Identification of thioaptamer ligand against E-selectin: potential application for inflamed vasculature targeting.

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Aman P Mann

Full Text Available Active targeting of a drug carrier to a specific target site is crucial to provide a safe and efficient delivery of therapeutics and imaging contrast agents. E-selectin expression is induced on the endothelial cell surface of vessels in response to inflammatory stimuli but is absent in the normal vessels. Thus, E-selectin is an attractive molecular target, and high affinity ligands for E-selectin could be powerful tools for the delivery of therapeutics and/or imaging agents to inflamed vessels. In this study, we identified a thiophosphate modified aptamer (thioaptamer, TA against E-selectin (ESTA-1 by employing a two-step selection strategy: a recombinant protein-based TA binding selection from a combinatorial library followed by a cell-based TA binding selection using E-selectin expressing human microvascular endothelial cells. ESTA-1 selectively bound to E-selectin with nanomolar binding affinity (K(D = 47 nM while exhibiting minimal cross reactivity to P- and L-selectin. Furthermore, ESTA-1 binding to E-selectin on the endothelial cells markedly antagonized the adhesion (over 75% inhibition of sLe(x positive HL-60 cells at nanomolar concentration. ESTA-1 also bound specifically to the inflamed tumor-associated vasculature of human carcinomas derived from breast, ovarian, and skin but not to normal organs, and this binding was highly associated with the E-selectin expression level. Similarly, intravenously injected ESTA-1 demonstrated distinct binding to the tumor vasculature in a breast cancer xenograft model. Together, our data substantiates the discovery of a thioaptamer (ESTA-1 that binds to E-selectin with high affinity and specificity, thereby highlighting the potential application of ESTA-1 for E-selectin targeted delivery.

Expression of E-selectin ligands on circulating tumor cells: cross-regulation with cancer stem cell regulatory pathways?

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Burdick, Monica M.; Henson, Karissa A.; Delgadillo, Luis F.; Choi, Young Eun; Goetz, Douglas J.; Tees, David F. J.; Benencia, Fabian

2012-01-01

Although significant progress has been made in the fight against cancer, successful treatment strategies have yet to be developed to combat those tumors that have metastasized to distant organs. Poor characterization of the molecular mechanisms of cancer spread is a major impediment to designing predictive diagnostics and effective clinical interventions against late stage disease. In hematogenous metastasis, it is widely suspected that circulating tumor cells (CTCs) express specific adhesion molecules that actively initiate contact with the vascular endothelium lining the vessel walls of the target organ. This “tethering” is mediated by ligands expressed by CTCs that bind to E-selectin expressed by endothelial cells. However, it is currently unknown whether expression of functional E-selectin ligands on CTCs is related to cancer stem cell regulatory or maintenance pathways, particularly epithelial-to-mesenchymal transition and the reverse, mesenchymal-to-epithelial transition. In this hypothesis and theory article, we explore the potential roles of these mechanisms on the dynamic regulation of selectin ligands mediating CTC trafficking during metastasis.

Coordinated and unique functions of the E-selectin ligand ESL-1 during inflammatory and hematopoietic recruitment in mice.

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Sreeramkumar, Vinatha; Leiva, Magdalena; Stadtmann, Anika; Pitