proteoglycan families are critically assessed in development, cancer and angiogenesis, and in various acquired and genetic diseases where their expression is aberrant. Copyright © 2015.

  8. The expression of glycosaminoglycans and proteoglycans in the uterine cervix of albino rats after local hyaluronidase infusion.

    Science.gov (United States)

    Souza, Guilherme Negrão; Camano, Luiz; Araujo Júnior, Edward; Nader, Helena Bonciani; Medeiros, Valquíria; Martins, João Roberto Maciel; Souza, Eduardo

    2014-06-01

    To assess the local effect of hyaluronidase injection on the expression of glycosaminoglycans (GAGs) and proteoglycans (PGs) in the extracellular matrix of the uterine cervix from pregnant albino rats. Ten pregnant rats were divided into two groups on day 18 of pregnancy. The experimental group (Gexp) of rats received an intracervical infusion of 0.02 mL of hyaluronidase diluted to 1 mL with distilled water, whereas the control group (Gc) received 1 mL of distilled water. On day 20 of pregnancy, the pregnant rats were sacrificed and the uterine cervixes from all rats were then dissected. The qualitative expression of hyaluronic acid (HA) was assessed by immunohistochemistry and quantified by sandwich ELISA. To compare the quantitative GAG values between groups, a Student's t-test for independent samples was performed. PGs were also assessed by immunohistochemical analysis. The electrophoretic profile of newly synthesized radioactively labeled GAGs degraded by specific enzymes showed that there were two predominant GAGs in both Gc and Gexp, i.e. heparan sulfate (HS) and a mixture of hondroitin sulfate (CS) and dermatan sulfate (DS). The concentrations of GAGs showed a significant reduction of CS/DS (p < 0.004) and HS (p < 0.005) relative to Gc. HA staining was less intense in the lamina propria and area surrounding the blood vessels in Gexp compared to Gc. The HA contents were also significantly reduced (p < 0.012). Intracervical hyaluronidase infusion promoted a significant reduction in the concentration of sulfated GAGs as assessed by both qualitative (histochemical) and quantitative (fluorometric) measurements of HA.

  9. Large proteoglycan complexes and disturbed collagen architecture in the corneal extracellular matrix of mucopolysaccharidosis type VII (Sly syndrome).

    Science.gov (United States)

    Young, Robert D; Liskova, Petra; Pinali, Christian; Palka, Barbara P; Palos, Michalis; Jirsova, Katerina; Hrdlickova, Enkela; Tesarova, Marketa; Elleder, Milan; Zeman, Jiri; Meek, Keith M; Knupp, Carlo; Quantock, Andrew J

    2011-08-24

    Deficiencies in enzymes involved in proteoglycan (PG) turnover underlie a number of rare mucopolysaccharidoses (MPS), investigations of which can considerably aid understanding of the roles of PGs in corneal matrix biology. Here, the authors analyze novel pathologic changes in MPS VII (Sly syndrome) to determine the nature of PG-collagen associations in stromal ultrastructure. Transmission electron microscopy and electron tomography were used to investigate PG-collagen architectures and interactions in a cornea obtained at keratoplasty from a 22-year-old man with MPS VII, which was caused by a compound heterozygous mutation in the GUSB gene. Transmission electron microscopy showed atypical morphology of the epithelial basement membrane and Bowman's layer in MPS VII. Keratocytes were packed with cytoplasmic vacuoles containing abnormal glycosaminoglycan (GAG) material, and collagen fibrils were thinner than in normal cornea and varied considerably throughout anterior (14-32 nm), mid (13-42 nm), and posterior (17-39 nm) regions of the MPS VII stroma. PGs viewed in three dimensions were striking in appearance in that they were significantly larger than PGs in normal cornea and formed highly extended linkages with multiple collagen fibrils. Cellular changes in the MPS VII cornea resemble those in other MPS. However, the wide range of collagen fibril diameters throughout the stroma and the extensive matrix presence of supranormal-sized PG structures appear to be unique features of this disorder. The findings suggest that the accumulation of stromal chondroitin-, dermatan-, and heparan-sulfate glycosaminoglycans in the absence of β-glucuronidase-mediated degradation can modulate collagen fibrillogenesis.

  10. Inhibition and enhancement of neural regeneration by chondroitin sulfate proteoglycans

    Directory of Open Access Journals (Sweden)

    Heikki Rauvala

    2017-01-01

    Full Text Available The current dogma in neural regeneration research implies that chondroitin sulfate proteoglycans (CSPGs inhibit plasticity and regeneration in the adult central nervous system (CNS. We argue that the role of the CSPGs can be reversed from inhibition to activation by developmentally expressed CSPG-binding factors. Heparin-binding growth-associated molecule (HB-GAM; also designated as pleiotrophin has been studied as a candidate molecule that might modulate the role of CSPG matrices in plasticity and regeneration. Studies in vitro show that in the presence of soluble HB-GAM chondroitin sulfate (CS chains of CSPGs display an enhancing effect on neurite outgrowth. Based on the in vitro studies, we suggest a model according to which the HB-GAM/CS complex binds to the neuron surface receptor glypican-2, which induces neurite growth. Furthermore, HB-GAM masks the CS binding sites of the neurite outgrowth inhibiting receptor protein tyrosine phosphatase sigma (PTPσ, which may contribute to the HB-GAM-induced regenerative effect. In vivo studies using two-photon imaging after local HB-GAM injection into prick-injury of the cerebral cortex reveal regeneration of dendrites that has not been previously demonstrated after injuries of the mammalian nervous system. In the spinal cord, two-photon imaging displays HB-GAM-induced axonal regeneration. Studies on the HB-GAM/CS mechanism in vitro and in vivo are expected to pave the way for drug development for injuries of brain and spinal cord.

  11. NCBI nr-aa BLAST: CBRC-TGUT-37-0500 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TGUT-37-0500 gb|EAW94995.1| heparan sulfate proteoglycan 2 (perlecan), isoform... CRA_b [Homo sapiens] gb|EAW94996.1| heparan sulfate proteoglycan 2 (perlecan), isoform CRA_b [Homo sapiens] EAW94995.1 4e-24 49% ...

  12. High sodium diet converts renal proteoglycans into pro-inflammatory mediators in rats

    NARCIS (Netherlands)

    Hijmans, Ryanne S.; Shrestha, Pragyi; Sarpong, Kwaku A.; Yazdani, Saleh; el Masri, Rana; de Jong, Wilhelmina H. A.; Navis, Gerjan; Vives, Romain R.; van den Born, Jacob

    2017-01-01

    Background High dietary sodium aggravates renal disease by affecting blood pressure and by its recently shown pro-inflammatory and pro-fibrotic effects. Moreover, pro-inflammatory modification of renal heparan sulfate (HS) can induce tissue remodeling. We aim to investigate if high sodium intake in

  13. Disease: H00493 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available wicz M Chondrodysplasias due to proteoglycan defects. Glycobiology 12:57R-68R (20... Carson DD Heparan sulfate proteoglycans: key players in cartilage biology. Crit Rev Eukaryot Gene Expr 15:2

  14. Heparan sulfate regulates ADAM12 through a molecular switch mechanism

    DEFF Research Database (Denmark)

    Sørensen, Hans P; Vives, Romain R; Manetopoulos, Christina

    2008-01-01

    The disintegrin and metalloproteases (ADAMs) are emerging as therapeutic targets in human disease, but specific drug design is hampered by potential redundancy. Unlike other metzincins, ADAM pro domains remain bound to the mature enzyme to regulate activity. Here ADAM12, a protease that promotes....... These data present a novel concept that might allow targeting of ADAM12 and suggest that other ADAMs may have specific regulatory activity embedded in their pro and catalytic domain structures....

  15. Heparan sulfates in skeletal muscle development and physiology.

    NARCIS (Netherlands)

    Jenniskens, G.J.; Veerkamp, J.H.; Kuppevelt, A.H.M.S.M. van

    2006-01-01

    Recent years have seen an emerging interest in the composition of the skeletal muscle extracellular matrix (ECM) and in the developmental and physiological roles of its constituents. Many cell surface-associated and ECM-embedded molecules occur in highly organized spatiotemporal patterns, suggesting

  16. Syndecan proteoglycans and cell adhesion

    DEFF Research Database (Denmark)

    Woods, A; Oh, E S; Couchman, J R

    1998-01-01

    It is now becoming clear that a family of transmembrane proteoglycans, the syndecans, have important roles in cell adhesion. They participate through binding of matrix ligand to their glycosaminoglycan chains, clustering, and the induction of signaling cascades to modify the internal microfilament...

  17. An introduction to proteoglycans and their localization

    DEFF Research Database (Denmark)

    Couchman, John R; Pataki, Andreea Csilla

    2012-01-01

    Proteoglycans comprise a core protein to which one or more glycosaminoglycan chains are covalently attached. Although a small number of proteins have the capacity to be glycanated and become proteoglycans, it is now realized that these macromolecules have a range of functions, dependent on type...... locations. Therefore, the participation of proteoglycans in disease is receiving increased attention. In this short review, proteoglycan structure, function, and localizations are summarized, with reference to accompanying reviews in this issue as well as other recent literature. Included are some remarks...... on proteoglycan and glycosaminoglycan localization techniques, with reference to the special physicochemical properties of these complex molecules....

  18. 78 FR 37554 - Government-Owned Inventions; Availability for Licensing

    Science.gov (United States)

    2013-06-21

    ... glypican 3 protein inhibits the growth of hepatocellular carcinoma in vitro. Int J Cancer 2011 May1;128(9...-3 Protein and Heparan Sulfate for Treatment of Cancer Description of Technology: Hepatocellular... therapy. Glypican-3 (GPC3) is a cell surface protein that is preferentially expressed on HCC cells, making...

  19. Polyelectrolyte properties of proteoglycan monomers

    Science.gov (United States)

    Li, Xiao; Reed, Wayne F.

    1991-03-01

    Light scattering measurements were made on proteoglycan monomers (PGM) over a wide range of ionic strengths Cs, and proteoglycan concentrations [PG]. At low Cs there were clear peaks in the angular scattering intensity curve I(q), which moved towards higher scattering wave numbers q, as [PG]1/3. This differs from the square root dependence of scattering peaks found by neutron scattering from more concentrated polyelectrolyte solutions. The peaks remained roughly fixed as Cs increased, but diminished in height, and superposed I(q) curves yielded a sort of isosbestic point. Under certain assumptions the static structure factor S(q) could be extracted from the measured I(q), and was found to retain a peak. A simple hypothesis concerning coexisting disordered and liquidlike correlated states is presented, which qualitatively accounts for the most salient features of the peaks. There was evidence of a double component scattering autocorrelation decay at low Cs, which, when resolved into two apparent diffusion coefficients, gave the appearance of simultaneous ``ordinary'' and ``extraordinary'' phases. The extraordinary phase was ``removable,'' however, by filtering. At higher Cs the proteoglycans appear to behave as random nonfree draining polyelectrolyte coils, with a near constant ratio of 0.67 between hydrodynamic radius and radius of gyration. The apparent persistence length varied as roughly the -0.50 power of ionic strength, similar to various linear synthetic and biological polyelectrolytes. Electrostatic excluded volume theory accounted well for the dependence of A2 on Cs.

  20. Proteoglycan expression correlates with the phenotype of malignant and non-malignant EBV-positive B-cell lines.

    Science.gov (United States)

    Tsidulko, Alexandra Y; Matskova, Liudmila; Astakhova, Lidiia A; Ernberg, Ingemar; Grigorieva, Elvira V

    2015-12-22

    The involvement of proteoglycans (PGs) in EBV-host interactions and lymphomagenesis remains poorly investigated. In this study, expression of major proteoglycans (syndecan-1, glypican-1, perlecan, versican, brevican, aggrecan, NG2, serglycin, decorin, biglycan, lumican, CD44), heparan sulphate (HS) metabolic system (EXT1/2, NDST1/2, GLCE, HS2ST1, HS3ST1/2, HS6ST1/2, SULF1/2, HPSE) and extracellular matrix (ECM) components (collagen 1A1, fibronectin, elastin) in primary B cells and EBV carrying cell lines with different phenotypes, patterns of EBV-host cell interaction and viral latency stages (type I-III) was investigated. Primary B cells expressed a wide repertoire of PGs (dominated by serglycin and CD44) and ECM components. Lymphoblastoid EBV+ B cell lines (LCLs) showed specific PG expression with down-regulation of CD44 and ECM components and up-regulation of serglycin and perlecan/HSPG2. For Burkitt's lymphoma cells (BL), serglycin was down-regulated in BL type III cells and perlecan in type I BL cells. The biosynthetic machinery for HS was active in all cell lines, with some tendency to be down-regulated in BL cells. 5'-aza-dC and/or Trichostatin A resulted in transcriptional upregulation of the genes, suggesting that low expression of ECM components, proteoglycan core proteins and HS biosynthetic system is due to epigenetic suppression in type I cells. Taken together, our data show that proteoglycans are expressed in primary B lymphocytes whereas they are not or only partly expressed in EBV-carrying cell lines, depending on their latency type program.

  1. Auxiliary and autonomous proteoglycan signaling networks.

    Science.gov (United States)

    Elfenbein, Arye; Simons, Michael

    2010-01-01

    Proteoglycans represent a structurally heterogeneous family of proteins that typically undergo extensive posttranslational modification with sulfated sugar chains. Although historically believed to affect signaling pathways exclusively as growth factor coreceptors, proteoglycans are now understood to initiate and modulate signal transduction cascades independently of other receptors. From within the extracellular matrix, proteoglycans are able to shield protein growth factors from circulating proteases and establish gradients that guide cell migration. Extracellular proteoglycans are also critical in the maintenance of growth factor stores and are thus instrumental in modulating paracrine signaling. At the cell membrane, proteoglycans stabilize ligand-receptor interactions, creating potentiated ternary signaling complexes that regulate cell proliferation, endocytosis, migration, growth factor sensitivity, and matrix adhesion. In some cases, proteoglycans are able to independently activate various signaling cascades, attenuate the signaling of growth factors, or orchestrate multimeric intracellular signaling complexes. Signaling between cells is also modulated by proteoglycan activity at the cell membrane, as exemplified by the proteoglycan requirement for effective synaptogenesis between neurons. Finally, proteoglycans are able to regulate signaling from intracellular compartments, particularly in the context of storage granule formation and maintenance. These proteoglycans are also major determinants of exocytic vesicle fate and other vesicular trafficking pathways. In contrast to the mechanisms underlying classical ligand-receptor signaling, proteoglycan signaling is frequently characterized by ligand promiscuity and low-affinity binding; likewise, these events commonly do not exhibit the same degree of reliance on intermolecular structure or charge configurations as other ligand-receptor interactions. Such unique features often defy conventional mechanisms of

  2. Reactivating the extracellular matrix synthesis of sulfated glycosaminoglycans and proteoglycans to improve the human skin aspect and its mechanical properties

    Directory of Open Access Journals (Sweden)

    Chajra H

    2016-12-01

    , and versican and a stimulation of their respective sGAGs, such as chondroitin sulfate and heparan sulfate, were found on skin explants. The biosynthesis of macromolecules seems to be correlated at the microscopic level to a better organization and quality of the dermis, with collagen fibrils having homogenous diameters. The dermis seems to be compacted as observed on images obtained by two-photon microscopy and ultrasound imaging. At the macroscopic level, this dermis organization shows a smoothed profile similar to a younger skin, with improved mechanical properties such as firmess. Conclusion: The obtained results demonstrate that the defined cosmetic composition induces the synthesis of sGAGs and proteoglycans, which contributes to the overall dermal reorganization. This activity in the dermis in turn impacts the surface and mechanical properties of the skin. Keywords: cosmetic composition, decorin, dermis, polysaccharide

  3. Endocan: a novel circulating proteoglycan.

    Science.gov (United States)

    Kali, Arunava; Shetty, K S Rathan

    2014-01-01

    Endocan is a novel endothelium derived soluble dermatan sulfate proteoglycan. It has the property of binding to a wide range of bioactive molecules associated with cellular signaling and adhesion and thus regulating proliferation, differentiation, migration, and adhesion of different cell types in health and disease. An increase in tissue expression or serum level of endocan reflects endothelial activation and neovascularization which are prominent pathophysiological changes associated with inflammation and tumor progression. Consequently, endocan has been used as a blood-based and tissue-based biomarker for various cancers and inflammation and has shown promising results.

  4. An introduction to proteoglycans and their localization.

    Science.gov (United States)

    Couchman, John R; Pataki, Csilla A

    2012-12-01

    Proteoglycans comprise a core protein to which one or more glycosaminoglycan chains are covalently attached. Although a small number of proteins have the capacity to be glycanated and become proteoglycans, it is now realized that these macromolecules have a range of functions, dependent on type and in vivo location, and have important roles in invertebrate and vertebrate development, maintenance, and tissue repair. Many biologically potent small proteins can bind glycosaminoglycan chains as a key part of their function in the extracellular matrix, at the cell surface, and also in some intracellular locations. Therefore, the participation of proteoglycans in disease is receiving increased attention. In this short review, proteoglycan structure, function, and localizations are summarized, with reference to accompanying reviews in this issue as well as other recent literature. Included are some remarks on proteoglycan and glycosaminoglycan localization techniques, with reference to the special physicochemical properties of these complex molecules.

  5. Preparation of Proteoglycan Mimetic Graft Copolymers.

    Science.gov (United States)

    Kipper, Matt J; Place, Laura W

    2016-01-01

    Proteoglycans are proteins with pendant glycosaminoglycan polysaccharide side chains. The method described here enables the preparation of graft copolymers with glycosaminoglycan side chains, which mimic the structure and composition of proteoglycans. By controlling the stoichiometry, graft copolymers can be obtained with a wide range of glycosaminoglycan side-chain densities. The method presented here uses a three-step reaction mechanism to first functionalize a hyaluronic acid backbone, followed by reductive amination to couple the glycosaminoglycan side chain to the backbone, by the reducing end. Proteoglycan mimics like the ones proposed here could be used to study the structure-property relationships of proteoglycans and to introduce the biochemical and biomechanical properties of proteoglycans into biomaterials and therapeutic formulations.

  6. Functions of proteoglycans at the cell surface

    DEFF Research Database (Denmark)

    Höök, M; Woods, A; Johansson, S

    1986-01-01

    Proteoglycans (primarily heparan sulphate proteoglycans) are found at the surface of most adherent eukaryotic cells. Earlier studies suggest that these molecules can be associated with the cell surface principally by two different mechanisms. Proteoglycans may occur as membrane......-intercalated glycoproteins, where the core protein of the proteoglycan is anchored in the lipid interior of the plasma membrane, or they may be bound via the polysaccharide components of the molecule to specific anchoring proteins present at the cell surface. A number of functions have been proposed for cell surface......-associated proteoglycans, including: regulation of cell-substrate adhesion; regulation of cell proliferation; participation in the binding and uptake of extracellular components; and participation in the regulation of extracellular matrix formation. Evidence is discussed suggesting that the cell-associated heparan...

  7. The identification of proteoglycans and glycosaminoglycans in archaeological human bones and teeth.

    Directory of Open Access Journals (Sweden)

    Yvette M Coulson-Thomas

    Full Text Available Bone tissue is mineralized dense connective tissue consisting mainly of a mineral component (hydroxyapatite and an organic matrix comprised of collagens, non-collagenous proteins and proteoglycans (PGs. Extracellular matrix proteins and PGs bind tightly to hydroxyapatite which would protect these molecules from the destructive effects of temperature and chemical agents after death. DNA and proteins have been successfully extracted from archaeological skeletons from which valuable information has been obtained; however, to date neither PGs nor glycosaminoglycan (GAG chains have been studied in archaeological skeletons. PGs and GAGs play a major role in bone morphogenesis, homeostasis and degenerative bone disease. The ability to isolate and characterize PG and GAG content from archaeological skeletons would unveil valuable paleontological information. We therefore optimized methods for the extraction of both PGs and GAGs from archaeological human skeletons. PGs and GAGs were successfully extracted from both archaeological human bones and teeth, and characterized by their electrophoretic mobility in agarose gel, degradation by specific enzymes and HPLC. The GAG populations isolated were chondroitin sulfate (CS and hyaluronic acid (HA. In addition, a CSPG was detected. The localization of CS, HA, three small leucine rich PGs (biglycan, decorin and fibromodulin and glypican was analyzed in archaeological human bone slices. Staining patterns were different for juvenile and adult bones, whilst adolescent bones had a similar staining pattern to adult bones. The finding that significant quantities of PGs and GAGs persist in archaeological bones and teeth opens novel venues for the field of Paleontology.

  8. Minireview

    DEFF Research Database (Denmark)

    Chung, Heesung; Multhaupt, Hinke A B; Oh, Eok-Soo

    2016-01-01

    Syndecans are transmembrane heparan sulfate proteoglycans, with roles in development, tumorigenesis and inflammation, and growing evidence for involvement in tissue regeneration. This is a fast developing field with the prospect of utilizing tissue engineering and biomaterials in novel therapies...

  9. Co-deposition of basement membrane components during the induction of murine splenic AA amyloid

    DEFF Research Database (Denmark)

    Lyon, A W; Narindrasorasak, S; Young, I D

    1991-01-01

    Past studies have demonstrated that during murine AA amyloid induction there is co-deposition of the AA amyloid peptide and the basement membrane form of heparan sulfate proteoglycan. The synthesis and accumulation of heparan sulfate proteoglycan does not usually occur in the absence of other...... basement membrane components, such as type IV collagen, laminin, and fibronectin. Using immunohistochemical techniques, the present experiments have demonstrated that in addition to the heparan sulfate proteoglycan, there are other basement membrane components present in splenic AA amyloid deposits...... and these are present as soon as AA amyloid deposits are detectable. The results indicate that within the time constraints imposed by the experiments, the basement membrane components, fibronectin, laminin, type IV collagen, and heparan sulfate proteoglycan are co-deposited 36 to 48 hours after the AgNO3 and amyloid...

  10. Altered fibroblast proteoglycan production in COPD.

    Science.gov (United States)

    Hallgren, Oskar; Nihlberg, Kristian; Dahlbäck, Magnus; Bjermer, Leif; Eriksson, Leif T; Erjefält, Jonas S; Löfdahl, Claes-Göran; Westergren-Thorsson, Gunilla

    2010-05-11

    Airway remodeling in COPD includes reorganization of the extracellular matrix. Proteoglycans play a crucial role in this process as regulators of the integrity of the extracellular matrix. Altered proteoglycan immunostaining has been demonstrated in COPD lungs and this has been suggested to contribute to the pathogenesis. The major cell type responsible for production and maintenance of ECM constituents, such as proteoglycans, are fibroblasts. Interestingly, it has been proposed that central airways and alveolar lung parenchyma contain distinct fibroblast populations. This study explores the hypothesis that altered depositions of proteoglycans in COPD lungs, and in particular versican and perlecan, is a result of dysregulated fibroblast proteoglycan production. Proliferation, proteoglycan production and the response to TGF-beta1 were examined in vitro in centrally and distally derived fibroblasts isolated from COPD patients (GOLD stage IV) and from control subjects. Phenotypically different fibroblast populations were identified in central airways and in the lung parenchyma. Versican production was higher in distal fibroblasts from COPD patients than from control subjects (p proteoglycan production in distally derived fibroblasts from COPD patients and control subjects. In contrast, centrally derived fibroblasts from COPD patients were less responsive to TGF-beta1 than those from control subjects. The results show that fibroblasts from COPD patients have alterations in proteoglycan production that may contribute to disease development. Distally derived fibroblasts from COPD patients have enhanced production of versican that may have a negative influence on the elastic recoil. In addition, a lower perlecan production in centrally derived fibroblasts from COPD patients may indicate alterations in bronchial basement membrane integrity in severe COPD.

  11. Interactions between bovine cornea proteoglycans and collagen.

    OpenAIRE

    Speziale, P.; Bardoni, A; Balduini, C.

    1980-01-01

    Two types of proteoglycan subunits were obtained from bovine cornea, the first mainly composed of proteochondroitin sulphate and the second of proteokeratan sulphate. These two fractions can be obtained from the tissue as an aggregate, and are able to recombine each other after separation, to re-form the original structure. In order to investigate collagen-proteoglycan interactions, type-I collagen was isolated from bovine cornea by pepsin digestion followed by 3.5% (w/v) NaCl precipitation, ...

  12. Altered fibroblast proteoglycan production in COPD

    Directory of Open Access Journals (Sweden)

    Erjefält Jonas S

    2010-05-01

    Full Text Available Abstract Background Airway remodeling in COPD includes reorganization of the extracellular matrix. Proteoglycans play a crucial role in this process as regulators of the integrity of the extracellular matrix. Altered proteoglycan immunostaining has been demonstrated in COPD lungs and this has been suggested to contribute to the pathogenesis. The major cell type responsible for production and maintenance of ECM constituents, such as proteoglycans, are fibroblasts. Interestingly, it has been proposed that central airways and alveolar lung parenchyma contain distinct fibroblast populations. This study explores the hypothesis that altered depositions of proteoglycans in COPD lungs, and in particular versican and perlecan, is a result of dysregulated fibroblast proteoglycan production. Methods Proliferation, proteoglycan production and the response to TGF-β1 were examined in vitro in centrally and distally derived fibroblasts isolated from COPD patients (GOLD stage IV and from control subjects. Results Phenotypically different fibroblast populations were identified in central airways and in the lung parenchyma. Versican production was higher in distal fibroblasts from COPD patients than from control subjects (p 1 triggered similar increases in proteoglycan production in distally derived fibroblasts from COPD patients and control subjects. In contrast, centrally derived fibroblasts from COPD patients were less responsive to TGF-β1 than those from control subjects. Conclusions The results show that fibroblasts from COPD patients have alterations in proteoglycan production that may contribute to disease development. Distally derived fibroblasts from COPD patients have enhanced production of versican that may have a negative influence on the elastic recoil. In addition, a lower perlecan production in centrally derived fibroblasts from COPD patients may indicate alterations in bronchial basement membrane integrity in severe COPD.

  13. Relation of glypican-3 and E-cadherin expressions to clinicopathological features and prognosis of mucinous and non-mucinous colorectal adenocarcinoma.

    Science.gov (United States)

    Foda, Abd Al-Rahman Mohammad; Mohammad, Mie Ali; Abdel-Aziz, Azza; El-Hawary, Amira Kamal

    2015-06-01

    Glypican-3 (GPC3) is a member of the membrane-bound heparin sulfate proteoglycans. E-cadherin is an adhesive receptor that is believed to act as a tumor suppressor gene. Many studies had investigated E-cadherin expressions in colorectal carcinoma (CRC) while only one study had investigated GPC3 expression in CRC. This study aims to investigate expression of GCP3 and E-cadherin in colorectal mucinous carcinoma (MA) and non-mucinous adenocarcinoma (NMA) using manual tissue microarray technique. Tumor tissue specimens are collected from 75 cases of MC and 75 cases of NMA who underwent radical surgery from Jan 2007 to Jan 2012 at the Gastroenterology Centre, Mansoura University, Egypt. Their clinicopathological parameters and survival data were revised and analyzed using established statistical methodologies. High-density manual tissue microarrays were constructed using modified mechanical pencil tip technique and immunohistochemistry for GPC3 and E-cadherin was done. NMA showed higher expression of GPC3 than MA with no statistically significant relation. NMA showed a significantly higher E-cadherin expression than MA. GPC3 and E-cadherin positivity rates were significantly interrelated in NMA, but not in MA, group. In NMA group, there was no significant relation between either GPC3 or E-cadherin expression and the clinicopathological features. In a univariate analysis, neither GPC3 nor E-cadherin expression showed a significant impact on disease-free survival (DFS) or overall survival (OS). GPC3 and E-cadherin expressions are not independent prognostic factors in CRC. However, expressions of both are significantly interrelated in NMA patients, suggesting an excellent interplay between both, in contrast to MA. Further molecular studies are needed to further explore the relationship between GCP3 and E-cadherin in colorectal carcinogenesis.

  14. Viscoelasticity of Concentrated Proteoglycan Solutions

    Science.gov (United States)

    Meechai, Nispa; Jamieson, Alex; Blackwell, John; Carrino, David

    2001-03-01

    Proteoglycan Aggregate (PGA) is the principal macromolecular component of the energy-absorbing matrix of cartilage and tendon. Its brush-like supramolecular structure consists of highly-ionic subunits, non-covalently bound to a hyaluronate chain. We report viscoelastic behavior of concentrated solutions of PGA, purified by column fractionation to remove free subunits. At physiological ionic strength, these preparations exhibit a sol-to-gel transition when the concentration is increased above molecular overlap. The strain dependence of concentrated solutions shows a pronounced non-linearity above a critical strain, at which the storage modulus decreases suddenly, and the loss modulus exhibits a maximum. This response is similar to that observed for close-packed dispersions of soft spheres, when the applied strain is sufficient to move a sphere past its neighbors. At low and high ionic strength, the elasticity of solutions near the overlap concentration decreases. The former is interpreted as due to a decrease in intramolecular and intermolecular electrostatic repulsions, because of strong trapping of counterions within the PGA brush, the latter to salt-induced brush collapse.

  15. Hippocampal proteoglycans brevican and versican are linked to spatial memory of Sprague-Dawley rats in the morris water maze.

    Science.gov (United States)

    Saroja, Sivaprakasam R; Sase, Ajinkya; Kircher, Susanne G; Wan, Jia; Berger, Johannes; Höger, Harald; Pollak, Arnold; Lubec, Gert

    2014-09-01

    Proteoglycans (PGs) are major constituents of the extracellular matrix and have recently been proposed to contribute to synaptic plasticity. Hippocampal PGs have not yet been studied or linked to memory. The aim of the study, therefore, was to isolate and characterize rat hippocampal PGs and determine their possible role in spatial memory. PGs were extracted from rat hippocampi by anion-exchange chromatography and analyzed by nano LC-MS/MS. Twenty male Sprague-Dawley rats were tested in the morris water maze. PGs agrin, amyloid beta A4 protein, brevican, glypican-1, neurocan, phosphacan, syndecan-4, and versican were identified in the hippocampi. Brevican and versican levels in the membrane fraction were higher in the trained group, correlating with the time spent in the target quadrant. α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor GluR1 was co-precipitated with brevican and versican. Levels for a receptor complex containing GluR1 was higher in trained while GluR2 and GluR3-containing complex levels were higher in yoked rats. The findings provide information about the PGs present in the rat hippocampus, demonstrating that versican and brevican are linked to memory retrieval in the morris water maze and that PGs interact with α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor GluR1, which is linked to memory retrieval. Proteoglycans (PGs) are major constituents of the extracellular matrix of the brain and were proposed to contribute to synaptic plasticity. This report addressed PGs in rat hippocampus and suggests that PGs brevican and versican are linked to spatial memory, and form a complex with the GluR1 subunit of the AMPA receptor, a key signaling molecule in memory mechanisms. © 2014 International Society for Neurochemistry.

  16. Recent developments in proteoglycan purification and analysis

    NARCIS (Netherlands)

    Didraga, Mihaela Alina; Barroso, B.; Bischoff, Rainer

    2006-01-01

    Proteoglycans are ubiquitous biomolecules in the body located in the extracellular matrix, on the cell surface and also within the cells. They contain at least one glycosaminoglycan (GAG) chain covalently attached to a core protein and may also present N- or O-linked glycans. The high structural

  17. Proteoglycans at the bone-implant interface.

    Science.gov (United States)

    Klinger, M M; Rahemtulla, F; Prince, C W; Lucas, L C; Lemons, J E

    1998-01-01

    The widespread success of clinical implantology stems from bone's ability to form rigid, load-bearing connections to titanium and certain bioactive coatings. Adhesive biomolecules in the extracellular matrix are presumably responsible for much of the strength and stability of these junctures. Histochemical and spectroscopic analyses of retrievals have been supplemented by studies of osteoblastic cells cultured on implant materials and of the adsorption of biomolecules to titanium powder. These data have often been interpreted to suggest that proteoglycans permeate a thin, collagen-free zone at the most intimate contact points with implant surfaces. This conclusion has important implications for the development of surface modifications to enhance osseointegration. The evidence for proteoglycans at the interface, however, is somewhat less than compelling due to the lack of specificity of certain histochemical techniques and to possible sectioning artifacts. With this caveat in mind, we have devised a working model to explain certain observations of implant interfaces in light of the known physical and biological properties of bone proteoglycans. This model proposes that titanium surfaces accelerate osseointegration by causing the rapid degradation of a hyaluronan meshwork formed as part of the wound-healing response. It further suggests that the adhesive strength of the thin, collagen-free zone is provided by a bilayer of decorin proteoglycans held in tight association by their overlapping glycosaminoglycan chains.

  18. The function of heparanase in diabetes and its complications.

    Science.gov (United States)

    Wang, Fang; Wan, Andrea; Rodrigues, Brian

    2013-10-01

    Heparan sulfate proteoglycans are ubiquitous glycoproteins that contain several heparan sulfate polysaccharide side chains attached to a core protein. They function not only as a primary structural component of the extracellular matrix, but also provide a storage depot for bioactive molecules, such as basic fibroblast growth factor, vascular endothelial growth factor and lipoprotein lipase. Heparanase is an endoglycosidase that specifically hydrolyzes heparan sulfate into oligosaccharides. Recent studies have indicated that heparanase is engaged in the initiation and progression of diabetes, in addition to its associated complications. This review focuses on the participation of heparanase in the cleavage of heparan sulfate proteoglycans in pancreatic islets promoting beta cell death, promotion of atherosclerosis, and its role in cardiac metabolic switching in the early stage of cardiomyopathy during diabetes. Understanding the mechanisms by which heparanase is regulated in diabetes could provide a drug target to prevent diabetes and its complications. Copyright © 2013. Published by Elsevier Inc.

  19. Bone Proteoglycan Changes During Skeletal Unloading

    Science.gov (United States)

    Yamauchi, M.; Uzawa, K.; Pornprasertsuk, S.; Arnaud, S.; Grindeland, R.; Grzesik, W.

    1999-01-01

    Skeletal adaptability to mechanical loads is well known since the last century. Disuse osteopenia due to the microgravity environment is one of the major concerns for space travelers. Several studies have indicated that a retardation of the mineralization process and a delay in matrix maturation occur during the space flight. Mineralizing fibrillar type I collagen possesses distinct cross-linking chemistries and their dynamic changes during mineralization correlate well with its function as a mineral organizer. Our previous studies suggested that a certain group of matrix proteoglycans in bone play an inhibitory role in the mineralization process through their interaction with collagen. Based on these studies, we hypothesized that the altered mineralization during spaceflight is due in part to changes in matrix components secreted by cells in response to microgravity. In this study, we employed hindlimb elevation (tail suspension) rat model to study the effects of skeletal unloading on matrix proteoglycans in bone.

  20. Immunoexpression of Heat Shock Protein 70, Glypican 3, Glutamine Synthetase, and Beta-Catenin in Hepatocellular Carcinoma After Liver Transplantation: Association Between Positive Glypican 3 and Beta-Catenin With the Presence of Larger Nodules.

    Science.gov (United States)

    Ataide, E C; Perales, S R; Silva, M G; Filho, F C; Sparapani, A C; Latuf Filho, P F; Stucchi, R S B; Vassallo, J; Escanhoela, C A F; Boin, I F S F

    2017-05-01

    Hepatocellular carcinoma (HCC) is the 6th leading cause of cancer worldwide. Its recurrence ranges from 6% to 26%. In the literature, many factors are associated with higher risk of recurrence, without a clear definition of the best method that could predict this highly lethal event. The aim of this study was to evaluate the immunoexpression of immunohistochemical markers: HSP70, glypican 3, glutamine synthetase, and beta-catenin, as well as studying their association with tumor characteristics and prognosis of patients undergoing liver transplantation for HCC. We studied 90 patients who underwent liver transplantation from 1998 to 2012. Afterwards we evaluated factors related to survival, tumor recurrence, and the correlation of expression of the immunohistochemical markers. Immunohistochemical marker glutamine synthetase showed a positive trend toward better survival. HSP70-positive patients had a higher prevalence of histologic grade III. Patients with positive glypican 3 showed larger lesions and a higher number with AFP >200 ng/mL. Patients with positive beta-catenin showed larger nodules and more with histologic grade III. The association between beta-catenin and glypican 3 showed positive association with larger nodules. Most of the markers studied had a correlation with at least one of the variables studied, confirming our hypothesis that these markers can indeed assist in assessing the prognosis of patients undergoing liver transplantation for HCC. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Proteoglycan degradation by the ADAMTS family of proteinases.

    Science.gov (United States)

    Stanton, Heather; Melrose, James; Little, Christopher B; Fosang, Amanda J

    2011-12-01

    Proteoglycans are key components of extracellular matrices, providing structural support as well as influencing cellular behaviour in physiological and pathological processes. The diversity of proteoglycan function reported in the literature is equally matched by diversity in proteoglycan structure. Members of the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) family of enzymes degrade proteoglycans and thereby have the potential to alter tissue architecture and regulate cellular function. In this review, we focus on ADAMTS enzymes that degrade the lectican and small leucine-rich repeat families of proteoglycans. We discuss the known ADAMTS cleavage sites and the consequences of cleavage at these sites. We illustrate our discussion with examples from the literature in which ADAMTS proteolysis of proteoglycans makes profound changes to tissue function. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. Preparation of proteoglycan from salmon nasal cartilage under nondenaturing conditions.

    Science.gov (United States)

    Tatara, Yota; Suto, Shinichiro; Sasaki, Yoshitaka; Endo, Masahiko

    2015-01-01

    Salmon nasal cartilage was micronized in ethanol using a rotor-stator homogenizer for the high yield of proteoglycan extraction. This procedure also brought about depressing the degradation of proteoglycan and the contamination of collagens. Proteoglycan was extracted by 4 M magnesium chloride and isolated by anion-exchange chromatography. The gel filtration HPLC and the antibody reactivity showed that the core protein was intact.

  3. Identification of chondroitin sulfate E proteoglycans and heparin proteoglycans in the secretory granules of human lung mast cells

    Energy Technology Data Exchange (ETDEWEB)

    Stevens, R.L.; Austen, K.F. (Brigham and Women' s Hospital, Boston, MA (USA)); Fox, C.C.; Lichtenstein, L.M. (Johns Hopkins School of Medicine, Baltimore, MD (USA))

    1988-04-01

    The predominant subclasses of mast cells in both the rat and the mouse can be distinguished from one another by their preferential synthesis of {sup 35}S-labeled proteoglycans that contain either heparin or oversulfated chondroitin sulfate glycosaminoglycans. Although ({sup 35}S)heparin proteoglycans have been isolated from human lung mast cells of 40-70% purity and from a skin biopsy specimen of a patient with urticaria pigmentosa, no highly sulfated chondroitin sulfate proteoglycan has been isolated from any enriched or highly purified population of human mast cells. The authors demonstrate that human lung mast cells of 96% purity incorporate ({sup 35}S)sulfate into separate heparin and chondroitin sulfate proteoglycans in an {approx}2:1 ratio. As assessed by HPLC of the chondroitinase ABC digests, the chondroitin ({sup 35}S)sulfate proteoglycans isolated from these human lung mast cells contain the same unusual chondroitin sulfate E disaccharide that is present in proteoglycans produced by interleukin 3-dependent mucosal-like mouse mast cells. Both the chondroitin ({sup 35}S)sulfate E proteoglycans and the ({sup 35}S)heparin proteoglycans were exocytosed from the ({sup 35}S)sulfate-labeled cells via perturbation of the IgE receptor, indicating that both types of {sup 35}S-labeled proteoglycans reside in the secretory granules of these human lung mast cells.

  4. A network of HSPG core proteins and HS modifying enzymes regulates netrin-dependent guidance of D-type motor neurons in Caenorhabditis elegans.

    Science.gov (United States)

    Gysi, Stephan; Rhiner, Christa; Flibotte, Stephane; Moerman, Donald G; Hengartner, Michael O

    2013-01-01

    Heparan sulfate proteoglycans (HSPGs) are proteins with long covalently attached sugar side chains of the heparan sulfate (HS) type. Depending on the cellular context HS chains carry multiple structural modifications such as sulfate residues or epimerized sugars allowing them to bind to a wide range of molecules. HSPGs have been found to play extremely diverse roles in animal development and were shown to interact with certain axon guidance molecules. In this study we describe the role of the Caenorhabditis elegans HSPG core proteins Syndecan (SDN-1) and Glypican (LON-2) and the HS modifying enzymes in the dorsal guidance of D-type motor axons, a process controlled mainly by the conserved axon guidance molecule UNC-6/Netrin. Our genetic analysis established the specific HS code relevant for this axon guidance event. Using two sensitized genetic backgrounds, we isolated novel components influencing D-type motor axon guidance with a link to HSPGs, as well as new alleles of several previously characterized axon guidance genes. Interestingly, the dorsal axon guidance defects induced by mutations in zfp-1 or lin-35 depended on the transgene oxIs12 used to visualize the D-type motor neurons. oxIs12 is a large multi-copy transgene that enlarges the X chromosome by approximately 20%. In a search for genes with a comparable phenotype we found that a mutation in the known dosage compensation gene dpy-21 showed similar axon guidance defects as zfp-1 or lin-35 mutants. Thus, derepression of genes on X, where many genes relevant for HS dependent axon guidance are located, might also influence axon guidance of D-type motor neurons.

  5. A network of HSPG core proteins and HS modifying enzymes regulates netrin-dependent guidance of D-type motor neurons in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Stephan Gysi

    Full Text Available Heparan sulfate proteoglycans (HSPGs are proteins with long covalently attached sugar side chains of the heparan sulfate (HS type. Depending on the cellular context HS chains carry multiple structural modifications such as sulfate residues or epimerized sugars allowing them to bind to a wide range of molecules. HSPGs have been found to play extremely diverse roles in animal development and were shown to interact with certain axon guidance molecules. In this study we describe the role of the Caenorhabditis elegans HSPG core proteins Syndecan (SDN-1 and Glypican (LON-2 and the HS modifying enzymes in the dorsal guidance of D-type motor axons, a process controlled mainly by the conserved axon guidance molecule UNC-6/Netrin. Our genetic analysis established the specific HS code relevant for this axon guidance event. Using two sensitized genetic backgrounds, we isolated novel components influencing D-type motor axon guidance with a link to HSPGs, as well as new alleles of several previously characterized axon guidance genes. Interestingly, the dorsal axon guidance defects induced by mutations in zfp-1 or lin-35 depended on the transgene oxIs12 used to visualize the D-type motor neurons. oxIs12 is a large multi-copy transgene that enlarges the X chromosome by approximately 20%. In a search for genes with a comparable phenotype we found that a mutation in the known dosage compensation gene dpy-21 showed similar axon guidance defects as zfp-1 or lin-35 mutants. Thus, derepression of genes on X, where many genes relevant for HS dependent axon guidance are located, might also influence axon guidance of D-type motor neurons.

  6. Proteoglycan interactions with Sonic Hedgehog specify mitogenic responses

    Science.gov (United States)

    Chan, Jennifer A.; Balasubramanian, Srividya; Witt, Rochelle M.; Nazemi, Kellie J.; Choi, Yoojin; Pazyra-Murphy, Maria F.; Walsh, Carolyn O.; Thompson, Margaret; Segal, Rosalind A.

    2009-01-01

    SUMMARY Sonic Hedgehog (Shh) has dual roles in vertebrate development, as it promotes progenitor cell proliferation and induces tissue patterning. Here we show mitogenic and patterning functions of Shh can be uncoupled from one another. Using a genetic approach to selectively inhibit Shh-proteoglycan interactions in a mouse model, we show binding of Shh to proteoglycans is required for proliferation of neural stem/precursor cells but not for tissue patterning. Shh-proteoglycan interactions regulate both spatial and temporal features of Shh signaling. Proteoglycans localize Shh to specialized mitogenic niches and also act at the single cell level to regulate the duration of Shh signaling, thereby promoting a gene expression program important for cell division. As activation of the Shh pathway is a feature of diverse human cancers, selective stimulation of proliferation by Shh-proteoglycan interactions may also figure prominently in neoplastic growth. PMID:19287388

  7. Glycosaminoglycan and proteoglycan in skin aging.

    Science.gov (United States)

    Lee, Dong Hun; Oh, Jang-Hee; Chung, Jin Ho

    2016-09-01

    Glycosaminoglycans (GAGs) and proteoglycans (PGs) are abundant structural components of the extracellular matrix in addition to collagen fibers. Hyaluronic acid (HA), one of GAGs, forms proteoglycan aggregates, which are large complexes of HA and HA-binding PGs. Their crosslinking to other matrix proteins such as the collagen network results in the formation of supermolecular structures and functions to increase tissue stiffness. Skin aging can be classified as intrinsic aging and photoaging based on the phenotypes and putative mechanism. While intrinsic aging is characterized by a thinned epidermis and fine wrinkles caused by advancing age, photoaging is characterized by deep wrinkles, skin laxity, telangiectasias, and appearance of lentigines and is mainly caused by chronic sun exposure. The major molecular mechanism governing skin aging processes has been attributed to the loss of mature collagen and increased matrix metalloproteinase expression. However, various strategies focusing on collagen turnover remain unsatisfactory for the reversal or prevention of skin aging. Although the expression of GAGs and PGs in the skin and their regulatory mechanisms are not fully understood, we and others have elucidated various changes in GAGs and PGs in aged skin, suggesting that these molecules are important contributors to skin aging. In this review, we focus on skin-abundant GAGs and PGs and their changes in human skin during the skin aging process. Copyright © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

  8. Platelet Factor 4 Binds to Vascular Proteoglycans and Controls Both Growth Factor Activities and Platelet Activation.

    Science.gov (United States)

    Lord, Megan S; Cheng, Bill; Farrugia, Brooke L; McCarthy, Simon; Whitelock, John M

    2017-03-10

    Platelet factor 4 (PF4) is produced by platelets with roles in both inflammation and wound healing. PF4 is stored in platelet α-granules bound to the glycosaminoglycan (GAG) chains of serglycin. This study revealed that platelet serglycin is decorated with chondroitin/dermatan sulfate and that PF4 binds to these GAG chains. Additionally, PF4 had a higher affinity for endothelial-derived perlecan heparan sulfate chains than serglycin GAG chains. The binding of PF4 to perlecan was found to inhibit both FGF2 signaling and platelet activation. This study revealed additional insight into the ways in which PF4 interacts with components of the vasculature to modulate cellular events. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. [Expression features of glypican-3 and its diagnostic and differential values in hepatocellular carcinoma].

    Science.gov (United States)

    Bian, Yin-zhu; Yao, Deng-fu; Zhang, Chong-guo; Li, Shan-shan; Wu, Wei; Dong, Zhi-zhen; Qiu, Li-wei; Yu, Dan-dan

    2011-04-01

    To investigate the expression features of glypican-3 (GPC-3) and its diagnostic and differential values in hepatocellular carcinoma (HCC). Rat hepatoma models were made and the dynamic expression features of GPC-3 protein and its gene were investigated by Western blotting and RT-PCR respectively. Liver specimens from 36 HCC patients were collected by self-control method and the expression and clinicopathological features of GPC-3 were analyzed by immunohistochemistry. Serum GPC-3 levels were quantitatively detected by ELISA and its efficiency for HCC diagnosis was evaluated in patients with liver diseases. The incidence of GPC-3 was 0% in control, 83.3% in degeneration, 100% in precancerosis and 100% in canceration during dynamic formation of rat hepatoma, respectively. The positive GPC-3 was brown granule- like staining localized in membrane and cytoplasm in human HCC. The GPC-3 positive rates were 80.6% in HCC, 41.7% in surrounding tissues and none in distal tissues (P differentiation grade or the number of tumor except of tumor size (Z = 2.941, P expression closely associated with HCC and might be useful for early diagnosis of HCC.

  10. Disease: H00493 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available THORS ... Farach-Carson MC, Hecht JT, Carson DD ... TITLE ... Heparan sulfate proteoglycans: key players in cartilage biology... ... Schwartz NB, Domowicz M ... TITLE ... Chondrodysplasias due to proteoglycan defects. ... JOURNAL ... Glycobiol...ogy 12:57R-68R (2002) DOI:10.1093/glycob/12.4.57R ... PMID:15831077 (description) ... AU

  11. Proteoglycans of uterine fibroids and keloid scars: similarity in their proteoglycan composition.

    Science.gov (United States)

    Carrino, David A; Mesiano, Sam; Barker, Nichole M; Hurd, William W; Caplan, Arnold I

    2012-04-15

    Fibrosis is the formation of excess and abnormal fibrous connective tissue as a result of either a reparative or reactive process. A defining feature of connective tissue is its extracellular matrix, which provides structural support and also influences cellular activity. Two common human conditions that result from fibrosis are uterine fibroids (leiomyomas) and keloid scars. Because these conditions share a number of similarities and because their growth is due primarily to excessive extracellular matrix deposition, we compared the proteoglycans of uterine fibroids and keloid scars with corresponding normal tissues. Our analysis indicates that uterine fibroids and keloid scars contain higher amounts of glycosaminoglycans relative to normal myometrium and normal adult skin respectively. Proteoglycan composition is also different in the fibrotic tissues. Compared with unaffected tissues, uterine fibroids and keloid scars contain higher relative amounts of versican and lower relative amounts of decorin. There is also evidence for a higher level of versican catabolism in the fibrotic tissues compared with unaffected tissues. These qualitative and quantitative proteoglycan differences may play a role in the expansion of these fibroses and in their excessive matrix deposition and matrix disorganization, due to effects on cell proliferation, TGF (transforming growth factor)-β signalling and/or collagen fibril formation.

  12. Dysplastic nodules with glypican-3 positive immunostaining: a risk for early hepatocellular carcinoma.

    Directory of Open Access Journals (Sweden)

    Li Gong

    Full Text Available Glypican-3 (GPC3 has been reported to be a novel serum and histochemical marker for HCC. The positivity or negativity for GPC3 in hepatic precancerous lesions, such as dysplastic nodules (DN, has also been described. Moreover, our previous studies have demonstrated that some DN in liver cirrhosis represent monoclonal hyperplasia, and confirmed their neoplastic nature. However, additional studies must be performed to investigate further the relationship between DN with GPC3 positivity and HCC. Thus, we first investigated the expression of GPC3 in 136 HCC and 103 small DN (less than 1 cm in diameter by immunohistochemical staining and determined the clonality of 81 DN from female patients using X-chromosome inactivation mosaicism and polymorphism of androgen receptor (AR gene. Then we examined these samples for chromosomal loss of heterozygosity (LOH at 11 microsatellite polymorphism sites. The results demonstrated that GPC3 immunoreactivity was detected in 103 of 136 HCC (75.7% and 19 of 103 DN (18.4%, and the positive ratio correlated with HBsAg positivity. Clonality assays showed that 15 GPC3-positive DN from female patients, including 12 high-grade DN (HGDN, and 28 (42.4% of 66 GPC3-negative DN, were monoclonal. In addition, among 19 GPC3-positive DN, chromosomal LOH was found at loci D6S1008 (100%, 19/19, D8S262 (52.6%, 10/19 and D11S1301 (57.9%, 11/19. However, the LOH frequency in GPC3-negative DN was 5.95% (5/84, 23.8% (20/84, and 4.76% (4/84 in three loci, respectively. Thus, we concluded that GPC3-positive DN, especially GPC3-positive HGDN, was really a late premalignant lesion of HCC.

  13. Recent Insights into Cell Surface Heparan Sulphate Proteoglycans and Cancer

    DEFF Research Database (Denmark)

    Couchman, John R; Multhaupt, Hinke; Sanderson, Ralph D

    2016-01-01

    A small group of cell surface receptors are proteoglycans, possessing a core protein with one or more covalently attached glycosaminoglycan chains. They are virtually ubiquitous and their chains are major sites at which protein ligands of many types interact. These proteoglycans can signal...... and regulate important cell processes, such as adhesion, migration, proliferation, and differentiation. Since many protein ligands, such as growth factors, morphogens, and cytokines, are also implicated in tumour progression, it is increasingly apparent that cell surface proteoglycans impact tumour cell...... behaviour. Here, we review some recent advances, emphasising that many tumour-related functions of proteoglycans are revealed only after their modification in processes subsequent to synthesis and export to the cell surface. These include enzymes that modify heparan sulphate structure, recycling of whole...

  14. Mycobacterial antigens stimulate rheumatoid mononuclear cells to cartilage proteoglycan depletion

    NARCIS (Netherlands)

    Wilbrink, B.; Bijlsma, J. W.; Huber-Bruning, O.; van Roy, J. L.; den Otter, W.; van Eden, W.

    1990-01-01

    In a coculture with porcine articular cartilage explants unstimulated blood mononuclear cells (BMC) from patients with rheumatoid arthritis (RA), but not from healthy controls, induced proteoglycan depletion of dead cartilage. Specific stimulation of the RA BMC with Mycobacterium tuberculosis (MT),

  15. MicroRNA regulation of proteoglycan function in cancer.

    Science.gov (United States)

    Ibrahim, Sherif A; Hassan, Hebatallah; Götte, Martin

    2014-11-01

    MicroRNAs are small noncoding RNAs acting as physiological regulators of gene expression at the post-transcriptional level. In cancer, the expression of microRNAs is dysregulated compared to healthy tissue, suggesting a mechanistic role in disease progression. Recent experimental evidence supports the important molecular role of proteoglycans as microRNA targets in this process. Misexpression of specific microRNAs results in aberrant expression patterns of proteoglycans, as well as their biosynthetic enzymes. Consequently, cell proliferation and apoptosis, adhesion, migration, invasiveness, epithelial-to-mesenchymal transition and cancer stem cell properties are affected as a result of the multifunctional properties of proteoglycans. A pharmacological targeting of the microRNA-proteoglycan axis emerges as a new therapeutic concept in cancer. © 2014 FEBS.

  16. Crystal packing in three related disaccharides: precursors to heparan sulfate oligosaccharides

    Directory of Open Access Journals (Sweden)

    Graeme J. Gainsford

    2015-06-01

    Full Text Available The three title compounds form part of a set of important precursor dissacharides which lead to novel therapeutics, in particular for Alzheimer's disease. All three crystallize as poorly diffracting crystals with one independent molecule in the asymmetric unit. Two of them are isostructural: 4-methoxyphenyl 4-O-[6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-(9-fluorenylmethyloxycarbonyl-α-d-glucopyranosyl]-2-O-benzoyl-3-O-benzyl-6-O-chloroacetyl-α-l-idopyranoside, C59H56ClN3O16, (I, the ido-relative of a reported gluco-disaccharide [Gainsford et al., 2013. Acta Cryst. C69, 679–682] and 4-methoxyphenyl 4-O-[6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-(9-fluorenylmethyloxycarbonyl-α-d-glucopyranosyl]-2-O-benzoyl-3-O-benzyl-6-O-methoxyacetyl-α-l-idopyranoside, C60H59N3O17, (II. Both exhibit similar conformational disorder of pendant groups. The third compound 4-methoxyphenyl 4-O-[6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-α-d-glucopyranosyl]-2-O-benzoyl-3-O-benzyl-6-O-methoxyoacetyl-β-d-glucopyranoside, C52H55N3O15, (III, illustrates that a slightly larger set of weak intermolecular interactions can result in a less disordered molecular arrangement. The molecules are bound by weak C—H...O(ether hydrogen bonds in (I and (II, augmented by C—H...π interactions in (III. The absolute configurations were determined, although at varying levels of significance from the limited observed data.

  17. Endothelial cell surface heparan sulfate (ESHS) and synthetic heparin derivatives as hemocompatible coating for biomaterials

    Energy Technology Data Exchange (ETDEWEB)

    Hoffmann, M.; Huppertz, B.; Horres, R.; Baumann, H. [RWTH, Aachen (Germany). Makromolekulare Chemie und Textilchemie, Haemokompatible und Biokompatible Biomaterialien; Keller, R. [Klinische Anstalten der Stadt Koeln (Germany)

    2001-02-01

    In the present overview a coating procedure, that has been developed in our working group for medical devices e.g. implants, which are exposed to permanent blood contact and therefore have to fulfill the highest standard of hemocompatibility is described. For this purpose an endothelial cell surface heparansulfate, which belongs to the class of glycosaminoglycans is used as coating substance. This substance can be isolated from endothelial cell culture, tissue extracts or organ perfusates. Alternatively chemical regio- and stereoselective modified derivatives of the structurally related anticoagulant heparin were brought to action. These substances are anchored covalently or ionically by application of a wide spectrum of immobilization techniques on many different material surfaces. Polymer materials modified as described here have been tested for hemocompatibility in invitro and in invivo experiments with Austrian sheep. The results show, that the described method is an advanced solution for the creation of long term hemocompatible artificial material surfaces. (orig.) [German] In dem vorliegenden Uebersichtsartikel wird ein in unserer Arbeitsgruppe entwickeltes athrombogenes und plaettcheninertes Beschichtungsverfahren fuer medizinische Werkstoffoberflaechen wie z.B. Implantate, die staendigem direktem Blutkontakt ausgesetzt sind und infolge dessen ein Hoechstmass an Haemokompatibilitaet aufweisen muessen, zusammenfassend beschrieben. Hierzu wird als Beschichtungssubstanz ein aus Zellkultur, Gewebeextrakten oder Organperfusaten isolierbares zur Klasse der Glycosaminoglycane zaehlendes Endothelzelloberflaechenparansulfat (ESHS) verwendet. Alternativ werden chemisch regio- und stereoselektiv modifizierte Derivate des strukturverwandten klassischen Antikoagulanzes Heparin als Beschichtungssubstanz eingesetzt. Diese Substanzen werden unter Anwendung eines breiten Spektrums von Immobilisierungstechniken auf verschiedensten Werkstoffoberflaechen kovalent oder ionisch verankert. Solchermassen modifizierte Polymermaterialien wurden sowohl in in-vitro, wie auch in in-vivo Versuchen an oesterreichischen Bergschafen auf ihre Blutvertraeglichkeit hin getestet. Die Ergebnisse zeigen, dass mit der hier beschriebenen Methodik eine zukunftsweisende Loesung zur Schaffung langzeitblutvertraeglicher kuenstlicher Werkstoffoberflaechen zur Verfuegung steht. (orig.)

  18. Immobilization of heparan sulfate on electrospun meshes to support embryonic stem cell culture and differentiation

    NARCIS (Netherlands)

    Meade, K.A.; White, K.J.; Pickford, C.E.; Holley, R.J.; Marson, A.; Tillotson, D.; Kuppevelt, A.H.M.S.M. van; Whittle, J.D.; Day, A.J.; Merry, C.L.

    2013-01-01

    As our understanding of what guides the behavior of multi- and pluripotent stem cells deepens, so too does our ability to utilize certain cues to manipulate their behavior and maximize their therapeutic potential. Engineered, biologically functionalized materials have the capacity to influence stem

  19. Increased deposition of glycosaminoglycans and altered structure of heparan sulfate in idiopathic pulmonary fibrosis

    NARCIS (Netherlands)

    Westergren-Thorsson, G.; Hedstrom, U.; Nybom, A.; Tykesson, E.; Ahrman, E.; Hornfelt, M.; Maccarana, M.; Kuppevelt, T.H. van; Dellgren, G.; Wildt, M.; Zhou, X.H.; Eriksson, L.; Bjermer, L.; Hallgren, O.

    2017-01-01

    Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant deposition of extracellular matrix (ECM) constituents, including glycosaminoglycans (GAGs), that may play a role in remodelling processes by influencing critical mediators such as growth factors. We hypothesize that GAGs may be altered

  20. Probing cleavage promiscuity of heparinase III towards chemoenzymatically synthetic heparan sulfate oligosaccharides.

    Science.gov (United States)

    Hu, Guixin; Shao, Meng; Gao, Xin; Wang, Fengshan; Liu, Chunhui

    2017-10-01

    An insightful investigation into specificity of bacterial heparinase III has been intriguingly difficult due to heterogeneity of polymeric substrates. Herein, we chemoenzymatically synthesized a tailored library of HS oligosaccharides as substrates. A ∼15-fold reactivity difference to heparinase III was found between trisaccharides bearing different primary cleavage sites. Variable glucosamine modification decreased reactivity of trisaccharides by >20-fold compared with their counterpart primary substrates, while iduronate-containing secondary linkage showed slightly less sensitivity. The 2-sulfated iduronate residue extremely reduced reactivity to its adjacent primary site at reducing end of oligosaccharides, but showed marginal influence on the non-reducing site. Moreover, oligosaccharide susceptibility to digestion was size-dependent and had an obvious preference for the internal linkages over those near to non-reducing/reducing ends. Surface plasmon resonance revealed cleavage promiscuity attributed to different affinities or incorrect binding of substrates to the enzyme. The attractive information on heparinase III will be valuable in characterizing heparin and HS. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. EDTA-insoluble, calcium-binding proteoglycan in bovine bone

    Science.gov (United States)

    Hashimoto, Y.; Lester, G. E.; Caterson, B.; Yamauchi, M.

    1995-01-01

    A calcium ion precipitable, trypsin-generated proteoglycan fragment has been isolated from the demineralized, EDTA-insoluble matrices of bone. The demineralized matrix was completely digested with trypsin, increasing concentrations of CaCl2 were added to the supernatant, and the resulting precipitates were analyzed. The amount of precipitate gradually increased with higher concentrations of calcium and was reversibly solubilized by EDTA. After molecular sieve and anion exchange chromatography, a proteoglycan-containing peak was obtained. Immunochemical analysis showed that this peak contained chondroitin 4-sulfate and possibly keratan sulfate. Amino acid analysis showed that this proteoglycan contained high amounts of aspartic acid/asparagine (Asx), serine (Ser), glutamic acid/glutamine (Glx), proline (Pro), and glycine (Gly); however, it contained little leucine (Leu) which suggests that it is not a member of the leucine-rich small proteoglycan family. In addition, significant amounts of phosphoserine (P-Ser) and hydroxyproline (Hyp) were identified in hydrolysates of this fraction. A single band (M(r) 59 kDa) was obtained on SDS-PAGE that stained with Stains-all but not with Coomassie Brilliant Blue R-250. If bone powder was trypsinized prior to demineralization, this proteoglycan-containing fraction was not liberated. Collectively, these results indicate that a proteoglycan occurs in the demineralized matrix that is precipitated with CaCl2 and is closely associated with both mineral and collagen matrices. Such a molecule might facilitate the structural network for the induction of mineralization in bone.

  2. EDTA-insoluble, calcium-binding proteoglycan in bovine bone.

    Science.gov (United States)

    Hashimoto, Y; Lester, G E; Caterson, B; Yamauchi, M

    1995-05-01

    A calcium ion precipitable, trypsin-generated proteoglycan fragment has been isolated from the demineralized, EDTA-insoluble matrices of bone. The demineralized matrix was completely digested with trypsin, increasing concentrations of CaCl2 were added to the supernatant, and the resulting precipitates were analyzed. The amount of precipitate gradually increased with higher concentrations of calcium and was reversibly solubilized by EDTA. After molecular sieve and anion exchange chromatography, a proteoglycan-containing peak was obtained. Immunochemical analysis showed that this peak contained chondroitin 4-sulfate and possibly keratan sulfate. Amino acid analysis showed that this proteoglycan contained high amounts of aspartic acid/asparagine (Asx), serine (Ser), glutamic acid/glutamine (Glx), proline (Pro), and glycine (Gly); however, it contained little leucine (Leu) which suggests that it is not a member of the leucine-rich small proteoglycan family. In addition, significant amounts of phosphoserine (P-Ser) and hydroxyproline (Hyp) were identified in hydrolysates of this fraction. A single band (M(r) 59 kDa) was obtained on SDS-PAGE that stained with Stains-all but not with Coomassie Brilliant Blue R-250. If bone powder was trypsinized prior to demineralization, this proteoglycan-containing fraction was not liberated. Collectively, these results indicate that a proteoglycan occurs in the demineralized matrix that is precipitated with CaCl2 and is closely associated with both mineral and collagen matrices. Such a molecule might facilitate the structural network for the induction of mineralization in bone.

  3. Distribution of individual components of basement membrane in human colon polyps and adenocarcinomas as revealed by monoclonal antibodies

    DEFF Research Database (Denmark)

    Ljubimov, A V; Bartek, J; Couchman, J R

    1992-01-01

    by the presence of fibrillar deposits of basement-membrane components, mainly of collagen type IV and/or heparan sulfate proteoglycan. This reaction was never observed in polyps and may be derived from myofibroblasts reported to accumulate in colon cancer stroma. The combined use of antibodies to basement......-membrane components (laminin, entactin/nidogen, collagen type IV and large heparan sulfate proteoglycan), as well as to keratin 8. In all adenocarcinomas, including mucinous, basement membranes were altered more at the invasive front than in the parenchyma. The degree of this alteration was inversely correlated...

  4. Host glycosaminoglycan confers susceptibility to bacterial infection in Drosophila melanogaster.

    Science.gov (United States)

    Baron, Miriam J; Wong, Sandra L; Nybakken, Kent; Carey, Vincent J; Madoff, Lawrence C

    2009-02-01

    Many pathogens engage host cell surface glycosaminoglycans, but redundancy in pathogen adhesins and host glycosaminoglycan-anchoring proteins (heparan sulfate proteoglycans) has limited the understanding of the importance of glycosaminoglycan binding during infection. The alpha C protein of group B streptococcus, a virulence determinant for this neonatal human pathogen, binds to host glycosaminoglycan and mediates the entry of bacteria into human cells. We studied alpha C protein-glycosaminoglycan binding in Drosophila melanogaster, whose glycosaminoglycan repertoire resembles that of humans but whose genome includes only three characterized membrane heparan sulfate proteoglycan genes. The knockdown of glycosaminoglycan polymerases or of heparan sulfate proteoglycans reduced the cellular binding of alpha C protein. The interruption of alpha C protein-glycosaminoglycan binding was associated with longer host survival and a lower bacterial burden. These data indicate that the glycosaminoglycan-alpha C protein interaction involves multiple heparan sulfate proteoglycans and impairs bacterial killing. Host glycosaminoglycans, anchored by multiple proteoglycans, thereby determine susceptibility to infection. Because there is homology between Drosophila and human glycosaminoglycan/proteoglycan structures and many pathogens express glycosaminoglycan-binding structures, our data suggest that interfering with glycosaminoglycan binding may protect against infections in humans.

  5. Proteoglycan: site mapping and site-directed mutagenesis.

    Science.gov (United States)

    Hagen, Fred K

    2012-01-01

    Identification of proteoglycan chain modification sites cannot yet be reliably predicted from primary amino acid sequence data. A number of studies have shown that serine is the predominant amino acid that is modified and it is frequently flanked by a C-terminal glycine and proximal N-terminal acidic amino acids; however, not all simple Ser-Gly motifs constitute a modification site. Here we present a rapid method for cloning small, defined segments of putative proteoglycan attachment sites and expressing them as a mini-reporter protein in an insect tissue culture system that is expandable to high throughput analysis. Reporter proteins with attached proteoglycans can be readily discerned from their unmodified form, by a simple gel-shift assay and Western blot detection for an epitope tag engineered into the reporter. Unmodified proteins are generated as a reference standard by treating cells with dsRNA to knock down the endogenous polypeptide xylose transferase, which is responsible for initiating proteoglycan site attachment. Examination of proteoglycan attachment by different metazoan organisms can be studied in the same cell line by cotransfecting a polypeptide xylose transferase expression plasmid and reporter construct from human, mouse, frog, or worm, for example. Reporter proteins engineer with point mutations can be rapidly generated with this system to pinpoint the exact residue that is glycosylated, to verify the mapping data.

  6. Proteoglycans support proper granule formation in pancreatic acinar cells.

    Science.gov (United States)

    Aroso, Miguel; Agricola, Brigitte; Hacker, Christian; Schrader, Michael

    2015-10-01

    Zymogen granules (ZG) are specialized organelles in the exocrine pancreas which allow digestive enzyme storage and regulated secretion. The molecular mechanisms of their biogenesis and the sorting of zymogens are still incompletely understood. Here, we investigated the role of proteoglycans in granule formation and secretion of zymogens in pancreatic AR42J cells, an acinar model system. Cupromeronic Blue cytochemistry and biochemical studies revealed an association of proteoglycans primarily with the granule membrane. Removal of proteoglycans by carbonate treatment led to a loss of membrane curvature indicating a supportive role in the maintenance of membrane shape and stability. Chemical inhibition of proteoglycan synthesis impaired the formation of normal electron-dense granules in AR42J cells and resulted in the formation of unusually small granule structures. These structures still contained the zymogen carboxypeptidase, a cargo molecule of secretory granules, but migrated to lighter fractions after density gradient centrifugation. Furthermore, the basal secretion of amylase was increased in AR42J cells after inhibitor treatment. In addition, irregular-shaped granules appeared in pancreatic lobules. We conclude that the assembly of a proteoglycan scaffold at the ZG membrane is supporting efficient packaging of zymogens and the proper formation of stimulus-competent storage granules in acinar cells of the pancreas.

  7. Intermittent hydrostatic compressive force stimulates exclusively the proteoglycan synthesis of osteoarthritic human cartilage

    NARCIS (Netherlands)

    Lafeber, F.; Veldhuijzen, J. P.; Vanroy, J. L.; Huber-Bruning, O.; Bijlsma, J. W.

    1992-01-01

    In paired observations the in vitro proteoglycan turnover was studied of human normal and osteoarthritic cartilage in the absence and presence of intermittent hydrostatic compressive force. Shortly after collection, osteoarthritic cartilage showed a higher proteoglycan synthesis rate than normal

  8. Characterization of a dermatan sulfate proteoglycan synthesized by murine parietal yolk sac (PYS-2) cells

    DEFF Research Database (Denmark)

    Couchman, J R; Woods, A; Höök, M

    1985-01-01

    A dermatan sulfate proteoglycan has been isolated from a murine parietal yolk sac cell line, which in culture synthesizes basement membrane components. The proteoglycan has a molecular weight of 200,000-300,000 with 10-15 dermatan sulfate chains of Mr = 14,000-16,000. The glycosaminoglycan chains......-polyacrylamide gel electrophoresis of chondroitinase ABC-treated 125I-labeled proteoglycan reveals two polypeptides with molecular weights of 34,000 and 27,000. Results from papain digestion of the proteoglycan suggest that most of the polysaccharide chains are clustered at a papain-resistant segment of the core...... protein (Mr = 8,000). This proteoglycan is distinctly different from the large cartilage proteoglycan in the smaller size of its core protein, and its relationship to other small chondroitin and dermatan sulfate proteoglycans and to the chondroitin sulfate proteoglycan recently located in rat tissue...

  9. Metabolism of Cartilage Proteoglycans in Health and Disease

    Science.gov (United States)

    Vynios, Demitrios H.

    2014-01-01

    Cartilage proteoglycans are extracellular macromolecules with complex structure, composed of a core protein onto which a variable number of glycosaminoglycan chains are attached. Their biosynthesis at the glycosaminoglycan level involves a great number of sugar transferases well-orchestrated in Golgi apparatus. Similarly, their degradation, either extracellular or intracellular in lysosomes, involves a large number of hydrolases. A deficiency or malfunction of any of the enzymes participating in cartilage proteoglycan metabolism may lead to severe disease state. This review summarizes the findings regarding this topic. PMID:25105124

  10. Metabolism of Cartilage Proteoglycans in Health and Disease

    Directory of Open Access Journals (Sweden)

    Demitrios H. Vynios

    2014-01-01

    Full Text Available Cartilage proteoglycans are extracellular macromolecules with complex structure, composed of a core protein onto which a variable number of glycosaminoglycan chains are attached. Their biosynthesis at the glycosaminoglycan level involves a great number of sugar transferases well-orchestrated in Golgi apparatus. Similarly, their degradation, either extracellular or intracellular in lysosomes, involves a large number of hydrolases. A deficiency or malfunction of any of the enzymes participating in cartilage proteoglycan metabolism may lead to severe disease state. This review summarizes the findings regarding this topic.

  11. Proteoglycan synthesis and Golgi organization in polarized epithelial cells.

    Science.gov (United States)

    Dick, Gunnar; Akslen-Hoel, Linn K; Grøndahl, Frøy; Kjos, Ingrid; Prydz, Kristian

    2012-12-01

    A large number of complex glycosylation mechanisms take place in the Golgi apparatus. In epithelial cells, glycosylated protein molecules are transported to both the apical and the basolateral surface domains. Although the prevailing view is that the Golgi apparatus provides the same lumenal environment for glycosylation of apical and basolateral cargo proteins, there are indications that proteoglycans destined for the two opposite epithelial surfaces are exposed to different conditions in transit through the Golgi apparatus. We will here review data relating proteoglycan and glycoprotein synthesis to characteristics of the apical and basolateral secretory pathways in epithelial cells.

  12. Local changes in proteoglycan synthesis during culture are different for normal and osteoarthritic cartilage

    NARCIS (Netherlands)

    Lafeber, F. P.; van der Kraan, P. M.; van Roy, H. L.; Vitters, E. L.; Huber-Bruning, O.; van den Berg, W. B.; Bijlsma, J. W.

    1992-01-01

    Proteoglycan synthesis of mild-to-moderate osteoarthritic human knee cartilage was compared with that of normal cartilage of the same donor. Immediately after cartilage was obtained, the synthesis rate of proteoglycans was higher for osteoarthritic cartilage than for normal cartilage. Proteoglycan

  13. Detailed expression profile of all six Glypicans and their modifying enzyme Notum during chick embryogenesis and their role in dorsal-ventral patterning of the neural tube.

    Science.gov (United States)

    Saad, Kawakeb; Otto, Anthony; Theis, Susanne; Kennerley, Niki; Munsterberg, Andrea; Luke, Graham; Patel, Ketan

    2017-04-20

    Vertebrate development is orchestrated by secreted signalling molecules that regulate cell behaviour and cell fate decisions during early embryogenesis. The activity of key signalling molecules including members of Hedgehog, Bone Morphogenetic Proteins and Wnt families are regulated by Glypicans, a family of GPI linked polypeptides. Glypicans either promote or inhibit the action of signalling molecules and add a layer of complexity that needs to be understood in order to fully decipher the processes that regulate early vertebrate development. Here we present a detailed expression profile of all six Glypicans and their modifying enzyme Notum during chick embryogenesis. Our results strongly suggest that these proteins have many as yet undiscovered roles to play during early embryogenesis. Finally, we have taken an experimental approach to investigate their role during the patterning of a key embryonic structure - the neural tube. In particular, we show that over-expression of Notum leads to the dorsalisation of this structure. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Wnt Signaling Cascades and the Roles of Syndecan Proteoglycans

    DEFF Research Database (Denmark)

    Pataki, Csilla A; Couchman, John R; Brábek, Jan

    2015-01-01

    /planar cell polarity and Wnt/calcium signaling. Syndecans are type I transmembrane proteoglycans with a long evolutionary history, being expressed in all Bilateria and in almost all cell types. Both Wnt pathways have been extensively studied over the past 30 years and shown to have roles during development...

  15. Mechanisms of axon regeneration: The significance of proteoglycans.

    Science.gov (United States)

    Sakamoto, Kazuma; Kadomatsu, Kenji

    2017-10-01

    Therapeutics specific to neural injury have long been anticipated but remain unavailable. Axons in the central nervous system do not readily regenerate after injury, leading to dysfunction of the nervous system. This failure of regeneration is due to both the low intrinsic capacity of axons for regeneration and the various inhibitors emerging upon injury. After many years of concerted efforts, however, these hurdles to axon regeneration have been partially overcome. This review summarizes the mechanisms regulating axon regeneration. We highlight proteoglycans, particularly because it has become increasingly clear that these proteins serve as critical regulators for axon regeneration. Studies on proteoglycans have revealed that glycans not only assist in the modulation of protein functions but also act as main players-e.g., as functional ligands mediating intracellular signaling through specific receptors on the cell surface. By regulating clustering of the receptors, glycans in the proteoglycan moiety, i.e., glycosaminoglycans, promote or inhibit axon regeneration. In addition, proteoglycans are involved in various types of neural plasticity, ranging from synaptic plasticity to experience-dependent plasticity. Although studies on proteins have progressively facilitated our understanding of the nervous system, glycans constitute a new frontier for further research and development in this field. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Mesenchymal stem cell therapy in proteoglycan induced arthritis

    NARCIS (Netherlands)

    Swart, J. F.; de Roock, S.; Hofhuis, F. M.; Rozemuller, H.; van den Broek, T.; Moerer, P.; Broere, F.|info:eu-repo/dai/nl/264075323; van Wijk, F.; Kuis, W.; Prakken, B. J.; Martens, a.c.m|info:eu-repo/dai/nl/375286063; Wulffraat, N. M.

    2015-01-01

    Objectives: To explore the immunosuppressive effect and mechanism of action of intraperitoneal (ip) and intra-articular (ia) mesenchymal stem cell (MSC) injection in proteoglycan induced arthritis (PGIA). Methods: MSC were administered ip or ia after establishment of arthritis. We used serial

  17. Evaluation of antiglypican-3 therapy as a promising target for amelioration of hepatic tissue damage in hepatocellular carcinoma.

    Science.gov (United States)

    Zaghloul, Randa A; El-Shishtawy, Mamdouh M; El Galil, Khaled H Abd; Ebrahim, Mohamed A; Metwaly, AbdelHamid A; Al-Gayyar, Mohammed M

    2015-01-05

    In Egypt, hepatocellular carcinoma (HCC) was predicted to continue to rise over the next few decades causing a national problem. Meanwhile, glypican-3 (GPC3), a highly expressed glypican, has emerged as a potential target for HCC immunotherapy. Therefore, we aimed to identify the impact of blocking GPC3 on liver damage in HCC as well as a possible mechanism. Fifty four HCC patients, 20 cirrhotic patients and 10 healthy subjects were recruited. Serum levels of GPC3, sulfatase-2 (SULF-2), heparan sulfate proteoglycan (HSPG), insulin-like growth factor-II (IGF-II) were measured by ELISA. In parallel, HCC was induced in 40 male Sprague-Dawley rats in presence/absence of antiGPC-3. Liver impairment was detected by investigating liver sections stained with hematoxylin/eosin and serum α-fetoprotein (AFP). Liver homogenates of GPC3, SULF-2, and HSPG were measured by ELISA. Gene expression of caspase-3 and IGF-II were assayed by RT-PCR. HCC patients showed significant elevated serum levels of GPC3, IGF-II and SULF-2 accompanied by decreased HSPG. However, treatment of HCC rats with antiGPC-3 significantly reduced serum AFP and showed nearly normal hepatocytes. In addition, antiGPC-3 significantly reduced elevated liver homogenates protein levels of GPC3 and SULF-2 and gene expression of IGF-II and caspase-3. antiGPC-3 restored the reduced hepatic HSPG. antiGPC-3 showed anti-tumor activity as well as hepatoprotective effects. antiGPC-3-chemoprotective effect can be explained by forced reduction of IGF-II expression, restoration of HSPGs, deactivation of SULF-2 and reduction of gene expression of caspase-3. Targeting GPC3 is a promising therapeutic approach for HCC. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Proteoglycan expression is influenced by mechanical load in TMJ discs.

    Science.gov (United States)

    Nakao, Y; Konno-Nagasaka, M; Toriya, N; Arakawa, T; Kashio, H; Takuma, T; Mizoguchi, I

    2015-01-01

    The expression and assembly of the extracellular matrix are profoundly associated with adaptive and pathological responses of the temporomandibular joint (TMJ). To better understand the adaptive responses of the TMJ disc to mechanical loading, we examined the expression of 2 modular proteoglycans and 10 small leucine-rich proteoglycans (SLRPs) at the mRNA and protein levels and determined the contents of proteoglycan-related glycosaminoglycans (GAGs) in rat TMJ discs in response to altered mechanical loading caused by an incisal bite plane. One hundred thirty 7-week-old male Wistar rats were assigned to control and bite plane groups. TMJ disc thickness and the intensity of toluidine blue staining of metachromasia increased in the posterior band after 2 weeks of wearing the bite plane. GAG content increased significantly in the bite plane group after 2 weeks. Quantitative real-time RT-PCR (reverse transcription polymerase chain reaction) analysis indicated that biglycan and chondroadherin mRNA levels increased after 2 weeks and that the level of decorin mRNA increased at 4 weeks. Versican mRNA levels increased after 3 weeks, particularly for the V0 and V1 versican isoforms, which carry more GAG attachment sites than do the V2 and V3 isoforms. Western analysis demonstrated a corresponding increase in the levels of versican, biglycan, and decorin core proteins at 4 weeks in the bite plane group. These results indicate that mechanical loading differentially influences proteoglycan mRNA expression and protein accumulation in the TMJ disc. The change in proteoglycan mRNA and protein levels may lead to the modulation of matrix-matrix and cell-matrix interactions and has important biological significance for adaptation to complicated biomechanical requirements and for tissue maintenance in the TMJ disc. © International & American Associations for Dental Research 2014.

  19. Proteoglycans maintain lung stability in an elastase-treated mouse model of emphysema.

    Science.gov (United States)

    Takahashi, Ayuko; Majumdar, Arnab; Parameswaran, Harikrishnan; Bartolák-Suki, Erzsébet; Suki, Béla

    2014-07-01

    Extracellular matrix remodeling and tissue rupture contribute to the progression of emphysema. Lung tissue elasticity is governed by the tensile stiffness of fibers and the compressive stiffness of proteoglycans. It is not known how proteoglycan remodeling affects tissue stability and destruction in emphysema. The objective of this study was to characterize the role of remodeled proteoglycans in alveolar stability and tissue destruction in emphysema. At 30 days after treatment with porcine pancreatic elastase, mouse lung tissue stiffness and alveolar deformation were evaluated under varying tonicity conditions that affect the stiffness of proteoglycans. Proteoglycans were stained and measured in the alveolar walls. Computational models of alveolar stability and rupture incorporating the mechanical properties of fibers and proteoglycans were developed. Although absolute tissue stiffness was only 24% of normal, changes in relative stiffness and alveolar shape distortion due to changes in tonicity were increased in emphysema (P proteoglycan stiffness, was higher in emphysema (P proteoglycan stiffness was increased. Consequently, this general network model explains why increasing proteoglycan deposition protects the alveolar walls from rupture in emphysema. Our results suggest that the loss of proteoglycans observed in human emphysema contributes to disease progression, whereas treatments that promote proteoglycan deposition in the extracellular matrix should slow the progression of emphysema.

  20. Immunological methods for the detection and determination of connective tissue proteoglycans

    DEFF Research Database (Denmark)

    Caterson, B; Baker, J R; Christner, J E

    1982-01-01

    In this paper we report the use of immunological methods for specifically detecting and determining proteoglycan in cartilage and other connective tissues. Antibodies (polyclonal and monoclonal) have been raised against specific components of cartilage proteoglycan aggregates (i.e., proteoglycan...... monomer and link protein). Radioimmunoassay procedures and immunohistochemical procedures have been developed and used to demonstrate the occurrence of cartilage-like proteoglycan and link protein in bovine aorta. Similarly, immunofluorescent studies have been used to analyze proteoglycan distribution...... in skin. Using antibodies specific for chondroitin-4-sulfated proteoglycan, their presence was demonstrated in dermal connective tissue and connective tissue surrounding nerve and muscle sheaths. However, chondroitin-4-sulfated proteoglycan was completely absent in the epidermis of skin and areas...

  1. Production and characterization of monoclonal antibodies directed against connective tissue proteoglycans

    DEFF Research Database (Denmark)

    Caterson, B; Christner, J E; Baker, J R

    1985-01-01

    Monoclonal antibodies have been raised against determinants present in cartilage proteoglycan. Characterization of the specificity of these antibodies indicated that they recognize determinants present in the keratan sulfate glycosaminoglycan chain and on chondroitin sulfate oligosaccharide stubs...... attached to the proteoglycan core protein after chondroitinase digestion of the proteoglycan (i.e., delta-unsaturated 4- and 6-sulfated and unsulfated chondroitin sulfate on the proteoglycan core). The antibody recognizing keratan sulfate has been used to demonstrate the presence of a keratan sulfate......-rich proteoglycan subpopulation that increases with increasing age of animal compared with chondroitin sulfate-rich proteoglycans. Monoclonal antibodies recognizing determinants on chondroitinase-treated proteoglycan have been used in immunohistochemical localization studies determining the differential...

  2. Syndecans as cell surface receptors: Unique structure equates with functional diversity

    DEFF Research Database (Denmark)

    Choi, Youngsil; Chung, Heesung; Jung, Heyjung

    2011-01-01

    An increasing number of functions for syndecan cell surface heparan sulfate proteoglycans have been proposed over the last decade. Moreover, aberrant syndecan regulation has been found to play a critical role in multiple pathologies, including cancers, as well as wound healing and inflammation. A...

  3. Immunohistochemical localization of basement membrane components during hair follicle morphogenesis

    DEFF Research Database (Denmark)

    Westgate, G E; Shaw, D A; Harrap, G J

    1984-01-01

    Specific antisera were used to investigate the distributions of several basement membrane zone (BMZ) components, namely, bullous pemphigoid antigen (BPA), heparan sulfate proteoglycan (HSPG), laminin, and type IV collagen, during the development of hair follicles in late embryo rats. BPA was not ...

  4. Antibody mapping and tissue localization of globular and cysteine-rich regions of perlecan domain III

    DEFF Research Database (Denmark)

    Couchman, J R; Ljubimov, A V; Sthanam, M

    1995-01-01

    Perlecan is the best-characterized basement membrane heparan sulfate proteoglycan. It has a large (approximately 400 KD) core protein consisting of five distinct domains. Domain III, a centrally located domain, contains three globular domains separated by cysteine-rich epidermal growth factor (EG...

  5. Disruption of glomerular basement membrane charge through podocyte-specific mutation of agrin does not alter glomerular permselectivity.

    NARCIS (Netherlands)

    Harvey, S.J.; Jarad, G.; Cunningham, J.; Rops, A.L.; Vlag, J. van der; Berden, J.H.M.; Moeller, M.J.; Holzman, L.B.; Burgess, R.W.; Miner, J.H.

    2007-01-01

    Glomerular charge selectivity has been attributed to anionic heparan sulfate proteoglycans (HSPGs) in the glomerular basement membrane (GBM). Agrin is the predominant GBM-HSPG, but evidence that it contributes to the charge barrier is lacking, because newborn agrin-deficient mice die from

  6. Are primed polymorphonuclear leukocytes contributors to the high heparanase levels in hemodialysis patients?

    NARCIS (Netherlands)

    Cohen-Mazor, M.; Sela, S.; Mazor, R.; Ilan, N.; Vlodavsky, I.; Rops, A.L.; Vlag, J. van der; Cohen, H.I.; Kristal, B.

    2008-01-01

    Patients on chronic hemodialysis (HD) are at high risk for developing atherosclerosis and cardiovascular complications. Heparanase, an endoglycosidase that cleaves heparan sulfate (HS) side chains of proteoglycans, is involved in extracellular matrix degradation and, as such, may be involved in the

  7. Disease: H01498 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available H01498 Multiple joint dislocations, short stature, craniofacial dysmorphism, and c...ongenital heart defects (JDSSDHD); Larsen-like syndrome Multiple joint dislocations, short stature, craniofa...ondroitin sulfate, and heparan sulfate proteoglycans. The affected individuals showed dysmorphic faces, bilateral dislocations

  8. Gene : CBRC-TGUT-37-0500 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TGUT-37-0500 Novel UN D UNKNOWN PGBM_HUMAN 3e-25 49% ref|NP_001001876.1| basemen...t membrane-specific heparan sulfate proteoglycan core protein [Gallus gallus] emb|CAE51322.1| basement mem

  9. Mutations in EXTL3 Cause Neuro-immuno-skeletal Dysplasia Syndrome

    NARCIS (Netherlands)

    Oud, M.M.; Tuijnenburg, P.; Hempel, M.; Vlies, N. van; Ren, Z.; Ferdinandusse, S.; Jansen, M.H.; Santer, R.; Johannsen, J.; Bacchelli, C.; Alders, M.; Li, R.; Davies, R.; Dupuis, L.; Cale, C.M.; Wanders, R.J.; Pals, S.T.; Ocaka, L.; James, C.; Muller, I.; Lehmberg, K.; Strom, T.; Engels, H.; Williams, H.J.; Beales, P.; Roepman, R.; Dias, P.; Brunner, H.G.; Cobben, J.M.; Hall, C.; Hartley, T.; Quesne Stabej, P. Le; Mendoza-Londono, R.; Davies, E.G.; Sousa, S.B.; Lessel, D.; Arts, H.H.; Kuijpers, T.W.

    2017-01-01

    EXTL3 regulates the biosynthesis of heparan sulfate (HS), important for both skeletal development and hematopoiesis, through the formation of HS proteoglycans (HSPGs). By whole-exome sequencing, we identified homozygous missense mutations c.1382C>T, c.1537C>T, c.1970A>G, and c.2008T>G in EXTL3 in

  10. Effects of glucosamine on proteoglycan loss by tendon, ligament and joint capsule explant cultures.

    Science.gov (United States)

    Ilic, M Z; Martinac, B; Samiric, T; Handley, C J

    2008-12-01

    To investigate the effect of glucosamine on the loss of newly synthesized radiolabeled large and small proteoglycans by bovine tendon, ligament and joint capsule. The kinetics of loss of (35)S-labeled large and small proteoglycans from explant cultures of tendon, ligament and joint capsule treated with 10mM glucosamine was investigated over a 10-day culture period. The kinetics of loss of (35)S-labeled small proteoglycans and the formation of free [(35)S]sulfate were determined for the last 10 days of a 15-day culture period. The proteoglycan core proteins were analyzed by gel electrophoresis followed by fluorography. The metabolism of tendon, ligament and joint capsule explants exposed to 10mM glucosamine was evaluated by incorporation of [(3)H]serine and [(35)S]sulfate into protein and glycosaminoglycans, respectively. Glucosamine at 10mM stimulated the loss of small proteoglycans from ligament explant cultures. This was due to the increased loss of both macromolecular and free [(35)S]sulfate to the medium indicating that glucosamine affected the release of small proteoglycans as well as their intracellular degradation. The degradation pattern of small proteoglycans in ligament was not affected by glucosamine. In contrast, glucosamine did not have an effect on the loss of large or small proteoglycans from tendon and joint capsule or large proteoglycans from ligament explant cultures. The metabolism of cells in tendon, ligament and joint capsule was not impaired by the presence of 10mM glucosamine. Glucosamine stimulated the loss of small proteoglycans from ligament but did not have an effect on small proteoglycan catabolism in joint capsule and tendon or large proteoglycan catabolism in ligament, tendon or synovial capsule. The consequences of glucosamine therapy at clinically relevant concentrations on proteoglycan catabolism in joint fibrous connective tissues need to be further assessed in an animal model.

  11. The Role of NG2 Proteoglycan in Glioma

    Directory of Open Access Journals (Sweden)

    Sridevi Yadavilli

    2016-02-01

    Full Text Available Neuron glia antigen-2 ((NG2, also known as chondroitin sulphate proteoglycan 4, or melanoma-associated chondroitin sulfate proteoglycan is a type-1 membrane protein expressed by many central nervous system (CNS cells during development and differentiation and plays a critical role in proliferation and angiogenesis. ‘NG2’ often references either the protein itself or the highly proliferative and undifferentiated glial cells expressing high levels of NG2 protein. NG2 glia represent the fourth major type of neuroglia in the mammalian nervous system and are classified as oligodendrocyte progenitor cells by virtue of their committed oligodendrocyte generation in developing and adult brain. Here, we discuss NG2 glial cells as well as NG2 protein and its expression and role with regards to CNS neoplasms as well as its potential as a therapeutic target for treating childhood CNS cancers.

  12. Characterization and N-terminal sequence of human platelet proteoglycan.

    Science.gov (United States)

    Périn, J P; Bonnet, F; Maillet, P; Jollès, P

    1988-01-01

    Human platelet proteoglycan (P.PG) was prepared from a 4 M-guanidinium chloride platelet extract in the presence of proteinase inhibitors. The purification procedure included CsCl-density-gradient centrifugation, DEAE-Sepharose CL-6B ion-exchange chromatography and f.p.l.c. on a Mono Q HR 5/5 column. P.PG was recovered as a polydisperse molecule, but the protein core appeared to be at least 90% homogeneous. This observation could be due to partial proteolysis of the core protein during extraction. The N-terminal sequence of the human P.PG core protein was determined up to residue 66 and was shown to be highly homologous to the propeptide of an embryonic rat yolk-sac tumour proteoglycan (PG19); the significance of this homology is discussed. Images Fig. 4. Fig. 5. PMID:3214420

  13. Glypican 6 Enhances N-Methyl-D-Aspartate Receptor Function in Human-Induced Pluripotent Stem Cell-Derived Neurons.

    Science.gov (United States)

    Sato, Kaoru; Takahashi, Kanako; Shigemoto-Mogami, Yukari; Chujo, Kaori; Sekino, Yuko

    2016-01-01

    The in vitro use of neurons that are differentiated from human induced pluripotent stem cells (hiPSC-neurons) is expected to improve the prediction accuracy of preclinical tests for both screening and safety assessments in drug development. To achieve this goal, hiPSC neurons are required to differentiate into functional neurons that form excitatory networks and stably express N-methyl-D-aspartate receptors (NMDARs). Recent studies have identified some astrocyte-derived factors that are important for the functional maturation of neurons. We therefore examined the effects of the astrocyte-derived factor glypican 6 (GPC6) on hiPSC-neurons. When we pharmacologically examined which receptor subtypes mediate L-glutamate (L-Glu)-induced changes in the intracellular Ca(2+) concentrations in hiPSC neurons using fura-2 Ca(2+) imaging, NMDAR-mediated responses were not detected through 7 days in vitro (DIV). These cells were also not vulnerable to excitotoxicity at 7 DIV. However, a 5-days treatment with GPC6 from 3 DIV induced an NMDAR-mediated Ca(2+) increase in hiPSC-neurons and increased the level of NMDARs on the cell surface. We also found that GPC6-treated hiPSC-neurons became responsive to excitotoxicity. These results suggest that GPC6 increases the level of functional NMDARs in hiPSC-neurons. Glial factors may play a key role in accelerating the functional maturation of hiPSC neurons for drug-development applications.

  14. Austromegabalanus psittacus barnacle shell structure and proteoglycan localization and functionality.

    Science.gov (United States)

    Fernández, M S; Arias, J I; Neira-Carrillo, A; Arias, J L

    2015-09-01

    Comparative analyzes of biomineralization models have being crucial for the understanding of the functional properties of biominerals and the elucidation of the processes through which biomacromolecules control the synthesis and structural organization of inorganic mineral-based biomaterials. Among calcium carbonate-containing bioceramics, egg, mollusk and echinoderm shells, and crustacean carapaces, have being fairly well characterized. However, Thoraceca barnacles, although being crustacea, showing molting cycle, build a quite stable and heavily mineralized shell that completely surround the animal, which is for life firmly cemented to the substratum. This makes barnacles an interesting model for studying processes of biomineralization. Here we studied the main microstructural and ultrastructural features of Austromegabalanus psittacus barnacle shell, characterize the occurrence of specific proteoglycans (keratan-, dermatan- and chondroitin-6-sulfate proteoglycans) in different soluble and insoluble organic fractions extracted from the shell, and tested them for their ability to crystallize calcium carbonate in vitro. Our results indicate that, in the barnacle model, proteoglycans are good candidates for the modification of the calcite crystal morphology, although the cooperative effect of some additional proteins in the shell could not be excluded. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Single-Molecule Imaging of Proteoglycans in the Pericellular Matrix.

    Science.gov (United States)

    Scrimgeour, Jan; McLane, Louis T; Chang, Patrick S; Curtis, Jennifer E

    2017-12-05

    The pericellular matrix is a robust, hyaluronan-rich polymer brush-like structure that controls access to the cell surface, and plays an important role in cell adhesion, migration, and proliferation. We report the observation of single bottlebrush proteoglycan dynamics in the pericellular matrix of living chondrocytes. Our investigations show that the pericellular matrix undergoes gross extension on the addition of exogenous aggrecan, and that this extension is significantly in excess of that observed in traditional particle exclusion assays. The mean-square displacement of single, bound proteoglycans increases with distance to cell surface, indicating reduced confinement by neighboring hyaluronan-aggrecan complexes. This is consistent with published data from quantitative particle exclusion assays that show openings in the pericellular matrix microstructure ranging from ∼150 nm near the cell surface to ∼400 nm near the cell edge. In addition, the mobility of tethered aggrecan drops significantly when the cell coat is enriched with bottlebrush proteoglycans. Single-molecule imaging in this thick polysaccharide matrix on living cells has significant promise in the drive to elucidate the role of the pericellular coat in human health. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  16. The collaggrecan: Synthesis and visualization of an artificial proteoglycan.

    Science.gov (United States)

    Raspanti, Mario; Caravà, Elena; Sgambato, Antonella; Natalello, Antonino; Russo, Laura; Cipolla, Laura

    2016-05-01

    An artificial aggrecan-like proteoglycan has been designed and synthesized in vitro. At variance with natural proteoglycans, whose glycosaminoglycan chains are always O-linked via a tetrasaccharide bridge to the serine residues of a specific protein core, the present structure consists of chondroitin-6-sulfate chains directly bound to the lysine and hydroxylysine residues of a collagen molecule backbone. The resulting macromolecule has been characterized by histochemistry, atomic force microscopy and FTIR. The number of variables involved (e.g., length and type of the collagen backbone, glycosaminoglycan species, sulfation type and pattern, molecular weight, number and length of side chains, etc.) makes possible to conceive an almost endless variety of artificial proteoglycans, each precisely tailored to a specific functional role. In addition to their use as biomaterials, glycated collagens interact with cells in complex ways and a previous study has already shown the ability of a glycated collagen to redirect fibroblastoma cells from proliferation to differentiation. The research is still underway. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Murine macrophage heparanase: inhibition and comparison with metastatic tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Savion, N.; Disatnik, M.H.; Nevo, Z.

    1987-01-01

    Circulating macrophages and metastatic tumor cells can penetrate the vascular endothelium and migrate from the circulatory system to extravascular compartments. Both activated murine macrophages and different metastatic tumor cells attach, invade, and penetrate confluent vascular endothelial cell monolayer in vitro, by degrading heparan sulfate proteoglycans in the subendothelial extracellular matrix. The sensitivity of the enzymes from the various sources degrading the heparan sulfate proteoglycan was challenged and compared by a series of inhibitors. Activated macrophages demonstrate a heparanase with an endoglycosidase activity that cleaves from the (/sup 35/S)O/sub 4//sup -/-labeled heparan sulfate proteoglycans of the extracellular matrix 10 kDa glycosaminoglycan fragments. The degradation of (/sup 35/S)O/sub 4//sup -/-labeled extracellular matrix proteoglycans by the macrophages' heparanase is significantly inhibited in the presence of heparan sulfate (10..mu..g/ml), arteparon (10..mu..g/ml), and heparin at a concentration of 3 ..mu..g/ml. Degradation of this heparan sulfate proteoglycan is a two-step sequential process involving protease activity followed by heparanase activity. B16-BL6 metastatic melanoma cell heparanase, which is also a cell-associated enzyme, was inhibited by heparin to the same extent as the macrophage haparanase. On the other hand, heparanase of the highly metastatic variant (ESb) of a methylcholanthrene-induced T lymphoma, which is an extracellular enzyme released by the cells to the incubation medium, was more sensitive to heparin and arteparon than the macrophages' heparanase. These results may indicate the potential use of heparin or other glycosaminoglycans as specific and differential inhibitors for the formation in certain cases of blood-borne tumor metastasis.

  18. Suppression of Glypican 3 Inhibits Growth of Hepatocellular Carcinoma Cells through Up-Regulation of TGF-β2

    Directory of Open Access Journals (Sweden)

    Chris K. Sun

    2011-08-01

    Full Text Available Glypican 3 (GPC3 is a valuable diagnostic marker and a potential therapeutic target in hepatocellular carcinoma (HCC. To evaluate the efficacy of targeting GPC3 at the translational level, we used RNA interference to examine the biologic and molecular effects of GPC3 suppression in HCC cells in vitro and in vivo. Transfection of Huh7 and HepG2 cells with GPC3-specific small interfering RNA (siRNA inhibited cell proliferation (P < .001 together with cell cycle arrest at the G1 phase, down-regulation of antiapoptotic protein (Bcl-2, Bcl-xL, and Mcl-1, and replicative senescence. Gene expression analysis revealed that GPC3 suppression significantly correlated with transforming growth factor beta receptor (TGFBR pathway (P = 4.57e-5 and upregulated TGF-β2 at both RNA and protein levels. The effects of GPC3 suppression by siRNA can be recapitulated by addition of human recombinant TGF-β2 to HCC cells in culture, suggesting the possible involvement of TGF-β2 in growth inhibition of HCC cells. Cotransfection of siRNA-GPC3 with siRNA-TGF-β2 partially attenuated the effects of GPC3 suppression on cell proliferation, cell cycle progression, apoptosis, and replicative senescence, confirming the involvement of TGF-β2 in siRNA-GPC3-mediated growth suppression. In vivo, GPC3 suppression significantly inhibited the growth of orthotopic xenografts of Huh7 and HepG2 cells (P < .05, accompanied by increased TGF-β2 expression, reduced cell proliferation (observed by proliferating cell nuclear antigen staining, and enhanced apoptosis (by TUNEL staining. In conclusion, molecular targeting of GPC3 at the translational level offers an effective option for the clinical management of GPC3-positive HCC patients.

  19. Insights into the key roles of proteoglycans in breast cancer biology and translational medicine

    DEFF Research Database (Denmark)

    Theocharis, Achilleas D.; Skandalis, Spyros S.; Neill, Thomas

    2015-01-01

    Proteoglycans control numerous normal and pathological processes, among which are morphogenesis, tissue repair, inflammation, vascularization and cancer metastasis. During tumor development and growth, proteoglycan expression is markedly modified in the tumor microenvironment. Altered expression...... of proteoglycans on tumor and stromal cell membranes affects cancer cell signaling, growth and survival, cell adhesion, migration and angiogenesis. Despite the high complexity and heterogeneity of breast cancer, the rapid evolution in our knowledge that proteoglycans are among the key players in the breast tumor...... microenvironment suggests their potential as pharmacological targets in this type of cancer. It has been recently suggested that pharmacological treatment may target proteoglycan metabolism, their utilization as targets for immunotherapy or their direct use as therapeutic agents. The diversity inherent...

  20. Immunological characterization of a basement membrane-specific chondroitin sulfate proteoglycan

    DEFF Research Database (Denmark)

    McCarthy, K J; Accavitti, M A; Couchman, J R

    1989-01-01

    Reichert's membrane, an extraembryonic membrane present in developing rodents, has been proposed as an in vivo model for the study of basement membranes. We have used this membrane as a source for isolation of basement membrane proteoglycans. Reichert's membranes were extracted in a guanidine/3......-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate buffer followed by cesium chloride density-gradient ultracentrifugation under dissociative conditions. The proteoglycans were subsequently purified from the two most dense fractions (greater than 1.3 g/ml) by ion-exchange chromatography. Mice were immunized...... with the proteoglycan preparation and four mAbs recognizing the core protein of a high-density, buoyant chondroitin sulfate proteoglycan were raised. Confirmation of antibody specificity was carried out by the preparation of affinity columns made from each of the mAbs. Chondroitin sulfate proteoglycans (CSPGs) were...

  1. Ultrastructure features of camel cornea--collagen fibril and proteoglycans.

    Science.gov (United States)

    Almubrad, Turki; Akhtar, Saeed

    2012-01-01

      The uniform distribution of collagen fibrils and proteoglycans maintain the transparency of normal cornea. We describe the ultrastructural features of camel cornea including collagen fibrils and proteoglycans (PGs).   Camel corneas (of 6-, 8-, and 10-month-old animals) were fixed in 2.5% glutaraldehyde containing cuprolinic blue in sodium acetate buffer and processed for electron microscopy. The 'AnalySIS LS Professional' program was used to analyze the collagen fibril diameter.   The camel cornea consists of four layers: the epithelium (227 μm), stroma (388 μm), Descemet's membrane (DM), and endothelium. The epithelium constituted 36% of the camel cornea, whereas corneal stroma constituted 62% of the corneal thickness (629 μm). The PGs in the posterior stroma were significantly larger in number and size compared with the anterior and middle stroma. The collagen fibril diameter was 25 nm and interfibrillar spacing 40 nm. Fibrillar structures are present throughout the DM.   The structure of the camel cornea is very different from human and other animals. The unique structure of the cornea might be an adaptation to help the camel to survive in a hot and dry climate. The camel cornea may also be a good model to study the effect of hot and dry climates on the cornea. © 2011 American College of Veterinary Ophthalmologists.

  2. Thyroid hormone excess stimulates the synthesis of proteoglycan in human skin fibroblasts in culture

    Energy Technology Data Exchange (ETDEWEB)

    Shishiba, Yoshimasa; Ozawa, Yasunori; Shimizu, Taeko (Division of Endocrinology and Endocrine Research Laboratory, Toranomon Hospital (Japan)); Takeuchi, Yasuhiro; Yokoi, Noriko (Okinaka Memorial Institute for Medical Research, Akasaka, Tokyo (Japan))

    1990-01-01

    We previously demonstrated that proteoglycan accumulated in the affected skin of circumscribed pretibial myxedema of Graves' disease. As an underlying mechanism responsible for the accumulation, we sought to determine whether excess thyroid hormone was partially responsible for the increase in proteoglycan synthesis. Human skin fibroblasts were cultured in Ham's F-10 medium containing 1% Nutridoma with graded doses of T{sub 3}(0.184 x 10{sup -9} to 46 x 10{sup -9} mol/l) and were labelled with ({sup 35}S)sulphate and ({sup 3}H)glucosamine. Proteoglycans were purified by Sephadex G-50, Q-Sepharose chromatography with NaCl-gradient and Sepharose CL-6B chromatography. {sup 35}S and {sup 3}H incorporated into dermatan sulphate proteoglycan and heparan sulphate proteoglycan and {sup 3}H incorporated into hyaluronan were measured. {sup 35}S and {sup 3}H incorporation into dermatan sulphate proteoglycan was minimum at a T{sub 3} concentration of 0.184 x 10{sup -9} mol/l, and increased with increasing doses of T{sub 3} up to 46 x 10{sup -9} mol/l. {sup 35}S and {sup 3}H incorporation into heparan sulphate proteoglycan also increased with increasing-doses of T{sub 3}. {sup 3}H incorporation into hyaluranan was not influenced at all by T{sub 3}. The increased incorporation of {sup 35}S into proteoglycan in high-T{sub 3} culture reflects the increased synthesis of proteoglycan because 1. the extent of sulphation of disaccharides examined by thin-layer chromatography was not altered by T{sub 3}; 2. the specific activity of ({sup 35}S)sulphate was not influenced by T{sub 3}, and 3. T{sub 3} did not decrease the degradation rate of cell-associated proteoglycan. (author).

  3. Keratan sulfate-containing proteoglycans in sheep brain with particular reference to phosphacan and synaptic vesicle proteoglycan isoforms.

    Science.gov (United States)

    Sinouris, Efstathios A; Skandalis, Spyros S; Kilia, Virginia; Theocharis, Achilleas D; Theocharis, Dimitrios A; Ravazoula, Panagiota; Vynios, Demitrios H; Papageorgakopoulou, Nickoletta

    2009-05-01

    Proteoglycans (PGs) are widely expressed in all areas of the brain. In this study, the keratan sulfate-containing PGs (KS-PGs) from cerebrum (CB), cerebellum (CL) and brainstem (BS) of young sheep brain were isolated, purified and characterized. The amount of KS-PGs in CL was significantly lower than that in CB and BS. KS-PGs were characterized by increased extent of glycosylation and heterogeneity of KS chains in CL. Western blot analyses demonstrated the presence of the KS-PGs phosphacan, SV2A and SV2B isoforms of synaptic vesicle proteoglycan in all three areas of the young sheep brain. Phosphacan predominated in BS and CB, showing significant molecular heterogeneity. SV2A and SV2B were found in two forms of high and low molecular sizes according to their extent of glycosylation in sheep brain. SV2A predominated in CL, where forms with very high molecular sizes were detected. Immunohistochemical examination revealed that SV2A was localized in the extracellular matrix of both gray and white matter. In contrast, phosphacan and SV2B were mainly localized in the white matter in all brain regions. The results of the present study demonstrated that KS-PGs are present in the three areas of the sheep brain, showing significant variations in their content, structure and localization among the distinct areas. These differences may be important for the physiology of the brain.

  4. Human papillomavirus species-specific interaction with the basement membrane-resident non-heparan sulfate receptor.

    Science.gov (United States)

    Richards, Kathleen F; Mukherjee, Santanu; Bienkowska-Haba, Malgorzata; Pang, Jia; Sapp, Martin

    2014-12-05

    Using a cell culture model where virus is bound to the extracellular matrix (ECM) prior to cell surface binding, we determined that human papillomavirus type 16 (HPV16) utilizes ECM resident laminin (LN) 332 as an attachment receptor for infectious entry. In presence of LN332, soluble heparin can function as ligand activator rather than competitive inhibitor of HPV16 infection. We also show that the ability to use LN332 binding as a productive attachment step for infectious entry is not conserved amongst HPV types. In the alpha genus, species 9 members (HPV16) attach to ECM via LN332, while members of species 7 (HPV18) are completely inhibited by heparin pre-incubation due to an inability to use LN332. Since HPV species 7 and 9 are preferentially associated with adenocarcinoma and squamous cell carcinoma of the cervix, respectively, our data provide first evidence that pre-entry events may contribute to the anatomical-site preference of HPV species.

  5. Host cell heparan sulfate glycosaminoglycans are ligands for OspF‐related proteins of the Lyme disease spirochete

    National Research Council Canada - National Science Library

    Lin, Yi‐Pin; Bhowmick, Rudra; Coburn, Jenifer; Leong, John M

    2015-01-01

    ...), a large family of plasmid‐encoded surface lipoproteins that are produced in the mammalian host, can be divided into the OspF ‐related, OspEF ‐leader peptide ( E lp) and OspE ‐related subfamilies...

  6. Human Papillomavirus Species-Specific Interaction with the Basement Membrane-Resident Non-Heparan Sulfate Receptor

    Directory of Open Access Journals (Sweden)

    Kathleen F. Richards

    2014-12-01

    Full Text Available Using a cell culture model where virus is bound to the extracellular matrix (ECM prior to cell surface binding, we determined that human papillomavirus type 16 (HPV16 utilizes ECM resident laminin (LN 332 as an attachment receptor for infectious entry. In presence of LN332, soluble heparin can function as ligand activator rather than competitive inhibitor of HPV16 infection. We also show that the ability to use LN332 binding as a productive attachment step for infectious entry is not conserved amongst HPV types. In the alpha genus, species 9 members (HPV16 attach to ECM via LN332, while members of species 7 (HPV18 are completely inhibited by heparin pre-incubation due to an inability to use LN332. Since HPV species 7 and 9 are preferentially associated with adenocarcinoma and squamous cell carcinoma of the cervix, respectively, our data provide first evidence that pre-entry events may contribute to the anatomical-site preference of HPV species.

  7. NO EVIDENCE FOR AN INDEPENDENT ROLE OF ANTI-HEPARAN SULFATE REACTIVITY APART FROM ANTI-DNA IN LUPUS NEPHRITIS

    NARCIS (Netherlands)

    HYLKEMA, MN; ZWET, IVD; KRAMERS, C; VANBRUGGEN, MCJ; SWAAK, AJG; BERDEN, JHM; SMEENK, RJT; Hylkema, Machteld

    The presence of anti-heparan sulphate (HS) reactivity in serum is closely related to the occurrence of nephritis in patients with systemic lupus erythematosus (SLE). Since patients with lupus nephritis in general also have high titres of anti-DNA antibodies, we wanted to clarify the relationship

  8. Heparan sulfate and dermatan sulfate derived disaccharides are sensitive markers for newborn screening for mucopolysaccharidoses types I, II and III

    DEFF Research Database (Denmark)

    de Ruijter, Jessica; de Ru, Minke H; Wagemans, Tom

    2012-01-01

    Mucopolysaccharidoses (MPSs) are a group of lysosomal storage disorders (LSDs) caused by a defect in the degradation of glycosaminoglycans (GAGs). The accumulation of GAGs in MPS patients results in extensive, severe and progressive disease. Disease modifying therapy is available for three...

  9. Heparan Sulfate-Binding Foot-and-Mouth Disease Virus Enters Cells Via Caveolae-Mediated Endocytosis

    Science.gov (United States)

    Foot-and-mouth disease virus (FMDV) utilizes different cell surface macromolecules to facilitate infection of cultured cells. Virus which is virulent for susceptible animals infects cells via four members of the alpha V subclass of cellular integrins. In contrast, tissue culture adaptation of some...

  10. The effect of OTR4120, a heparan sulfate glycosaminoglycan memetic on improving acute and impaired wound healing in rats

    NARCIS (Netherlands)

    M. Tong (Miao)

    2012-01-01

    markdownabstract__Abstract__ Dating back to the prehistoric times, wounds have been common with mankind. The treatment of wounds is an art as old as humanity. Today, wounds are of increasing concern in our society in terms of their prevalence and costs. In the developed countries, patients

  11. Insights into the key roles of proteoglycans in breast cancer biology and translational medicine.

    Science.gov (United States)

    Theocharis, Achilleas D; Skandalis, Spyros S; Neill, Thomas; Multhaupt, Hinke A B; Hubo, Mario; Frey, Helena; Gopal, Sandeep; Gomes, Angélica; Afratis, Nikos; Lim, Hooi Ching; Couchman, John R; Filmus, Jorge; Sanderson, Ralph D; Schaefer, Liliana; Iozzo, Renato V; Karamanos, Nikos K

    2015-04-01

    Proteoglycans control numerous normal and pathological processes, among which are morphogenesis, tissue repair, inflammation, vascularization and cancer metastasis. During tumor development and growth, proteoglycan expression is markedly modified in the tumor microenvironment. Altered expression of proteoglycans on tumor and stromal cell membranes affects cancer cell signaling, growth and survival, cell adhesion, migration and angiogenesis. Despite the high complexity and heterogeneity of breast cancer, the rapid evolution in our knowledge that proteoglycans are among the key players in the breast tumor microenvironment suggests their potential as pharmacological targets in this type of cancer. It has been recently suggested that pharmacological treatment may target proteoglycan metabolism, their utilization as targets for immunotherapy or their direct use as therapeutic agents. The diversity inherent in the proteoglycans that will be presented herein provides the potential for multiple layers of regulation of breast tumor behavior. This review summarizes recent developments concerning the biology of selected proteoglycans in breast cancer, and presents potential targeted therapeutic approaches based on their novel key roles in breast cancer. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Evaluation of influence of proteoglycans on hydration of articular cartilage with the use of ultrasound

    Directory of Open Access Journals (Sweden)

    Yi-yi YANG

    2015-04-01

    Full Text Available Objective To monitor the changes in hydration behaviour of articular cartilage induced by degradation of proteoglycans, and to explore the effect of proteoglycans on hydration behaviour of articular cartilage by using high-frequency ultrasound. Methods Twelve porcine patellae with smooth cartilage surface were prepared and equally divided into two groups: normal group without any enzyme treatment, and trypsin group they were treated with 0.25% trypsin for 8h to digest proteoglycan in the cartilage. The hydration behaviour of the cartilage tissue was scanned by high-frequency ultrasound system with a central frequency of 25MHz. Parameters including cartilage hydration strain and cartilage thickness were measured. The histopathological changes in the articular cartilage were observed under a light microscope. Results It took approximately 20min to reach equilibrium during the hydration process in the normal cartilages, while proteoglycan-degraded cartilage took only about 5min to achieve equilibrium. The equilibrium strain of normal cartilage was 3.5%±0.5%. The degradation of proteoglycans induced a significant decrease in equilibrium strain (1.8%±0.2%, P0.05. Conclusion Proteoglycans play an important role in hydration behaviour of articular cartilage. The degradation of proteoglycans could induce degeneration of cartilage structure and decrease in hydration behaviour after dehydration. DOI: 10.11855/j.issn.0577-7402.2015.03.03

  13. The Motile Breast Cancer Phenotype Roles of Proteoglycans/Glycosaminoglycans

    Directory of Open Access Journals (Sweden)

    Dragana Nikitovic

    2014-01-01

    Full Text Available The consecutive stages of cancer growth and dissemination are obligatorily perpetrated through specific interactions of the tumor cells with their microenvironment. Importantly, cell-associated and tumor microenvironment glycosaminoglycans (GAGs/proteoglycan (PG content and distribution are markedly altered during tumor pathogenesis and progression. GAGs and PGs perform multiple functions in specific stages of the metastatic cascade due to their defined structure and ability to interact with both ligands and receptors regulating cancer pathogenesis. Thus, GAGs/PGs may modulate downstream signaling of key cellular mediators including insulin growth factor receptor (IGFR, epidermal growth factor receptor (EGFR, estrogen receptors (ERs, or Wnt members. In the present review we will focus on breast cancer motility in correlation with their GAG/PG content and critically discuss mechanisms involved. Furthermore, new approaches involving GAGs/PGs as potential prognostic/diagnostic markers or as therapeutic agents for cancer-related pathologies are being proposed.

  14. Expression of NG2 proteoglycan in the degenerated intervertebral disc in dachshunds.

    Science.gov (United States)

    Abdel-Hakiem, Mohammed; Yamashita, Ayuko; Atiba, Ayman; Okamura, Yasuhiko; Katayama, Masaaki; Youssef, Haroun; Isomura, Hiroshi; Uzuka, Yuji

    2016-01-01

    The pathogenesis of intervertebral disc (IVD) degeneration is not fully understood. The biomolecular signaling pathways involved in the IVD degeneration require further investigation. The aim of this study was to investigate the expression of NG2 proteoglycan in the degenerated IVD. IVD samples were obtained from 16 Dachshunds that were confirmed to have IVD herniation and subsequently underwent hemilaminectomy. The samples were subjected to histological and immunohistochemical (IHC) examinations. IHC revealed positive results for the expression of NG2 proteoglycan in all examined samples. The results showed the expression of NG2 proteoglycan by the degenerated IVDs.

  15. Effect of retinoic acid on proteoglycan turnover in bovine articular cartilage cultures

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, M.A.; Handley, C.J.

    1987-10-01

    This paper describes proteoglycan catabolism by adult bovine articular cartilage treated with retinoic acid as a means of stimulating the loss of this macromolecule from the extracellular matrix of cartilage. Addition of retinoic acid (10(-12)-10(-6) M) to adult bovine articular cartilage which had been labeled with (/sup 35/S)sulfate for 6 h after 5 days in culture, resulted in a dose-dependent increase in the rate of loss of /sup 35/S-labeled proteoglycans from the matrix of the tissue. Concomitant with this loss was a decrease in the proteoglycan content of the tissue. Incubation of cultures treated with 1 microM retinoic acid, at 4 degrees C, or with 0.5 mM cycloheximide, resulted in a significant decrease in the rate of retinoic acid-induced loss of proteoglycans and demonstrated cellular involvement in this process. Analysis of the /sup 35/S-labeled proteoglycans remaining in the matrix showed that the percentage of radioactivity associated with the small proteoglycan species extracted from the matrix of articular cartilage explants labeled with (/sup 35/S)sulfate after 5 days in culture was 15% and this increased to 22% in tissue maintained in medium alone. In tissue treated with 1 microM retinoic acid for 6 days, the percentage of radioactivity associated with the small proteoglycan was 58%. Approximately 93% of the /sup 35/S-labeled proteoglycans released into the medium of control and retinoic acid-treated cultures was recovered in high density fractions after CsCl gradient centrifugation and eluted on Sepharose CL-2B as a broad peak with a Kav of 0.30-0.37. Less than 17% of these proteoglycans was capable of aggregating with hyaluronate. These results indicate that in both control and retinoic acid-treated cultures the larger proteoglycan species is lost to the medium at a greater rate than the small proteoglycan species. The effect of retinoic acid on proteoglycan turnover was shown to be reversible.

  16. The degradation of human endothelial cell-derived perlecan and release of bound basic fibroblast growth factor by stromelysin, collagenase, plasmin, and heparanases.

    Science.gov (United States)

    Whitelock, J M; Murdoch, A D; Iozzo, R V; Underwood, P A

    1996-04-26

    Perlecan is a modular heparan sulfate proteoglycan that is localized to cell surfaces and within basement membranes. Its ability to interact with basic fibroblast growth factor (bFGF) suggests a central role in angiogenesis during development, wound healing, and tumor invasion. In the present study we investigated, using domain specific anti-perlecan monoclonal antibodies, the binding site of bFGF on human endothelial perlecan and its cleavage by proteolytic and glycolytic enzymes. The heparan sulfate was removed from perlecan by heparitinase treatment, and the approximately 450-kDa protein core was digested with various proteases. Plasmin digestion resulted in a large fragment of approximately 300 kDa, whereas stromelysin and rat collagenase cleaved the protein core into smaller fragments. All three proteases removed immunoreactivity toward the anti-domain I antibody. We showed also that perlecan bound bFGF specifically by the heparan sulfate chains located on the amino-terminal domain I. Once bound, the growth factor was released very efficiently by stromelysin, rat collagenase, plasmin, heparitinase I, platelet extract, and heparin. Interestingly, heparinase I, an enzyme with a substrate specificity for regions of heparan sulfate similar to those that bind bFGF, released only small amounts of bFGF. Our findings provide direct evidence that bFGF binds to heparan sulfate sequences attached to domain I and support the hypothesis that perlecan represents a major storage site for this growth factor in the blood vessel wall. Moreover, the concerted action of proteases that degrade the protein core and heparanases that remove the heparan sulfate may modulate the bioavailability of the growth factor.

  17. Analysis of proteoglycans derived sulphated disaccharides by liquid chromatography/mass spectrometry

    NARCIS (Netherlands)

    Barroso, B.; Didraga, Mihaela Alina; Bischoff, Rainer

    2005-01-01

    A method has been developed for the identification and quantitative determination of sulphated disaccharides derived from chondroitin sulphate (CS) and dermatan sulphate (DS) chains attached to proteoglycans (PGs). After digestion with Chondroitinase ABC, the pool of disaccharides can be directly

  18. Keratan sulfate and dermatan sulfate proteoglycans associate with type VI collagen in fetal rabbit cornea

    National Research Council Canada - National Science Library

    Takahashi, T; Cho, HI; Kublin, CL; Cintron, C

    1993-01-01

    .... Because certain cytochemical data suggested that proteoglycans are associated with type VI collagen in the fetal rabbit cornea, we developed polyclonal antibodies specific to the core proteins of rabbit corneal KSPG...

  19. Proteoglycans in health and disease: the multiple roles of syndecan shedding

    DEFF Research Database (Denmark)

    Manon-Jensen, Tina; Itoh, Yoshifumi; Couchman, John R

    2010-01-01

    Proteolytic processes in the extracellular matrix are a major influence on cell adhesion, migration, survival, differentiation and proliferation. The syndecan cell-surface proteoglycans are important mediators of cell spreading on extracellular matrix and respond to growth factors and other...

  20. Synergistic effect of intervention of glypican-3 gene transcription combined with antitumor drugs in inhibiting hepatoma cell proliferation

    Directory of Open Access Journals (Sweden)

    YANG Jie

    2016-12-01

    Full Text Available ObjectiveTo investigate the inhibitory effect of intervention of glypican-3 (GPC3 gene transcription combined with antitumor drugs on hepatoma cell proliferation. MethodsFour types of GPC3-shRNA plasmids were established and transfected into HepG2 hepatoma cells. Quantitative real-time PCR and Western blot were used to measure the mRNA and protein expression of GPC3 to analyze its association with hepatoma cell proliferation and apoptosis. The independent samples t-test was used for comparison of continuous data between any two groups, and a one-way analysis of variance was used for comparison between multiple groups. ResultsAmong these four plasmids, shRNA1 had a transfection efficiency of >85% in the transfection of HepG2 cells and a silence efficiency of 89.3% at the mRNA level, and the protein expression of GPC3 was significantly inhibited(P<0.01). At 72 hours, the GPC3-shRNA1 co-intervention group had an HepG2 cell inhibition rate of 71.1%, significantly different from that in the negative group (t=18.092, P<0.001, an inhibition rate of migration of 89.1%, significantly lower than that in the negative group (t=8.326, P<0.001, and inhibition rates of HepG2 cell movement and invasion of 53.6% and 60.1%, which were significantly different from those in the negative group (t=52.400 and 48.245, both P<0.001. The GPC3-shRNA1 co-intervention group had a β-catenin mRNA inhibition rate of 46.9% and a Gli1 mRNA upregulation rate of 7.4%, significantly different from those in the negative group (t=30.108 and -3.551, P<0.001 and P=0.009. At 24 hours, 10 μmol/L sorafenib combined with shRNA1 had an inhibition rate of tumor cells of 52.6% and 100 μmol/L sorafenib combined with shRNA1 had an inhibition rate of tumor cells of 79.5%, which were significantly different from that in the control group (t=23.314 and 50.352, both P<0.001. The half-maximal inhibitory concentrations of sorafenib, rapamycin, and erlotinib for HepG2 were 4.67±1

  1. Anti-aging effects of high molecular weight proteoglycan from salmon nasal cartilage in hairless mice.

    Science.gov (United States)

    Goto, Masashi; Yamazaki, Shota; Kato, Yoji; Yamamoto, Kazushi; Katagata, Yohtaro

    2012-05-01

    Proteoglycans comprise a family of complex macromolecules consisting of a core protein with covalently attached glycosaminoglycan (GAG) chains. The skin anti-aging effects of oral administration of proteoglycan fractions with different molecular weights from salmon nasal cartilage were investigated in a hairless mouse model of skin aging; aging was caused by repeated ultraviolet B (UVB) irradiation. Three proteoglycan fractions of different molecular weights were prepared from salmon nasal cartilage water extract by ion-exchange column chromatography and gel filtration column chromatography. Physiological and histological analysis of the skin indicated that oral administration of high molecular weight proteoglycan inhibited UVB-induced skin aging, defined as increased erythema, increased transepidermal water loss (TEWL), decreased hydration, and epidermal and dermal hypertrophies. The serum and dorsal skin inflammatory cytokine levels indicated that high molecular weight proteoglycan acts on gut immunity and improves skin by inhibiting surplus inflammatory cytokines produced by UVB irradiation. These results suggest that high molecular weight proteoglycan from salmon nasal cartilage is effective in preventing skin aging.

  2. Xylose phosphorylation functions as a molecular switch to regulate proteoglycan biosynthesis.

    Science.gov (United States)

    Wen, Jianzhong; Xiao, Junyu; Rahdar, Meghdad; Choudhury, Biswa P; Cui, Jixin; Taylor, Gregory S; Esko, Jeffrey D; Dixon, Jack E

    2014-11-04

    Most eukaryotic cells elaborate several proteoglycans critical for transmitting biochemical signals into and between cells. However, the regulation of proteoglycan biosynthesis is not completely understood. We show that the atypical secretory kinase family with sequence similarity 20, member B (Fam20B) phosphorylates the initiating xylose residue in the proteoglycan tetrasaccharide linkage region, and that this event functions as a molecular switch to regulate subsequent glycosaminoglycan assembly. Proteoglycans from FAM20B knockout cells contain a truncated tetrasaccharide linkage region consisting of a disaccharide capped with sialic acid (Siaα2-3Galβ1-4Xylβ1) that cannot be further elongated. We also show that the activity of galactosyl transferase II (GalT-II, B3GalT6), a key enzyme in the biosynthesis of the tetrasaccharide linkage region, is dramatically increased by Fam20B-dependent xylose phosphorylation. Inactivating mutations in the GALT-II gene (B3GALT6) associated with Ehlers-Danlos syndrome cause proteoglycan maturation defects similar to FAM20B deletion. Collectively, our findings suggest that GalT-II function is impaired by loss of Fam20B-dependent xylose phosphorylation and reveal a previously unappreciated mechanism for regulation of proteoglycan biosynthesis.

  3. Tgfβ-Smad and MAPK signaling mediate scleraxis and proteoglycan expression in heart valves.

    Science.gov (United States)

    Barnette, Damien N; Hulin, Alexia; Ahmed, A S Ishtiaq; Colige, Alain C; Azhar, Mohamad; Lincoln, Joy

    2013-12-01

    Mature heart valves are complex structures consisting of three highly organized extracellular matrix layers primarily composed of collagens, proteoglycans and elastin. Collectively, these diverse matrix components provide all the necessary biomechanical properties for valve function throughout life. In contrast to healthy valves, myxomatous valve disease is the most common cause of mitral valve prolapse in the human population and is characterized by an abnormal abundance of proteoglycans within the valve tri-laminar structure. Despite the clinical significance, the etiology of this phenotype is not known. Scleraxis (Scx) is a basic-helix-loop-helix transcription factor that we previously showed to be required for establishing heart valve structure during remodeling stages of valvulogenesis. In this study, we report that remodeling heart valves from Scx null mice express decreased levels of proteoglycans, particularly chondroitin sulfate proteoglycans (CSPGs), while overexpression in embryonic avian valve precursor cells and adult porcine valve interstitial cells increases CSPGs. Using these systems we further identify that Scx is positively regulated by canonical Tgfβ2 signaling during this process and this is attenuated by MAPK activity. Finally, we show that Scx is increased in myxomatous valves from human patients and mouse models, and overexpression in human mitral valve interstitial cells modestly increases proteoglycan expression consistent with myxomatous mitral valve phenotypes. Together, these studies identify an important role for Scx in regulating proteoglycans in embryonic and mature valve cells and suggest that imbalanced regulation could influence myxomatous pathogenesis. © 2013.

  4. A novel approach for the characterisation of proteoglycans and biosynthetic enzymes in a snail model.

    Science.gov (United States)

    Gesteira, Tarsis F; Coulson-Thomas, Vivien Jane; Ogata, Fernando T; Farias, Eduardo H C; Cavalheiro, Renan P; de Lima, Marcelo A; Cunha, Gabriel L A; Nakayasu, Ernesto S; Almeida, Igor C; Toma, Leny; Nader, Helena B

    2011-12-01

    Proteoglycans encompass a heterogeneous group of glycoconjugates where proteins are substituted with linear, highly negatively charged glycosaminoglycan chains. Sulphated glycosaminoglycans are ubiquitous to the animal kingdom of the Eukarya domain. Information on the distribution and characterisation of proteoglycans in invertebrate tissues is limited and restricted to a few species. By the use of multidimensional protein identification technology and immunohistochemistry, this study shows for the first time the presence and tissue localisation of different proteoglycans, such as perlecan, aggrecan, and heparan sulphate proteoglycan, amongst others, in organs of the gastropoda Achatina fulica. Through a proteomic analysis of Golgi proteins and immunohistochemistry of tissue sections, we detected the machinery involved in glycosaminoglycan biosynthesis, related to polymer formation (polymerases), as well as secondary modifications (sulphation and uronic acid epimerization). Therefore, this work not only identifies both the proteoglycan core proteins and glycosaminoglycan biosynthetic enzymes in invertebrates but also provides a novel method for the study of glycosaminoglycan and proteoglycan evolution. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. Fragile X mental retardation protein regulates trans-synaptic signaling in Drosophila

    Directory of Open Access Journals (Sweden)

    Samuel H. Friedman

    2013-11-01

    Fragile X syndrome (FXS, the most common inherited determinant of intellectual disability and autism spectrum disorders, is caused by loss of the fragile X mental retardation 1 (FMR1 gene product (FMRP, an mRNA-binding translational repressor. A number of conserved FMRP targets have been identified in the well-characterized Drosophila FXS disease model, but FMRP is highly pleiotropic in function and the full spectrum of FMRP targets has yet to be revealed. In this study, screens for upregulated neural proteins in Drosophila fmr1 (dfmr1 null mutants reveal strong elevation of two synaptic heparan sulfate proteoglycans (HSPGs: GPI-anchored glypican Dally-like protein (Dlp and transmembrane Syndecan (Sdc. Our recent work has shown that Dlp and Sdc act as co-receptors regulating extracellular ligands upstream of intracellular signal transduction in multiple trans-synaptic pathways that drive synaptogenesis. Consistently, dfmr1 null synapses exhibit altered WNT signaling, with changes in both Wingless (Wg ligand abundance and downstream Frizzled-2 (Fz2 receptor C-terminal nuclear import. Similarly, a parallel anterograde signaling ligand, Jelly belly (Jeb, and downstream ERK phosphorylation (dpERK are depressed at dfmr1 null synapses. In contrast, the retrograde BMP ligand Glass bottom boat (Gbb and downstream signaling via phosphorylation of the transcription factor MAD (pMAD seem not to be affected. To determine whether HSPG upregulation is causative for synaptogenic defects, HSPGs were genetically reduced to control levels in the dfmr1 null background. HSPG correction restored both (1 Wg and Jeb trans-synaptic signaling, and (2 synaptic architecture and transmission strength back to wild-type levels. Taken together, these data suggest that FMRP negatively regulates HSPG co-receptors controlling trans-synaptic signaling during synaptogenesis, and that loss of this regulation causes synaptic structure and function defects characterizing the FXS disease state.

  6. Comparing prothrombin induced by vitamin K absence-II (PIVKA-II) with the oncofetal proteins glypican-3, Alpha feto protein and carcinoembryonic antigen in diagnosing hepatocellular carcinoma among Egyptian patients.

    Science.gov (United States)

    Abd El Gawad, Iman A; Mossallam, Ghada I; Radwan, Noha H; Elzawahry, Heba M; Elhifnawy, Niveen M

    2014-06-01

    Hepatocellular carcinoma (HCC) is usually asymptomatic in the early stage and does not show elevated alpha-feto protein (AFP). AFP shows 60-80% sensitivity in diagnosing HCC. Glypican3 (GPC-3) is an oncofetal protein that is only detected in HCC cells but not in benign liver tissues, while Carcinoembryonic antigen (CEA) is expressed in various neoplasms including HCC. Although, it is not specific for HCC. Prothrombin induced by vitamin K absence-II (PIVKA-II) is an abnormal prothrombin protein that is increased in the serum of HCC patients. It has higher sensitivity and specificity compared to AFP. The aim of this study is to compare the clinical utility of PIVKA-II with GPC-3, AFP and CEA in diagnosing HCC. This study included 40 patients with HCC, 10 patients with cirrhosis as a benign control group, and 10 apparently healthy volunteers as normal controls. Serum samples were subjected to routine laboratory investigations, measurement of CEA, AFP using MEIA technique (Axsym), glypican3, and PIVKA-II using ELISA technique in the sera of all patients and controls. All markers showed the highest results in the HCC group. Higher concentrations of PIVKA-II were detected in patients with splenomegaly, and in tumors with size (>3cm). Combination of Glypican-3 and PIVKA-II showed the highest sensitivity, while GPC-3 alone and combination of GPC-3 and AFP showed the highest specificity to differentiate HCC from liver cirrhosis and normal controls. GPC-3, PIVKAII, and combination of both showed the highest sensitivity, while GPC-3 alone showed the highest specificity to differentiate HCC from liver cirrhosis. Glypican-3 is the only oncofetal antigen that showed comparable high diagnostic accuracy as PIVKA-II in diagnosing HCC among Egyptian patients. Copyright © 2014. Production and hosting by Elsevier B.V.

  7. Theranostic impact of NG2/CSPG4 proteoglycan in cancer.

    Science.gov (United States)

    Nicolosi, Pier Andrea; Dallatomasina, Alice; Perris, Roberto

    2015-01-01

    NG2/CSPG4 is an unusual cell-membrane integral proteoglycan widely recognized to be a prognostic factor, a valuable tool for ex vivo and non-invasive molecular diagnostics and, by virtue of its tight association with malignancy, a tantalizing therapeutic target in several tumour types. Although the biology behind its involvement in cancer progression needs to be better understood, implementation of NG2/CSPG4 in the routine clinical practice is attainable and has the potential to contribute to an improved individualized management of cancer patients. In this context, its polymorphic nature seems to be particularly valuable in the effort to standardize informative diagnostic procedures and consolidate forcible immunotherapeutic treatment strategies. We discuss here the underpinnings for this potential and highlight the benefits of taking advantage of the intra-tumour and inter-patient variability in the regulation of NG2/CSPG4 expression. We envision that NG2/CSPG4 may effectively be exploited in therapeutic interventions aimed at averting resistance to target therapy agents and at interfering with secondary lesion formation and/or tumour recurrence.

  8. The use of mushroom glucans and proteoglycans in cancer treatment.

    Science.gov (United States)

    Kidd, P M

    2000-02-01

    Immunoceuticals can be considered as substances having immunotherapeutic efficacy when taken orally. More than 50 mushroom species have yielded potential immunoceuticals that exhibit anticancer activity in vitro or in animal models and of these, six have been investigated in human cancers. All are non-toxic and very well tolerated. Lentinan and schizophyllan have little oral activity. Active Hexose Correlated Compound (AHCC) is poorly defined but has shown early clinical promise. Maitake D-Fraction has limited proof of clinical efficacy to date, but controlled research is underway. Two proteoglycans from Coriolus versicolor - PSK (Polysaccharide-K) and PSP (Polysaccharide-Peptide - have demonstrated the most promise. In Japanese trials since 1970, PSK significantly extended survival at five years or beyond in cancers of the stomach, colon-rectum, esophagus, nasopharynx, and lung (non-small cell types), and in a HLA B40-positive breast cancer subset. PSP was subjected to Phase II and Phase III trials in China. In double-blind trials, PSP significantly extended five-year survival in esophageal cancer. PSP significantly improved quality of life, provided substantial pain relief, and enhanced immune status in 70-97 percent of patients with cancers of the stomach, esophagus, lung, ovary, and cervix. PSK and PSP boosted immune cell production, ameliorated chemotherapy symptoms, and enhanced tumor infiltration by dendritic and cytotoxic T-cells. Their extremely high tolerability, proven benefits to survival and quality of life, and compatibility with chemotherapy and radiation therapy makes them well suited for cancer management regimens.

  9. Global analysis of neuronal phosphoproteome regulation by chondroitin sulfate proteoglycans.

    Directory of Open Access Journals (Sweden)

    Panpan Yu

    Full Text Available Chondroitin sulfate proteoglycans (CSPGs are major components of the extracellular matrix which mediate inhibition of axonal regeneration after injury to the central nervous system (CNS. Several neuronal receptors for CSPGs have recently been identified; however, the signaling pathways by which CSPGs restrict axonal growth are still largely unknown. In this study, we applied quantitative phosphoproteomics to investigate the global changes in protein phosphorylation induced by CSPGs in primary neurons. In combination with isobaric Tags for Relative and Absolute Quantitation (iTRAQ labeling, strong cation exchange chromatography (SCX fractionation, immobilized metal affinity chromatography (IMAC and LC-MS/MS, we identified and quantified 2214 unique phosphopeptides corresponding to 1118 phosphoproteins, with 118 changing significantly in abundance with CSPG treatment. The proteins that were regulated by CSPGs included key components of synaptic vesicle trafficking, axon guidance mediated by semaphorins, integrin signaling, cadherin signaling and EGF receptor signaling pathways. A significant number of the regulated proteins are cytoskeletal and related proteins that have been implicated in regulating neurite growth. Another highly represented protein category regulated by CSPGs is nucleic acid binding proteins involved in RNA post-transcriptional regulation. Together, by screening the overall phosphoproteome changes induced by CSPGs, this data expand our understanding of CSPG signaling, which provides new insights into development of strategies for overcoming CSPG inhibition and promoting axonal regeneration after CNS injury.

  10. Targeting Common but Complex Proteoglycans on Breast Cancer Cells and Stem Cells Using Evolutionary Refined Malaria Proteins

    Science.gov (United States)

    2015-11-01

    in hepatocellular carcinoma (Iozzo and Sanderson, 2011). GPC5 Glypican 5 Yes Binds ECM proteins and growth factors. Overexpressed in several...women, parasite-infected red blood cells express a protein that binds to a distinct sugar structure present only on certain cells of the placenta...sufficient amounts of recombinant protein for this project. We are currently trying to move the expression system into insect cells and are in the process of

  11. Proteoglycan fragmentation and respiratory mechanics in mechanically ventilated healthy rats.

    Science.gov (United States)

    Moriondo, Andrea; Pelosi, Paolo; Passi, Alberto; Viola, Manuela; Marcozzi, Cristiana; Severgnini, Paolo; Ottani, Vittoria; Quaranta, Marilisa; Negrini, Daniela

    2007-09-01

    This research investigated whether stretching of lung tissue due to increased positive alveolar pressure swings during mechanical ventilation (MV) at various tidal volumes (V(T)) might affect the composition and/or structure of the glycosaminoglycan (GAG) components of pulmonary extracellular proteoglycans. Experiments were performed in 30 healthy rats: 1) anesthetized and immediately killed (controls, C-0); 2) anesthetized and spontaneously breathing for 4 h (C-4h); and 3) anesthetized, paralyzed, and mechanically ventilated for 4 h with air at 0-cmH(2)O end-expiratory pressure and V(T) of 8 ml/kg (MV-1), 16 ml/kg (MV-2), 24 ml/kg (MV-3), or 32 ml/kg (MV-4), adjusting respiratory rates at a minute ventilation of 270 ml/min. Compared with C-0 and C-4h, a significant reduction of dynamic and static compliance of the respiratory system and of the lung was observed only in MV-4, while extravascular lung water significantly increased in MV-3 and MV-4, but not in MV-1 and MV-2. However, even in MV-1, MV induced a significant fragmentation of pulmonary GAGs. Extraction of covalently bound GAGs and wash out of loosely bound or fragmented GAGs progressively increased with increasing V(T) and was associated with increased expression of local (matrix metalloproteinase-2) and systemic (matrix metalloproteinase-9) activated metalloproteases. We conclude that 1) MV, even at "physiological" low V(T), severely affects the pulmonary extracellular architecture, exposing the lung parenchyma to development of ventilator-induced lung injury; and 2) respiratory mechanics is not a reliable clinical tool for early detection of lung injury.

  12. Benoxaprofen stimulates proteoglycan synthesis in normal canine knee cartilage in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Palmoski, M.J.; Brandt, K.D.

    1983-06-01

    Several nonsteroidal antiinflammatory drugs which are cyclooxygenase inhibitors (e.g., salicylates, fenoprofen, ibuprofen) have been shown to suppress proteoglycan synthesis by normal joint cartilage in vitro. We examined the effect of benoxaprofen, a long-acting proprionic acid derivative which inhibits lipoxygenase in addition to causing moderate cyclooxygenase inhibition. When added to the culture medium in concentrations comparable with those obtainable in serum of patients treated with the drug (e.g., 10 and 50 micrograms/ml), benoxaprofen increased proteoglycan synthesis in slices of normal canine knee cartilage to 126% and 135%, respectively, of control levels. These concentrations of the drug augmented net protein synthesis to 154% and 123%, respectively, of control levels. Incorporation of /sup 3/H glucosamine into 9-aminoacridine precipitable material was increased by benoxaprofen, showing that it stimulates net proteoglycan synthesis, and not merely sulfation. At concentrations of either 10 or 50 micrograms/ml, the drug had no effect on proteoglycan catabolism or on the ability of proteoglycans to interact with cartilage hyaluronic acid to form macromolecular aggregates. Nordihydroguaiaretic acid, a free radical scavenger which, like benoxaprofen, inhibits the lipoxygenase as well as cyclooxygenase pathways of arachidonic acid metabolism, also increased /sup 35/S glycosaminoglycan synthesis in cartilage slices. The stimulation of glycosaminoglycan and protein synthesis by benoxaprofen suggests that its action on the chondrocyte may be different from that of most other nonsteroidal antiinflammatory drugs.

  13. Cell-matrix interactions: focus on proteoglycan-proteinase interplay and pharmacological targeting in cancer.

    Science.gov (United States)

    Theocharis, Achilleas D; Gialeli, Chrisostomi; Bouris, Panagiotis; Giannopoulou, Efstathia; Skandalis, Spyros S; Aletras, Alexios J; Iozzo, Renato V; Karamanos, Nikos K

    2014-11-01

    Proteoglycans are major constituents of extracellular matrices, as well as cell surfaces and basement membranes. They play key roles in supporting the dynamic extracellular matrix by generating complex structural networks with other macromolecules and by regulating cellular phenotypes and signaling. It is becoming evident, however, that proteolytic enzymes are required partners for matrix remodeling and for modulating cell signaling via matrix constituents. Proteinases contribute to all stages of diseases, particularly cancer development and progression, and contextually participate in either the removal of damaged products or in the processing of matrix molecules and signaling receptors. The dynamic interplay between proteoglycans and proteolytic enzymes is a crucial biological step that contributes to the pathophysiology of cancer and inflammation. Moreover, proteoglycans are implicated in the expression and secretion of proteolytic enzymes and often modulate their activities. In this review, we describe the emerging biological roles of proteoglycans and proteinases, with a special emphasis on their complex interplay. We critically evaluate this important proteoglycan-proteinase interactome and discuss future challenges with respect to targeting this axis in the treatment of cancer. © 2014 FEBS.

  14. Proteoglycans in host–pathogen interactions: molecular mechanisms and therapeutic implications

    Science.gov (United States)

    Bartlett, Allison H.; Park, Pyong Woo

    2015-01-01

    Many microbial pathogens subvert proteoglycans for their adhesion to host tissues, invasion of host cells, infection of neighbouring cells, dissemination into the systemic circulation, and evasion of host defence mechanisms. Where studied, specific virulence factors mediate these proteoglycan–pathogen interactions, which are thus thought to affect the onset, progression and outcome of infection. Proteoglycans are composites of glycosaminoglycan (GAG) chains attached covalently to specific core proteins. Proteoglycans are expressed ubiquitously on the cell surface, in intracellular compartments, and in the extracellular matrix. GAGs mediate the majority of ligand-binding activities of proteoglycans, and many microbial pathogens elaborate cell-surface and secreted factors that interact with GAGs. Some pathogens also modulate the expression and function of proteoglycans through known virulence factors. Several GAG-binding pathogens can no longer attach to and invade host cells whose GAG expression has been reduced by mutagenesis or enzymatic treatment. Furthermore, GAG antagonists have been shown to inhibit microbial attachment and host cell entry in vitro and reduce virulence in vivo. Together, these observations underscore the biological significance of proteoglycan–pathogen interactions in infectious diseases. PMID:20113533

  15. 1980 Volvo award in basic science. Proteoglycans in experimental intervertebral disc degeneration.

    Science.gov (United States)

    Lipson, S J; Muir, H

    1981-01-01

    An animal model of intervertebral disc degeneration induced surgically by ventral nuclear herniation in the rabbit produces morphologic changes of disc degeneration. Histologic characteristics and proteoglycan changes have been studied at various times after herniation. After injury, there was metaplasia into fibrocartilage originating from the cells along the margins of the annular wound, with proliferation of cells changing almost the entire disc space into fibrocartilage. A vertebral osteophyte occurred through an endochondral ossification sequence. Aggregating proteoglycans had two periods of repletion in the early course of degeneration. The water content of the disc was rapidly but only transiently restored in the first two days after herniation, whilst the changes in the total proteoglycan content of the disc paralleled these changes. Hyaluronic acid content decreased rapidly after herniation, but the size of the proteoglycan monomers did not change with degeneration. It is suggested that loss of confined fluid mechanics signals an abortive repair attempt rather than that of biochemical changes in proteoglycans initiate disc degeneration.

  16. Small-angle neutron scattering studies from solutions of bovine nasal cartilage proteoglycan

    Science.gov (United States)

    Patel, A.; Stivala, S. S.; Damle, S. P.; Gregory, J. D.; Bunick, G. J.; Uberbacher, E. C.

    1986-02-01

    Small-angle neutron scattering, SANS, of the proteoglycan subunit of bovine nasal cartilage in 0.15 N LiC1 at 25°C yielded the radius of gyration, R g, radius of gyration of the cross-section, R q, persistence length, a *, and the molecular weight, M. The following values were obtained: M = 3.9 × 10 6, R g = 745 Å, R q = 34.6 Å and a * = 35.2 Å. These values compare favorably with those that were obtained from small angle X-ray scattering, SAXS, of a similar extract. The scattering curve of the proteoglycan subunit in D 2O showed a characteristic broad peak in the specified angular range similar to that observed from SAXS, thus confirming the polyelectrolyte nature of the proteoglycan.

  17. Combined alcian blue and silver staining of subnanogram quantities of proteoglycans and glycosaminoglycans in sodium dodecyl sulfate-polyacrylamide gels

    DEFF Research Database (Denmark)

    Møller, H J; Heinegård, D; Poulsen, J H

    1993-01-01

    Proteoglycans stain weakly in polyacrylamide gels by traditional protein stains such as coomassie brilliant blue or silver. In the present work preparations of large aggregating proteoglycan from human articular cartilage were used to evaluate a convenient staining method based on successive stai...

  18. Chronic pulsatile shear stress impacts synthesis of proteoglycans by endothelial cells: effect on platelet aggregation and coagulation.

    Science.gov (United States)

    Elhadj, Selim; Mousa, Shaker A; Forsten-Williams, Kimberly

    2002-01-01

    Endothelial-derived proteoglycans are important regulators of the coagulation-pathway in vivo and our primary objective of this study was to determine whether chronic shear stress affected the synthesis, release, and activity of proteoglycans from bovine aortic endothelial cells (BAEC). BAEC were cultured under shear and proteoglycans were purified from BAEC conditioned media and analyzed using both anionic exchange and size exclusion chromatography. The overall amount of proteoglycans produced per cell was significantly greater for the high shear-treated samples compared to the low shear-treated samples indicating that the shear magnitude did impact cell responsiveness. While overall size and composition of the proteoglycans and glycosaminoglycan (GAG) side chains were not altered by shear, the relative proportion of the high and low molecular weight species was inversely related to shear and differed significantly from that found under static tissue culture conditions. Moreover, a unique proteoglycan peak was identified from low shear stress (5 +/- 2 dynes/cm(2)) conditioned media when compared to high shear conditions (23 +/- 8 dynes/cm(2)) via anionic exchange chromatography, suggesting that subtle changes in the GAG structures may impact activity of these molecules. In order to characterize whether these changes impacted proteoglycan function, we studied the effects of shear specific proteoglycans on the inhibition of thrombin-induced human platelet aggregation as well as on platelet-fibrin clot dynamics. Proteoglycans from high shear-treated samples were less effective inhibitors of both platelet aggregation and blood coagulation inhibition than proteoglycans from low shear-treated samples and both were less effective than proteoglycans isolated from static tissue culture samples. However, due to changes in the overall proteoglycan synthesis and release rate, the high and low shear-treated sample had essentially identical effects on these activities

  19. A Bifurcated Proteoglycan Binding Small Molecule Carrier for siRNA Delivery

    Science.gov (United States)

    Gooding, Matt; Adigbli, Derick; Edith Chan, A W; Melander, Roberta J; MacRobert, Alexander J; Selwood, David L

    2014-01-01

    A wider application of siRNA- and miRNA- based therapeutics is restricted by the currently available delivery systems. We have designed a new type of small molecule carrier (SMoC) system for siRNA modeled to interact with cell surface proteoglycans. This bifurcated SMoC has similar affinity for the model proteoglycan heparin to an equivalent polyarginine peptide and exhibits significant mRNA knockdown of protein levels comparable to lipofectamine and the previously reported linear SMoC. PMID:24472581

  20. Rat mesangial cells in vitro synthesize a spectrum of proteoglycan species including those of the basement membrane and interstitium

    DEFF Research Database (Denmark)

    Thomas, G J; Shewring, L; McCarthy, K J

    1995-01-01

    including laminin, fibronectin, type IV collagen and the basement membrane heparan sulphate proteoglycan (BM-HSPG) known as perlecan. In addition, using Mab 2B5 we demonstrate that RMC synthesize a specific basement membrane chondroitin sulfate (BM-CSPG), a matrix component that in normal animals...... is localized in the mesangium but is not found in the pericapillary glomerular basement membrane (GBM). Further characterization of the proteoglycans synthesized by RMC in vitro revealed: (i) a second large CSPG, identified as versican; (ii) two small dermatan sulphate proteoglycans identified as biglycan...... and decorin, which together account for the majority of the proteoglycans; (iii) a large HSPG-I, probably related to perlecan; and (iv) a small HSPG-II. The cell layer proteoglycans can be sub-divided into a class that are probably free in the membrane, and a class of anchored molecules of the extracellular...

  1. Mapping of matrix metalloproteinase cleavage sites on syndecan-1 and syndecan-4 ectodomains

    DEFF Research Database (Denmark)

    Manon-Jensen, Tina; Multhaupt, Hinke A B; Couchman, John R

    2013-01-01

    Syndecans are transmembrane heparan sulfate proteoglycans with roles in cell proliferation, differentiation, adhesion, and migration. They have been associated with multiple functions in tumour progression, through their ability to interact with a wide range of ligands as well as other receptors......, which makes them key effectors in the pericellular microenvironment. Extracellular shedding of syndecans by tumour-associated matrix metalloproteinases (MMPs) may have an important role in tumour progression. Such ectodomain shedding generates soluble ectodomains that may function as paracrine...

  2. Structural and cell adhesion properties of zebrafish syndecan-4 are shared with higher vertebrates

    DEFF Research Database (Denmark)

    Whiteford, James; Ko, Sunggeon; Lee, Weontae

    2008-01-01

    The syndecan proteoglycans are an ancient class of receptor, bearing heparan sulfate chains that interact with numerous potential ligands including growth factors, morphogens, and extracellular matrix molecules. The single syndecan of invertebrates appears not to have cell adhesion roles, but the......-4 are consistent across the vertebrate spectrum and reflect an early acquisition of specialization after syndecan gene duplication events at the invertebrate/early chordate boundary....

  3. Dual roles of heparanase in vascular calcification associated with human carotid atherosclerosis

    DEFF Research Database (Denmark)

    Aldi, S.; Eriksson, L.; Kronqvist, M.

    2017-01-01

    Vascular intimal calcification is a hallmark of advanced atherosclerosis and an active process akin to bone remodeling. Heparanase (HPSE) is an endo-β-glucuronidase, which cleaves glycosaminoglycan chains of heparan sulfate proteoglycans. The role of heparanase in osteogenesis and bone remodeling...... is controversial. Previously, we have reported the upregulation of HPSE in human carotid endarterectomies from symptomatic patients and showed that the HPSE expression levels correlated with markers of inflammation and increased thrombogenicity....

  4. Glycan Sulfation Modulates Dendritic Cell Biology and Tumor Growth

    Directory of Open Access Journals (Sweden)

    Roland El Ghazal

    2016-05-01

    Full Text Available In cancer, proteoglycans have been found to play roles in facilitating the actions of growth factors, and effecting matrix invasion and remodeling. However, little is known regarding the genetic and functional importance of glycan chains displayed by proteoglycans on dendritic cells (DCs in cancer immunity. In lung carcinoma, among other solid tumors, tumor-associated DCs play largely subversive/suppressive roles, promoting tumor growth and progression. Herein, we show that targeting of DC glycan sulfation through mutation in the heparan sulfate biosynthetic enzyme N-deacetylase/N-sulfotransferase-1 (Ndst1 in mice increased DC maturation and inhibited trafficking of DCs to draining lymph nodes. Lymphatic-driven DC migration and chemokine (CCL21-dependent activation of a major signaling pathway required for DC migration (as measured by phospho-Akt were sensitive to Ndst1 mutation in DCs. Lewis lung carcinoma tumors in mice deficient in Ndst1 were reduced in size. Purified CD11c+ cells from the tumors, which contain the tumor-infiltrating DC population, showed a similar phenotype in mutant cells. These features were replicated in mice deficient in syndecan-4, the major heparan sulfate proteoglycan expressed on the DC surface: Tumors were growth-impaired in syndecan-4–deficient mice and were characterized by increased infiltration by mature DCs. Tumors on the mutant background also showed greater infiltration by NK cells and NKT cells. These findings indicate the genetic importance of DC heparan sulfate proteoglycans in tumor growth and may guide therapeutic development of novel strategies to target syndecan-4 and heparan sulfate in cancer.

  5. Essential contribution of tumor-derived perlecan to epidermal tumor growth and angiogenesis

    DEFF Research Database (Denmark)

    Jiang, Xinnong; Multhaupt, Hinke; Chan, En

    2004-01-01

    As a major heparan sulfate proteoglycan (PG) in basement membranes, perlecan has been linked to tumor invasion, metastasis, and angiogenesis. Here we produced epidermal tumors in immunocompromised rats by injection of mouse RT101 tumor cells. Tumor sections stained with species-specific perlecan...... factor. In vivo, antisense perlecan-transfected cells generated no tumors, whereas untransfected and vector-transfected cells formed tumors with obvious neovascularization, suggesting that tumor perlecan rather than host perlecan controls tumor growth and angiogenesis....

  6. α2,3 and α2,6 N-Linked Sialic Acids Facilitate Efficient Binding and Transduction by Adeno-Associated Virus Types 1 and 6

    OpenAIRE

    Wu, Zhijian; Miller, Edward; Agbandje-McKenna, Mavis; Samulski, Richard Jude

    2006-01-01

    Recombinant adeno-associated viruses (AAVs) are promising vectors in the field of gene therapy. Different AAV serotypes display distinct tissue tropism, believed to be related to the distribution of their receptors on target cells. Of the 11 well-characterized AAV serotypes, heparan sulfate proteoglycan and sialic acid have been suggested to be the attachment receptors for AAV type 2 and types 4 and 5, respectively. In this report, we identify the receptor for the two closely related serotype...

  7. IGF-I regulates HT1080 fibrosarcoma cell migration through a syndecan-2/Erk/ezrin signaling axis.

    Science.gov (United States)

    Mytilinaiou, Maria; Nikitovic, Dragana; Berdiaki, Aikaterini; Papoutsidakis, Antonis; Papachristou, Dionysios J; Tsatsakis, Aristidis; Tzanakakis, George N

    2017-12-01

    Fibrosarcoma is a tumor of mesenchymal origin, originating from fibroblasts. IGF-I is an anabolic growth factor which exhibits significant involvement in cancer progression. In this study, we investigated the possible participation of syndecan-2 (SDC-2), a cell membrane heparan sulfate (HS) proteoglycan on IGF-I dependent fibrosarcoma cell motility. Our results demonstrate that SDC-2-deficient HT1080 cells exhibit attenuated IGF-I-dependent chemotactic migration (p fibrosarcoma cell migration. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Change in Long-Spacing Collagen in Descemet's Membrane of Diabetic Goto-Kakizaki Rats and Its Suppression by Antidiabetic Agents

    OpenAIRE

    Yoshihiro Akimoto; Hajime Sawada; Mica Ohara-Imaizumi; Shinya Nagamatsu; Hayato Kawakami

    2008-01-01

    We examined changes in the ultrastructure and localization of major extracellular matrix components, including 5 types of collagen (type I, III, IV, VI, and VIII), laminin, fibronectin, and heparan sulfate proteoglycan in Descemet's membrane of the cornea of diabetic GK rats. In the cornea of diabetic GK rats, more long-spacing collagen fibrils were observed in Descemet's membrane than in the membrane of the nondiabetic Wistar rats. Both GK and Wistar rats showed an age-dependent increase in...

  9. Perlecan and tumor angiogenesis

    DEFF Research Database (Denmark)

    Jiang, Xinnong; Couchman, John R

    2003-01-01

    Perlecan is a major heparan sulfate proteoglycan (HSPG) of basement membranes (BMs) and connective tissues. The core protein of perlecan is divided into five domains based on sequence homology to other known proteins. Commonly, the N-terminal domain I of mammalian perlecan is substituted with thr...... have unwanted promoting effects on tumor cell proliferation and tumor angiogenesis. Understanding of these attributes at the molecular level may offer opportunities for therapeutic intervention....

  10. Syndecans, signaling, and cell adhesion

    DEFF Research Database (Denmark)

    Couchman, J R; Woods, A

    1996-01-01

    Syndecans are transmembrane proteoglycans which can participate in diverse cell surface interactions, involving extracellular matrix macromolecules, growth factors, protease inhibitors, and even viral entry. Currently, all extracellular interactions are believed to be mediated by distinct...... structures within the heparan sulfate chains, leaving the roles of chondroitin sulfate chains and extracellular portion of the core proteins to be elucidated. Evidence that syndecans are a class of receptor involved in cell adhesion is mounting, and their small cytoplasmic domains may link...

  11. Immune Recognition of Citrullinated Proteoglycan Aggrecan Epitopes in Mice with Proteoglycan-Induced Arthritis and in Patients with Rheumatoid Arthritis.

    Science.gov (United States)

    Markovics, Adrienn; Ocskó, Tímea; Katz, Robert S; Buzás, Edit I; Glant, Tibor T; Mikecz, Katalin

    2016-01-01

    Rheumatoid arthritis (RA) is an autoimmune inflammatory disease affecting the joints. Anti-citrullinated protein antibodies (ACPA) are frequently found in RA. Previous studies identified a citrullinated epitope in cartilage proteoglycan (PG) aggrecan that elicited pro-inflammatory cytokine production by RA T cells. We recently reported the presence of ACPA-reactive (citrullinated) PG in RA cartilage. Herein, we sought to identify additional citrullinated epitopes in human PG that are recognized by T cells or antibodies from RA patients. We used mice with PG-induced arthritis (PGIA) as a screening tool to select citrulline (Cit)-containing PG peptides that were more immunogenic than the arginine (R)-containing counterparts. The selected peptide pairs were tested for induction of pro-inflammatory T-cell cytokine production in RA and healthy control peripheral blood mononuclear cell (PBMC) cultures using ELISA and flow cytometry. Anti-Cit and anti-R peptide antibodies were detected by ELISA. Splenocytes from mice with PGIA exhibited greater T-cell cytokine secretion in response to the Cit than the R version of PG peptide 49 (P49) and anti-P49 antibodies were found in PGIA serum. PBMC from ACPA+ and ACPA- RA patients, but not from healthy controls, responded to Cit49 with robust cytokine production. High levels of anti-Cit49 antibodies were found in the plasma of a subset of ACPA+ RA patients. Another PG peptide (Cit13) similar to the previously described T-cell epitope induced greater cytokine responses than R13 by control (but not RA) PBMC, however, anti-Cit13 antibodies were rarely detected in human plasma. We identified a novel citrullinated PG epitope (Cit49) that is highly immunogenic in mice with PGIA and in RA patients. We also describe T-cell and antibody reactivity with Cit49 in ACPA+ RA. As citrullinated PG might be present in RA articular cartilage, Cit PG epitope-induced T-cell activation or antibody deposition may occur in the joints of RA patients.

  12. Immune Recognition of Citrullinated Proteoglycan Aggrecan Epitopes in Mice with Proteoglycan-Induced Arthritis and in Patients with Rheumatoid Arthritis.

    Directory of Open Access Journals (Sweden)

    Adrienn Markovics

    Full Text Available Rheumatoid arthritis (RA is an autoimmune inflammatory disease affecting the joints. Anti-citrullinated protein antibodies (ACPA are frequently found in RA. Previous studies identified a citrullinated epitope in cartilage proteoglycan (PG aggrecan that elicited pro-inflammatory cytokine production by RA T cells. We recently reported the presence of ACPA-reactive (citrullinated PG in RA cartilage. Herein, we sought to identify additional citrullinated epitopes in human PG that are recognized by T cells or antibodies from RA patients.We used mice with PG-induced arthritis (PGIA as a screening tool to select citrulline (Cit-containing PG peptides that were more immunogenic than the arginine (R-containing counterparts. The selected peptide pairs were tested for induction of pro-inflammatory T-cell cytokine production in RA and healthy control peripheral blood mononuclear cell (PBMC cultures using ELISA and flow cytometry. Anti-Cit and anti-R peptide antibodies were detected by ELISA.Splenocytes from mice with PGIA exhibited greater T-cell cytokine secretion in response to the Cit than the R version of PG peptide 49 (P49 and anti-P49 antibodies were found in PGIA serum. PBMC from ACPA+ and ACPA- RA patients, but not from healthy controls, responded to Cit49 with robust cytokine production. High levels of anti-Cit49 antibodies were found in the plasma of a subset of ACPA+ RA patients. Another PG peptide (Cit13 similar to the previously described T-cell epitope induced greater cytokine responses than R13 by control (but not RA PBMC, however, anti-Cit13 antibodies were rarely detected in human plasma.We identified a novel citrullinated PG epitope (Cit49 that is highly immunogenic in mice with PGIA and in RA patients. We also describe T-cell and antibody reactivity with Cit49 in ACPA+ RA. As citrullinated PG might be present in RA articular cartilage, Cit PG epitope-induced T-cell activation or antibody deposition may occur in the joints of RA patients.

  13. Testican-3: a brain-specific proteoglycan member of the BM-40/SPARC/osteonectin family.

    Science.gov (United States)

    Hartmann, Ursula; Hülsmann, Hanni; Seul, Judith; Röll, Sandra; Midani, Heven; Breloy, Isabelle; Hechler, Daniel; Müller, Regina; Paulsson, Mats

    2013-05-01

    The testicans are a three-member family of secreted proteoglycans structurally related to the BM-40/secreted protein acidic and rich in cystein (SPARC) osteonectin family of extracellular calcium-binding proteins. In vitro studies have indicated that testicans are involved in the regulation of extracellular protease cascades and in neuronal function. Here, we describe the biochemical characterization and tissue distribution of mouse testican-3 as well as the inactivation of the corresponding gene. The expression of testican-3 in adult mice is restricted to the brain, where it is located diffusely within the extracellular matrix, as well as associated with cells. Brain-derived testican-3 is a heparan sulphate proteoglycan. In cell culture, the core protein is detected in the supernatant and the extracellular matrix, whereas the proteoglycan form is restricted to the supernatant. This indicates possible interactions of the testican-3 core protein with components of the extracellular matrix which are blocked by addition of the glycosaminoglycan chains. Mice deficient in testican-3 are viable and fertile and do not show an obvious phenotype. This points to a functional redundancy among the different members of the testican family or between testican-3 and other brain heparan sulphate proteoglycans. © 2013 International Society for Neurochemistry.

  14. A Proteoglycan-Like Molecule Offers Insights Into Ground Substance Changes During Holothurian Intestinal Regeneration.

    Science.gov (United States)

    Vázquez-Vélez, Gabriel E; Rodríguez-Molina, José F; Quiñones-Frías, Mónica C; Pagán, María; García-Arrarás, José E

    2016-06-01

    Extracellular matrix remodeling is an essential component of regenerative processes in metazoans. Among these animals, holothurians (sea cucumbers) are distinguished by their great regenerative capacities. We have previously shown that fibrous collagen as well as other fibrous components disappear from the connective tissue (CT) early during intestinal regeneration, and later return as the organ primordia form. We now report on changes of the nonfibrous component of the CT. We have used Alcian Blue staining and an antibody, Proteoglycan Like-1 (PGL-1), that recognizes a proteoglycan-like antigen to identify the presence of proteoglycans in normal and regenerating intestines. Our results show that early in regeneration, the ground substance resembles that of the mesentery, the structure from where the new intestine originates. As regeneration proceeds, Alcian Blue staining and PGL-1 labeling reorganize, so that by 4 weeks the normal intestinal CT pattern is achieved. Together with our previous findings, the data suggest that CT components that might be detrimental to regeneration disappear early on, while those that might be beneficial to regeneration, such as proteoglycans, are present throughout the regenerative process. © 2016 The Histochemical Society.

  15. Age-related decrease in proteoglycan synthesis of human articular chondrocytes: the role of nonenzymatic glycation

    NARCIS (Netherlands)

    Degroot, J.; Verzijl, N.; Bank, R. A.; Lafeber, F. P.; Bijlsma, J. W.; TeKoppele, J. M.

    1999-01-01

    To examine the effect of nonenzymatic glycation of cartilage extracellular matrix on the synthetic activity of chondrocytes. The proteoglycan-synthesis rate (35SO4(2-) incorporation) and levels of advanced nonenzymatic glycation (determined by high-performance liquid chromatography measurement of

  16. Increased proteoglycan synthesis by the cardiovascular system of coarctation hypertensive rats

    DEFF Research Database (Denmark)

    Lipke, D W; Couchman, J R

    1991-01-01

    Proteoglycan (PG) synthesis in the cardiovascular system of coarctation hypertensive rats was examined by in vivo and in vitro labeling of glycosaminoglycans with 35SO4 in rats made hypertensive for short (4 days) and longer (14 days) durations. With in vivo labeling, only tissues directly exposed...

  17. Enhanced cellular uptake of antisecretory peptide AF-16 through proteoglycan binding.

    Science.gov (United States)

    Matson Dzebo, Maria; Reymer, Anna; Fant, Kristina; Lincoln, Per; Nordén, Bengt; Rocha, Sandra

    2014-10-21

    Peptide AF-16, which includes the active site of Antisecretory Factor protein, has antisecretory and anti-inflammatory properties, making it a potent drug candidate for treatment of secretory and inflammatory diseases such as diarrhea, inflammatory bowel diseases, and intracranial hypertension. Despite remarkable physiological effects and great pharmaceutical need for drug discovery, very little is yet understood about AF-16 mechanism of action. In order to address interaction mechanisms, we investigated the binding of AF-16 to sulfated glycosaminoglycan, heparin, with focus on the effect of pH and ionic strength, and studied the influence of cell-surface proteoglycans on cellular uptake efficiency. Confocal laser scanning microscopy and flow cytometry experiments on wild type and proteoglycan-deficient Chinese hamster ovary cells reveal an endocytotic nature of AF-16 cellular uptake that is, however, less efficient for the cells lacking cell-surface proteoglycans. Isothermal titration calorimetry provides quantitative thermodynamic data and evidence for that the peptide affinity to heparin increases at lower pH and ionic strength. Experimental data, supported by theoretical modeling, of peptide-glycosaminoglycan interaction indicate that it has a large electrostatic contribution, which will be enhanced in diseases accompanied by decreased pH and ionic strength. These observations show that cell-surface proteoglycans are of general and crucial importance for the antisecretory and anti-inflammatory activities of AF-16.

  18. Effects of electromagnetic fields on proteoglycan metabolism of bovine articular cartilage explants

    NARCIS (Netherlands)

    De Mattei, M; Pasello, M; Pellati, A; Stabellini, G; Massari, L; Gemmati, D; Caruso, A

    2003-01-01

    Electromagnetic field (EMF) exposure has been proposed for the treatment of osteoarthritis. In this study, we investigated the effects of EMF (75 Hz, 2,3 mT) on proteoglycan (PG) metabolism of bovine articular cartilage explants cultured in vitro, both under basal conditions and in the presence of

  19. Actions of Calcium Channel Blockers on Vascular Proteoglycan Synthesis: Relationship to Atherosclerosis

    Science.gov (United States)

    Survase, Soniya; Ivey, Melanie E; Nigro, Julie; Osman, Narin; Little, Peter J

    2005-01-01

    Calcium channel blockers (CCBs) are a widely used group of antihypertensive agents. CCBs are efficacious in the reduction of blood pressure but the extent to which they manifest beneficial effects on cardiovascular disease is variable. Clinical studies indicate that pleiotropic actions make significant contributions to the efficacy of agents aimed at preventing atherosclerosis. The “response to retention” hypothesis implicates the binding and retention of lipoproteins by glycosaminoglycan chains on proteoglycans as an initiating step in atherogenesis. Atherogenic factors act as agonists and several classes of drugs including peroxisome proliferating-activated receptor (PPAR)-α and -γ ligands act as antagonists in this model. Initial data have demonstrated that high concentrations of CCBs inhibit proteoglycan synthesis. Newer preliminary data show that the action is very modest at reasonable concentrations and appears to be independent of calcium channel blocking activity. We have reviewed the role of cardiovascular drugs acting on vascular smooth muscle proteoglycan synthesis and considered the potential action of CCBs in this model. We conclude that the inhibition of proteoglycan synthesis by CCBs does not play a role in the attenuation of atherosclerosis; however, the antihypertensive efficacy and alternative beneficial actions provide support for the use of CCBs in the therapy of cardiovascular disease. PMID:17319105

  20. Snorc is a novel cartilage specific small membrane proteoglycan expressed in differentiating and articular chondrocytes

    DEFF Research Database (Denmark)

    Heinonen, J; Taipaleenmäki, H; Roering, P

    2011-01-01

    -tag was expressed in Cos7 cells, and the cell lysate was studied for putative glycosaminoglycan attachment by digestion with chondroitinase ABC and Western blotting. RESULTS: The predicted molecule is a small, 121 amino acids long type I single-pass transmembrane chondroitin sulfate proteoglycan, that contains ER...

  1. Age-related decrease in proteoglycan synthesis of human articular chondrocytes: The role of nonenzymatic glycation

    NARCIS (Netherlands)

    Groot, J. de; Verzijl, N.; Bank, R.A.; Lafeber, F.P.J.G.; Bijlsma, J.W.J.; TeKoppele, J.M.

    1999-01-01

    Objective. To examine the effect of nonenzymatic glycation of cartilage extracellular matrix on the synthetic activity of chondrocytes. Methods. The proteoglycan-synthesis rate (35SO42- incorporation) and levels of advanced nonenzymatic glycation (determined by high-performance liquid chromatography

  2. Cartilage proteoglycan aggrecan epitopes induce proinflammatory autoreactive T-cell responses in rheumatoid arthritis and osteoarthritis.

    NARCIS (Netherlands)

    Jong, H. de; Berlo, S.E.; Hombrink, P.; Otten, H.G.; Eden, W. van; Lafeber, F.P.J.G.; Heurkens, A.H.M.; Bijlsma, J.W.J.; Glant, T.T.; Prakken, B.J.

    2010-01-01

    OBJECTIVES: To explore potential T-cell epitopes of the core protein of human cartilage proteoglycan aggrecan (PG) in patients with rheumatoid arthritis (RA) or osteoarthritis. METHODS: Peptide-specific T-cell proliferation and cytokine/chemokine production in response to PG-specific peptides were

  3. Cloning and sequence analysis of a partial cDNA for chicken cartilage proteoglycan core protein.

    Science.gov (United States)

    Sai, S; Tanaka, T; Kosher, R A; Tanzer, M L

    1986-01-01

    A chicken embryo sternal cartilage cDNA library, created in the plasmid expression vector pUC9, was screened for sequences coding for immunologically detectable core protein of the large, major proteoglycan of cartilage. A 1229-base-pair cDNA clone was isolated that contained only one extended open reading frame, which had sequences coding for a polypeptide of 379 amino acid residues. These deduced sequences corresponded to those anticipated from current models of proteoglycan structure; a deduced sequence encompassing 21 amino acids was almost identical to a known sequence of bovine nasal cartilage proteoglycan. Significant homology was found between the deduced amino acid sequence of the proteoglycan and two regions of a chicken hepatic lectin. Immunoprecipitation of the products of cell-free translation yielded a component of about 340 kDa, and transfer blot hybridization of sternal cartilage RNA showed a single mRNA of about 8.1 kilobases. Hybridizable mRNA sequences were readily detectable by dot-blot analyses of the cytoplasm of cartilaginous tissues of the chicken embryo, whereas similar analyses of prechondrogenic limb mesenchymal cells did not demonstrate such hybridizable mRNA signals. Images PMID:3460082

  4. Genistein inhibits PDGF-stimulated proteoglycan synthesis in vascular smooth muscle without blocking PDGFβ receptor phosphorylation.

    Science.gov (United States)

    Little, Peter J; Getachew, Robel; Rezaei, Hossein Babaahmadi; Sanchez-Guerrero, Estella; Khachigian, Levon M; Wang, Haitao; Liao, Sufen; Zheng, Wenhua; Ballinger, Mandy L; Osman, Narin

    2012-09-01

    The signaling pathways that regulate the synthesis and structure of proteoglycans secreted by vascular smooth muscle cells are potential therapeutic targets for preventing lipid deposition in the early stage of atherosclerosis. PDGF stimulates both core protein expression and elongation of glycosaminoglycan (GAG) chains on proteoglycans. In this study we investigated the effects of the tyrosine kinase inhibitor genistein on PDGF mediated receptor phosphorylation and proteoglycan synthesis in human vascular smooth muscle cells. We demonstrate that genistein does not block phosphorylation of the activation site of the PDGF receptor at Tyr(857) and two other downstream sites Tyr(751) and Tyr(1021). Genistein blocked PDGF-mediated proteoglycan core protein synthesis however it had no effect on GAG chain elongation. These results differ markedly to two other tyrosine kinase inhibitors, imatinib and Ki11502, that block PDGF receptor phosphorylation and PDGF mediated GAG elongation. We conclude that the action of genistein on core protein synthesis does not involve the PDGF receptor and that PDGF mediates GAG elongation via the PDGF receptor. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. Serum Amyloid A, but Not C-Reactive Protein, Stimulates Vascular Proteoglycan Synthesis in a Pro-Atherogenic Manner

    Science.gov (United States)

    Wilson, Patricia G.; Thompson, Joel C.; Webb, Nancy R.; de Beer, Frederick C.; King, Victoria L.; Tannock, Lisa R.

    2008-01-01

    Inflammatory markers serum amyloid A (SAA) and C-reactive protein (CRP) are predictive of cardiac disease and are proposed to play causal roles in the development of atherosclerosis, in which the retention of lipoproteins by vascular wall proteoglycans is critical. The purpose of this study was to determine whether SAA and/or CRP alters vascular proteoglycan synthesis and lipoprotein retention in a pro-atherogenic manner. Vascular smooth muscle cells were stimulated with either SAA or CRP (1 to 100 mg/L) and proteoglycans were then isolated and characterized. SAA, but not CRP, increased proteoglycan sulfate incorporation by 50 to 100% in a dose-dependent manner (P proteoglycans; P proteoglycan synthesis in vivo, ApoE−/− mice were injected with an adenovirus expressing human SAA-1, a null virus, or saline. Mice that received adenovirus expressing SAA had increased TGF-β concentrations in plasma and increased aortic biglycan content compared with mice that received either null virus or saline. Thus, SAA alters vascular proteoglycans in a pro-atherogenic manner via the stimulation of TGF-β and may play a causal role in the development of atherosclerosis. PMID:18974302

  6. Proteoglycan biosynthesis by human corneas from patients with types 1 and 2 macular corneal dystrophy

    Energy Technology Data Exchange (ETDEWEB)

    Midura, R.J.; Hascall, V.C.; MacCallum, D.K.; Meyer, R.F.; Thonar, E.J.; Hassell, J.R.; Smith, C.F.; Klintworth, G.K. (National Institute of Dental Research, Bethesda, MD (USA))

    1990-09-15

    Corneal buttons were obtained from patients with types 1 and 2 macular corneal dystrophy (MCD) and from control patients with Fuchs' dystrophy or keratoconus. Buttons were incubated for 20 h in the presence of (3H)glucosamine or (2-3H)mannose. Radiolabeled proteoglycans and lactosaminoglycan-glycoproteins (L-GPs) were purified using chromatography on Q-Sepharose, Superose 6, and octyl-Sepharose. They were identified using chondroitinase ABC, keratanase or endo-beta-galactosidase digestion, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis or Superose 6 chromatography. This study confirms previous reports that type 1 MCD corneas synthesize a normal dermatan sulfate-proteoglycan (DS-PG) and an abnormal keratan sulfate-proteoglycan (KS-PG). The data indicate that typ 1 MCD corneas synthesize L-GP instead of KS-PG. This L-GP has a core protein of similar hydrophobicity (elution from octyl-Sepharose) and nearly similar mass (42 kDa) as the core protein of the KS-PG. It has identical glycoconjugates as those of the KS-PG except that they lack sulfate. Thus, type 1 MCD fails to synthesize keratan sulfate as a result of a defect in a sulfotransferase specific for sulfating lactosaminoglycans. Further, proteoglycans synthesized by a cornea from a patient with type 2 MCD were studied. This cornea synthesized a normal ratio of KS-PG to DS-PG although net synthesis of proteoglycans was approximately 30% below normal. The KS-PG appeared normal whereas the DS-PG had dermatan sulfate chains that were approximately 40% shorter than normal.

  7. Effects of compression on the loss of newly synthesized proteoglycans and proteins from cartilage explants

    Energy Technology Data Exchange (ETDEWEB)

    Sah, R.L.; Doong, J.Y.; Grodzinsky, A.J.; Plaas, A.H.; Sandy, J.D. (Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology Harvard-M.I.T., Cambridge (United States))

    1991-04-01

    The effects of mechanical compression of calf cartilage explants on the catabolism and loss into the medium of proteoglycans and proteins radiolabeled with (35S)sulfate and (3H)proline were examined. A single 2- or 12-h compression of 3-mm diameter cartilage disks from a thickness of 1.25 to 0.50 mm, or slow cyclic compression (2 h on/2 h off) from 1.25 mm to 1.00, 0.75, or 0.50 mm for 24 h led to transient alterations and/or sustained increases in loss of radiolabeled macromolecules. The effects of imposing or removing loads were consistent with several compression-induced physical mediators including fluid flow, diffusion, and matrix disruption. Cyclic compression induced convective fluid flow and enhanced the loss of 35S- and 3H-labeled macromolecules from tissue into medium. In contrast, prolonged static compression induced matrix consolidation and appeared to hinder the diffusional transport and loss of 35S- and 3H-labeled macromolecules. Since high amplitude cyclic compression led to a sustained increase in the rate of loss of 3H- and 35S-labeled macromolecules that was accompanied by an increase in the rate of loss of (3H)hydroxyproline residues and an increase in tissue hydration, such compression may have caused disruption of the collagen meshwork. The 35S-labeled proteoglycans lost during such cyclic compression were of smaller average size than those from controls, but contained a similarly low proportion (approximately 15%) that could form aggregates with excess hyaluronate and link protein. The size distribution and aggregability of the remaining tissue proteoglycans and 35S-labeled proteoglycans were not markedly affected. The loss of tissue proteoglycan paralleled the loss of 35S-labeled macromolecules.

  8. X-ray diffraction study of structural stability of giant proteoglycan molecules of mucus

    Science.gov (United States)

    Vazina, A. A.; Lanina, N. F.; Vasilieva, A. A.; Korneev, V. N.; Zabelin, A. V.; Polyakova, E. P.

    2009-05-01

    X-ray diffraction study of various native and modified gastrointestinal mucins was carried out using synchrotron radiation. The mucus X-ray patterns of mammals and invertebrates are very similar and display a large number of sharp diffraction rings at the spacing of about 4.65 nm, which are due to the helical packing of polysaccharide chains covalently connected to the protein core. A comparative analysis of the X-ray patterns obtained earlier by us from various samples of mucus and biological tissues showed that the 4.65(±0.15) nm spacing is a nanoscale structural invariant of giant proteoglycan molecules of both the mucus and the extracellular matrix of tissues. A role of structural dynamics of proteoglycan scaffolding of biological systems in mechanism of modifying adaptation of organisms to significant changes of temperature is discussed.

  9. X-ray diffraction study of structural stability of giant proteoglycan molecules of mucus

    Energy Technology Data Exchange (ETDEWEB)

    Vazina, A.A. [Institute of Theoretical and Experimental Biophysics, RAS, Institutskaya St. 3, 142290 Pushchino, Moscow Region (Russian Federation); Russian Research Center ' Kurchatov Institute' , 123182 Moscow (Russian Federation)], E-mail: vazina@iteb.ru; Lanina, N.F.; Vasilieva, A.A. [Institute of Theoretical and Experimental Biophysics, RAS, Institutskaya St. 3, 142290 Pushchino, Moscow Region (Russian Federation); Korneev, V.N. [Institute of Cell Biophysics, RAS, 142290 Pushchino (Russian Federation); Zabelin, A.V. [Russian Research Center ' Kurchatov Institute' , 123182 Moscow (Russian Federation); Polyakova, E.P. [Timiryazev Moscow Agricultural Academy, 127550 Moscow (Russian Federation)

    2009-05-11

    X-ray diffraction study of various native and modified gastrointestinal mucins was carried out using synchrotron radiation. The mucus X-ray patterns of mammals and invertebrates are very similar and display a large number of sharp diffraction rings at the spacing of about 4.65 nm, which are due to the helical packing of polysaccharide chains covalently connected to the protein core. A comparative analysis of the X-ray patterns obtained earlier by us from various samples of mucus and biological tissues showed that the 4.65({+-}0.15) nm spacing is a nanoscale structural invariant of giant proteoglycan molecules of both the mucus and the extracellular matrix of tissues. A role of structural dynamics of proteoglycan scaffolding of biological systems in mechanism of modifying adaptation of organisms to significant changes of temperature is discussed.

  10. Collagens and proteoglycans of the cornea: importance in transparency and visual disorders.

    Science.gov (United States)

    Massoudi, Dawiyat; Malecaze, Francois; Galiacy, Stephane D

    2016-02-01

    The cornea represents the external part of the eye and consists of an epithelium, a stroma and an endothelium. Due to its curvature and transparency this structure makes up approximately 70% of the total refractive power of the eye. This function is partly made possible by the particular organization of the collagen extracellular matrix contained in the corneal stroma that allows a constant refractive power. The maintenance of such an organization involves other molecules such as type V collagen, FACITs (fibril-associated collagens with interrupted triple helices) and SLRPs (small leucine-rich proteoglycans). These components play crucial roles in the preservation of the correct organization and function of the cornea since their absence or modification leads to abnormalities such as corneal opacities. Thus, the aim of this review is to describe the different corneal collagens and proteoglycans by highlighting their importance in corneal transparency as well as their implication in corneal visual disorders.

  11. Concentration profiles of collagen and proteoglycan in articular cartilage by Fourier transform infrared imaging and principal component regression

    Science.gov (United States)

    Yin, Jianhua; Xia, Yang; Lu, Mei

    2012-03-01

    Fourier-transform infrared imaging (FT-IRI) technique with the principal component regression (PCR) method was used to quantitatively determine the 2D images and the depth-dependent concentration profiles of two principal macromolecular components (collagen and proteoglycan) in articular cartilage. Ten 6 μm thick sections of canine humeral cartilage were imaged at a pixel size of 6.25 μm in FT-IRI. The infrared spectra extracted from FT-IRI experiments were imported into a PCR program to calculate the quantitative distributions of both collagen and proteoglycan in dry cartilage, which were subsequently converted into the wet-weight based concentration profiles. The proteoglycan profiles by FT-IRI and PCR significantly correlated in linear regression with the proteoglycan profiles by the non-destructive μMRI (the goodness-of-fit 0.96 and the Pearson coefficient 0.98). Based on these concentration relationships, the concentration images of collagen and proteoglycan in both healthy and lesioned articular cartilage were successfully constructed two dimensionally. The simultaneous construction of both collagen and proteoglycan concentration images demonstrates that this combined imaging and chemometrics approach could be used as a sensitive tool to accurately resolve and visualize the concentration distributions of macromolecules in biological tissues.

  12. Imatinib inhibits vascular smooth muscle proteoglycan synthesis and reduces LDL binding in vitro and aortic lipid deposition in vivo

    Science.gov (United States)

    Ballinger, Mandy L; Osman, Narin; Hashimura, Kazuhiko; de Haan, Judy B; Jandeleit-Dahm, Karin; Allen, Terri; Tannock, Lisa R; Rutledge, John C; Little, Peter J

    2010-01-01

    Abstract The ‘response to retention’ hypothesis of atherogenesis proposes that proteoglycans bind and retain low-density lipoproteins (LDL) in the vessel wall. Platelet-derived growth factor (PDGF) is strongly implicated in atherosclerosis and stimulates proteoglycan synthesis. Here we investigated the action of the PDGF receptor inhibitor imatinib on PDGF-mediated proteoglycan biosynthesis in vitro, lipid deposition in the aortic wall in vivo and the carotid artery ex vivo. In human vSMCs, imatinib inhibited PDGF mediated 35S-SO4 incorporation into proteoglycans by 31% (P proteoglycans from PDGF stimulated cells in the presence of imatinib was approximately 2.5-fold higher than for PDGF treatment alone. In high fat fed ApoE−/– mice, imatinib reduced total lipid staining area by ∼31% (P proteoglycans and reduces LDL binding in vitro and in vivo and this effect is mediated via the PDGF receptor. These findings validate a novel mechanism to prevent cardiac disease. PMID:19754668

  13. Structural interactions between collagen and proteoglycans are elucidated by three-dimensional electron tomography of bovine cornea.

    Science.gov (United States)

    Lewis, Philip N; Pinali, Christian; Young, Robert D; Meek, Keith M; Quantock, Andrew J; Knupp, Carlo

    2010-02-10

    Interactions between collagens and proteoglycans help define the structure and function of extracellular matrices. The cornea, which contains proteoglycans with keratan sulphate or chondroitin/dermatan sulphate glycosaminoglycan chains, is an excellent model system in which to study collagen-proteoglycan structures and interactions. Here, we present the first three-dimensional electron microscopic reconstructions of the cornea, and these include corneas from which glycosaminoglycans have been selectively removed by enzymatic digestion. Our reconstructions show that narrow collagen fibrils associate with sulphated proteoglycans that appear as extended, variable-length linear structures. The proteoglycan network appears to tether two or more collagen fibrils, and thus organize the matrix with enough spatial specificity to fulfill the requirements for corneal transparency. Based on the data, we propose that the characteristic pseudohexagonal fibril arrangement in cornea is controlled by the balance of a repulsive force arising from osmotic pressure and an attractive force due to the thermal motion of the proteoglycans. Copyright 2010 Elsevier Ltd. All rights reserved.

  14. Quantification of collagen and proteoglycan deposition in a murine model of airway remodelling

    Science.gov (United States)

    Reinhardt, Alistair K; Bottoms, Stephen E; Laurent, Geoffrey J; McAnulty, Robin J

    2005-01-01

    Background Sub-epithelial extracellular matrix deposition is a feature of asthmatic airway remodelling associated with severity of disease, decline in lung function and airway hyperresponsiveness. The composition of, and mechanisms leading to, this increase in subepithelial matrix, and its importance in the pathogenesis of asthma are unclear. This is partly due to limitations of the current models and techniques to assess airway remodelling. Methods In this study we used a modified murine model of ovalbumin sensitisation and challenge to reproduce features of airway remodelling, including a sustained increase in sub-epithelial matrix deposition. In addition, we have established techniques to accurately and specifically measure changes in sub-epithelial matrix deposition, using histochemical and immunohistochemical staining in conjunction with digital image analysis, and applied these to the measurement of collagen and proteoglycans. Results 24 hours after final ovalbumin challenge, changes similar to those associated with acute asthma were observed, including inflammatory cell infiltration, epithelial cell shedding and goblet cell hyperplasia. Effects were restricted to the bronchial and peribronchial regions with parenchymal lung of ovalbumin sensitised and challenged mice appearing histologically normal. By 12 days, the acute inflammatory changes had largely resolved and increased sub-epithelial staining for collagen and proteoglycans was observed. Quantitative digital image analysis confirmed the increased deposition of sub-epithelial collagen (33%, p proteoglycans (32%, p proteoglycan deposition in an animal model of airway remodelling. This model will be useful for measurement of other matrix components, as well as for assessment of the molecular mechanisms contributing to, and agents to modulate airway remodelling. PMID:15819978

  15. 2-Arachidonoylglycerol Reduces Proteoglycans and Enhances Remyelination in a Progressive Model of Demyelination.

    Science.gov (United States)

    Feliú, Ana; Bonilla Del Río, Itziar; Carrillo-Salinas, Francisco Javier; Hernández-Torres, Gloria; Mestre, Leyre; Puente, Nagore; Ortega-Gutiérrez, Silvia; López-Rodríguez, Maria L; Grandes, Pedro; Mecha, Miriam; Guaza, Carmen

    2017-08-30

    The failure to undergo remyelination is a critical impediment to recovery in multiple sclerosis. Chondroitin sulfate proteoglycans (CSPGs) accumulate at demyelinating lesions creating a nonpermissive environment that impairs axon regeneration and remyelination. Here, we reveal a new role for 2-arachidonoylglycerol (2-AG), the major CNS endocannabinoid, in the modulation of CSPGs deposition in a progressive model of multiple sclerosis, the Theiler's murine encephalomyelitis virus-induced demyelinating disease. Treatment with a potent reversible inhibitor of the enzyme monoacylglycerol lipase, which accounts for 85% of the 2-AG degradation in the mouse CNS, modulates neuroinflammation and reduces CSPGs accumulation and astrogliosis around demyelinated lesions in the spinal cord of Theiler's murine encephalomyelitis virus-infected mice. Inhibition of 2-AG hydrolysis augments the number of mature oligodendrocytes and increases MBP, leading to remyelination and functional recovery of mice. Our findings establish a mechanism for 2-AG promotion of remyelination with implications in axonal repair in CNS demyelinating pathologies.SIGNIFICANCE STATEMENT The deposition of chondroitin sulfate proteoglycans contributes to the failure in remyelination associated with multiple sclerosis. Here we unveil a new role for 2-arachidonoylglycerol, the major CNS endocannabinoid, in the modulation of chondroitin sulfate proteoglycan accumulation in Theiler's murine encephalomyelitis virus-induced demyelinating disease. The treatment during the chronic phase with a potent reversible inhibitor of the enzyme monoacylglycerol lipase, which accounts for 85% of the 2-arachidonoylglycerol degradation in the mouse CNS, modulates neuroinflammation and reduces chondroitin sulfate proteoglycan deposition around demyelinated lesions in the spinal cord of Theiler's murine encephalomyelitis virus-infected mice. The increased 2-arachidonoylglycerol tone promotes remyelination in a model of progressive

  16. Ultrastructural immunocytochemical localization of chondroitin sulfate proteoglycan in Bruch's membrane of the rat

    DEFF Research Database (Denmark)

    Lin, W L; Essner, E; McCarthy, K J

    1992-01-01

    Two monoclonal antibodies (Mab 4D5 and 2D6) raised against the core protein of a basement membrane chondroitin sulfate proteoglycan from Reichert's membrane of the rat, were used for ultrastructural immunoperoxidase localization of this protein in Bruch's membrane of the rat. Immunoreactivity for...... for both antibodies was found in the basal lamina (basement membrane) of the choriocapillary endothelium and retinal pigment epithelium, in collagen fibers in the collagenous zones, and surrounding the elastic layer....

  17. Chondroitin-6-sulfate-containing proteoglycan: a new component of human skin dermoepidermal junction

    DEFF Research Database (Denmark)

    Fine, J D; Couchman, J R

    1988-01-01

    as 54 gestational days. Indirect immunoelectron microscopy and NaCl-split skin studies were performed to ultrastructurally localize the antigen; immune deposits were detectable within the lamina densa in chondroitinase-treated skin. These findings demonstrate that chondroitin sulfate proteoglycan...... is present within all skin basement membranes; that it is present in the region of the lamina densa; and that similar to some other ubiquitous basement membrane antigens, it is present early in the developing fetus....

  18. Roles of the Small Leucine-rich Repeat Proteoglycans OMD and PRELP in Development and Cancer

    OpenAIRE

    Papadaki, V.

    2014-01-01

    Osteomodulin (OMD) and Proline/arginine-rich and Leucine-rich Repeat protein (PRELP) belong to the small leucine-rich repeat proteoglycan (SLRP) family and as extracellular matrix components have the ability to influence various cellular functions, including cell growth, migration and proliferation, while their mutation or aberrant expression can cause developmental disorders and cancer. This thesis extends previous work in further understanding the roles of OMD and PRELP in cancer and also d...

  19. Preservation of the Structure of Enzymatically-Degraded Bovine Vitreous Using Synthetic Proteoglycan Mimics

    Science.gov (United States)

    Zhang, Qianru; Filas, Benjamen A.; Roth, Robyn; Heuser, John; Ma, Nan; Sharma, Shaili; Panitch, Alyssa; Beebe, David C.; Shui, Ying-Bo

    2014-01-01

    Purpose. Vitreous liquefaction and subsequent posterior vitreous detachment can lead to several sight-threatening diseases, including retinal detachment, macular hole and macular traction syndrome, nuclear cataracts, and possibly, open-angle glaucoma. In this study, we tested the ability of three novel synthetic chondroitin sulfate proteoglycan mimics to preserve the structure and physical properties of enzymatically-degraded bovine vitreous. Methods. Chondroitin sulfate proteoglycan mimics, designed to bind to type II collagen, hyaluronic acid, or both, were applied to trypsin- or collagenase-treated bovine vitreous in situ and in vitro. Rheology and liquefaction tests were performed to determine the physical properties of the vitreous, while Western blots were used to detect the presence and degradation of soluble collagen II (α1). Deep-etch electron microscopy (DEEM) identified the ultrastructure of mimic-treated and untreated enzyme-degraded bovine vitreous. Results. Proteoglycan mimics preserved the physical properties of trypsin-degraded bovine vitreous and protected against vitreous liquefaction. Although the collagen-binding mimic maintained the physical properties of collagenase-treated vitreous, liquefaction still occurred. Western blots indicated that the mimic provided only marginal protective ability against soluble collagen degradation. Deep-etch electron microscopy, however, showed increased density and isotropy of microstructural components in mimic-treated vitreous, supporting the initial result that vitreous structure was preserved. Conclusions. Proteoglycan mimics preserved bovine vitreous physical properties after enzymatic degradation. These compounds may be useful in delaying or preventing the pathological effects of age-related, or enzymatically-induced, degradation of the vitreous body. PMID:25342623

  20. Proteoglycan 4: a dynamic regulator of skeletogenesis and parathyroid hormone skeletal anabolism.

    Science.gov (United States)

    Novince, Chad M; Michalski, Megan N; Koh, Amy J; Sinder, Benjamin P; Entezami, Payam; Eber, Matthew R; Pettway, Glenda J; Rosol, Thomas J; Wronski, Thomas J; Kozloff, Ken M; McCauley, Laurie K

    2012-01-01

    Proteoglycan 4 (Prg4), known for its lubricating and protective actions in joints, is a strong candidate regulator of skeletal homeostasis and parathyroid hormone (PTH) anabolism. Prg4 is a PTH-responsive gene in bone and liver. Prg4 null mutant mice were used to investigate the impact of proteoglycan 4 on skeletal development, remodeling, and PTH anabolic actions. Young Prg4 mutant and wild-type mice were administered intermittent PTH(1-34) or vehicle daily from 4 to 21 days. Young Prg4 mutant mice had decreased growth plate hypertrophic zones, trabecular bone, and serum bone formation markers versus wild-type mice, but responded with a similar anabolic response to PTH. Adult Prg4 mutant and wild-type mice were administered intermittent PTH(1-34) or vehicle daily from 16 to 22 weeks. Adult Prg4 mutant mice had decreased trabecular and cortical bone, and blunted PTH-mediated increases in bone mass. Joint range of motion and animal mobility were lower in adult Prg4 mutant versus wild-type mice. Adult Prg4 mutant mice had decreased marrow and liver fibroblast growth factor 2 (FGF-2) mRNA and reduced serum FGF-2, which were normalized by PTH. A single dose of PTH decreased the PTH/PTHrP receptor (PPR), and increased Prg4 and FGF-2 to a similar extent in liver and bone. Proteoglycan 4 supports endochondral bone formation and the attainment of peak trabecular bone mass, and appears to support skeletal homeostasis indirectly by protecting joint function. Bone- and liver-derived FGF-2 likely regulate proteoglycan 4 actions supporting trabeculae formation. Blunted PTH anabolic responses in adult Prg4 mutant mice are associated with altered biomechanical impact secondary to joint failure. Copyright © 2012 American Society for Bone and Mineral Research.

  1. Vascular wall proteoglycan synthesis and structure as a target for the prevention of atherosclerosis

    Directory of Open Access Journals (Sweden)

    Peter J Little

    2007-03-01

    Full Text Available Peter J Little1, 2, 3, Mandy L. Ballinger1, Narin Osman1,31Cell Biology of Diabetes Laboratory, Baker Heart Research Institute, Melbourne, Australia; Monash University, Departments of 2Medicine and 3Immunology, Central and Eastern Clinical School, Alfred Hospital, Melbourne, AustraliaAbstract: Atherosclerosis is the underlying pathology of most cardiovascular disease and it represents the major cause of premature death in modern societies. Current therapies target risk factors being hypertension, hypercholesterolemia, hypertriglyceridemia and hyperglycemia when diabetes is present however the maximum efficacy of these strategies is often 30% or less. Areas of vascular biology that may lead to the development of a complementary vascular wall directed therapy are: inflammation, oxidation, endothelial dysfunction, diabetes-specific factors —hyperglycemia and advanced glycation endproducts and lipid retention by vascular matrix specifically proteoglycans. The major structural features of proteoglycans that determine low-density lipoprotein (LDL binding are the length and sulfation pattern on the glycosaminoglycan (GAG chains. Emerging data discussed in this review indicates that these structural properties are subject to considerable regulation by vasoactive substances possibly using novel signaling pathways. For example, GAG elongation stimulated by platelet-derived growth factor is not blocked by the receptor tyrosine kinase antagonist, genistein suggesting that there may be a previously unknown signaling pathway involved in this response. Thus, modifying proteoglycan synthesis and structure may represent a prime target to prevent LDL binding and entrapment in the vessel wall and thus prevent the development and progression of atherosclerosis.Keywords: proteoglycans, signaling, lipoproteins, atherosclerosis

  2. Salmon cartilage proteoglycan suppresses mouse experimental colitis through induction of Foxp3{sup +} regulatory T cells

    Energy Technology Data Exchange (ETDEWEB)

    Mitsui, Toshihito [Department of Microbiology and Immunology, Hirosaki University Graduate School of Medicine, Zaifu-cho 5, Hirosaki, Aomori 036-8562 (Japan); Department of Digestive Surgery, Hirosaki University Graduate School of Medicine, Hirosaki, Aomori 036-8562 (Japan); Sashinami, Hiroshi [Department of Microbiology and Immunology, Hirosaki University Graduate School of Medicine, Zaifu-cho 5, Hirosaki, Aomori 036-8562 (Japan); Sato, Fuyuki; Kijima, Hiroshi [Department of Pathology and Bioscience, Hirosaki University Graduate School of Medicine, Hirosaki, Aomori 036-8562 (Japan); Ishiguro, Yoh; Fukuda, Shinsaku [Department of Digestive Internal Medicine, Hirosaki University Graduate School of Medicine, Hirosaki, Aomori 036-8562 (Japan); Yoshihara, Shuichi [Department of Glycomedicine, Hirosaki University Graduate School of Medicine, Hirosaki, Aomori 036-8562 (Japan); Hakamada, Ken-Ichi [Department of Digestive Surgery, Hirosaki University Graduate School of Medicine, Hirosaki, Aomori 036-8562 (Japan); Nakane, Akio, E-mail: a27k03n0@cc.hirosaki-u.ac.jp [Department of Microbiology and Immunology, Hirosaki University Graduate School of Medicine, Zaifu-cho 5, Hirosaki, Aomori 036-8562 (Japan)

    2010-11-12

    Research highlights: {yields} Salmon proteoglycan suppresses IL-10{sup -/-} cell transfer-induced colitis progression. {yields} Salmon proteoglycan suppresses Th1- and Th17-related factors in colitis mice. {yields} Salmon proteoglycan enhances Foxp3 expression. -- Abstract: Proteoglycans (PGs) are complex glycohydrates which are widely distributed in extracellular matrix (ECM). PGs are involved in the construction of ECM, cell proliferation and differentiation. ECM components are involved in transduction of proinflammatory responses, but it is still unknown whether PGs are involved in inflammatory response. In this study, we investigated the effect of PG extracted from salmon cartilage on the progression of experimental colitis-induced in severe combined immunodeficiency mice by cell transfer from interleukin-10 (IL-10){sup -/-} mice. IL-10{sup -/-} cell-transferred mice showed weight loss, colon shortening and histological appearance of mild colitis. Daily oral administration of PG attenuated the clinical progression of colitis in a dose-dependent manner. Colitis-induced mice showed the elevated expression of IFN-{gamma}, IL-12, TNF-{alpha}, IL-21, IL-23p19, IL-6, IL-17A and retinoic acid-related orphan receptor {gamma}t (ROR{gamma}t) in lamina propria mononuclear cells (LPMCs) and oral administration of PG suppressed the expression of these factors. Conversely, expression of Foxp3 that induces CD4{sup +}CD25{sup +} regulatory T cells in LPMCs was enhanced by PG administration. These findings suggested that salmon PG attenuated the progression of colitis due to suppression of inflammatory response by enhancement of regulatory T cell induction.

  3. Vascular wall proteoglycan synthesis and structure as a target for the prevention of atherosclerosis

    Science.gov (United States)

    Little, Peter J; Ballinger, Mandy L; Osman, Narin

    2007-01-01

    Atherosclerosis is the underlying pathology of most cardiovascular disease and it represents the major cause of premature death in modern societies. Current therapies target risk factors being hypertension, hypercholesterolemia, hypertriglyceridemia and hyperglycemia when diabetes is present however the maximum efficacy of these strategies is often 30% or less. Areas of vascular biology that may lead to the development of a complementary vascular wall directed therapy are: inflammation, oxidation, endothelial dysfunction, diabetes-specific factors—hyperglycemia and advanced glycation endproducts and lipid retention by vascular matrix specifically proteoglycans. The major structural features of proteoglycans that determine low-density lipoprotein (LDL) binding are the length and sulfation pattern on the glycosaminoglycan (GAG) chains. Emerging data discussed in this review indicates that these structural properties are subject to considerable regulation by vasoactive substances possibly using novel signaling pathways. For example, GAG elongation stimulated by platelet-derived growth factor is not blocked by the receptor tyrosine kinase antagonist, genistein suggesting that there may be a previously unknown signaling pathway involved in this response. Thus, modifying proteoglycan synthesis and structure may represent a prime target to prevent LDL binding and entrapment in the vessel wall and thus prevent the development and progression of atherosclerosis. PMID:17583182

  4. T-cell adhesion induced by proteoglycan-immobilized cytokine MIP-1 beta.

    Science.gov (United States)

    Tanaka, Y; Adams, D H; Hubscher, S; Hirano, H; Siebenlist, U; Shaw, S

    1993-01-07

    Lymphocyte migration from blood into tissue depends on integrin-mediated adhesion to endothelium. Adhesion requires not only integrin ligands on the endothelium, but also activation signals because T-cell integrins cannot bind well until they are activated. The physiological 'triggers' for T-cell adhesion are unknown, but cytokines may be good candidates as they are released during inflammation and trigger adhesion in neutrophils and monocytes. We have identified a cytokine, macrophage inflammatory protein-1 beta (MIP-1 beta), that induces both chemotaxis and adhesion of T cells; MIP-1 beta is most effective at augmenting adhesion of CD8+ T cells to the vascular cell adhesion molecule VCAM-1. We reasoned that, as cytokines in vivo will be rapidly washed away, MIP-1 beta might be bound to endothelial surfaces and so induce adhesion in its immobilized form. Here we show that: (1) MIP-1 beta is present on lymph node endothelium; (2) immobilized MIP-1 beta induces binding of T cells to VCAM-1 in vitro. MIP-1 beta was immobilized by binding to proteoglycan: a conjugate of heparin with bovine serum albumin and cellular proteoglycan CD44 were both effective. We propose that MIP-1 beta and other cytokines with glycosaminoglycan-binding sites will bind to and be presented by endothelial proteoglycans to trigger adhesion selectively not only of lymphocyte subsets, but also of other cell types.

  5. Vascular wall proteoglycan synthesis and structure as a target for the prevention of atherosclerosis.

    Science.gov (United States)

    Little, Peter J; Ballinger, Mandy L; Osman, Narin

    2007-01-01

    Atherosclerosis is the underlying pathology of most cardiovascular disease and it represents the major cause of premature death in modern societies. Current therapies target risk factors being hypertension, hypercholesterolemia, hypertriglyceridemia and hyperglycemia when diabetes is present however the maximum efficacy of these strategies is often 30% or less. Areas of vascular biology that may lead to the development of a complementary vascular wall directed therapy are: inflammation, oxidation, endothelial dysfunction, diabetes-specific factors--hyperglycemia and advanced glycation endproducts and lipid retention by vascular matrix specifically proteoglycans. The major structural features of proteoglycans that determine low-density lipoprotein (LDL) binding are the length and sulfation pattern on the glycosaminoglycan (GAG) chains. Emerging data discussed in this review indicates that these structural properties are subject to considerable regulation by vasoactive substances possibly using novel signaling pathways. For example, GAG elongation stimulated by platelet-derived growth factor is not blocked by the receptor tyrosine kinase antagonist, genistein suggesting that there may be a previously unknown signaling pathway involved in this response. Thus, modifying proteoglycan synthesis and structure may represent a prime target to prevent LDL binding and entrapment in the vessel wall and thus prevent the development and progression of atherosclerosis.

  6. Genes encoding proteoglycans are associated with the risk of anterior cruciate ligament ruptures.

    Science.gov (United States)

    Mannion, Sasha; Mtintsilana, Asanda; Posthumus, Michael; van der Merwe, Willem; Hobbs, Hayden; Collins, Malcolm; September, Alison V

    2014-12-01

    Genetic variants within genes involved in fibrillogenesis have previously been implicated in anterior cruciate ligament (ACL) injury susceptibility. Proteoglycans also have important functions in fibrillogenesis and maintaining the structural integrity of ligaments. Genes encoding proteoglycans are plausible candidates to be investigated for associations with ACL injury susceptibility; polymorphisms within genes encoding the proteoglycans aggrecan (ACAN), biglycan (BGN), decorin (DCN), fibromodulin (FMOD) and lumican (LUM) were examined. A case-control genetic association study was conducted. 227 participants with surgically diagnosed ACL ruptures (ACL group) and 234 controls without any history of ACL injury were genotyped for 10 polymorphisms in 5 proteoglycan genes. Inferred haplotypes were constructed for specific regions. The G allele of ACAN rs1516797 was significantly under-represented in the controls (p=0.024; OR=0.72; 95% CI 0.55 to 0.96) compared with the ACL group. For DCN rs516115, the GG genotype was significantly over-represented in female controls (p=0.015; OR=9.231; 95%CI 1.16 to 73.01) compared with the ACL group and the AA genotype was significantly under-represented in controls (p=0.013; OR=0.33; 95% CI 0.14 to 0.78) compared with the female non-contact ACL injury subgroup. Haplotype analyses implicated regions overlapping ACAN (rs2351491 C>T-rs1042631 T>C-rs1516797 T>G), BGN (rs1126499 C>T-rs1042103 G>A) and LUM-DCN (rs2268578 T>C-rs13312816 A>T-rs516115 A>G) in ACL injury susceptibility. These independent associations and haplotype analyses suggest that regions within ACAN, BGN, DCN and a region spanning LUM-DCN are associated with ACL injury susceptibility. Taking into account the functions of these genes, it is reasonable to propose that genetic sequence variability within the genes encoding proteoglycans may potentially modulate the ligament fibril properties. Published by the BMJ Publishing Group Limited. For permission to use (where not

  7. Degenerative suspensory ligament desmitis as a systemic disorder characterized by proteoglycan accumulation

    Directory of Open Access Journals (Sweden)

    Yoon Jung

    2006-04-01

    Full Text Available Abstract Background Degenerative suspensory ligament desmitis (DSLD is a debilitating disorder thought to be limited to suspensory ligaments of Peruvian Pasos, Peruvian Paso crosses, Arabians, American Saddlebreds, American Quarter Horses, Thoroughbreds, and some European breeds. It frequently leads to persistent, incurable lameness and need to euthanize affected horses. The pathogenesis remains unclear, though the disease appears to run in families. Treatment and prevention are empirical and supportive, and not effective in halting the progression of the disease. Presently, the presumptive diagnosis of DSLD is obtained from patient signalment and history, clinical examination, and ultrasonographic examination of clinically affected horses, and is confirmed at post mortem examination. Presently, there are no reliable methods of diagnosing DSLD in asymptomatic horses. The goal of this study was to characterize and define the disorder in terms of tissue involvement at the macroscopic and microscopic levels. Results We examined tissues and organs from 28 affected horses (22 Peruvian Pasos, 6 horses of other breeds and from 8 control horses. Histopathological examination revealed the presence of excessive amounts of proteoglycans in the following tissues removed from DSLD-affected horses: suspensory ligaments, superficial and deep digital flexor tendons, patellar and nuchal ligaments, cardiovascular system, and sclerae. Electron microscopy demonstrated changes in diameters of collagen fibrils in the tendon, and in smooth muscle cells of the media of the aorta compatible with increased cell permeability in DSLD-affected cells. Separation of tendon extracts by gel chromatography revealed the presence of additional proteoglycan(s in extracts from affected, but not control extracts. Conclusion This study demonstrates for the first time that DSLD, a disease process previously thought to be limited to the suspensory ligaments of the distal limbs of

  8. Structure of corneal layers, collagen fibrils, and proteoglycans of tree shrew cornea.

    Science.gov (United States)

    Almubrad, Turki; Akhtar, Saeed

    2011-01-01

    The stroma is the major part of the cornea, in which collagen fibrils and proteoglycans are distributed uniformly. We describe the ultrastructure of corneal layers, collagen fibrils (CF), and proteoglycans (PGs) in the tree shrew cornea. Tree shrew corneas (5, 6, and 10 week old animals) and normal human corneas (24, 25, and 54 years old) were fixed in 2.5% glutaraldehyde containing cuprolinic blue in a sodium acetate buffer. The tissue was processed for electron microscopy. The 'iTEM Olympus Soft Imaging Solutions GmbH' program was used to measure the corneal layers, collagen fibril diameters and proteoglycan areas. The tree shrew cornea consists of 5 layers: the epithelium, Bowman's layer, stroma, Descemet's membrane, and endothelium. The epithelium was composed of squamous cells, wing cells and basal cells. The Bowman's layer was 5.5±1.0 µm thick and very similar to a normal human Bowman's layer. The stroma was 258±7.00 µm thick and consisted of collagen fibril lamellae. The lamellae were interlaced with one another in the anterior stroma, but ran parallel to one another in the middle and posterior stroma. Collagen fibrils were decorated with proteoglycan filaments with an area size of 390 ±438 nm(2). The collagen fibril had a minimum diameter of 39±4.25 nm. The interfibrillar spacing was 52.91±6.07 nm. Within the collagen fibrils, very small electron-dense particles were present. The structure of the tree shrew cornea is very similar to that of the normal human cornea. As is the case with the human cornea, the tree shrew cornea had a Bowman's layer, lamellar interlacing in the anterior stroma and electron-dense particles within the collagen fibrils. The similarities of the tree shrew cornea with the human cornea suggest that it could be a good structural model to use when studying changes in collagen fibrils and proteoglycans in non-genetic corneal diseases, such as ectasia caused after LASIK (laser-assisted in situ keratomileusis).

  9. Structure of corneal layers, collagen fibrils, and proteoglycans of tree shrew cornea

    Science.gov (United States)

    Almubrad, Turki

    2011-01-01

    Purpose The stroma is the major part of the cornea, in which collagen fibrils and proteoglycans are distributed uniformly. We describe the ultrastructure of corneal layers, collagen fibrils (CF), and proteoglycans (PGs) in the tree shrew cornea. Methods Tree shrew corneas (5, 6, and 10 week old animals) and normal human corneas (24, 25, and 54 years old) were fixed in 2.5% glutaraldehyde containing cuprolinic blue in a sodium acetate buffer. The tissue was processed for electron microscopy. The ‘iTEM Olympus Soft Imaging Solutions GmbH’ program was used to measure the corneal layers, collagen fibril diameters and proteoglycan areas. Results The tree shrew cornea consists of 5 layers: the epithelium, Bowman’s layer, stroma, Descemet’s membrane, and endothelium. The epithelium was composed of squamous cells, wing cells and basal cells. The Bowman’s layer was 5.5±1.0 µm thick and very similar to a normal human Bowman’s layer. The stroma was 258±7.00 µm thick and consisted of collagen fibril lamellae. The lamellae were interlaced with one another in the anterior stroma, but ran parallel to one another in the middle and posterior stroma. Collagen fibrils were decorated with proteoglycan filaments with an area size of 390 ±438 nm2. The collagen fibril had a minimum diameter of 39±4.25 nm. The interfibrillar spacing was 52.91±6.07 nm. Within the collagen fibrils, very small electron-dense particles were present. Conclusions The structure of the tree shrew cornea is very similar to that of the normal human cornea. As is the case with the human cornea, the tree shrew cornea had a Bowman's layer, lamellar interlacing in the anterior stroma and electron-dense particles within the collagen fibrils. The similarities of the tree shrew cornea with the human cornea suggest that it could be a good structural model to use when studying changes in collagen fibrils and proteoglycans in non-genetic corneal diseases, such as ectasia caused after LASIK (laser

  10. Syndecan-4 and integrins: combinatorial signaling in cell adhesion

    DEFF Research Database (Denmark)

    Couchman, J R; Woods, A

    1999-01-01

    It is now becoming clear that additional transmembrane components can modify integrin-mediated adhesion. Syndecan-4 is a transmembrane heparan sulfate proteoglycan whose external glycosaminoglycan chains can bind extracellular matrix ligands and whose core protein cytoplasmic domain can signal...... during adhesion. Two papers in this issue of JCS demonstrate, through transfection studies, that syndecan-4 plays roles in the formation of focal adhesions and stress fibers. Overexpression of syndecan-4 increases focal adhesion formation, whereas a partially truncated core protein that lacks the binding...... site for protein kinase C(&agr;) and phosphatidylinositol 4, 5-bisphosphate acts as a dominant negative inhibitor of focal adhesion formation. Focal adhesion induction does not require interaction between heparan sulfate glycosaminoglycan and ligand but can occur when non-glycanated core protein...

  11. Selective inhibition of proteoglycan and hyaluronate synthesis in chondrocyte cultures by cyclofenil diphenol, a non-steroidal weak oestrogen.

    Science.gov (United States)

    Mason, R M; Lineham, J D; Phillipson, M A; Black, C M

    1984-10-15

    Cyclofenil diphenol, a weak non-steroidal oestrogen, binds to albumin. In the presence of concentrations of albumin just sufficient to keep cyclofenil diphenol in solution, the compound inhibited the synthesis of [35S]proteoglycans, [3H]glycoproteins, [3H]hyaluronate and [3H]proteins in primary cultures of chondrocytes from the Swarm rat chondrosarcoma in a dose-dependent manner. When excess albumin was present, conditions were found (90 micrograms of cyclofenil diphenol and 4 mg of albumin per ml of culture medium) which completely inhibited [35S]proteoglycan and [3H]hyaluronate synthesis but had little effect on [3H]protein or [3H]glycoprotein synthesis. The time of onset of inhibition of [35S]proteoglycan synthesis by cyclofenil diphenol was very rapid (t1/2 less than 25 min) and incompatible with an action mediated through suppression of proteoglycan core protein synthesis. Cyclofenil diphenol inhibited the synthesis of [35S]chondroitin sulphate chains onto p-nitrophenyl beta-D-xyloside in the cultures. Cyclofenil diphenol had little effect on the secretion from chondrocytes of [35S]proteoglycans synthesized immediately prior to treatment. Chondrocyte cultures treated with cyclofenil diphenol recovered their biosynthetic activities almost completely within 3 h of removing the compound from the culture medium. Cyclofenil diphenol had a similar inhibitory action on the synthesis of [35S]proteoglycans in secondary cultures of human dermal fibroblasts from both normal subjects and patients with systemic sclerosis. It is proposed that cyclofenil diphenol inhibits the synthesis of [35S]proteoglycans by interfering with the formation of the glycosaminoglycan side chains of these molecules in the Golgi apparatus of cells. The action may be due to disturbance of Golgi membrane organization by the compound.

  12. Concentration determination of collagen and proteoglycan in bovine nasal cartilage by Fourier transform infrared imaging and PLS

    Science.gov (United States)

    Zhang, Xuexi; Xiao, Zhi-Yan; Yin, Jianhua; Xia, Yang

    2014-09-01

    Fourier transform infrared imaging (FTIRI) combined with chemometrics can be used to detect the structure of bio-macromolecule, measure the concentrations of some components, and so on. In this study, FTIRI with Partial Least-Squares (PLS) regression was applied to study the concentration of two main components in bovine nasal cartilage (BNC), collagen and proteoglycan. An infrared spectrum library was built by mixing the collagen and chondroitin 6-sulfate (main of proteoglycan) at different ratios. Some pretreatments are needed for building PLS model. FTIR images were collected from BNC sections at 6.25μm and 25μm pixel size. The spectra extracted from BNC-FTIR images were imported into the PLS regression program to predict the concentrations of collagen and proteoglycan. These PLS-determined concentrations are agreed with the result in our previous work and biochemical analytical results. The prediction shows that the concentrations of collagen and proteoglycan in BNC are comparative on the whole. However, the concentration of proteoglycan is a litter higher than that of collagen, to some extent.

  13. Evidence of glycoproteins and sulphated proteoglycan-like presence in extracellular polymeric substance from anaerobic granular sludge.

    Science.gov (United States)

    Bourven, Isabelle; Bachellerie, Guillaume; Costa, Guy; Guibaud, Gilles

    2015-01-01

    The protein fraction of extracellular polymeric substance (EPS) from two anaerobic granular sludge samples was characterized with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and a far western blotting method. SDS-PAGE was used with various staining applications to obtain a protein (silver), glycoprotein [periodic acid-Shiff's (PAS)] or proteoglycan-like (Alcian blue at pH 2.5 (carboxylic group) or 1 (sulphated group)) fingerprint. The fingerprints of the EPS denatured protein from the two sludge samples differed. Some proteins are specific to Soluble (S) or Bound (B)-EPS (20-100 kDa). Denatured proteins with a polysaccharide moieties characterization are more present in B-EPS. Glycoproteins with α-d-mannosyl and/or α-d-glucosyl (90, 50, 40 kDa) were detected. Proteoglycan-like and sulphated proteoglycan-like substances are also detected, mainly in B-EPS. A 68 kDa sulphated proteoglycan-like substance contains two glucidic residue types: α-d-mannosyl and/or α-d-glucosyl and N-acetyl-β-d-glucosamine. Such heteroproteins are present around the membrane as well as the surface-layer from Archaea and from some bacteria. The glycoprotein and sulphated proteoglycan-like substance are assumed to contribute to anaerobic granule strength, thanks to their ability to perform interactions of various nature (ionic, hydrophobic, Ca(2+) as divalent cation bridging, etc.).

  14. The effects of proteoglycan and type II collagen on T1rho relaxation time of articular cartilage

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Won Seok; Yoo, Hye Jin; Hong, Sung Hwan; Choi, Ja Young [Dept. of Radiology and Institute of Radiation Medicine, Seoul National University Hospital, Seoul National University College of Medicine, Seoul (Korea, Republic of)

    2015-02-15

    To evaluate the effects of proteoglycan and type II collagen within articular cartilage on T1rho relaxation time of articular cartilage. This study was exempted by the institutional and animal review boards, and informed consent was not required. Twelve porcine patellae were assigned to three groups of control, trypsin-treated (proteoglycan-degraded), or collagenase-treated (collagen-degraded). The T1rho images were obtained with a 3 tesla magnetic resonance imaging scanner with a single loop coil. Statistical differences were detected by analysis of variance to evaluate the effects of the enzyme on T1rho relaxation time. Safranin-O was used to stain proteoglycan in the articular cartilage and immunohistochemical staining was performed for type II collagen. Mean T1rho values of the control, trypsin-treated, and collagenase-treated groups were 37.72 +/- 5.82, 57.53 +/- 8.24, and 45.08 +/- 5.31 msec, respectively (p < 0.001). Histology confirmed a loss of proteoglycan and type II collagen in the trypsin- and collagenase-treated groups. Degradation of proteoglycans and collagen fibers in the articular cartilage increased the articular cartilage T1rho value.

  15. Chemical Biology in the Embryo: In Situ Imaging of Sulfur Biochemistry in Normal and Proteoglycan-Deficient Cartilage Matrix.

    Science.gov (United States)

    Hackett, Mark J; George, Graham N; Pickering, Ingrid J; Eames, B Frank

    2016-05-03

    Proteoglycans (PGs) are heavily glycosylated proteins that play major structural and biological roles in many tissues. Proteoglycans are abundant in cartilage extracellular matrix; their loss is a main feature of the joint disease osteoarthritis. Proteoglycan function is regulated by sulfation-sulfate ester formation with specific sugar residues. Visualization of sulfation within cartilage matrix would yield vital insights into its biological roles. We present synchrotron-based X-ray fluorescence imaging of developing zebrafish cartilage, providing the first in situ maps of sulfate ester distribution. Levels of both sulfur and sulfate esters decrease as cartilage develops through late phase differentiation (maturation or hypertrophy), suggesting a functional link between cartilage matrix sulfur content and chondrocyte differentiation. Genetic experiments confirm that sulfate ester levels were due to cartilage proteoglycans and support the hypothesis that sulfate ester levels regulate chondrocyte differentiation. Surprisingly, in the PG synthesis mutant, the total level of sulfur was not significantly reduced, suggesting sulfur is distributed in an alternative chemical form during lowered cartilage proteoglycan production. Fourier transform infrared imaging indicated increased levels of protein in the mutant fish, suggesting that this alternative sulfur form might be ascribed to an increased level of protein synthesis in the mutant fish, as part of a compensatory mechanism.

  16. Synthetic Site-Selectively Mono-6-O-Sulfated Heparan Sulfate Dodecasaccharide Shows Anti-Angiogenic Properties In Vitro and Sensitizes Tumors to Cisplatin In Vivo.

    Directory of Open Access Journals (Sweden)

    Egle Avizienyte

    Full Text Available Heparan sulphate (HS, a ubiquitously expressed glycosaminoglycan (GAG, regulates multiple cellular functions by mediating interactions between numerous growth factors and their cell surface cognate receptors. However, the structural specificity of HS in these interactions remains largely undefined. Here, we used completely synthetic, structurally defined, alternating N-sulfated glucosamine (NS and 2-O-sulfated iduronate (IS residues to generate dodecasaccharides ([NSIS]6 that contained no, one or six glucosamine 6-O-sulfates (6S. The aim was to address how 6S contributes to the potential of defined HS dodecasaccharides to inhibit the angiogenic growth factors FGF2 and VEGF165, in vitro and in vivo. We show that the addition of a single 6S at the non-reducing end of [NSIS]6, i.e. [NSIS6S]-[NSIS]5, significantly augments the inhibition of FGF2-dependent endothelial cell proliferation, migration and sprouting in vitro when compared to the non-6S variant. In contrast, the fully 6-O-sulfated dodecasaccharide, [NSIS6S]6, is not a potent inhibitor of FGF2. Addition of a single 6S did not significantly improve inhibitory properties of [NSIS]6 when tested against VEGF165-dependent endothelial cell functions.In vivo, [NSIS6S]-[NSIS]5 blocked FGF2-dependent blood vessel formation without affecting tumor growth. Reduction of non-FGF2-dependent ovarian tumor growth occurred when [NSIS6S]-[NSIS]5 was combined with cisplatin. The degree of inhibition by [NSIS6S]-[NSIS]5 in combination with cisplatin in vivo equated with that induced by bevacizumab and sunitinib when administered with cisplatin. Evaluation of post-treatment vasculature revealed that [NSIS6S]-[NSIS]5 treatment had the greatest impact on tumor blood vessel size and lumen formation. Our data for the first time demonstrate that synthetic, structurally defined oligosaccharides have potential to be developed as active anti-angiogenic agents that sensitize tumors to chemotherapeutic agents.

  17. Cleavage of group 1 coronavirus spike proteins: how furin cleavage is traded off against heparan sulfate binding upon cell culture adaptation

    NARCIS (Netherlands)

    Haan, de C.A.M.; Haijema, B.J.; Schellen, P.; Wichgers Schreur, P.J.; Lintelo, te E.; Vennema, H.; Rottier, P.J.M.

    2008-01-01

    A longstanding enigmatic feature of the group 1 coronaviruses is the uncleaved phenotype of their spike protein, an exceptional property among class I fusion proteins. Here, however, we show that some group 1 coronavirus spike proteins carry a furin enzyme recognition motif and can actually be

  18. A Simple Method for Discovering Druggable, Specific Glycosaminoglycan-Protein Systems. Elucidation of Key Principles from Heparin/Heparan Sulfate-Binding Proteins.

    Directory of Open Access Journals (Sweden)

    Aurijit Sarkar

    Full Text Available Glycosaminoglycans (GAGs affect human physiology and pathology by modulating more than 500 proteins. GAG-protein interactions are generally assumed to be ionic and nonspecific, but specific interactions do exist. Here, we present a simple method to identify the GAG-binding site (GBS on proteins that in turn helps predict high specific GAG-protein systems. Contrary to contemporary thinking, we found that the electrostatic potential at basic arginine and lysine residues neither identifies the GBS consistently, nor its specificity. GBSs are better identified by considering the potential at neutral hydrogen bond donors such as asparagine or glutamine sidechains. Our studies also reveal that an unusual constellation of ionic and non-ionic residues in the binding site leads to specificity. Nature engineers the local environment of Asn45 of antithrombin, Gln255 of 3-O-sulfotransferase 3, Gln163 and Asn167 of 3-O-sulfotransferase 1 and Asn27 of basic fibroblast growth factor in the respective GBSs to induce specificity. Such residues are distinct from other uncharged residues on the same protein structure in possessing a significantly higher electrostatic potential, resultant from the local topology. In contrast, uncharged residues on nonspecific GBSs such as thrombin and serum albumin possess a diffuse spread of electrostatic potential. Our findings also contradict the paradigm that GAG-binding sites are simply a collection of contiguous Arg/Lys residues. Our work demonstrates the basis for discovering specifically interacting and druggable GAG-protein systems based on the structure of protein alone, without requiring access to any structure-function relationship data.

  19. Serglycin proteoglycan is required for multiple myeloma cell adhesion, in vivo growth, and vascularization.

    Science.gov (United States)

    Purushothaman, Anurag; Toole, Bryan P

    2014-02-28

    Recently, it was discovered that serglycin, a hematopoietic cell proteoglycan, is the major proteoglycan expressed and constitutively secreted by multiple myeloma (MM) cells. High levels of serglycin are present in the bone marrow aspirates of at least 30% of newly diagnosed MM patients. However, its contribution to the pathophysiology of MM is unknown. Here, we show that serglycin knockdown (by ∼85% compared with normal levels), using lentiviral shRNA, dramatically attenuated MM tumor growth in mice with severe combined immunodeficiency. Tumors formed from cells deficient in serglycin exhibited diminished levels of hepatocyte growth factor expression and impaired development of blood vessels, indicating that serglycin may affect tumor angiogenesis. Furthermore, knockdown of serglycin significantly decreased MM cell adhesion to bone marrow stromal cells and collagen I. Even though serglycin proteoglycan does not have a transmembrane domain, flow cytometry showed that serglycin is present on the MM cell surface, and attachment to the cell surface is, at least in part, dependent on its chondroitin sulfate side chains. Co-precipitation of serglycin from conditioned medium of MM cells using a CD44-Fc chimera suggests that CD44 is the cell surface-binding partner for serglycin, which therefore may serve as a major ligand for CD44 at various stages during myeloma progression. Finally, we demonstrate that serglycin mRNA expression in MM cells is up-regulated by activin, a predominant cytokine among those increased in MM patients with osteolytic lesions. These studies provide direct evidence for a critical role for serglycin in MM pathogenesis and show that targeting serglycin may provide a novel therapeutic approach for MM.

  20. The study of optical properties and proteoglycan content of tendons by PS-OCT

    Science.gov (United States)

    Yang, Ying; Rupani, Asha; Weightman, Alan; Wimpenny, Ian; Bagnaninchi, Pierre; Ahearne, Mark

    2011-03-01

    Tendons are load-bearing collagenous tissues consisting mainly of type I collagen and various proteoglycans (PGs) including decorin and versican. It is widely accepted that highly orientated collagen fibers in tendons a play critical role for transferring tensile stress and demonstrate birefringent optical properties. However, the influence that proteoglycans have on the optical properties of tendons is yet to be fully elucidated. Tendinopathy (defined as a syndrome of tendon pain, tenderness and swelling that affects the normal function of the tissue) is a common disease associated with sporting injuries or degeneration. PG's are the essential components of the tendon extracellular matrix; changes in their quantities and compositions have been associated with tendinopathy. In this study, polarization sensitive optical coherence tomography (PS-OCT) has been used to reveal the relationship between proteoglycan content/location and birefringent properties of tendons. Tendons dissected from freshly slaughtered chickens were imaged at regular intervals by PS-OCT and polarizing light microscope during the extraction of PGs or glycosaminoglycans using established protocols (guanidine hydrochloride (GuHCl) or proteinase K solution). The macroscopic and microscopic time lapsed images are complimentary; mutually demonstrating that there was a higher concentration of PG's in the outer sheath region than in the fascicles; and the integrity of the sheath affected extraction process and the OCT birefringence bands. Extraction of PGs using GuHCl disturbed the organization of local collagen bundles, which corresponded to a reduction in the frequency of birefringence bands and the band width by PS-OCT. The feature of OCT penetration depth helped us to define the heterogeneous distribution of PG's in tendon, which was complimented by polarizing light microscopy. The results provide new insight of tendon structure and also demonstrate a great potential for using PS-OCT as a

  1. In vivo contribution of amino acid sulfur to cartilage proteoglycan sulfation

    Science.gov (United States)

    Pecora, Fabio; Gualeni, Benedetta; Forlino, Antonella; Superti-Furga, Andrea; Tenni, Ruggero; Cetta, Giuseppe; Rossi, Antonio

    2006-01-01

    Cytoplasmic sulfate for sulfation reactions may be derived either from extracellular fluids or from catabolism of sulfur-containing amino acids and other thiols. In vitro studies have pointed out the potential relevance of sulfur-containing amino acids as sources for sulfation when extracellular sulfate concentration is low or when its transport is impaired such as in DTDST [DTD (diastrophic dysplasia) sulfate transporter] chondrodysplasias. In the present study, we have considered the contribution of cysteine and cysteine derivatives to in vivo macromolecular sulfation of cartilage by using the mouse model of DTD we have recently generated [Forlino, Piazza, Tiveron, Della Torre, Tatangelo, Bonafe, Gualeni, Romano, Pecora, Superti-Furga et al. (2005) Hum. Mol. Genet. 14, 859–871]. By intraperitoneal injection of [35S]cysteine in wild-type and mutant mice and determination of the specific activity of the chondroitin 4-sulfated disaccharide in cartilage, we demonstrated that the pathway by which sulfate is recruited from the intracellular oxidation of thiols is active in vivo. To check whether cysteine derivatives play a role, sulfation of cartilage proteoglycans was measured after treatment for 1 week of newborn mutant and wild-type mice with hypodermic NAC (N-acetyl-L-cysteine). The relative amount of sulfated disaccharides increased in mutant mice treated with NAC compared with the placebo group, indicating an increase in proteoglycan sulfation due to NAC catabolism, although pharmacokinetic studies demonstrated that the drug was rapidly removed from the bloodstream. In conclusion, cysteine contribution to cartilage proteoglycan sulfation in vivo is minimal under physiological conditions even if extracellular sulfate availability is low; however, the contribution of thiols to sulfation becomes significant by increasing their plasma concentration. PMID:16719839

  2. A jasmonic acid derivative improves skin healing and induces changes in proteoglycan expression and glycosaminoglycan structure.

    Science.gov (United States)

    Henriet, Elodie; Jäger, Sibylle; Tran, Christian; Bastien, Philippe; Michelet, Jean-François; Minondo, Anne-Marie; Formanek, Florian; Dalko-Csiba, Maria; Lortat-Jacob, Hugues; Breton, Lionel; Vivès, Romain R

    2017-09-01

    Jasmonates are plant hormones that exhibit anti-cancer and anti-inflammatory properties and have therefore raised interest for human health applications. The molecular basis of these activities remains poorly understood, although increasing evidence suggests that a variety of mechanisms may be involved. Recently, we have reported that a jasmonate derivative (JAD) displayed anti-aging effects on human skin by inducing extracellular matrix (ECM) remodeling. Based on this observation, we have investigated here the effects of JAD on proteoglycans and glycosaminoglycan (GAG) polysaccharides, which are major cell-surface/ECM components and are involved in a multitude of biological processes. In parallel, we have examined the ability of JAD to promote growth factor activities and improve skin wound healing. Proteoglycan expression was analyzed on epidermal primary keratinocytes and reconstituted skin epidermis, using electron/immunofluorescence microscopy, western blotting and flow cytometry. GAG composition was determined by disaccharide analysis. Finally, biological activities of JAD were assessed in cellulo, in FGF-7 induced migration/proliferation assays, as well as in vivo, using a suction blister model performed on 24 healthy volunteers. JAD was found to induce expression of major skin proteoglycans and to induce subtle changes in GAG structure. In parallel, we showed that JAD promoted FGF-7 and improved skin healing by accelerating epithelial repair in vivo. This study highlights JAD as a promising compound for investigating GAG structure-function relationships and for applications in skin cosmetic /corrective strategies. We propose here a novel mechanism, by which jasmonate derivatives may elicit biological activities in mammals. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Changes of proteoglycan and collagen II of the adjacent intervertebral disc in the cervical instability models.

    Science.gov (United States)

    Wu, Bin; Meng, Chunyang; Wang, Haibin; Jia, Cunling; Zhao, Yifeng

    2016-12-01

    Post-operation of cervical decompression fusion and internal fixation (CDF) accelerated adjacent segment disc degeneration (ASD). It is not clear that whether instability of one single segmental accelerates the degeneration of adjacent segment disc. This study aims to explore the effect of cervical instability on the change of morphology and biochemistry in adjective segment (above) in the L5/6 cervical instability rabbit models. Thirty-two mature New Zealand white rabbits (3000±250g) were randomly divided into two groups, control group (n=8) and model group (n=24). The animal models were established by destruction of partly annulus fibrosus and suction of nucleus pulposus. ASD was detected by X-ray after 4, 8 or 12 weeks surgery (8 model rabbits of each time). Animals were then euthanatized for cervical intervertebral disc tissue samples separation. Histomorphology, proteoglycan and collagen II of samples were detected. Histomorphology data showed that notochord cells were decreased in C4/5 cervical nucleus pulposus and were replaced by fibroblast-like cells; a small amount cartilage cells were emerged; intervertebral disc anulus fibrosus becomes rough, disorganized, hyaline degeneration and pigmentation, in which contained fibrocartilage cells and cracks between the inner and outer layers. Proteoglycan content of nucleus pulposus was significantly decreased. Meanwhile, type II collagen of nucleus pulposus and annulus was also apparently reduced. Cervical instability can alter morphology and reduce the content of proteoglycan and collagen II in adjacent intervertebral disc, thereby contributes adjacent intervertebral disc degeneration. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  4. Effect of articular cartilage proteoglycan depletion on high frequency ultrasound backscatter.

    Science.gov (United States)

    Pellaumail, B; Watrin, A; Loeuille, D; Netter, P; Berger, G; Laugier, P; Saïed, A

    2002-07-01

    To study the effect of variations of articular cartilage proteoglycans (PG) on high-frequency ultrasound backscatter. The study was performed on patellar cartilages of immature and mature rats (N=36). The variation of PG content was induced by enzyme digestion. Control and treated cartilages were explored in vitro using a 55MHz scanning acoustic microscopy, then assessed by histology for the fibrillar collagen organization analysis. The variations of proteoglycan and collagen content were evaluated. Thickness measurements performed on both B-scan images and histologic sections were compared. Ultrasonic radio-frequency signals reflected by the cartilage surface and backscattered from its internal matrix were processed to estimate the integrated reflection coefficient (IRC) and apparent integrated backscatter (AIB). Although hyaluronidase treatment of immature and mature cartilages removed approximately 50% of the proteoglycans, the echogenicity level of ultrasound images of degraded cartilages was similar to that of controls. IRC and AIB parameters did not significantly vary. Histologic sections of degraded cartilage displayed no change in collagen fiber organization. The thickness mean values measured by ultrasound in PG-depleted groups were significantly higher than in controls, whereas no significant difference in thickness was detected by histological measurement. The increase in cartilage thickness may potentially be explained by a decrease of speed of sound in PG-depleted cartilages that is more likely subsequent to an increase of water content. Current results indicate that PG depletion has no significant effect on high frequency ultrasound backscattered from rat patellar cartilage. Ultrasound may provide information about variations of PG content via speed of sound measurement. Copyright 2002 OsteoArthritis Research Society International. Published by Elsevier Science Ltd. All rights reserved.

  5. Studies on the hyaluronate binding properties of newly synthesized proteoglycans purified from articular chondrocyte cultures

    Energy Technology Data Exchange (ETDEWEB)

    Sandy, J.D.; Plaas, A.H.

    1989-06-01

    Primary cultures of rabbit articular chondrocytes have been maintained for 10 days and labeled with (35S)sulfate, (3H)leucine, and (35S)cysteine in pulse-chase protocols to study the structure and hyaluronate binding properties of newly synthesized proteoglycan monomers. Radiolabeled monomers were purified from medium and cell-layer fractions by dissociative CsCl gradient centrifugation with bovine carrier monomer, and analyzed for hyaluronate binding affinity on Sepharose CL-2B in 0.5 M Na acetate, 0.1% Triton X-100, pH 6.8. Detergent was necessary to prevent self-association of newly synthesized monomers during chromatography. Monomers secreted during a 30-min pulse labeling with (35S)sulfate had a low affinity relative to carrier. Those molecules released into the medium during the first 12 h of chase remained in the low affinity form whereas those retained by the cell layer rapidly acquired high affinity. In cultures where more than 90% of the preformed cell-layer proteoglycan was removed by hyaluronidase digestion before radiolabeling the newly synthesized low affinity monomers also rapidly acquired high affinity if retained in the cell layer. Cultures labeled with amino acid precursors were used to establish the purity of monomer preparations and to isolate core proteins for study. Leucine- or cysteine-labeled core proteins derived from either low or high affinity monomer preparations migrated as a single major species on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with electrophoretic mobility very similar to that of core protein derived from extracted proteoglycan monomer. Purified low affinity monomers were converted to the high affinity form by treatment at pH 8.6; however, this change was prevented by guanidinium-HCl at concentrations above 0.8 M.

  6. Atheroprotective remodelling of vascular dermatan sulphate proteoglycans in response to hypercholesterolaemia in a rat model

    Science.gov (United States)

    Oberkersch, Roxana; Maccari, Francesca; Bravo, Alicia I; Volpi, Nicola; Gazzaniga, Silvina; Calabrese, Graciela C

    2014-01-01

    Proteoglycan accumulation within the arterial intima has been implicated in atherosclerosis progression in humans. Nevertheless, hypercholesterolaemia is unable to induce intimal thickening and atheroma plaque development in rats. The study was performed to analyse proteoglycans modifications in rats fed with a high-cholesterol diet to understand whether vascular wall remodelling protects against lesions. Sections obtained from rat aortas showed normal features, in intimal-to-media ratio and lipid accumulation. However, focal endothelial hyperplasia and neo-intima rearrangement were observed in high-cholesterol animals. Besides, hypercholesterolaemia induced an inflammatory microenviroment. We determined the expression of different proteoglycans from aortic cells by Western blot and observed a diminished production of decorin and biglycan in high-cholesterol animals compared with control (P < 0.01 and P < 0.05, respectively). Versican was increased in high-cholesterol animals (P < 0.05), whereas perlecan production showed no differences. No modification of the total content of glycosaminoglycans (GAGs) was found between the two experimental groups. In contrast, the chondroitin sulphate/dermatan sulphate ratio was increased in the high-cholesterol group as compared to the control (0.56 and 0.34, respectively). Structural alterations in the disaccharide composition of galactosaminoglycans were also detected by HPLC, as the ratio of 6-sulphate to 4-sulphate disaccharides was increased in high-cholesterol animals (P < 0.05). Our results suggest that attenuation of decorin and biglycan expression might be an effective strategy to inhibit the first step in atherogenesis, although specific GAG structural modification associated with the development of vascular disease took place. Results emphasize the potential application of therapies based on vascular matrix remodelling to treat atherosclerosis. PMID:24602133

  7. Cell-surface proteoglycan in sea urchin primary mesenchyme cell migration

    Energy Technology Data Exchange (ETDEWEB)

    Lane, M.C.

    1989-01-01

    Early in the development of the sea urchin embryo, the primary mesenchyme cells (PMC) migrate along the basal lamina of the blastocoel. Migration is inhibited in L. pictus embryos cultured in sulfate-free seawater and in S. purpuratus embryos exposed to exogenous {beta}-D-xylosides. An in vitro assay was developed to test the migratory capacity of normal PMC on normal and treated blastocoelic matrix. Sulfate deprivation and exposure to exogenous xyloside render PMC nonmotile on either matrix. Materials removed from the surface of normal PMC by treatment with 1 M urea restored migratory ability to defective cells, whereas a similar preparation isolated from the surface of epithelial cells at the same stage did not. Migration also resumed when cells were removed from the xyloside or returned to normal seawater. The urea extract was partially purified and characterized by radiolabeling, gel electrophoresis, fluorography, ion exchange chromatography, and western blotting. The PMC synthesize a large chondroitin sulfate/dermatan sulfate proteoglycan that is present in an active fraction isolated by chromatography. Chondroitinase ABC digestion of live cells blocked migration reversibly, further supporting the identification of the chondroitin sulfate/dermatan sulfate proteoglycan as the active component in the urea extract. Much of the incorporated sulfate was distributed along the filopodia in {sup 35}SO{sub 4}-labelled PMC by autoradiography. The morphology of normal and treated S. purpuratus PMC was examined by scanning electron microscopy, and differences in spreading, particularly of the extensive filopodia present on the cells, was observed. A model for the role of the chondroitin sulfate/dermatan sulfate proteoglycan in cell detachment during migration is proposed.

  8. Multitasking Human Lectin Galectin-3 Interacts with Sulfated Glycosaminoglycans and Chondroitin Sulfate Proteoglycans.

    Science.gov (United States)

    Talaga, Melanie L; Fan, Ni; Fueri, Ashli L; Brown, Robert K; Bandyopadhyay, Purnima; Dam, Tarun K

    2016-08-16

    Glycosaminoglycan (GAG) binding proteins (GAGBPs), including growth factors, cytokines, morphogens, and extracellular matrix proteins, interact with both free GAGs and those covalently linked to proteoglycans. Such interactions modulate a variety of cellular and extracellular events, such as cell growth, metastasis, morphogenesis, neural development, and inflammation. GAGBPs are structurally and evolutionarily unrelated proteins that typically recognize internal sequences of sulfated GAGs. GAGBPs are distinct from the other major group of glycan binding proteins, lectins. The multifunctional human galectin-3 (Gal-3) is a β-galactoside binding lectin that preferentially binds to N-acetyllactosamine moieties on glycoconjugates. Here, we demonstrate through microcalorimetric and spectroscopic data that Gal-3 possesses the characteristics of a GAGBP. Gal-3 interacts with unmodified heparin, chondroitin sulfate-A (CSA), -B (CSB), and -C (CSC) as well as chondroitin sulfate proteoglycans (CSPGs). While heparin, CSA, and CSC bind with micromolar affinity, the affinity of CSPGs is nanomolar. Significantly, CSA, CSC, and a bovine CSPG were engaged in multivalent binding with Gal-3 and formed noncovalent cross-linked complexes with the lectin. Binding of sulfated GAGs was completely abolished when Gal-3 was preincubated with β-lactose. Cross-linking of Gal-3 by CSA, CSC, and the bovine CSPG was reversed by β-lactose. Both observations strongly suggest that GAGs primarily occupy the lactose/LacNAc binding site of Gal-3. Hill plot analysis of calorimetric data reveals that the binding of CSA, CSC, and a bovine CSPG to Gal-3 is associated with progressive negative cooperativity effects. Identification of Gal-3 as a GAGBP should help to reveal new functions of Gal-3 mediated by GAGs and proteoglycans.

  9. Proteoglycan and proteome profiling of central human pulmonary fibrotic tissue utilizing miniaturized sample preparation

    DEFF Research Database (Denmark)

    Malmström, Johan; Larsen, Kristoffer; Hansson, Lennart

    2002-01-01

    The objective of this study was to isolate fibrotic cells from human lung biopsies taken from different central pulmonary locations. A comparison was made of cell morphology, proteoglycan- and protein-expression in mesenchymal cell cultures obtained from human bronchial biopsies from patients...... with asthmatic-like disorders. We isolated viable cells from 10 out of the 12 biopsies. The fibroblast-like cells were positive for the biomarker a-smooth muscle actin, indicating that the cells were in an activated state. Two different types of fibroblast-like cells were observed from human pulmonary connective...

  10. Characterization of a large glycoprotein proteoglycan by size-exclusion chromatography combined with light and X-ray scattering methods.

    Science.gov (United States)

    Watanabe, Yasushi; Inoko, Yoji

    2013-08-16

    The molecular weight and chain conformation of a proteoglycan derived from shark cartilage in solution were characterized by size-exclusion chromatography combined with low-angle laser light scattering and small-angle X-ray scattering methods. The total molecular weight of the proteoglycan was 3.9±0.2 million and the molecular weight of the main component was about 2.0±0.2 million. The X-ray scattering data revealed that the main components of the proteoglycan are nearly equal to a chain with excluded volume and their persistence lengths range from 13.5 to 16.4nm. These results show that size-exclusion chromatography combined with low-angle laser light scattering and small-angle X-ray scattering measurements are complementarily useful for characterization of large biopolymers in solution. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Human skin basement membrane-associated heparan sulphate proteoglycan: distinctive differences in ultrastructural localization as a function of developmental age

    DEFF Research Database (Denmark)

    Horiguchi, Y; Fine, J D; Couchman, J R

    1991-01-01

    at different developmental ages using two monoclonal antibodies to a well-characterized basement membrane-associated heparan sulphate proteoglycan. A series of foetal skin specimens (range, 54-142 gestational days) were examined using an immunoperoxidase immunoelectron microscopic technique. In specimens...... representing very early developmental ages, very diffuse immunoreaction products were detected. However, by approximately 76 gestational days, some accentuation of heparan sulphate proteoglycan was noted along the lamina densa, and by 142 gestational days, the distribution of heparan sulphate proteoglycan...... was identical to that observed in neonatal and adult human skin. These findings demonstrate that active remodelling of the dermo-epidermal junction occurs during at least the first two trimesters, and affects not only basement membrane-associated structures but also specific antigens....

  12. Apolipoprotein AV Accelerates Plasma Hydrolysis OfTriglyceride-Rich Lipoproteins By Interaction With Proteoglycan BoundLipoprotein Lipase

    Energy Technology Data Exchange (ETDEWEB)

    Merkel, Martin; Loeffler, Britta; Kluger, Malte; Fabig, Nathalie; Geppert, Gesa; Pennacchio, Len A.; Laatsch, Alexander; Heeren, Joerg

    2005-02-22

    Apolipoprotein A5 (APOA5) is associated with differences intriglyceride levels and familial combined hyperlipidemia. In genetically engineered mice, apoAV plasma levels are inversely correlated with plasmatriglycerides. To elucidate the mechanism by which apoAV influences plasma triglycerides, metabolic studies and in vitro assays resembling physiological conditions were performed. In hAPOA5 transgenic mice(hAPOA5tr), catabolism of chylomicrons and VLDL was accelerated due to a faster plasma hydrolysis of triglycerides by lipoprotein lipase (LPL).Hepatic VLDL and intestinal chylomicron production were not affected. The functional interplay between apoAV and LPL was further investigated by crossbreeding a human LPL transgene with the apoa5 knockout, and the hAPOA5tr to an LPL deficient background. Increased LPL activity completely normalized hypertriglyceridemia of apoa5 deficient mice,however, over expression of human apoAV modulated triglyceride levels only slightly when LPL was reduced. To reflect the physiological situation in which LPL is bound to cell surface proteoglycans, we examined hydrolysis in the presence or absence of proteoglycans. Without proteoglycans, apoAV derived either from triglyceride-rich lipoproteins, hAPOA5tr HDL, or a recombinant source did not alter the LPL hydrolysis rate. In the presence of proteoglycans, however, apoAV led to a significant and dose-dependent increase in LPL mediated hydrolysis of VLDL triglycerides. These results were confirmed in cell culture using a proteoglycan-deficient cell line.A direct interaction between LPL and apoAV was found by ligand blotting.It is proposed, that apoAV reduces triglyceride levels by guiding VLDL and chylomicrons to proteoglycans bound LPL for lipolysis.

  13. Stromal Edema in Klf4 Conditional Null Mouse Cornea is Associated with Altered Collagen Fibril Organization and Reduced Proteoglycans

    Science.gov (United States)

    Young, Robert D.; Swamynathan, Shivalingappa K.; Boote, Craig; Mann, Mary; Quantock, Andrew J.; Piatigorsky, Joram; Funderburgh, James L.; Meek, Keith M.

    2009-01-01

    Purpose Klf4, one of the highly expressed transcription factors in mouse cornea, plays an important role in maturation and maintenance of the ocular surface. Here, the authors examined the structure and proteoglycan composition of the Klf4 conditional null (Klf4CN) corneal stroma, to further characterize the previously reported Klf4CN stromal edema. Methods Collagen fibril spacing and diameter were calculated from scattering intensity profiles from small angle synchrotron X-ray scattering patterns obtained across the cornea along a vertical meridian at 0.5mm intervals. Collagen fibril organization and proteoglycans were visualised by electron microscopy (EM) with or without the cationic dye Cuprolinic blue. Proteoglycans and glycosaminoglycans were further analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE) and immunoblots. Q-RT-PCR was used to measure the transcript levels. Results In the central cornea the average collagen interfibrillar Bragg spacing increased from 44.5nm (SD +/-1.8nm) in wild type to 66.5nm (SD +/-2.3nm) in Klf4CN, as measured by X-ray scattering and confirmed by EM. Mean collagen fibril diameter increased from 32nm (SD+/-0.4nm) in wild type to 42.3nm (SD+/-4.8nm) in Klf4CN corneal stroma. Downregulation of proteoglycans detected by EM in the Klf4CN stroma was confirmed by FACE and immunoblots. Q-RT-PCR showed that while the Klf4CN corneal proteoglycan transcript levels remained unchanged, matrix metalloproteinase (MMP) transcript levels were significantly upregulated. Conclusions The Klf4CN corneal stromal edema is characterized by increased collagen interfibrillar spacing and increased diameter of individual fibrils. The stroma also exhibits reduced interfibrillar proteoglycans throughout the corneal stroma, which is possibly caused by increased expression of MMPs. PMID:19387067

  14. Detection of coronary atherosclerotic plaques with superficial proteoglycans and foam cells using real-time intrinsic fluorescence spectroscopy.

    Science.gov (United States)

    Angheloiu, George O; Haka, Abigail S; Georgakoudi, Irene; Arendt, Joseph; Müller, Markus G; Scepanovic, Obrad R; Evanko, Stephen P; Wight, Thomas N; Mukherjee, Prasun; Waldeck, David H; Dasari, Ramachandra R; Fitzmaurice, Maryann; Kramer, John R; Feld, Michael S

    2011-03-01

    The protein components of low-density lipoprotein (LDL), oxidized LDL and proteoglycans such as versican contain tryptophan, an amino acid with characteristic fluorescence features at 308 nm excitation wavelength. We hypothesize that intrinsic fluorescence spectroscopy at 308 nm excitation wavelength IFS308, a method suitable for clinical use, can identify coronary artery lesions with superficial foam cells (SFCs) and/or proteoglycans. We subjected 119 human coronary artery specimens to in vitro fluorescence and reflectance spectroscopy. We used 5 basis spectra to model IFS308, and extracted their contributions to each individual IFS308 spectrum. A diagnostic algorithm using the contributions of Total Tryptophan and fibrous cap to IFS308 was built to identify specimens with SFCs and/or proteoglycans in their top 50 μm. We detected SFCs and/or proteoglycans, such as versican or the glycosaminoglycan hyaluronan, in 24 fibrous cap atheromas or pathologic intimal thickening (PIT) lesions. An algorithm using the contributions of Total Tryptophan and fibrous cap to IFS308 was able to identify these segments with 92% sensitivity and 80% specificity. We were able to establish a set of characteristic LDL, oxidized LDL, versican and hyaluronan fluorescence spectra, ready to be used for real-time diagnosis. The IFS(308) technique detects SFCs and/or proteoglycans in fibrous cap atheromas and PIT lesions. SFCs and proteoglycans are histological markers of vulnerable plaques, and this study is a step further in developing an invasive clinical tool to detect the vulnerable atherosclerotic plaque. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  15. Stromal edema in klf4 conditional null mouse cornea is associated with altered collagen fibril organization and reduced proteoglycans.

    Science.gov (United States)

    Young, Robert D; Swamynathan, Shivalingappa K; Boote, Craig; Mann, Mary; Quantock, Andrew J; Piatigorsky, Joram; Funderburgh, James L; Meek, Keith M

    2009-09-01

    Klf4, one of the highly expressed transcription factors in the mouse cornea, plays an important role in maturation and maintenance of the ocular surface. In this study, the structure and proteoglycan composition of the Klf4 conditional null (Klf4CN) corneal stroma was investigated, to further characterize the previously reported Klf4CN stromal edema. Collagen fibril spacing and diameter were calculated from scattering intensity profiles from small angle synchrotron x-ray scattering patterns obtained across the cornea along a vertical meridian at 0.5-mm intervals. Collagen fibril organization and proteoglycans were visualized by electron microscopy (EM), with or without the cationic dye cuprolinic blue. Proteoglycans and glycosaminoglycans were further analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE) and immunoblot analysis. Q-RT-PCR was used to measure the transcript levels. In the central cornea, the average collagen interfibrillar Bragg spacing increased from 44.5 nm (SD +/-1.8) in wild-type to 66.5 nm (SD +/-2.3) in Klf4CN, as measured by x-ray scattering and confirmed by EM. Mean collagen fibril diameter increased from 32 nm (SD +/-0.4) in wild-type to 42.3 nm (SD +/-4.8) in Klf4CN corneal stroma. Downregulation of proteoglycans detected by EM in the Klf4CN stroma was confirmed by FACE and immunoblot analysis. Q-RT-PCR showed that, whereas the Klf4CN corneal proteoglycan transcript levels remained unchanged, matrix metalloproteinase (MMP) transcript levels were significantly upregulated. The Klf4CN corneal stromal edema is characterized by increased collagen interfibrillar spacing and increased diameter of individual fibrils. The stroma also exhibits reduced interfibrillar proteoglycans throughout, which is possibly caused by increased expression of MMPs.

  16. Receptor tyrosine phosphatase beta is expressed in the form of proteoglycan and binds to the extracellular matrix protein tenascin

    DEFF Research Database (Denmark)

    Barnea, G; Grumet, M; Milev, P

    1994-01-01

    The extracellular domain of receptor type protein tyrosine phosphatase beta (RPTP beta) exhibits striking sequence similarity with a soluble, rat brain chondroitin sulfate proteoglycan (3F8 PG). Immunoprecipitation experiments of cells transfected with RPTP beta expression vector and metabolically...... labeled with [35S]sulfate and [35S]methionine indicate that the transmembrane form of RPTP beta is indeed a chondroitin sulfate proteoglycan. The 3F8 PG is therefore a variant form composed of the entire extracellular domain of RPTP beta probably generated by alternative RNA splicing. Previous...

  17. Immunofluorescent localization of collagen types I, III, IV, V, fibronectin, laminin, entactin, and heparan sulphate proteoglycan in human immature placenta.

    Science.gov (United States)

    Rukosuev, V S

    1992-03-15

    The distribution of eight components of the extracellular matrix in immature human placenta was studied by an indirect immunofluorescence method with monospecific antibodies. In the stroma of the term chorionic villi, collagen types I, III, IV, V, and fibronectin formed a mesh of fibers and conglomerates. Heparan sulphate proteoglycan formed multiple conglomerates, whereas laminin comprised small, scanty, discrete granules. Collagen type IV, laminin, entactin, and heparan sulphate proteoglycan were confined to the basement membrane of the trophoblast. Sometimes, only collagen type IV was identified in fetal vascular basement membrane.

  18. Epiphycan from salmon nasal cartilage is a novel type of large leucine-rich proteoglycan.

    Science.gov (United States)

    Tatara, Yota; Kakizaki, Ikuko; Kuroda, Yoshiyuki; Suto, Shinichiro; Ishioka, Haruna; Endo, Masahiko

    2013-08-01

    Chum salmon (Oncorhynchus keta) nasal cartilage was examined by next-generation DNA sequencing and mass spectrometric analyses, and 14 types of proteoglycans including epiphycan (EPY) were found. A cDNA encoding EPY was cloned and sequenced. The cDNA encoded 589 amino acids comprised a glycosaminoglycan (GAG) domain containing 55 potential GAG-modified sites (Ser-Gly and/or Gly-Ser), a cysteine cluster and 6 leucine-rich repeats. EPY was purified from salmon nasal cartilage and the structure of the GAG was characterized. As a result of unsaturated disaccharide analysis, GAG was found to be composed of chondroitin 6-sulfate (58.0%), chondroitin 4-sulfate (26.5%) and non-sulfated chondroitin (15.3%). The average molecular weight of GAG was estimated to be 3.0 × 10(4). Ser-100 and Ser-103 were identified as serine residues substituted by GAG chains by chemical modification and mass spectrometric analysis. More than 50 serine residues were assumed to be substituted by GAG chains. EPY is heavily substituted by chondroitin sulfate, giving an overall molecular weight of just under 2 × 10(6). EPY from salmon nasal cartilage is a novel type of large leucine-rich proteoglycan.

  19. Serglycin proteoglycan deletion induces defects in platelet aggregation and thrombus formation in mice

    Science.gov (United States)

    Woulfe, Donna S.; Lilliendahl, Joanne Klimas; August, Shelley; Rauova, Lubica; Kowalska, M. Anna; Åbrink, Magnus; Pejler, Gunnar; White, James G.

    2008-01-01

    Serglycin (SG), the hematopoietic cell secretory granule proteoglycan, is crucial for storage of specific secretory proteins in mast cells, neutrophils, and cytotoxic T lymphocytes. We addressed the role of SG in platelets using SG−/− mice. Wild-type (WT) but not SG−/− platelets contained chondroitin sulfate proteoglycans. Electron microscopy revealed normal α-granule structure in SG−/− platelets. However, SG−/− platelets and megakaryocytes contained unusual scroll-like membranous inclusions, and SG−/− megakaryocytes showed extensive emperipolesis of neutrophils. SG−/− platelets had reduced ability to aggregate in response to low concentrations of collagen or PAR4 thrombin receptor agonist AYPGKF, and reduced fibrinogen binding after AYPGKF, but aggregated normally to ADP. 3H-serotonin and ATP secretion were greatly reduced in SG−/− platelets. The α-granule proteins platelet factor 4, β-thromboglobulin, and platelet-derived growth factor were profoundly reduced in SG−/− platelets. Exposure of P-selectin and αIIb after thrombin treatment was similar in WT and SG−/− platelets. SG−/− mice exhibited reduced carotid artery thrombus formation after exposure to FeCl3. This study demonstrates that SG is crucial for platelet function and thrombus formation. We propose that SG−/− platelet function deficiencies are related to inadequate packaging and secretion of selected α-granule proteins and reduced secretion of dense granule contents critical for platelet activation. PMID:18094327

  20. Biomimetic Proteoglycan Interactions with Type I Collagen Investigated via 2D and 3D TEM

    Science.gov (United States)

    Moorehead, Carli

    Collagen is one of the leading components in extracellular matrix (ECM), providing durability, structural integrity, and functionality for many tissues. Regulation of collagen fibrillogenesis and degradation is important in the treatment of a number of diseases from orthopedic injuries to genetic deficiencies. Recently, novel, biocompatible, semi-synthetic biomimetic proteoglycans (BPGs) were developed, which consist of an enzymatically resistant synthetic polymer core and natural chondroitin sulfate bristles. It was demonstrated that BPGs affect type I collagen fibrillogenesis in vitro, as reflected by their impact delaying the kinetic formation of gels similar to native PGs. This indicates that the morphology of collagen scaffolds as well as endogenous ECM could also be modulated by these proteoglycan mimics. However, the imaging modality used previously, reflectance confocal microscopy, did not yield the resolution necessary to spatially localize BPGs within the collagen network or investigate the effect of BPGs on the quality of collagen fibrils produced in an in vitro fibrillogenesis model which is important for understanding the method of interaction. Consequently, a histological technique, electron tomography, was adapted and utilized to 3D image the nano-scale structures within this simplified tissue model. BPGs were found to aid in lateral growth and enhance fibril banding periodicity resulting in structures more closely resembling those in tissue, in addition to attaching to the collagen surface despite the lack of a protein core.

  1. Small leucine rich proteoglycans are differently distributed in normal and pathological endometrium.

    Science.gov (United States)

    Lucariello, Angela; Trabucco, Elisabetta; Boccia, Olga; Perna, Angelica; Sellitto, Carmine; Castaldi, Maria A; De Falco, Maria; De Luca, Antonio; Cobellis, Luigi

    2015-01-01

    During the woman's fertile period, the non-pregnant uterus is subject to constant cyclic changes. The complex mechanisms that control the balance among proliferation, differentiation, cell death and the structural remodeling of the extracellular matrix can contribute to the benign or malignant endometrial pathological state. The small leucine-rich proteoglycans (SLRPs) are important components of cell surface and extracellular matrices. Using immunohistochemistry, we showed that the distribution patterns of SLRPs were completely modified in the pathological compared to normal endometrium. The expression of SLRPs was low/absent in all endometrial pathologies examined compared to normal endometrium. We observed an increase of lumican from proliferative to secretory phase of the endometrium and a decrease of fibromodulin, biglycan and decorin. In menopause endometrial tissue, the level of expression of fibromodulin, biglycan, decorin and lumican dramatically decreased. The results revealed the prominence and importance of proteoglycans in the tissue architecture and extracellular matrix organization. Copyright © 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  2. 1 kHz vibration increases proteoglycan production in ATDC5 chondrocytes

    Science.gov (United States)

    Argadine, Heather M.; Kinnick, Randall R.; Greenleaf, James F.; Bolander, Mark E.

    2005-04-01

    In vitro studies have shown that treatment with 1.5 MHz ultrasound signal (160 mW/cm2) at a 200 μs tone burst repeating at 1 kHz increases proteoglycan synthesis in chondrocytes [J. Parvisi et al., J. Orthop. Res. 17, 488-494 (1999)]. It was hypothesized that a continuous 1 kHz signal would be similar to the pulsed 1.5 MHz signal in stimulating chondrocytes to produce proteoglycan, which may cause accelerated fracture healing. In vitro experiments were performed with ATDC5 cells, a chondrogenic clonal cell line, plated in 6-well plates for 3 to 7 days before receiving ultrasound treatments. Cells were treated with either 1.5 MHz pulsed signal or 1 kHz signal for 20 minutes per day for 9 to 11 days. The signals were calibrated so that the bottom of the 6-well plate moved 10 nm for each condition. After the final treatment, cell layers were stained with Alcian blue, which stains cartilage nodules providing a measure of chondrogenesis. Both 1.5 MHz and 1 kHz led to a highly significant increase in chondrogenesis compared to control. Quantitative image analysis of stained wells showed that treatments with either signal increased number of nodules 2.3-fold (p<0.02) and total area of nodules 3-fold (p<0.02) compared to controls.

  3. Silver nanoparticles alter proteoglycan expression in the promotion of tendon repair.

    Science.gov (United States)

    Kwan, Karen H L; Yeung, Kelvin W K; Liu, Xuelai; Wong, Kenneth K Y; Shum, Ho Cheung; Lam, Yun Wah; Cheng, Shuk Han; Cheung, Kenneth M C; To, Michael K T

    2014-10-01

    This study demonstrates a novel method of using silver nanoparticles for Achilles tendon injury healing. In vitro results indicated a stimulatory effect on cell proliferation and collagen synthesis with silver nanoparticles. Biomechanical test on the 42-day post operation Achilles tendon sample exhibited a significant improvement in tensile modulus when compared to the untreated group. Histology suggested that silver nanoparticles promoted cell alignment and proteoglycan synthesis. The collagen deposition was also improved. An alleviation of tumor necrosis factor α, and an increase in fibromodulin and proliferating cell nuclear antigen expression were seen in silver nanoparticles group by immunohistochemistry. This study further corroborates the finding of our previous study that silver nanoparticles help to restore the functionality of injured connective tissues. We believe that the anti-inflammatory nature of silver nanoparticles has an important role in accelerating the healing process and reducing scarring, leading to better functional outcome. From the clinical editor: Tendon healing after surgeries remains a slow and tedious process, typically requiring several weeks of recovery time and gradual introduction of physical therapy. There are no currently utilized methods that could promote tendon healing. In this study, silver nanoparticles are reported to facilitate Achilles tendon repair in a model system, through increased proteoglycan and collagen synthesis, paving the way to potential clinical applications in the future. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Lectican proteoglycans, their cleaving metalloproteinases, and plasticity in the central nervous system extracellular microenvironment

    Science.gov (United States)

    Howell, Matthew D.; Gottschall, Paul E.

    2013-01-01

    The extracellular matrix in the central nervous system actively orchestrates and modulates changes in neural structure and function in response to experience, after injury, during disease, and with changes in neuronal activity. A component of the multi-protein, extracellular matrix aggregate in brain, the chondroitin sulfate-bearing proteoglycans known as lecticans, inhibit neurite outgrowth, alter dendritic spine shape, elicit closure of critical period plasticity, and block target reinnervation and functional recovery after injury as the major component of a glial scar. While removal of the chondroitin sulfate chains from lecticans with chondroitinase ABC improves plasticity, proteolytic cleavage of the lectican core protein may change the conformation of the matrix aggregate and also modulate neural plasticity. This review centers on the roles of the lecticans and the endogenous metalloproteinase families that proteolytically cleave lectican core proteins, the matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs), in neural plasticity. These extracellular metalloproteinases modulate structural neural plasticity—including changes in neurite outgrowth and dendritic spine remodeling—and synaptic plasticity. Some of these actions have been demonstrated to occur via cleavage of the proteoglycan core protein. Other actions of the proteases include cleavage of non-matrix substrate proteins, whereas still other actions may occur directly at the cell surface without proteolytic cleavage. The data convincingly demonstrate that metalloproteinases modulate physiological and pathophysiological neural plasticity. PMID:22626649

  5. Analysis of the chondroitin sulfate proteoglycan core protein (CSPGCP) gene in achondroplasia and pseudoachondroplasia.

    Science.gov (United States)

    Finkelstein, J E; Doege, K; Yamada, Y; Pyeritz, R E; Graham, J M; Moeschler, J B; Pauli, R M; Hecht, J T; Francomano, C A

    1991-01-01

    Achondroplasia and pseudoachondroplasia are autosomal dominant skeletal dysplasias resulting in short-limbed dwarfism. Histologic and ultrastructural studies of the cartilage in pseudoachondroplasia and in homozygous achondroplasia have suggested a structural abnormality in chondroitin sulfate proteoglycan (CSPG), a major structural protein in the extra-cellular matrix. The gene encoding CSPG core protein (CSPGCP) is thus a logical "candidate gene" for analysis in these conditions. cDNA probes encoding CSPGCP were used to identify restriction fragment length polymorphisms (RFLPs) in DNA from a panel of control individuals. No gross alterations at the CSPGCP locus were noted in DNA from 37 individuals with achondroplasia and 5 individuals with pseudoachondroplasia. In addition, allelic frequencies of the RFLPs were not significantly different among controls and patients with either condition. In one three-generation family with achondroplasia, close linkage of the CSPGCP locus and the skeletal dysplasia was excluded using a Bgl II polymorphism. Similarly, in a three-generation family with pseudoachondroplasia, the CSPGCP gene was not tightly linked to the disease phenotype. These results indicate that mutations at the chondroitin sulfate proteoglycan core protein locus do not cause achondroplasia or pseudoachondroplasia in these families. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:1670752

  6. New SPECT tracers: Example of tracers of proteoglycans and melanin; Nouveaux traceurs TEMP: exemple des traceurs des proteoglycanes et de la melanine

    Energy Technology Data Exchange (ETDEWEB)

    Cachin, F.; Mestas, D.; Kelly, A.; Merlin, C.; Veyre, A.; Maublant, J. [CRLCC Jean-Perrin, Service de Medecine Nucleaire, 63 - Clermont-Ferrand (France); Cachin, F.; Chezal, J.M.; Miot-Noirault, E.; Moins, N.; Auzeloux, P.; Vidal, A.; Bonnet-Duquennoy, M.; Boisgard, S.; D' Incan, M.; Madelmont, J.C.; Maublant, J. [Universite d' Auvergne, EA 4231, 63 - Clermont-Ferrand (France); Boisgard, S. [CHRU Gabriel-Montpied, Service d' Orthopedie, 63 - Clermont-Ferrand (France); D' Incan, M. [CHRU Gabriel-Montpied, Service de Dermatologie, 63 - Clermont-Ferrand (France); Redini, F. [Inserm, U957-EA3822, Faculte de Medecine, 44 - Nantes (France); Filaire, M. [Universite d' Auvergne, Lab. d' Anatomie, 63 - Clermont-Ferrand (France)

    2009-02-15

    The majority of research program on new radiopharmaceuticals turn to tracers used for positron emission tomography (PET). Only a few teams work on new non fluorine labeled tracers. However, the coming of SPECT/CT gamma cameras, the arrival of semi-conductors gamma cameras should boost the development of non-PET tracers. We exhibit in this article the experience acquired by our laboratory in the conception and design of two new non fluorine labelled compounds. The {sup 99m}Tc-N.T.P. 15-5 (N.T.P. 15-5 for N-[tri-ethyl-ammonium]-3-propyl-[15]ane-N5) which binds to proteoglycans could be used for the diagnosis and staging of osteoarthritis and chondrosarcoma. The iodo benzamides, specific to the melanin, are nowadays compared to {sup 18}F-fluorodeoxyglucose in a phase III clinical trial for the diagnosis and detection of melanoma metastasis. Our last development focus on N-[2-(diethyl-amino)ethyl]-4 and 2-iodo benzamides respectively B.Z.A. and B.Z.A.2 hetero-aromatic analogues usable for melanoma treatment. (authors)

  7. Best paper NASS 2013: link-N can stimulate proteoglycan synthesis in the degenerated human intervertebral discs.

    Science.gov (United States)

    Gawri, Rahul; Antoniou, John; Ouellet, Jean; Awwad, Waleed; Steffen, Thomas; Roughley, Peter; Haglund, Lisbet; Mwale, Fackson

    2013-09-11

    Intervertebral disc (IVD) degeneration is the most common cause of back pain. Presently there is no medical treatment, leaving surgery as the only offered option. Here we evaluate the potential of Link-N to promote extracellular matrix regeneration in human IVDs. Human disc cells cultured in alginate and intact human discs were exposed to a combination of Link-N and ³⁵SO₄ in the presence or absence of interleukin (IL)-1, and the effect on proteoglycan synthesis was evaluated. In addition, message levels of aggrecan, matrix metalloproteinase (MMP)-3, MMP-13, a Disintegrin And Metalloproteinase with Thrombospondin Motifs (ADAMTS)-4 and ADAMTS-5 were evaluated in alginate cultures. Human disc cells responded in a dose dependent manner with maximal proteoglycan synthesis at 1 µg/mL Link-N. Link-N treatment also induced proteoglycan synthesis in intact human discs, and a prolonged effect was found up to one week after Link-N treatment. Message levels of proteinases were decreased by Link-N in the presence of IL-1. Thus, Link-N can promote proteoglycan synthesis and deplete proteinase expression in adult human discs. Link-N could therefore be a promising candidate for biologically-induced disc repair, and could provide an alternative to surgical intervention for early stage disc degeneration.

  8. Influence of Proteoglycan on Time-Dependent Mechanical Behaviors of Articular Cartilage under Constant Total Compressive Deformation

    Science.gov (United States)

    Murakami, Teruo; Sakai, Nobuo; Sawae, Yoshinori; Tanaka, Koji; Ihara, Maki

    Articular cartilage has biphasic property based on high water content. It is generally believed that the proteoglycan supports the compressive load, but the detailed loading mechanism has not yet been clarified. In this study, first we observed the changes in compressive stress and strain of articular cartilage under constant total compressive deflection. We evaluated the changes in modulus of elasticity, which was estimated from the stress-strain relation in equilibrium state. To examine the role of proteoglycan in compressed articular cartilage, we compared the time-dependent viscoelastic behaviors in both the intact cartilage and the cartilage treated with chondoroitinase ABC under constant total compressive deformation. We could confirm that the peak stress after compression and the modulus of elasticity at equilibrium were reduced after the digestion of proteoglycan. Next, we observed the changes in local strain in both articular cartilage specimens with and without chondroitinase treatment by monitoring the position of stained chondrocyte in the confocal laser scanning microscope. These visualized images indicated that the local strain changed time-dependently and depth-dependently. The digested cartilage showed the quicker change in movement and larger thinning in surface layer than the intact cartilage. These results indicate that the proteoglycan contributes to the compressive load-carrying capacity and controls the permeability.

  9. Oral administration of the NADPH-oxidase inhibitor apocynin partially restores diminished cartilage proteoglycan synthesis and reduces inflammation in mice.

    NARCIS (Netherlands)

    Hougee, S.; Hartog, A.; Sanders, A.; Graus, Y.M.; Hoijer, M.A.; Garssen, J.; Berg, W.B. van den; Beuningen, H.M. van; Smit, H.F.

    2006-01-01

    Apocynin, an inhibitor of NADPH-oxidase, is known to partially reverse the inflammation-mediated cartilage proteoglycan synthesis in chondrocytes. More recently, it was reported that apocynin prevents cyclooxygenase (COX)-2 expression in monocytes. The present study aimed to investigate whether

  10. High-oleic canola oil consumption enriches LDL particle cholesteryl oleate content and reduces LDL proteoglycan binding in humans.

    Science.gov (United States)

    Jones, Peter J H; MacKay, Dylan S; Senanayake, Vijitha K; Pu, Shuaihua; Jenkins, David J A; Connelly, Philip W; Lamarche, Benoît; Couture, Patrick; Kris-Etherton, Penny M; West, Sheila G; Liu, Xiaoran; Fleming, Jennifer A; Hantgan, Roy R; Rudel, Lawrence L

    2015-02-01

    Oleic acid consumption is considered cardio-protective according to studies conducted examining effects of the Mediterranean diet. However, animal models have shown that oleic acid consumption increases LDL particle cholesteryl oleate content which is associated with increased LDL-proteoglycan binding and atherosclerosis. The objective was to examine effects of varying oleic, linoleic and docosahexaenoic acid consumption on human LDL-proteoglycan binding in a non-random subset of the Canola Oil Multi-center Intervention Trial (COMIT) participants. COMIT employed a randomized, double-blind, five-period, cross-over trial design. Three of the treatment oil diets: 1) a blend of corn/safflower oil (25:75); 2) high oleic canola oil; and 3) DHA-enriched high oleic canola oil were selected for analysis of LDL-proteoglycan binding in 50 participants exhibiting good compliance. LDL particles were isolated from frozen plasma by gel filtration chromatography and LDL cholesteryl esters quantified by mass-spectrometry. LDL-proteoglycan binding was assessed using surface plasmon resonance. LDL particle cholesterol ester fatty acid composition was sensitive to the treatment fatty acid compositions, with the main fatty acids in the treatments increasing in the LDL cholesterol esters. The corn/safflower oil and high-oleic canola oil diets lowered LDL-proteoglycan binding relative to their baseline values (p = 0.0005 and p = 0.0012, respectively). At endpoint, high-oleic canola oil feeding resulted in lower LDL-proteoglycan binding than corn/safflower oil (p = 0.0243) and DHA-enriched high oleic canola oil (p = 0.0249), although high-oleic canola oil had the lowest binding at baseline (p = 0.0344). Our findings suggest that high-oleic canola oil consumption in humans increases cholesteryl oleate percentage in LDL, but in a manner not associated with a rise in LDL-proteoglycan binding. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  11. Multi-scale modeling of soft fibrous tissues based on proteoglycan mechanics.

    Science.gov (United States)

    Linka, Kevin; Khiêm, Vu Ngoc; Itskov, Mikhail

    2016-08-16

    Collagen in the form of fibers or fibrils is an essential source of strength and structural integrity in most organs of the human body. Recently, with the help of complex experimental setups, a paradigm change concerning the mechanical contribution of proteoglycans (PGs) took place. Accordingly, PG connections protect the surrounding collagen fibrils from over-stretching rather than transmitting load between them. In this paper, we describe the reported PG mechanics and incorporate it into a multi-scale model of soft fibrous tissues. To this end, a nano-to-micro model of a single collagen fiber is developed by taking the entropic-energetic transition on the collagen molecule level into account. The microscopic damage occurring inside the collagen fiber is elucidated by sliding of PGs as well as by over-stretched collagen molecules. Predictions of this two-constituent-damage model are compared to experimental data available in the literature. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Chondroitin Sulfate Proteoglycan 4 and Its Potential As an Antibody Immunotherapy Target across Different Tumor Types

    Directory of Open Access Journals (Sweden)

    Kristina M. Ilieva

    2018-01-01

    Full Text Available Overexpression of the chondroitin sulfate proteoglycan 4 (CSPG4 has been associated with the pathology of multiple types of such as melanoma, breast cancer, squamous cell carcinoma, mesothelioma, neuroblastoma, adult and pediatric sarcomas, and some hematological cancers. CSPG4 has been reported to exhibit a role in the growth and survival as well as in the spreading and metastasis of tumor cells. CSPG4 is overexpressed in several malignant diseases, while it is thought to have restricted and low expression in normal tissues. Thus, CSPG4 has become the target of numerous anticancer treatment approaches, including monoclonal antibody-based therapies. This study reviews key potential anti-CSPG4 antibody and immune-based therapies and examines their direct antiproliferative/metastatic and immune activating mechanisms of action.

  13. Prostate cancer cells specifically reorganize epithelial cell-fibroblast communication through proteoglycan and junction pathways.

    Science.gov (United States)

    Suhovskih, Anastasia V; Kashuba, Vladimir I; Klein, George; Grigorieva, Elvira V

    2017-01-02

    Microenvironment and stromal fibroblasts are able to inhibit tumor cell proliferation both through secreted signaling molecules and direct cell-cell interactions but molecular mechanisms of these effects remain unclear. In this study, we investigated a role of cell-cell contact-related molecules (protein ECM components, proteoglycans (PGs) and junction-related molecules) in intercellular communications between the human TERT immortalized fibroblasts (BjTERT fibroblasts) and normal (PNT2) or cancer (LNCaP, PC3, DU145) prostate epithelial cells. It was shown that BjTERT-PNT2 cell coculture resulted in significant decrease of both BjTERT and PNT2 proliferation rates and reorganization of transcriptional activity of cell-cell contact-related genes in both cell types. Immunocytochemical staining revealed redistribution of DCN and LUM in PNT2 cells and significant increase of SDC1 at the intercellular contact zones between BjTERT and PNT2 cells, suggesting active involvement of the PGs in cell-cell contacts and contact inhibition of cell proliferation. Unlike to PNT2 cells, PC3 cells did not respond to BjTERT in terms of PGs expression, moderately increased transcriptional activity of junctions-related genes (especially tight junction) and failed to establish PC3-BjTERT contacts. At the same time, PC3 cells significantly down-regulated junctions-related genes (especially focal adhesions and adherens junctions) in BjTERT fibroblasts resulting in visible preference for homotypic PC3-PC3 over heterotypic PC3-BjTERT contacts and autonomous growth of PC3 clones. Taken together, the results demonstrate that an instructing role of fibroblasts to normal prostate epithelial cells is revoked by cancer cells through deregulation of proteoglycans and junction molecules expression and overall disorganization of fibroblast-cancer cell communication.

  14. Proteoglycans in Leiomyoma and Normal Myometrium: Abundance, Steroid Hormone Control, and Implications for Pathophysiology.

    Science.gov (United States)

    Barker, Nichole M; Carrino, David A; Caplan, Arnold I; Hurd, William W; Liu, James H; Tan, Huiqing; Mesiano, Sam

    2016-03-01

    Uterine leiomyoma are a common benign pelvic tumors composed of modified smooth muscle cells and a large amount of extracellular matrix (ECM). The proteoglycan composition of the leiomyoma ECM is thought to affect pathophysiology of the disease. To test this hypothesis, we examined the abundance (by immunoblotting) and expression (by quantitative real-time polymerase chain reaction) of the proteoglycans biglycan, decorin, and versican in leiomyoma and normal myometrium and determined whether expression is affected by steroid hormones and menstrual phase. Leiomyoma and normal myometrium were collected from women (n = 17) undergoing hysterectomy or myomectomy. In vitro studies were performed on immortalized leiomyoma (UtLM) and normal myometrial (hTERT-HM) cells with and without exposure to estradiol and progesterone. In leiomyoma tissue, abundance of decorin messenger RNA (mRNA) and protein were 2.6-fold and 1.4-fold lower, respectively, compared with normal myometrium. Abundance of versican mRNA was not different between matched samples, whereas versican protein was increased 1.8-fold in leiomyoma compared with myometrium. Decorin mRNA was 2.4-fold lower in secretory phase leiomyoma compared with proliferative phase tissue. In UtLM cells, progesterone decreased the abundance of decorin mRNA by 1.3-fold. Lower decorin expression in leiomyoma compared with myometrium may contribute to disease growth and progression. As decorin inhibits the activity of specific growth factors, its reduced level in the leiomyoma cell microenvironment may promote cell proliferation and ECM deposition. Our data suggest that decorin expression in leiomyoma is inhibited by progesterone, which may be a mechanism by which the ovarian steroids affect leiomyoma growth and disease progression. © The Author(s) 2015.

  15. Cell binding fragments from a sponge proteoglycan-like aggregation factor.

    Science.gov (United States)

    Misevic, G N; Jumblatt, J E; Burger, M M

    1982-06-25

    The marine sponge Microciona prolifera aggregation factor (MAF) is a 2 X 10(7) dalton proteoglycan. MAF mediates species-specific cell-cell recognition through two functionally different sites: a Ca2+-independent species-specific cell binding site and a Ca2+-dependent MAF-MAF binding site. Dissociation procedures combined with protease treatment were used to produce cell-binding pieces from the large complex. The seven different sized fragments produced were all uronic acid-rich glycoproteins of the apparent molecular weights: 15 X 10(6), 2.5 X 10(5), 1.2 X 10(5), 7 X 10(4), 2.7 X 10(4), 5 X 10(3), and 3.6 X 10(3). Each of the fragments retained species-specific binding to Microciona cells and was also capable of inhibiting MAF-promoted cell aggregation. However, the fragments were unable to bind to MAF-conjugated agarose beads in the presence or absence of CA2+ ions. These three properties are those expected for the cell binding site of MAF. Since the binding affinity decreased linearly with decreasing molecular weight of the fragments, we believe that the cell binding sites in MAF may be highly polyvalent, although to fully support such a concept, a detailed chemical characterization of each of the fragments is needed. A high valency of cell binding sites would overcome a relatively low Ka for the single site and would thereby not only guarantee specificity but also explain the need for the large size of the proteoglycan complex found to mediate species-specific sponge aggregation.

  16. Chondroitin sulfate proteoglycan serglycin influences protein cargo loading and functions of tumor-derived exosomes.

    Science.gov (United States)

    Purushothaman, Anurag; Bandari, Shyam K; Chandrashekar, Darshan S; Jones, Richard J; Lee, Hans C; Weber, Donna M; Orlowski, Robert Z

    2017-09-26

    Tumor cells produce and utilize exosomes to promote tumor growth and metastasis. Tumor-cell-derived exosomes deliver cargos that partially mimic the contents of the parent cell to nearby or distant normal or abnormal cells, thereby reprogramming the recipient cells to support tumor progression. Mechanisms by which tumor-derived exosomes subserve the tumor are under intense investigation. Here we demonstrate a critical role of the chondroitin sulfate proteoglycan serglycin in regulating the protein cargo and functions of myeloma cell-derived exosomes. Previous studies have shown that serglycin, the only known intracellular proteoglycan, functions mainly in the storage of basically charged components within the intracellular granules/vesicles via serglycin's densely clustered, negatively charged glycosaminoglycan chains. Here we demonstrate that serglycin plays a critical role in the protein cargo loading of tumor-derived exosomes. Serglycin was detected in exosomes derived from cell culture supernatants of human myeloma cell lines and serum of myeloma patients. Mass spectrometry analysis of exosomal proteins identified significantly fewer protein components within exosomes derived from serglycin-knockdown myeloma cells than within exosomes from control cells. On gene ontology analysis, exosomes derived from serglycin-knockdown cells, but not from control cells, lacked many proteins that are required for mediating different cellular processes. In functional assays, exosomes from serglycin-knockdown cells failed to induce an invasive phenotype in myeloma cells and failed to promote migration of macrophages. These findings reveal that serglycin plays an important role in maintaining the protein cargo in tumor-derived exosomes and suggest that targeting serglycin may temper the influence of these exosomes on cancer progression.

  17. Expression of lumican, a small leucine-rich proteoglycan with antitumour activity, in human malignant melanoma.

    Science.gov (United States)

    Brézillon, S; Venteo, L; Ramont, L; D'Onofrio, M-F; Perreau, C; Pluot, M; Maquart, F-X; Wegrowski, Y

    2007-07-01

    The family of small leucine-rich proteoglycans (SLRPs), which includes decorin, lumican, biglycan and fibromodulin, constitutes an abundant component of the skin extracellular matrix. We previously demonstrated that human lumican inhibits melanoma growth and progression in a mouse experimental model, by regulating cell migration, proliferation and apoptosis. The aim of this study was to investigate the expression of lumican and decorin in human malignant melanoma and adjacent peritumoral tissue, to understand better their role in the control of growth and invasion of human melanoma. Expression of both proteoglycans was studied by immunohistochemistry using specific antibodies in 34 malignant melanomas, 12 Hutchinson's melanotic freckles and 4 cutaneous metastatic melanomas. We showed that lumican and decorin are located in the dermis and in the peritumoral stroma of malignant melanoma, but are not found in melanoma cells or dense tumour tissue. In the healthy dermis, distant from the tumour, the increasing ratio of lumican to decorin was inversely correlated with the proliferation of the tumour cells (P = 0.035). The comparison of the level of expression of lumican protein in superficial vs. nodular subtypes of malignant melanomas showed a decrease of lumican but not decorin in the peritumoral stroma of nodular subtypes. In the peritumoral stroma, the level of expression of lumican but not decorin decreased significantly (P = 0.016) with increasing Clark levels. In addition, immunocytochemical and reverse transcription PCR analyses of malignant melanoma cell lines (A-375, HT-144) and of MRC-5 and dermal fibroblasts from healthy donors in vitro confirmed that dermal fibroblasts are responsible for lumican and decorin synthesis in skin. CONCLUSIONS. Lumican may regulate vertical progression of human malignant melanoma, but further study is necessary to clarify the antitumour mechanism and the downstream signal transduction pathways involved.

  18. Polymorphisms in C-Reactive Protein and Glypican-5 Are Associated with Lung Cancer Risk and Gartrokine-1 Influences Cisplatin-Based Chemotherapy Response in a Chinese Han Population

    Directory of Open Access Journals (Sweden)

    Shuo Zhang

    2015-01-01

    Full Text Available The role of genetics in progression of cancer is an established fact, and susceptibility risk and difference in outcome to chemotherapy may be caused by the variation in low-penetrance alleles of risk genes. We selected seven genes (CRP, GPC5, ACTA2, AGPHD1, SEC14L5, RBMS3, and GKN1 that previously reported link to lung cancer (LC and genotyped single nucleotide polymorphisms (SNPs of these genes in a case-control study. A protective allele “C” was found in rs2808630 of the C-reactive protein (CRP. Model association analysis found genotypes “T/C” and “C/C” in the dominant model and genotype “T/C” in the overdominant model of rs2808630 associated with reduced LC risk. Gender-specific analysis in each model showed that genotypes “T/T” and “C/C” in rs2352028 of the Glypican 5 (GPC5 were associated with increased LC risk in males. Logistic regression analysis showed “C/T” genotype carriers of rs4254535 in the Gastrokine 1 (GKN1 had less likelihood to have chemotherapy response. Our results suggest a potential association between CRP and GPC5 variants with LC risk; variation in GKN1 is associated with chemotherapy response in the Chinese Han population.

  19. Altered Liver Proteoglycan/Glycosaminoglycan Structure as a Manifestation of Extracellular Matrix Remodeling upon BCG-induced Granulomatosis in Mice.

    Science.gov (United States)

    Kim, L B; Shkurupy, V A; Putyatina, A N

    2017-01-01

    Experimental BCG-induced granulomatosis in mice was used to study changes in the dynamics of individual liver proteoglycan components reflecting phasic extracellular matrix remodeling, determined by the host-parasite interaction and associated with granuloma development. In the early BCG-granulomatosis period, the increase in individual proteoglycan components promotes granuloma formation, providing conditions for mycobacteria adhesion to host cells, migration of phagocytic cells from circulation, and cell-cell interaction leading to granuloma development and fibrosis. Later, reduced reserve capacity of the extracellular matrix, development of interstitial fibrosis and granuloma fibrosis can lead to trophic shortage for cells within the granulomas, migration of macrophages out of them, and development of spontaneous necrosis and apoptosis typical of tuberculosis.

  20. Age-Related Changes in the System Metalloproteinases/Tissue Metalloproteinase Inhibitors and Proteoglycan Components in Mouse Organs.

    Science.gov (United States)

    Kim, L B; Shkurupy, V A; Putyatina, A N

    2016-05-01

    Activity of MMP and content of TIMP and some proteoglycan components were measured in the liver, lungs, and spleen of BALB/c mice aging 2, 6, and 12 months. The increase in the content of proteoglycan components in mouse organs was associated with age-related changes in MMP activity and TIMP-1 and TIMP-2 levels. The ratio between MMP activity and content of TIMP-1 and TIMP-2 differed in the studied organs. The levels of TIMP-1 and TIMP-2 in mouse blood serum and their concentration in studied organs changed asynchronously with age. They are important regulators that determine MMP activity in the analyzed age periods in mice, which can be determined as periods of growth, development, and ageing.

  1. 1988 Volvo award in basic science. Proteoglycan synthesis in the human intervertebral disc. Variation with age, region and pathology.

    Science.gov (United States)

    Bayliss, M T; Johnstone, B; O'Brien, J P

    1988-09-01

    Slices of human annulus fibrosus were cultured under conditions that controlled their hydration and prevented loss of proteoglycans from the extracellular matrix. A quantitative analysis of proteoglycan synthesis was carried out. Both the absolute rate of synthesis and the topographical variation in chondrocyte activity changed with age; the most active cells in the adult were found in the mid-annulus region, whereas in the fetal disc the cells in the inner annulus were the most active. The conditions under which the tissue was stored, and changes in hydration during culture, had considerable effects on synthesis. Pathological discs had a wide range of biological activity that reflected the heterogeneous properties of these specimens. It is suggested that this culture method provides a means of investigating the way in which the synthesis of the macromolecular components of the intervertebral disc are coordinated and subsequently incorporated into the extracellular matrix.

  2. Glycomic analyses of ovarian follicles during development and atresia

    Science.gov (United States)

    Hatzirodos, Nicholas; Nigro, Julie; Irving-Rodgers, Helen F.; Vashi, Aditya V.; Hummitzsch, Katja; Caterson, Bruce; Sullivan, Thomas R.; Rodgers, Raymond J.

    2012-01-01

    To examine the detailed composition of glycosaminoglycans during bovine ovarian follicular development and atresia, the specialized stromal theca layers were separated from the stratified epithelial granulosa cells of healthy (n = 6) and atretic (n = 6) follicles in each of three size ranges: small (3–5 mm), medium (6-9 mm) and large (10 mm or more) (n = 29 animals). Fluorophore-assisted carbohydrate electrophoresis analyses (on a per cell basis) and immunohistochemistry (n = 14) were undertaken. We identified the major disaccharides in thecal layers and the membrana granulosa as chondroitin sulfate-derived ∆uronic acid with 4-sulfated N-acetylgalactosamine and ∆uronic acid with 6-sulfated N-acetylgalactosamine and the heparan sulfate-derived Δuronic acid with N-acetlyglucosamine, with elevated levels in the thecal layers. Increasing follicle size and atresia was associated with increased levels of some disaccharides. We concluded that versican contains 4-sulfated N-acetylgalactosamine and it is the predominant 4-sulfated N-acetylgalactosamine proteoglycan in antral follicles. At least one other non- or 6-sulfated N-acetylgalactosamine proteoglycan(s), which is not decorin or an inter-α-trypsin inhibitor family member, is present in bovine antral follicles and associated with hitherto unknown groups of cells around some larger blood vessels. These areas stained positively for chondroitin/dermatan sulfate epitopes [antibodies 7D4, 3C5, and 4C3], similar to stem cell niches observed in other tissues. The sulfation pattern of heparan sulfate glycosaminoglycans appears uniform across follicles of different sizes and in healthy and atretic follicles. The heparan sulfate products detected in the follicles are likely to be associated with perlecan, collagen XVIII or betaglycan. PMID:22057033

  3. Changes of Proteoglycan Expression and Glycosaminoglycan Constituents in the Intervillous Space of the Pregnancy-Induced Hypertension Placenta

    OpenAIRE

    Matsukura, Daisuke; Yokoyama, Yoshihito; Tanaka, Kanji; Ozaki, Takashi; Mizunuma, Hideki

    2008-01-01

    The changes in proteoglycan (PG) expression and glycosaminoglycan (GAG) constituents in theintervillous space of the pregnancy-induced hypertension (PIR) placenta were investigated. PGs and GAGs werepurified from the extract of the placental intervillous space by the DEAE-Sephacel column and salt-concentrationgradient method. and the GAG sugar chains were released by the actinase and cellulase treatments. Thesugar chains from the placentas of normal pregnancy and PIR were compared by cellulos...

  4. Proteoglycan abnormalities in olfactory epithelium tissue from subjects diagnosed with schizophrenia.

    Science.gov (United States)

    Pantazopoulos, Harry; Boyer-Boiteau, Anne; Holbrook, Eric H; Jang, Woochan; Hahn, Chang-Gyu; Arnold, Steven E; Berretta, Sabina

    2013-11-01

    Emerging evidence points to proteoglycan abnormalities in the pathophysiology of schizophrenia (SZ). In particular, markedly abnormal expression of chondroitin sulfate proteoglycans (CSPGs), key components of the extracellular matrix, was observed in the medial temporal lobe. CSPG functions, including regulation of neuronal differentiation and migration, are highly relevant to the pathophysiology of SZ. CSPGs may exert similar functions in the olfactory epithelium (OE), a continuously regenerating neural tissue that shows cell and molecular abnormalities in SZ. We tested the hypothesis that CSPG expression in OE may be altered in SZ. CSPG-positive cells in postmortem OE from non-psychiatric control (n=9) and SZ (n=10) subjects were counted using computer-assisted light microscopy. 'Cytoplasmic' CSPG (c-CSPG) labeling was detected in sustentacular cells and some olfactory receptor neurons (c-CSPG+ORNs), while 'pericellular' CSPG (p-CSPG) labeling was found in basal cells and some ORNs (p-CSPG+ORNs). Dual labeling for CSPG and markers for mature and immature ORNs suggests that c-CSPG+ORNs correspond to mature ORNs, and p-CSPG+ORNs to immature ORNs. Previous studies in the same cohort demonstrated that densities of mature ORNs were unaltered (Arnold et al., 2001). In the present study, numerical densities of c-CSPG+ORNs were significantly decreased in SZ (p<0.025; 99.32% decrease), suggesting a reduction of CSPG expression in mature ORNs. Previous studies showed a striking increase in the ratios of immature neurons with respect to basal cells. In this study, we find that the ratio of p-CSPG+ORNs/CSPG+basal cells was significantly increased (p=0.03) in SZ, while numerical density changes of p-CSPG+ORNs (110.71% increase) or CSPG+basal cells (53.71% decrease), did not reach statistical significance. Together, these results indicate that CSPG abnormalities are present in the OE of SZ and specifically point to a reduction of CSPG expression in mature ORNs in SZ. Given the

  5. Syndecan-3 in limb skeletal development.

    Science.gov (United States)

    Kosher, R A

    1998-10-15

    Syndecan-3 is a member of a family of heparan sulfate proteoglycans that function as extracellular matrix receptors and as co-receptors for growth factors and signalling molecules. A variety of studies indicate that syndecan-3 is involved in several aspects of limb morphogenesis and skeletal development. Syndecan-3 participates in limb outgrowth and proliferation in response to the apical ectodermal ridge; mediates cell-matrix and/or cell-cell interactions involved in regulating the onset of chondrogenesis; may be involved in regulating the onset of osteogenesis and joint formation and, plays a role in regulating the proliferation of epiphyseal chondrocytes during endochondral ossification.

  6. A sharp end to sugary Wingless travels.

    Science.gov (United States)

    Droujinine, Ilia A; Yan, Dong; Perrimon, Norbert

    2014-09-29

    Drosophila melanogaster follicle stem cells are controlled by Wingless (Wg) ligands secreted 50 µm away, raising the question of how long-distance Wg spreading occurs. In this issue of JCB, Wang and Page-McCaw (2014. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201403084) demonstrate a potential mechanism by which the heparan sulfate proteoglycan Dally-like (Dlp) promotes Wg travel, whereas matrix Mmp2 (Metalloproteinase 2) impedes it by inactivating Dlp. © 2014 Droujinine et al.

  7. Systemic Administration of Proteoglycan Protects BALB/c Retired Breeder Mice from Experimental Arthritis

    Directory of Open Access Journals (Sweden)

    Larissa Lumi Watanabe Ishikawa

    2016-01-01

    Full Text Available This study was undertaken to evaluate the prophylactic potential of proteoglycan (PG administration in experimental arthritis. Female BALB/c retired breeder mice received two (2xPG50 and 2xPG100 groups or three (3xPG50 group intraperitoneal doses of bovine PG (50 μg or 100 μg every three days. A week later the animals were submitted to arthritis induction by immunization with three i.p. doses of bovine PG associated with dimethyldioctadecylammonium bromide adjuvant at intervals of 21 days. Disease severity was daily assessed after the third dose by score evaluation. The 3xPG50 group showed significant reduction in prevalence and clinical scores. This protective effect was associated with lower production of IFN-γ and IL-17 and increased production of IL-5 and IL-10 by spleen cells restimulated in vitro with PG. Even though previous PG administration restrained dendritic cells maturation this procedure did not alter the frequency of regulatory Foxp3+ T cells. Lower TNF-α and IL-6 levels and higher expression of ROR-γ and GATA-3 were detected in the paws of protected animals. A delayed-type hypersensitivity reaction confirmed specific tolerance induction. Taken together, these results indicate that previous PG inoculation determines a specific tolerogenic effect that is able to decrease severity of subsequently induced arthritis.

  8. NG2 Proteoglycan-Dependent Contributions of Pericytes and Macrophages to Brain Tumor Vascularization and Progression.

    Science.gov (United States)

    Stallcup, William B; You, Weon-Kyoo; Kucharova, Karolina; Cejudo-Martin, Pilar; Yotsumoto, Fusanori

    2016-02-01

    The NG2 proteoglycan promotes tumor growth as a component of both tumor and stromal cells. Using intracranial, NG2-negative B16F10 melanomas, we have investigated the importance of PC and Mac NG2 in brain tumor progression. Reduced melanoma growth in Mac-NG2ko and PC-NG2ko mice demonstrates the importance of NG2 in both stromal compartments. In each genotype, the loss of PC-endothelial cell interaction diminishes the formation of endothelial junctions and assembly of the basal lamina. Tumor vessels in Mac-NG2ko mice have smaller diameters, reduced patency, and increased leakiness compared to PC-NG2ko mice, thus decreasing tumor blood supply and increasing hypoxia. While the reduced PC interaction with endothelial cells in PC-NG2ko mice results from the loss of PC activation of β1 integrin signaling in endothelial cells, reduced PC-endothelial cell interaction in Mac-NG2ko mice results from 90% reduced Mac recruitment. The absence of Mac-derived signals in Mac-NG2ko mice causes the loss of PC association with endothelial cells. Reduced Mac recruitment may be due to diminished activation of integrins in the absence of NG2, causing decreased Mac interaction with endothelial adhesion molecules that are needed for extravasation. These results reflect the complex interplay that occurs between Mac, PC, and endothelial cells during tumor vascularization. © 2015 John Wiley & Sons Ltd.

  9. Roles of chondroitin sulfate proteoglycan 4 in fibrogenic/adipogenic differentiation in skeletal muscle tissues.

    Science.gov (United States)

    Takeuchi, Shiho; Nakano, Shin-Ichi; Nakamura, Katsuyuki; Ozoe, Atsufumi; Chien, Peggie; Yoshihara, Hidehito; Hakuno, Fumihiko; Matsuwaki, Takashi; Saeki, Yasushi; Takahashi, Shin-Ichiro; Yamanouchi, Keitaro; Nishihara, Masugi

    2016-10-01

    Intramuscular adipose tissue and fibrous tissue are observed in some skeletal muscle pathologies such as Duchenne muscular dystrophy and sarcopenia, and affect muscle strength and myogenesis. They originate from common fibrogenic/adipogenic cells in the skeletal muscle. Thus, elucidating the regulatory mechanisms underlying fibrogenic/adipogenic cell differentiation is an important step toward the mediation of these disorders. Previously, we established a highly adipogenic progenitor clone, 2G11, from rat skeletal muscle and showed that basic fibroblast growth factor (bFGF) is pro-adipogenic in these cells. Here, we demonstrated that 2G11 cells give rise to fibroblasts upon transforming growth factor (TGF)-β1 stimulation, indicating that they possess mesenchymal progenitor cells (MPC)-like characteristics. The previously reported MPC marker PDGFRα is expressed in other cell populations. Accordingly, we produced monoclonal antibodies that specifically bind to 2G11 cell surface antigens and identified chondroitin sulfate proteoglycan 4 (CSPG4) as a potential MPC marker. Based on an RNA interference analysis, we found that CSPG4 is involved in both the pro-adipogenic effect of bFGF and in TGF-β-induced alpha smooth muscle actin expression and stress fiber formation. By establishing an additional marker for MPC detection and characterizing its role in fibrogenic/adipogenic differentiation, these results will facilitate the development of effective treatments for skeletal muscle pathologies. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Study of optical properties and proteoglycan content of tendons by polarization sensitive optical coherence tomography

    Science.gov (United States)

    Yang, Ying; Rupani, Asha; Bagnaninchi, Pierre; Wimpenny, Ian; Weightman, Alan

    2012-08-01

    The highly orientated collagen fibers in tendons play a critical role for transferring tensile stress, and they demonstrate birefringent optical properties. However, the influence that proteoglycans (PGs) have on the optical properties of tendons is yet to be fully elucidated. PGs are the essential components of the tendon extracellular matrix; the changes in their quantities and compositions have been associated with tendinopathies. In this study, polarization sensitive optical coherence tomography (PS-OCT) has been used to reveal the relationship between PG content/location and birefringence properties of tendons. Fresh chicken tendons were imaged at regular intervals by PS-OCT and polarization light microscopy during the extraction of PGs, using guanidine hydrochloride (GuHCl). Complementary time-lapsed images taken from the two modalities mutually demonstrated that the extraction of PGs disturbed the local organization of collagen bundles. This corresponded with a decrease in birefringence and associated banding pattern observed by PS-OCT. Furthermore, this study revealed there was a higher concentration of PGs in the outer sheath region than in the fascicles, and therefore the change in birefringence was reduced when extraction was performed on unsheathed tendons. The results provide new insights of tendon structure and the role of PGs on the structural stability of tendons, which also demonstrates the great potential for using PS-OCT as a diagnostic tool to examine tendon pathology.

  11. Fourier transform infrared spectroscopic imaging and multivariate regression for prediction of proteoglycan content of articular cartilage.

    Directory of Open Access Journals (Sweden)

    Lassi Rieppo

    Full Text Available Fourier Transform Infrared (FT-IR spectroscopic imaging has been earlier applied for the spatial estimation of the collagen and the proteoglycan (PG contents of articular cartilage (AC. However, earlier studies have been limited to the use of univariate analysis techniques. Current analysis methods lack the needed specificity for collagen and PGs. The aim of the present study was to evaluate the suitability of partial least squares regression (PLSR and principal component regression (PCR methods for the analysis of the PG content of AC. Multivariate regression models were compared with earlier used univariate methods and tested with a sample material consisting of healthy and enzymatically degraded steer AC. Chondroitinase ABC enzyme was used to increase the variation in PG content levels as compared to intact AC. Digital densitometric measurements of Safranin O-stained sections provided the reference for PG content. The results showed that multivariate regression models predict PG content of AC significantly better than earlier used absorbance spectrum (i.e. the area of carbohydrate region with or without amide I normalization or second derivative spectrum univariate parameters. Increased molecular specificity favours the use of multivariate regression models, but they require more knowledge of chemometric analysis and extended laboratory resources for gathering reference data for establishing the models. When true molecular specificity is required, the multivariate models should be used.

  12. Fibroblast growth factor-2 promotes keratan sulfate proteoglycan expression by keratocytes in vitro

    Science.gov (United States)

    Long, C. J.; Roth, M. R.; Tasheva, E. S.; Funderburgh, M.; Smit, R.; Conrad, G. W.; Funderburgh, J. L.

    2000-01-01

    Keratocytes of the corneal stroma produce a specialized extracellular matrix responsible for corneal transparency. Corneal keratan sulfate proteoglycans (KSPG) are unique products of keratocytes that are down-regulated in corneal wounds and in vitro. This study used cultures of primary bovine keratocytes to define factors affecting KSPG expression in vitro. KSPG metabolically labeled with [(35)S]sulfate decreased during the initial 2-4 days of culture in quiescent cultures with low serum concentrations (0.1%). Addition of fetal bovine serum, fibroblast growth factor-2 (FGF-2), transforming growth factor beta, or platelet derived growth factor all stimulated cell division, but only FGF-2 stimulated KSPG secretion. Combined with serum, FGF-2 also prevented serum-induced KSPG down-regulation. KSPG secretion was lost during serial subculture with or without FGF-2. Expression of KSPG core proteins (lumican, mimecan, and keratocan) was stimulated by FGF-2, and steady state mRNA pools for these proteins, particularly keratocan, were significantly increased by FGF-2 treatment. KSPG expression therefore is supported by exogenous FGF-2 and eliminated by subculture of the cells in presence of serum. FGF-2 stimulates KSPG core protein expression primarily through an increase in mRNA pools.

  13. Role of moderate exercising on Achilles tendon collagen crimping patterns and proteoglycans.

    Science.gov (United States)

    Franchi, Marco; Torricelli, Paola; Giavaresi, Gianluca; Fini, Milena

    2013-01-01

    In this study, the morphological and morphometric changes in the collagen crimping pattern of Achilles tendon and metabolism/expression of tenocytes explanted from tendons of running (RUN) and sedentary (SED) rats were investigated to assess the effects of 12 weeks moderate running exercise. The number, the top angle width and the base length of each crimp in three different regions (proximal, central and distal) of RUN and SED tendons were measured with a polarized light microscope. The most significant morphometric differences in the crimps were detectable in the central region of the RUN tendons. In this region, crimps were fewer, larger and more flattened than those of other regions as a consequence of a functional adaptation of extracellular matrix to running, in order to increase tendon stiffness and force transmission efficiency. Conversely, the top angle width of the crimps reduced in proximal and distal regions of the RUN tendons, suggesting that these crimps might act as more reactive mechanical springs, able to store and improve the release of the stored strain energy in most loaded regions. Tenocytes explanted from Achilles tendons of both RUN and SED groups were cultured. Running influenced tenocytes which showed a significant increase in collagen type-I synthesis and proteoglycans production, suggesting enhancement of the loading transmission efficiency and facilitate inter-fibril and inter-fiber sliding.

  14. Sulphated glycosaminoglycans and proteoglycans in the developing vertebral column of juvenile Atlantic salmon (Salmo salar).

    Science.gov (United States)

    Hannesson, Kirsten O; Ytteborg, Elisabeth; Takle, Harald; Enersen, Grethe; Bæverfjord, Grete; Pedersen, Mona E

    2015-08-01

    In the present study, the distribution of sulphated glycosaminoglycans (GAGs) in the developing vertebral column of Atlantic salmon (Salmo salar) at 700, 900, 1100 and 1400 d° was examined by light microscopy. The mineralization pattern was outlined by Alizarin red S and soft structures by Alcian blue. The temporal and spatial distribution patterns of different types of GAGs: chondroitin-4-sulphate/dermatan sulphate, chondroitin-6-sulphate, chondroitin-0-sulphate and keratan sulphate were addressed by immunohistochemistry using monoclonal antibodies against the different GAGs. The specific pattern obtained with the different antibodies suggests a unique role of the different GAG types in pattern formation and mineralization. In addition, the distribution of the different GAG types in normal and malformed vertebral columns from 15 g salmon was compared. A changed expression pattern of GAGs was found in the malformed vertebrae, indicating the involvement of these molecules during the pathogenesis. The molecular size of proteoglycans (PGs) in the vertebrae carrying GAGs was analysed with western blotting, and mRNA transcription of the PGs aggrecan, decorin, biglycan, fibromodulin and lumican by real-time qPCR. Our study reveals the importance of GAGs in development of vertebral column also in Atlantic salmon and indicates that a more comprehensive approach is necessary to completely understand the processes involved.

  15. Purification, structural characterization and bioactivity evaluation of a novel proteoglycan produced by Corbicula fluminea.

    Science.gov (United States)

    Yan, Jing-Kun; Wang, Yao-Yao; Qiu, Wen-Yi; Wu, Li-Xia; Ding, Zhi-Chao; Cai, Wu-Dan

    2017-11-15

    A novel proteoglycan, named CFPS-11, was isolated from Corbicula fluminea, which is a food source of freshwater bivalve mollusk. CFPS-11 had an average molecular weight of 807.7kDa and consisted of d-glucose and d-glucosamine in a molar ratio of 12.2:1.0. The protein moiety (∼5%) of CFPS-11 was covalently bonded to the polysaccharide chain in O-linkage type through both serine and thereonine residues. The polysaccharide chain of CFPS-11 was composed of (1→4)-α-d-glucopyranosyl and (1→3,6)-α-d-glucopyranosyl residues, which branched at O-6. The branch chain consisted of (1→)-α-d-glucopyranosyl and (1→)-α-d-N-acetylglucosamine residues. CFPS-11 exhibited significant antioxidant activity in a dose-dependent manner and remarkable inhibition activities against α-amylase and α-glucosidase by in vitro assays. These findings indicated that the CFPS-11 from C. fluminea has the potential for development as a health food ingredient. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Ultrastructure Organization of Collagen Fibrils and Proteoglycans of Stingray and Shark Corneal Stroma

    Directory of Open Access Journals (Sweden)

    Saud A. Alanazi

    2015-01-01

    Full Text Available We report here the ultrastructural organization of collagen fibrils (CF and proteoglycans (PGs of the corneal stroma of both the stingray and the shark. Three corneas from three stingrays and three corneas from three sharks were processed for electron microscopy. Tissues were embedded in TAAB 031 resin. The corneal stroma of both the stingray and shark consisted of parallel running lamellae of CFs which were decorated with PGs. In the stingray, the mean area of PGs in the posterior stroma was significantly larger than the PGs of the anterior and middle stroma, whereas, in the shark, the mean area of PGs was similar throughout the stroma. The mean area of PGs of the stingray was significantly larger compared to the PGs, mean area of the shark corneal stroma. The CF diameter of the stingray was significantly smaller compared to the CF diameter in the shark. The ultrastructural features of the corneal stroma of both the stingray and the shark were similar to each other except for the CFs and PGs. The PGs in the stingray and shark might be composed of chondroitin sulfate (CS/dermatan sulfate (DS PGs and these PGs with sutures might contribute to the nonswelling properties of the cornea of the stingray and shark.

  17. Collagen Fibrils and Proteoglycans of Macular Dystrophy Cornea: Ultrastructure and 3D Transmission Electron Tomography.

    Science.gov (United States)

    Akhtar, Saeed; Alkatan, Hind M; Kirat, Omar; Khan, Adnan A; Almubrad, Turki

    2015-06-01

    We report the ultrastructure and 3D transmission electron tomography of collagen fibrils (CFs), proteoglycans (PGs), and microfibrils within the CF of corneas of patients with macular corneal dystrophy (MCD). Three normal corneas and three MCD corneas from three Saudi patients (aged 25, 31, and 49 years, respectively) were used for this study. The corneas were processed for light and electron microscopy studies. 3D images were composed from a set of 120 ultrastructural images using the program "Composer" and visualized using the program "Visuliser Kai". 3D image analysis of MCD cornea showed a clear organization of PGs around the CF at very high magnification and degeneration of the microfibrils within the CF. Within the MCD cornea, the PG area in the anterior stroma was significantly larger than in the middle and posterior stroma. The PG area in the MCD cornea was significantly larger compared with the PG area in the normal cornea. The CF diameter and inter-fibrillar spacing of the MCD cornea were significantly smaller compared with those of the normal cornea. Ultrastructural 3D imaging showed that the production of unsulfated keratin sulfate (KS) may lead to the degeneration of micro-CFs within the CFs. The effect of the unsulfated KS was higher in the anterior stroma compared with the posterior stroma.

  18. Pathophysiological Significance of Dermatan Sulfate Proteoglycans Revealed by Human Genetic Disorders

    Directory of Open Access Journals (Sweden)

    Shuji Mizumoto

    2017-03-01

    Full Text Available The indispensable roles of dermatan sulfate-proteoglycans (DS-PGs have been demonstrated in various biological events including construction of the extracellular matrix and cell signaling through interactions with collagen and transforming growth factor-β, respectively. Defects in the core proteins of DS-PGs such as decorin and biglycan cause congenital stromal dystrophy of the cornea, spondyloepimetaphyseal dysplasia, and Meester-Loeys syndrome. Furthermore, mutations in human genes encoding the glycosyltransferases, epimerases, and sulfotransferases responsible for the biosynthesis of DS chains cause connective tissue disorders including Ehlers-Danlos syndrome and spondyloepimetaphyseal dysplasia with joint laxity characterized by skin hyperextensibility, joint hypermobility, and tissue fragility, and by severe skeletal disorders such as kyphoscoliosis, short trunk, dislocation, and joint laxity. Glycobiological approaches revealed that mutations in DS-biosynthetic enzymes cause reductions in enzymatic activities and in the amount of synthesized DS and also disrupt the formation of collagen bundles. This review focused on the growing number of glycobiological studies on recently reported genetic diseases caused by defects in the biosynthesis of DS and DS-PGs.

  19. Changes in glycosaminoglycan structure on differentiation of human embryonic stem cells towards mesoderm and endoderm lineages.

    Science.gov (United States)

    Gasimli, Leyla; Hickey, Anne Marie; Yang, Bo; Li, Guoyun; dela Rosa, Mitche; Nairn, Alison V; Kulik, Michael J; Dordick, Jonathan S; Moremen, Kelley W; Dalton, Stephen; Linhardt, Robert J

    2014-06-01

    Proteoglycans are found on the cell surface and in the extracellular matrix, and serve as prime sites for interaction with signaling molecules. Proteoglycans help regulate pathways that control stem cell fate, and therefore represent an excellent tool to manipulate these pathways. Despite their importance, there is a dearth of data linking glycosaminoglycan structure within proteoglycans with stem cell differentiation. Human embryonic stem cell line WA09 (H9) was differentiated into early mesoderm and endoderm lineages, and the glycosaminoglycanomic changes accompanying these transitions were studied using transcript analysis, immunoblotting, immunofluorescence and disaccharide analysis. Pluripotent H9 cell lumican had no glycosaminoglycan chains whereas in splanchnic mesoderm lumican was glycosaminoglycanated. H9 cells have primarily non-sulfated heparan sulfate chains. On differentiation towards splanchnic mesoderm and hepatic lineages N-sulfo group content increases. Differences in transcript expression of NDST1, HS6ST2 and HS6ST3, three heparan sulfate biosynthetic enzymes, within splanchnic mesoderm cells compared to H9 cells correlate to changes in glycosaminoglycan structure. Differentiation of embryonic stem cells markedly changes the proteoglycanome. The glycosaminoglycan biosynthetic pathway is complex and highly regulated, and therefore, understanding the details of this pathway should enable better control with the aim of directing stem cell differentiation. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Cartilage integrity and proteoglycan turnover are comparable in canine experimentally induced and human joint degeneration

    Directory of Open Access Journals (Sweden)

    Femke Intema

    2010-10-01

    Full Text Available The value of experimental models of osteoarthritis (OA largely depends on the ability to translate observations to human OA. Surprisingly, direct comparison of characteristics of human and experimental OA is scarce. In the present study, cartilage integrity and matrix turnover in a canine model of joint degeneration were compared to human clinical OA. In 23 Beagle dogs, joint degeneration was induced in one knee, the contra-lateral knee served as a control. For comparison, human osteoarthritic and healthy knee cartilage were obtained at arthroplasty (n=14 and post-mortem (n=13. Cartilage was analyzed by histology and biochemistry. Values for cartilage integrity and proteoglycan (PG synthesis showed species specific differences; GAG content of healthy cartilage was 2-fold higher in canine cartilage and PG synthesis even 8-fold. However, the relative decrease in PG content between healthy and OA cartilage was similar for humans and canines (-17% vs. -15%, respectively, as was the histological damage (+7.0 vs. +6.1, respectively and the increase of PG synthesis (+100% vs. +70%, respectively. Remarkably, the percentage release of total and of newly formed PGs in human and canine controls was similar, as was the increase due to degeneration (+65% vs. +81% and +91% vs. +52%, respectively. Despite differences in control conditions, the observed changes in characteristics of cartilage integrity and matrix turnover are similar in a canine model of joint degeneration and human clinical OA. The canine Groove model shows that its characteristics reflect those of human OA which makes the model appropriate for studying human OA.

  1. Full-Length Recombinant Human Proteoglycan 4 Interacts with Hyaluronan to Provide Cartilage Boundary Lubrication.

    Science.gov (United States)

    Abubacker, Saleem; Dorosz, Samuel G; Ponjevic, Dragana; Jay, Gregory D; Matyas, John R; Schmidt, Tannin A

    2016-04-01

    Proteoglycan 4 (PRG4) is a mucin-like glycoprotein present in synovial fluid and at the surface of articular cartilage. The objectives of this study were to (1) assess the articular cartilage surface adsorption and in vitro cartilage boundary lubricating ability of full-length recombinant human PRG4 (rhPRG4), and (2) cartilage boundary lubricating ability of purified rhPRG4, both alone and in combination with hyaluronan (HA). rhPRG4 adsorption onto articular cartilage explants was assessed by immunohistochemistry and dot blot. An in vitro cartilage-cartilage friction test was used to assess rhPRG4's cartilage boundary lubricating ability compared to bovine PRG4, and that of purified rhPRG4 both alone and in combination with HA. rhPRG4 was able to adsorb to the articular surface, as well as the cut surface, of cartilage explants. The kinetic coefficient of friction of rhPRG4 was similar to that of PRG4 (p = 0.16) and lower than phosphate-buffered saline (p < 0.05), while that of purified rhPRG4 + HA was significantly lower than rhPRG4 alone (p < 0.05). This study demonstrates that rhPRG4 can adsorb to an intact articular cartilage surface and functions as an effective boundary lubricant, both alone and with HA, and provides the foundation for in vivo evaluation of this clinically relevant full-length rhPRG4 for treatment of osteoarthritis.

  2. Antibody recognizing 4-sulfated chondroitin sulfate proteoglycans restores memory in tauopathy-induced neurodegeneration.

    Science.gov (United States)

    Yang, Sujeong; Hilton, Sam; Alves, João Nuno; Saksida, Lisa M; Bussey, Timothy; Matthews, Russell T; Kitagawa, Hiroshi; Spillantini, Maria Grazia; Kwok, Jessica C F; Fawcett, James W

    2017-11-01

    Chondroitin sulfate proteoglycans (CSPGs) are the main active component of perineuronal nets (PNNs). Digestion of the glycosaminoglycan chains of CSPGs with chondroitinase ABC or transgenic attenuation of PNNs leads to prolongation of object recognition memory and activation of various forms of plasticity in the adult central nervous system. The inhibitory properties of the CSPGs depend on the pattern of sulfation of their glycosaminoglycans, with chondroitin 4-sulfate (C4S) being the most inhibitory form. In this study, we tested a number of candidates for functional blocking of C4S, leading to selection of an antibody, Cat316, which specifically recognizes C4S and blocks its inhibitory effects on axon growth. It also partly blocks binding of semaphorin 3A to PNNs and attenuates PNN formation. We asked whether injection of Cat316 into the perirhinal cortex would have the same effects on memory as chondroitinase ABC treatment. We found that masking C4S with the Cat316 antibody extended long-term object recognition memory in normal wild-type mice to 24 hours, similarly to chondroitinase or transgenic PNN attenuation. We then tested Cat316 for restoration of memory in a neurodegeneration model. Mice expressing tau with the P301S mutation showed profound loss of object recognition memory at 4 months of age. Injection of Cat316 into the perirhinal cortex normalized object recognition at 3 hours in P301S mice. These data indicate that Cat316 binding to C4S in the extracellular matrix can restore plasticity and memory in the same way as chondroitinase ABC digestion. Our results suggest that antibodies to C4S could be a useful therapeutic to restore memory function in neurodegenerative disorders. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Mapping the Differential Distribution of Proteoglycan Core Proteins in the Adult Human Retina, Choroid, and Sclera

    Science.gov (United States)

    Keenan, Tiarnan D. L.; Clark, Simon J.; Unwin, Richard D.; Ridge, Liam A.; Day, Anthony J.; Bishop, Paul N.

    2012-01-01

    Purpose. To examine the presence and distribution of proteoglycan (PG) core proteins in the adult human retina, choroid, and sclera. Methods. Postmortem human eye tissue was dissected into Bruch's membrane/choroid complex, isolated Bruch's membrane, or neurosensory retina. PGs were extracted and partially purified by anion exchange chromatography. Trypsinized peptides were analyzed by tandem mass spectrometry and PG core proteins identified by database search. The distribution of PGs was examined by immunofluorescence microscopy on human macular tissue sections. Results. The basement membrane PGs perlecan, agrin, and collagen-XVIII were identified in the human retina, and were present in the internal limiting membrane, blood vessel walls, and Bruch's membrane. The hyalectans versican and aggrecan were also detected. Versican was identified in Bruch's membrane, while aggrecan was distributed throughout the retina, choroid, and sclera. The cartilage link protein HAPLN1 was abundant in the interphotoreceptor matrix and sclera, while HAPLN4 (brain link protein 2) was found throughout the retina and choroid. The small leucine-rich repeat PG (SLRP) family members biglycan, decorin, fibromodulin, lumican, mimecan, opticin, and prolargin were present, with different patterns of distribution in the retina, choroid, and sclera. Conclusions. A combination of proteomics and immunohistochemistry approaches has provided for the first time a comprehensive analysis of the presence and distribution of PG core proteins throughout the human retina, choroid, and sclera. This complements our knowledge of glycosaminoglycan chain distribution in the human eye, and has important implications for understanding the structure and functional regulation of the eye in health and disease. PMID:23074202

  4. Crystal structure of syndesmos and its interaction with Syndecan-4 proteoglycan

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Heeyoun [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, 120-749 (Korea, Republic of); Yoo, Jiho [Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul, 120-749 (Korea, Republic of); Lee, Inhwan [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, 120-749 (Korea, Republic of); Kang, Ying Jin [Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul, 120-749 (Korea, Republic of); Cho, Hyun-Soo, E-mail: hscho8@yonsei.ac.kr [Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul, 120-749 (Korea, Republic of); Lee, Weontae, E-mail: wlee@spin.yonsei.ac.kr [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, 120-749 (Korea, Republic of)

    2015-08-07

    Syndesmos, nucleoside diphosphate linked moiety X (nudix)-type motif 16-like 1 (Nudt16l1), is evolutionarily divergent from the Nudt16 family. Syndesmos, which is co-localized with syndecan-4 cytoplasmic domain (Syn4{sup cyto}) in focal contacts, interacts with various cell adhesion adaptor proteins to control cell signaling. We determined the X-ray crystal structure of syndesmos; it is composed of seven α-helices and seven β-strands. Although syndesmos has a molecular topology similar to that of nudix hydrolase proteins, the structure of the nudix motif differs from that of X29. The dimeric interface of syndesmos is composed of α-helix 4, 7 and β-strand 2, 7, which primarily form hydrophobic interactions. The binding interaction between syndesmos and syn4{sup cyto} was characterized as a low-affinity interaction (K{sub d} = 62 μM) by surface plasmon resonance (SPR) and nuclear magnetic resonance (NMR). The NMR resonances of Lys (177, 178, 179), Gly182, and Ser183 in the C1 region and Lys193 and Lys194 in the V region of syndecan-4 are perturbed upon syndesmos binding. Our results provide structural insight into the molecular function of syndesmos in the regulation of cell signaling via binding to syndecan-4. - Highlights: • Crystal structure of syndesmos has been determined as a dimer at 2.01 Å resolution. • The molecular topology of syndesmos resembles that of the Nudix hydrolase protein. • The structure of the Nudix motif of syndesmos is quite different from that of X29. • Syndesmos binds cytoplasmic domain of syndecan-4 proteoglycan with low affinity.

  5. Up-regulation of proteoglycan 4 in temporomandibular osteoarthritic synovial cells by hyaluronic acid.

    Science.gov (United States)

    Guo, Huilin; Fang, Wei; Li, Yingjie; Ke, Jin; Deng, Mohong; Meng, Qinggong; Li, Jian; Long, Xing

    2015-09-01

    Hyaluronic acid (HA) injection is widely used in the treatment of temporomandibular joint (TMJ) osteoarthritis (OA). Proteoglycan 4 (PRG4) is another joint lubricant that protects surface of articular cartilage. But few studies had explored the role of HA in regulation of PRG4 expression in TMJ OA. In this study, the effects of HA on the expression of PRG4 in osteoarthritic TMJ synovial cells were investigated in hypoxia, which was similar to the TMJ physiologically. Synovial cells were isolated from the TMJ OA patients and were treated with or without HA under normoxia or hypoxia for indicated time periods. The proliferation of synovial cells was measured using Cell Counting Kit-8 (CCK-8). The gene expression of HAS2, VEGF, and PRG4 was detected by quantitative real-time PCR, and the secretion of PRG4 and VEGF was assayed by enzyme-linked immunosorbent assay (ELISA). Immunofluorescence was used to examine the protein expression of hypoxia-induced factor-1α (HIF-1α). Hyaluronic acid markedly increased the proliferation of osteoarthritic synovial cells in hypoxia. The expression of HAS2 and PRG4 mRNA of osteoarthritic synovial cells under hypoxia was enhanced by HA treatment. However, HA had no effect on reducing the VEGF and HIF-1α expression in synovial cells in hypoxia. Hyaluronic acid could promote the expression of HAS2 and PRG4, but could not modulate HIF-1α and VEGF expression of TMJ osteoarthritic synovial cells in hypoxia. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  6. Enrichment of committed human nucleus pulposus cells expressing chondroitin sulfate proteoglycans under alginate encapsulation.

    Science.gov (United States)

    Sun, Y; Lv, M; Zhou, L; Tam, V; Lv, F; Chan, D; Wang, H; Zheng, Z; Cheung, K M C; Leung, V Y L

    2015-07-01

    Intervertebral disc (IVD) degeneration is associated with a malfunction of the nucleus pulposus (NP). Alginate culturing provides a favorable microenvironment for the phenotypic maintenance of chondrocyte-like NP cells. However, NP cells are recently evidenced to present heterogeneous populations, including progenitors, fibroblastic cells and primitive NP cells. The aim of this study is to profile the phenotypic changes of distinct human NP cells populations and describe the dynamic expression of chondroitin sulfate glycosaminoglycans (CS-GAGs) in extended alginate encapsulation. Non-degenerated (ND-NPC) and degenerated (D-NPC) NP cells were expanded in monolayers, and subject to 28-day culture in alginate after serial passaging. CS-GAG compositional expression in monolayer-/alginate-cultured NP cells was evaluated by carbohydrate electrophoresis. Cellular phenotypic changes were assessed by immunologic detection and gene expression analysis. Relative to D-NPC, ND-NPC displayed remarkably higher expression levels of chondroitin-4-sulfate GAGs over the 28-day culture. Compared with monolayer culture, ND-NPC showed increased NP marker expression of KRT18, KRT19, and CDH2, as well as chondrocyte markers SOX9 and MIA in alginate culture. In contrast, expression of fibroblastic marker COL1A1, COL3A1, and FN1 were reduced. Interestingly, ND-NPC showed a loss of Tie2+ but gain in KRT19+/CD24+ population during alginate culture. In contrast, D-NPC showed more consistent expression levels of NP surface markers during culture. We demonstrate for the first time that extended alginate culture selectively enriches the committed NP cells and favors chondroitin-4-sulfate proteoglycan production. These findings suggest its validity as a model to investigate IVD cell function. Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  7. Differences in gene expression of human xylosyltransferases and determination of acceptor specificities for various proteoglycans

    Energy Technology Data Exchange (ETDEWEB)

    Roch, Christina; Kuhn, Joachim; Kleesiek, Knut [Institut fuer Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum NRW, Universitaetsklinik der Ruhr-Universitaet Bochum, 32545 Bad Oeynhausen (Germany); Goetting, Christian, E-mail: cgoetting@hdz-nrw.de [Institut fuer Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum NRW, Universitaetsklinik der Ruhr-Universitaet Bochum, 32545 Bad Oeynhausen (Germany)

    2010-01-01

    The xylosyltransferase (XT) isoforms XT-I and XT-II initiate the posttranslational glycosaminoglycan (GAG) synthesis. Here, we determined the relative expression of both isoforms in 33 human cell lines. The majority of tested cell lines showed dominant XYLT2 gene expression, while only in 23132/87, JAR, NCI-H510A and THP-1 was the XT-I mRNA expression higher. Nearly equal expression levels were detected in six cell lines. Additionally, to shed light on putative differences in acceptor specificities the acceptor properties of potential acceptor sequences were determined. Peptides were expressed as glutathione-S-transferase fusion proteins containing putative or known GAG attachment sites of in vivo proteoglycans. Kinetic analysis showed that K{sub m} and V{sub max} values for XT-I mediated xylosylation were slightly higher than those for XT-II, and that XT-I showed a lesser stringency concerning the acceptor sequence. Mutagenesis of the bikunin peptide sequence in the G-S-G attachment site and flanking regions generated potential acceptor molecules. Here, mutations on the N-terminal side and the attachment site were found to be more susceptible to a loss of acceptor function than mutations in the C-terminus. Altogether the known consensus sequence a-a-a-a-G-S-G-a-a/G-a ('a' representing Asp or Glu) for XT-I mediated xylosylation could be approved and additionally extended to apply to XT-II as well.

  8. Proteoglycan 4 and hyaluronan as boundary lubricants for model contact lens hydrogels.

    Science.gov (United States)

    Samsom, Michael; Iwabuchi, Yuno; Sheardown, Heather; Schmidt, Tannin A

    2017-07-07

    Clinical data show that in vitro contact lens friction is related to in vivo comfort. Solutions of biological lubricants hyaluronan (HA) and proteoglycan 4 (PRG4, also known as lubricin) reduce friction at a cornea-polydimethylsiloxane (PDMS) interface. The purpose of this study was to (1) determine if PRG4 can sorb to and lubricate model contact lens materials and (2) assess the boundary lubricating ability of PRG4 and HA compared to saline on model contact lens materials. PRG4 was obtained from bovine cartilage culture and suspended in saline at 300 µg/mL. N,N-Dimethylacrylamidetris (trimethylsiloxy) silane, (DMAA/TRIS) and methacryloxypropyltris (trimethylsiloxy) silane (pHEMA/TRIS) silicone hydrogels were prepared. A previously described in vitro eyelid-hydrogel and cornea-hydrogel biomechanical friction test was used to determine boundary lubricant effect. PRG4 sorption to the hydrogels was assessed using a soak-rinse protocol and western blotting. PRG4 effectively lubricated both silicone hydrogel materials and HA effectively lubricated pHEMA/TRIS, as indicated by a statistically significant reduction in friction compared to the saline control lubricant. An HA and PRG4 combination showed a synergistic effect for pHEMA/TRIS and effectively lubricated DMAA/TRIS. Biological boundary lubricants HA and PRG4 were shown to effectively lubricate silicone hydrogels when in solution. Additionally, HA and PRG4 showed synergistic lubrication for pHEMA/TRIS. The purpose of this study was not to replicate the friction coefficients of contact lenses, but rather to investigate lubricant-surface interactions for common contact lens constituents. These findings contribute to the potential development of biomolecule based lubricant drops for contact lens wearers. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2017. © 2017 Wiley Periodicals, Inc.

  9. Effects of sodium hyaluronate and methylprednisolone acetate on proteoglycan synthesis in equine articular cartilage explants.

    Science.gov (United States)

    Doyle, Aimie J; Stewart, Allison A; Constable, Peter D; Eurell, Jo Ann C; Freeman, David E; Griffon, Dominique J

    2005-01-01

    To determine effects of sodium hyaluronate (HA) on corticosteroid-induced cartilage matrix catabolism in equine articular cartilage explants. 30 articular cartilage explants from fetlock joints of 5 adult horses without joint disease. Articular cartilage explants were treated with control medium or medium containing methylprednisolone acetate (MPA; 0.05, 0.5, or 5.0 mg/mL), HA (0.1, 1.0, or 1.5 mg/mL), or both. Proteoglycan (PG) synthesis was measured by incorporation of sulfur 35-labeled sodium sulphate into PGs, and PG degradation was measured by release of radiolabeled PGs into the medium. Total glycosaminoglycan (GAG) content in media and explants and total explant DNA were determined. Methylprednisolone acetate caused a decrease in PG synthesis, whereas HA had no effect. Only the combination of MPA at a concentration of 0.05 mg/mL and HA at a concentration of 1.0 mg/mL increased PG synthesis, compared with control explants. Methylprednisolone acetate increased degradation of newly synthesized PGs into the medium, compared with control explants, and HA alone had no effect. Hyaluronate had no effect on MPA-induced PG degradation and release into media. Neither MPA alone nor HA alone had an effect on total cartilage GAG content. Methylprednisolone acetate caused an increase in release of GAG into the medium at 48 and 72 hours after treatment. In combination, HA had no protective effect on MPA-induced GAG release into the medium. Total cartilage DNA content was not affected by treatments. Our results indicate that HA addition has little effect on corticosteroid-induced cartilage matrix PG catabolism in articular cartilage explants.

  10. Chondroitin sulfate proteoglycans negatively regulate the positioning of mitochondria and endoplasmic reticulum to distal axons.

    Science.gov (United States)

    Sainath, Rajiv; Armijo-Weingart, Lorena; Ketscheck, Andrea; Xu, Zhuxuan; Li, Shuxin; Gallo, Gianluca

    2017-12-01

    Chondroitin sulfate proteoglycans (CSPGs) are components of the extracellular matrix that inhibit the extension and regeneration of axons. However, the underlying mechanism of action remains poorly understood. Mitochondria and endoplasmic reticulum (ER) are functionally inter-linked organelles important to axon development and maintenance. We report that CSPGs impair the targeting of mitochondria and ER to the growth cones of chicken embryonic sensory axons. The effect of CSPGs on the targeting of mitochondria is blocked by inhibition of the LAR receptor for CSPGs. The regulation of the targeting of mitochondria and ER to the growth cone by CSPGs is due to attenuation of PI3K signaling, which is known to be downstream of LAR receptor activation. Dynactin is a required component of the dynein motor complex that drives the normally occurring retrograde evacuation of mitochondria from growth cones. CSPGs elevate the levels of p150 Glu dynactin found in distal axons, and inhibition of the interaction of dynactin with dynein increased axon lengths on CSPGs. CSPGs decreased the membrane potential of mitochondria, and pharmacological inhibition of mitochondria respiration at the growth cone independent of manipulation of mitochondria positioning impaired axon extension. Combined inhibition of dynactin and potentiation of mitochondria respiration further increased axon lengths on CSPGs relative to inhibition of dynactin alone. These data reveal that the regulation of the localization of mitochondria and ER to growth cones is a previously unappreciated aspect of the effects of CSPGs on embryonic axons. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1351-1370, 2017. © 2017 Wiley Periodicals, Inc.

  11. Proteoglycans as Target for an Innovative Therapeutic Approach in Chondrosarcoma: Preclinical Proof of Concept.

    Science.gov (United States)

    Peyrode, Caroline; Weber, Valérie; Voissière, Aurélien; Maisonial-Besset, Aurélie; Vidal, Aurélien; Auzeloux, Philippe; Gaumet, Vincent; Borel, Michèle; Dauplat, Marie-Mélanie; Quintana, Mercedes; Degoul, Françoise; Rédini, Françoise; Chezal, Jean-Michel; Miot-Noirault, Elisabeth

    2016-11-01

    To date, surgery remains the only option for the treatment of chondrosarcoma, which is radio- and chemoresistant due in part to its large extracellular matrix (ECM) and poor vascularity. In case of unresectable locally advanced or metastatic diseases with a poor prognosis, improving the management of chondrosarcoma still remains a challenge. Our team developed an attractive approach of improvement of the therapeutic index of chemotherapy by targeting proteoglycan (PG)-rich tissues using a quaternary ammonium (QA) function conjugated to melphalan (Mel). First of all, we demonstrated the crucial role of the QA carrier for binding to aggrecan by surface plasmon resonance. In the orthotopic model of Swarm rat chondrosarcoma, an in vivo biodistribution study of Mel and its QA derivative (Mel-QA), radiolabeled with tritium, showed rapid radioactivity accumulation in healthy cartilaginous tissues and tumor after [3H]-Mel-QA injection. The higher T/M ratio of the QA derivative suggests some advantage of QA-active targeting of chondrosarcoma. The antitumoral effects were characterized by tumor volume assessment, in vivo 99mTc-NTP 15-5 scintigraphic imaging of PGs, 1H-HRMAS NMR spectroscopy, and histology. The conjugation of a QA function to Mel did not hamper its in vivo efficiency and strongly improved the tolerability of Mel leading to a significant decrease of side effects (hematologic analyses and body weight monitoring). Thus, QA conjugation leads to a significant improvement of the therapeutic index, which is essential in oncology and enable repeated cycles of chemotherapy in patients with chondrosarcoma. Mol Cancer Ther; 15(11); 2575-85. ©2016 AACR. ©2016 American Association for Cancer Research.

  12. Glucosamine exposure reduces proteoglycan synthesis in primary human endothelial cells in vitro

    Directory of Open Access Journals (Sweden)

    Trine M. Reine

    2016-09-01

    Full Text Available Purpose: Glucosamine (GlcN supplements are promoted for medical reasons, for example, for patients with arthritis and other joint-related diseases. Oral intake of GlcN is followed by uptake in the intestine, transport in the circulation and thereafter delivery to chondrocytes. Here, it is postulated to have an effect on synthesis and turnover of extracellular matrix constituents expressed by these cells. Following uptake in the intestine, serum levels are transiently increased, and the endothelium is exposed to increased levels of GlcN. We investigated the possible effects of GlcN on synthesis of proteoglycans (PGs, an important matrix component, in primary human endothelial cells. Methods: Primary human endothelial cells were cultured in vitro in medium with 5 mM glucose and 0–10 mM GlcN. PGs were recovered and analysed by western blotting, or by SDS-PAGE, gel chromatography or ion-exchange chromatography of 35S-PGs after 35S-sulphate labelling of the cells. Results: The synthesis and secretion of 35S-PGs from cultured endothelial cells were reduced in a dose- and time-dependent manner after exposure to GlcN. PGs are substituted with sulphated glycosaminoglycan (GAG chains, vital for PG function. The reduction in 35S-PGs was not related to an effect on GAG chain length, number or sulphation, but rather to the total expression of PGs. Conclusion: Exposure of endothelial cells to GlcN leads to a general decrease in 35S-PG synthesis. These results suggest that exposure to high levels of GlcN can lead to decreased matrix synthesis, contrary to what has been claimed by supporters of such supplements.

  13. Proteoglycan depletion and size reduction in lesions of early grade chondromalacia of the patella.

    Science.gov (United States)

    Väätäinen, U; Häkkinen, T; Kiviranta, I; Jaroma, H; Inkinen, R; Tammi, M

    1995-01-01

    OBJECTIVE--To determine the content and molecular size of proteoglycans (PGs) in patellar chondromalacia (CM) and control cartilages as a first step in investigating the role of matrix alterations in the pathogenesis of this disease. METHODS--Chondromalacia tissue from 10 patients was removed with a surgical knife. Using identical techniques, apparently healthy cartilage of the same site was obtained from 10 age matched cadavers (mean age 31 years in both groups). Additional pathological cartilage was collected from 67 patients with grades II-IV CM (classified according to Outerbridge) using a motorised shaver under arthroscopic control. The shaved cartilage chips were collected with a dense net from the irrigation fluid of the shaver. The content of tissue PGs was determined by Safranin O precipitation or uronic acid content, and the molecular size by mobility on agarose gel electrophoresis. RESULTS--The mean PG content of the CM tissue samples with a knife was dramatically reduced, being only 15% of that in controls. The cartilage chips collected from shaving operations of grades II, III, and IV CM showed a decreasing PG content: 9%, 5%, and 1% of controls, respectively. Electrophoretic analysis of PGs extracted with guanidium chloride from the shaved tissue samples suggested a significantly reduced size of aggrecans in the mild (grade II) lesions. CONCLUSION--These data show that there is already a dramatic and progressive depletion of PGs in CM grade II lesions. This explains the softening of cartilage, a typical finding in the arthroscopic examination of CM. The PG size reduction observed in grade II implicates proteolytic attack as a factor in the pathogenesis of CM. Images PMID:7492223

  14. Biosynthesis of promatrix metalloproteinase-9/chondroitin sulphate proteoglycan heteromer involves a Rottlerin-sensitive pathway.

    Directory of Open Access Journals (Sweden)

    Nabin Malla

    Full Text Available BACKGROUND: Previously we have shown that a fraction of the matrix metalloproteinase-9 (MMP-9 synthesized by the macrophage cell line THP-1 was bound to a chondroitin sulphate proteoglycan (CSPG core protein as a reduction sensitive heteromer. Several biochemical properties of the enzyme were changed when it was bound to the CSPG. METHODOLOGY/PRINCIPAL FINDINGS: By use of affinity chromatography, zymography, and radioactive labelling, various macrophage stimulators were tested for their effect on the synthesis of the proMMP-9/CSPG heteromer and its components by THP-1 cells. Of the stimulators, only PMA largely increased the biosynthesis of the heteromer. As PMA is an activator of PKC, we determined which PKC isoenzymes were expressed by performing RT-PCR and Western Blotting. Subsequently specific inhibitors were used to investigate their involvement in the biosynthesis of the heteromer. Of the inhibitors, only Rottlerin repressed the biosynthesis of proMMP-9/CSPG and its two components. Much lower concentrations of Rottlerin were needed to reduce the amount of CSPG than what was needed to repress the synthesis of the heteromer and MMP-9. Furthermore, Rottlerin caused a minor reduction in the activation of the PKC isoenzymes δ, ε, θ and υ (PKD3 in both control and PMA exposed cells. CONCLUSIONS/SIGNIFICANCE: The biosynthesis of the proMMP-9/CSPG heteromer and proMMP-9 in THP-1 cells involves a Rottlerin-sensitive pathway that is different from the Rottlerin sensitive pathway involved in the CSPG biosynthesis. MMP-9 and CSPGs are known to be involved in various physiological and pathological processes. Formation of complexes may influence both the specificity and localization of the enzyme. Therefore, knowledge about biosynthetic pathways and factors involved in the formation of the MMP-9/CSPG heteromer may contribute to insight in the heteromers biological function as well as pointing to future targets for therapeutic agents.

  15. Extracellular processing of the cartilage proteoglycan aggregate and its effect on CD44-mediated internalization of hyaluronan.

    Science.gov (United States)

    Danielson, Ben T; Knudson, Cheryl B; Knudson, Warren

    2015-04-10

    In many cells hyaluronan receptor CD44 mediates the endocytosis of hyaluronan and its delivery to endosomes/lysosomes. The regulation of this process remains largely unknown. In most extracellular matrices hyaluronan is not present as a free polysaccharide but often is found in complex with other small proteins and macromolecules such as proteoglycans. This is especially true in cartilage, where hyaluronan assembles into an aggregate structure with the large proteoglycan termed aggrecan. In this study when purified aggrecan was added to FITC-conjugated hyaluronan, no internalization of hyaluronan was detected. This suggested that the overall size of the aggregate prevented hyaluronan endocytosis and furthermore that proteolysis of the aggrecan was a required prerequisite for local, cell-based turnover of hyaluronan. To test this hypothesis, limited C-terminal digestion of aggrecan was performed to determine whether a size range of aggrecan exists that permits hyaluronan endocytosis. Our data demonstrate that only limited degradation of the aggrecan monomer was required to allow for hyaluronan internalization. When hyaluronan was combined with partially degraded, dansyl chloride-labeled aggrecan, blue fluorescent aggrecan was also visualized within intracellular vesicles. It was also determined that sonicated hyaluronan of smaller molecular size was internalized more readily than high molecular mass hyaluronan. However, the addition of intact aggrecan to hyaluronan chains sonicated for 5 and 10 s reblocked their endocytosis, whereas aggregates containing 15-s sonicated hyaluronan were internalized. These data suggest that hyaluronan endocytosis is regulated in large part by the extracellular proteolytic processing of hyaluronan-bound proteoglycan. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. The dermatan sulfate proteoglycan decorin modulates α2β1 integrin and the vimentin intermediate filament system during collagen synthesis.

    Directory of Open Access Journals (Sweden)

    Oliver Jungmann

    Full Text Available Decorin, a small leucine-rich proteoglycan harboring a dermatan sulfate chain at its N-terminus, is involved in regulating matrix organization and cell signaling. Loss of the dermatan sulfate of decorin leads to an Ehlers-Danlos syndrome characterized by delayed wound healing. Decorin-null (Dcn(-/- mice display a phenotype similar to that of EDS patients. The fibrillar collagen phenotype of Dcn(-/- mice could be rescued in vitro by decorin but not with decorin lacking the glycosaminoglycan chain. We utilized a 3D cell culture model to investigate the impact of the altered extracellular matrix on Dcn(-/- fibroblasts. Using 2D gel electrophoresis followed by mass spectrometry, we identified vimentin as one of the proteins that was differentially upregulated by the presence of decorin. We discovered that a decorin-deficient matrix leads to abnormal nuclear morphology in the Dcn(-/- fibroblasts. This phenotype could be rescued by the decorin proteoglycan but less efficiently by the decorin protein core. Decorin treatment led to a significant reduction of the α2β1 integrin at day 6 in Dcn(-/- fibroblasts, whereas the protein core had no effect on β1. Interestingly, only the decorin core induced mRNA synthesis, phosphorylation and de novo synthesis of vimentin indicating that the proteoglycan decorin in the extracellular matrix stabilizes the vimentin intermediate filament system. We could support these results in vivo, because the dermis of wild-type mice have more vimentin and less β1 integrin compared to Dcn(-/-. Furthermore, the α2β1 null fibroblasts also showed a reduced amount of vimentin compared to wild-type. These data show for the first time that decorin has an impact on the biology of α2β1 integrin and the vimentin intermediate filament system. Moreover, our findings provide a mechanistic explanation for the reported defects in wound healing associated with the Dcn(-/- phenotype.

  17. Selective inhibition of proteoglycan and hyaluronate synthesis in chondrocyte cultures by cyclofenil diphenol, a non-steroidal weak oestrogen.

    OpenAIRE

    Mason, R.M.; Lineham, J D; Phillipson, M A; Black, C M

    1984-01-01

    Cyclofenil diphenol, a weak non-steroidal oestrogen, binds to albumin. In the presence of concentrations of albumin just sufficient to keep cyclofenil diphenol in solution, the compound inhibited the synthesis of [35S]proteoglycans, [3H]glycoproteins, [3H]hyaluronate and [3H]proteins in primary cultures of chondrocytes from the Swarm rat chondrosarcoma in a dose-dependent manner. When excess albumin was present, conditions were found (90 micrograms of cyclofenil diphenol and 4 mg of albumin p...

  18. [The effects of core proteoglycan on the expressions of I and III collagen in human renal tubular epithelial cell induced by TGFbeta1 in vitro].

    Science.gov (United States)

    Cheng, Xue-Qin; Bao, Hua-Ying; Pan, Xiao-Qin; Fei, Li; Huang, Song-Ming; Zhang, Wei-Zhen

    2008-12-01

    To explore the roles of core proteoglycan and TGFbeta1 on the expressions of I and III collagen in human renal tubular epithelial cell line(HK-2) in vitro. Confluent HK-2 cells were exposed to TGFbeta1 and core proteoglycan for up to 48 h. The cells were divided into four groups. Group (1), negative control group; group(2), single 10 microg/L TGFbeta1 treated group; group (3), 10 microg/L TGFbeta1+10 microg/L core proteoglycan group; group (4), 10 microg/L TGFbeta1+100 microg/L core proteoglycan group. Morphologic characterization of HK-2 cells was shown by invertmicroscope; Precise amounts of I and III collagen mRNA were measured by RT-PCR. After 48 h, morphology of (1) group cells had no changes, most cells were normal shape; (2) group cells took great changes, most cells converted into spindle shape, like fibroblast, (3) and (4) groups, spindle shape cells reduced significantly. In contrast to (1) group, the expressions of I collagen in (2) group from mRNA significant increased by 27.86-fold. The expressions of III collagen increased by 21.83-fold. Comparing (3) and (4) groups to(2) group, the expressions of I collagen from mRNA effectively decreased 36.39% and 53.36%. III collagen expressions increased 26.35% and 47.96%èP<0.05érespectively. But, neither (3) group nor (4) group alone could regulate I and III collagen mRNA to normal levels. Core proteoglycan can inhibit the expressions of I and III collagen in HK-2 cells induced by TGFbeta1 in vitro. Possibly, suggest core proteoglycan contribute to the regulation of renal fibrosis.

  19. The epidermal growth factor-like domain of the human cartilage large aggregating proteoglycan, aggrecan: increased serum concentration in rheumatoid arthritis

    DEFF Research Database (Denmark)

    Møller, H J; Ingemann-Hansen, T; Poulsen, J H

    1994-01-01

    The large aggregating proteoglycan from human cartilage, aggrecan, has recently been shown to possess an immunologically detectable domain with close homology to epidermal growth factor (EGF), that is variably expressed by alternative mRNA splicing. Using a competitive ELISA we detected this domain...... in sera from both patients with RA and normal controls. The EGF-like domain could only be detected after digestion of sera with chondroitinase ABC, which demonstrates its proteoglycan origin. The concentration of the aggrecan EGF-like domain was considerably elevated in sera from patients with RA...

  20. Effect of disulfide bonding and multimerization on proteoglycan 4's cartilage boundary lubricating ability and adsorption.

    Science.gov (United States)

    Abubacker, Saleem; Ponjevic, Dragana; Ham, Hyun O; Messersmith, Phillip B; Matyas, John R; Schmidt, Tannin A

    2016-01-01

    The objectives of this study were to assess the cartilage boundary lubricating ability of (1) nonreduced (NR) disulfide-bonded proteoglycan 4 (PRG4) multimers versus PRG4 monomers and (2) NR versus reduced and alkylated (R/A) PRG4 monomers and to assess (3) the ability of NR PRG4 multimers versus monomers to adsorb to an articular cartilage surface. PRG4 was separated into two preparations, PRG4 multimer enriched (PRG4Multi+) and PRG4 multimer deficient (PRG4Multi-), using size exclusion chromatography (SEC) and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The cartilage boundary lubricating ability of PRG4Multi+ and PRG4Multi- was compared at a physiological concentration (450 μg/mL) and assessed over a range of concentrations (45, 150, and 450 μg/mL). R/A and NR PRG4Multi- were evaluated at 450 μg/mL. Immunohistochemistry with anti-PRG4 antibody 4D6 was performed to visualize the adsorption of PRG4 preparations to the surface of articular cartilage explants. Separation into enriched populations of PRG4Multi+ and PRG4Multi- was achieved using SEC and was confirmed by SDS-PAGE. PRG4Multi+ and PRG4Multi- both functioned as effective friction-reducing cartilage boundary lubricants at 450 μg/mL, with PRG4Multi+ being more effective than PRG4Multi-. PRG4Multi+ lubricated in a dose-dependent manner, however, PRG4Multi- did not. R/A PRG4Multi- lubricated similar to NR PRG4Multi-. PRG4-containing solutions showed 4D6 immunoreactivity at the articular surface; the immunoreactive intensity of PRG4Multi+ appeared to be similar to SF, whereas PRG4Multi- appeared to have less intensity. These results demonstrate that the intermolecular disulfide-bonded multimeric structure of PRG4 is important for its ability to adsorb to a cartilage surface and function as a boundary lubricant. These findings contribute to a greater understanding of the molecular basis of cartilage boundary lubrication of PRG4. Elucidating the PRG4 structure

  1. Characterization and flocculability of a novel proteoglycan produced by Talaromyces trachyspermus OU5.

    Science.gov (United States)

    Fang, Di; Shi, Cuicui

    2016-01-01

    A filamentous fungus strain OU5 was isolated from a soil sample for its ability to produce rich exopolymers (EPS), with high flocculation capability towards kaolin suspension and swine wastewater, at low-carbon source conditions. EPS from strain OU5 was extracted and characterized to determine its flocculating behavior and active constituents involved in the flocculation. Strain OU5 was identified as Talaromyces trachyspermus by 18S rDNA-ITS gene sequencing and morphological observation. The extracted EPS was a novel proteoglycan (designated as BF-OU5) composed of 84.6% (w/w) polysaccharides and 15.2% (w/w) proteins. The enzymatic digestion tests revealed that the polysaccharides in BF-OU5, composed of 67% glucose, 16.4% mannose, 8.6% xylose and 8% galactose, contributed to 99.7% of flocculating capacity and were the major active ingredients in the flocculation. By contrast, the proteins in BF-OU5 only had minor roles in the flocculation. The presence of hydroxyl, amide, carboxyl and methoxyl functional groups in BF-OU5, and the high molecular weight (1.053 × 10(5)-2.970 × 10(5) Da) as well as the structure of a spherical conformation with inner pores and channels made of cross-linked netted textures contributed to the flocculation. A dosage of 20 mg/l BF-OU5 initiated more than 92.5% of flocculating efficiency towards kaolin suspension without any added coagulants; its flocculability was stable over a wide range of pH (4.0-8.0) and temperature (20°C-100°C). Treatment of swine wastewater using BF-OU5 achieved 52.1% flocculating removal for chemical oxygen demand, 39.7% for Kjeldahl nitrogen, 18.6% for NH4(+)-N, 21.5% for total phosphorus, and 75% for turbidity. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  2. Diagnostic performance of alpha-fetoprotein, lens culinaris agglutinin-reactive alpha-fetoprotein, des-gamma carboxyprothrombin, and glypican-3 for the detection of hepatocellular carcinoma: a systematic review and meta-analysis protocol.

    Science.gov (United States)

    Huang, Ting-Shuo; Shyu, Yu-Chiau; Turner, Robin; Chen, Huang-Yang; Chen, Pei-Jer

    2013-06-06

    Diagnosis of early-stage hepatocellular carcinoma (HCC) followed by curative resection or liver transplantation offers the best chance for long-term patient survival. Clinically, ultrasonography has suboptimal sensitivity for detecting early-stage HCC. Several serological tests including alpha-fetoprotein (AFP), the ratio of lens culinaris agglutinin-reactive alpha-fetoprotein to total AFP (AFP-L3/AFP), des-gamma carboxyprothrombin (DCP), and glypican-3 (GPC-3) have been widely investigated as diagnostic biomarkers for early-stage HCC in at-risk populations. However, these tests are not recommended for routine HCC screening. Our objective is to determine the diagnostic performance of AFP, AFP-L3/AFP, DCP, and GPC-3 for the detection of HCC, particularly early-stage tumors meeting the Milan criteria. We will include cross-sectional studies that consecutively or randomly recruit target populations. We will search the Cochrane Library, Medline, Embase, Science Citation Index, and the Chinese National Knowledge Infrastructure. We will also search the MEDION and ARIF databases to identify diagnostic systematic reviews that include primary studies. Reference lists of relevant reviews will be searched for additional trials. Language restrictions will not be applied. Two reviewers will independently screen study eligibility and extract data. Methodological quality will be assessed according to the revised tool for the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2). Two authors will apply the QUADAS-2 assessment to all the included studies, and any discrepancies will be resolved by the third author. The following test characteristics will be extracted into 2 × 2 tables for all included studies: true positives, false positives, true negatives, and false negatives. Study-specific estimates of sensitivity and specificity with 95% confidence intervals will be displayed in forest plots. When possible, we will use the bivariate random-effects model or the Rutter

  3. The NG2 Proteoglycan Protects Oligodendrocyte Precursor Cells against Oxidative Stress via Interaction with OMI/HtrA2.

    Science.gov (United States)

    Maus, Frank; Sakry, Dominik; Binamé, Fabien; Karram, Khalad; Rajalingam, Krishnaraj; Watts, Colin; Heywood, Richard; Krüger, Rejko; Stegmüller, Judith; Werner, Hauke B; Nave, Klaus-Armin; Krämer-Albers, Eva-Maria; Trotter, Jacqueline

    2015-01-01

    The NG2 proteoglycan is characteristically expressed by oligodendrocyte progenitor cells (OPC) and also by aggressive brain tumours highly resistant to chemo- and radiation therapy. Oligodendrocyte-lineage cells are particularly sensitive to stress resulting in cell death in white matter after hypoxic or ischemic insults of premature infants and destruction of OPC in some types of Multiple Sclerosis lesions. Here we show that the NG2 proteoglycan binds OMI/HtrA2, a mitochondrial serine protease which is released from damaged mitochondria into the cytosol in response to stress. In the cytosol, OMI/HtrA2 initiates apoptosis by proteolytic degradation of anti-apoptotic factors. OPC in which NG2 has been downregulated by siRNA, or OPC from the NG2-knockout mouse show an increased sensitivity to oxidative stress evidenced by increased cell death. The proapoptotic protease activity of OMI/HtrA2 in the cytosol can be reduced by the interaction with NG2. Human glioma expressing high levels of NG2 are less sensitive to oxidative stress than those with lower NG2 expression and reducing NG2 expression by siRNA increases cell death in response to oxidative stress. Binding of NG2 to OMI/HtrA2 may thus help protect cells against oxidative stress-induced cell death. This interaction is likely to contribute to the high chemo- and radioresistance of glioma.

  4. Heparanase affects food intake and regulates energy balance in mice.

    Directory of Open Access Journals (Sweden)

    Linda Karlsson-Lindahl

    Full Text Available Mutation of the melanocortin-receptor 4 (MC4R is the most frequent cause of severe obesity in humans. Binding of agouti-related peptide (AgRP to MC4R involves the co-receptor syndecan-3, a heparan sulfate proteoglycan. The proteoglycan can be structurally modified by the enzyme heparanase. Here we tested the hypothesis that heparanase plays a role in food intake behaviour and energy balance regulation by analysing body weight, body composition and food intake in genetically modified mice that either lack or overexpress heparanase. We also assessed food intake and body weight following acute central intracerebroventricular administration of heparanase; such treatment reduced food intake in wildtype mice, an effect that was abolished in mice lacking MC4R. By contrast, heparanase knockout mice on a high-fat diet showed increased food intake and maturity-onset obesity, with up to a 40% increase in body fat. Mice overexpressing heparanase displayed essentially the opposite phenotypes, with a reduced fat mass. These results implicate heparanase in energy balance control via the central melanocortin system. Our data indicate that heparanase acts as a negative modulator of AgRP signaling at MC4R, through cleavage of heparan sulfate chains presumably linked to syndecan-3.

  5. Differential effects of transforming growth factors on localization of adhesion complex proteins following corneal epithelial cell wounding.

    Science.gov (United States)

    Gassner, H L; Esco, M; Smithson, M W; Kurpakus, M A

    1997-04-01

    The differential effects of transforming growth factor (TGF) alpha, beta 1 and beta 2 on the de novo localization of heparan sulfate proteoglycan, collagen type VII and laminin-1 to the adhesion complex were analyzed using an in vitro model of corneal epithelial cell wound healing. Bovine corneal explants were maintained in culture media containing either no growth factor or 1, 5, or 10 ng/ml TGF alpha, TGF beta 1 or TGF beta 2. After 24 or 48 hours in culture, cryostat sections of explants were processed for immunofluorescence microscopy using antibodies directed against heparan sulfate proteoglycan, collagen type VII or laminin-1. A comparison of antibody labeling patterns and relative fluorescence intensity of antibody labeling to controls suggested that TGF alpha inhibits the spatial polarization of proteins into the reforming adhesion complex during early stages of wound healing. Both TGF beta 1 and beta 2 enhanced the linear localization of the three proteins to the site of the reforming adhesion complex. However, in our model TGF beta isoforms did not have identical functions. TGF beta 2 accelerated the temporal localization of collagen type VII to the adhesion complex, an effect which was not observed with TGF beta 1. TGF beta, but not TGF alpha, may play an important role in corneal epithelial cell wound healing by accelerating the reformation of the adhesion complex and subsequent epithelial cell-extracellular matrix adhesion.

  6. Syndecan regulates cell migration and axon guidance in C. elegans.

    Science.gov (United States)

    Rhiner, Christa; Gysi, Stephan; Fröhli, Erika; Hengartner, Michael O; Hajnal, Alex

    2005-10-01

    During nervous system development, axons that grow out simultaneously in the same extracellular environment are often sorted to different target destinations. As there is only a restricted set of guidance cues known, regulatory mechanisms are likely to play a crucial role in controlling cell migration and axonal pathfinding. Heparan sulfate proteoglycans (HSPGs) carry long chains of differentially modified sugar residues that have been proposed to encode specific information for nervous system development. Here, we show that the cell surface proteoglycan syndecan SDN-1 functions autonomously in neurons to control the neural migration and guidance choices of outgrowing axons. Epistasis analysis suggests that heparan sulfate (HS) attached to SDN-1 can regulate guidance signaling by the Slit/Robo pathway. Furthermore, SDN-1 acts in parallel with other HSPG core proteins whose HS side chains are modified by the C5-epimerase HSE-5, and/or the 2O-sulfotransferase HST-2, depending on the cellular context. Taken together, our experiments show that distinct HS modification patterns on SDN-1 are involved in regulating axon guidance and cell migration in C. elegans.

  7. Heparanase isoform expression and extracellular matrix remodeling in intervertebral disc degenerative disease

    Directory of Open Access Journals (Sweden)

    Luciano Miller Reis Rodrigues

    2011-01-01

    Full Text Available OBJECTIVE: To determine the molecules involved in extracellular matrix remodeling and to identify and quantify heparanase isoforms present in herniated and degenerative discs. INTRODUCTION: Heparanase is an endo-beta-glucuronidase that specifically acts upon the heparan sulfate chains of proteoglycans. However, heparanase expression in degenerative intervertebral discs has not yet been evaluated. Notably, previous studies demonstrated a correlation between changes in the heparan sulfate proteoglycan pattern and the degenerative process associated with intervertebral discs. METHODS: Twenty-nine samples of intervertebral degenerative discs, 23 samples of herniated discs and 12 samples of non-degenerative discs were analyzed. The expression of both heparanase isoforms (heparanase-1 and heparanase-2 was evaluated using immunohistochemistry and real-time RT-PCR analysis. RESULTS: Heparanase-1 and heparanase-2 expression levels were significantly higher in the herniated and degenerative discs in comparison to the control tissues, suggesting a possible role of these proteins in the intervertebral degenerative process. CONCLUSION: The overexpression of heparanase isoforms in the degenerative intervertebral discs and the herniated discs suggests a potential role of both proteins in the mediation of inflammatory processes and in extracellular matrix remodeling. The heparanase-2 isoform may be involved in normal metabolic processes, as evidenced by its higher expression in the control intervertebral discs relative to the expression of heparanase-1.

  8. Anti–citrullinated protein antibody positivity correlates with cartilage damage and proteoglycan levels in patients with rheumatoid arthritis in the hand joints.

    Science.gov (United States)

    Renner, Nina; Krönke, Gerhard; Rech, Jürgen; Uder, Michael; Janka, Rolf; Lauer, Lars; Paul, Dominik; Herz, Barbara; Schlechtweg, Philipp; Hennig, Friedrich Frank; Schett, Georg; Welsch, Götz

    2014-12-01

    Objective: To investigate the factors associated with cartilage proteoglycan content in patients with rheumatoid arthritis (RA) Methods: 32 RA patients received high-field 3 Tesla Gadolinium-Enhanced MRI of Cartilage (dGEMRIC) for determining cartilage proteoglycan content. Measurements were performed in three individual cartilage regions (medial, central, lateral) of the metacarpophalangeal joints 2 and 3. dGEMRIC values were then related to disease duration, disease activity, anti-citrullinated protein antibody (ACPA) status, rheumatoid factor status and C-reactive protein level. Results: dGEMRIC values were not significantly different between the MCP2 and MCP3 joint. Inter-class correlations were high (>0.92) for all three (medial, central and lateral) cartilage compartments. dGEMRIC values were significantly lower in RA patients with longer disease duration (≥3 years) and those with ACPA positivity than those with a short disease duration (proteoglycan content and disease activity, C-reactive protein level and rheumatoid factor status was found. Conclusion: Decreased cartilage proteoglycan content in RA patients is associated with disease duration and ACPA positivity but not with the actual disease activity, CRP level or rheumatoid factor status. These data suggest that the cumulative burden of inflammation as well as ACPA are the determinants for cartilage damage in RA.

  9. Gold nanoparticles coated with a pyruvated trisaccharide epitope of the extracellular proteoglycan of Microciona prolifera as potential tools to explore carbohydrate-mediated cell recognition

    NARCIS (Netherlands)

    de Souza, A.C.; Vliegenthart, J.F.G.|info:eu-repo/dai/nl/06785267X; Kamerling, J.P.|info:eu-repo/dai/nl/070433941

    2008-01-01

    The species-specific cell adhesion in the marine sponge Microciona prolifera involves the interaction of an extracellular proteoglycan-like macromolecular complex, otherwise known as aggregation factor. In the interaction, two highly polyvalent functional domains play a role: a cell-binding and a

  10. ELISA for the core protein of the cartilage large aggregating proteoglycan, aggrecan: comparison with the concentrations of immunogenic keratan sulphate in synovial fluid, serum and urine

    DEFF Research Database (Denmark)

    Møller, H J; Larsen, F S; Ingemann-Hansen, T

    1994-01-01

    Immunological assays for fragments of the cartilage large aggregating proteoglycan, aggrecan, have been widely used to monitor cartilage turnover. These assays have commonly employed the monoclonal keratan sulphate antibody, 5D4. Keratan sulphate, however, is present in many tissues and 5D4 affin...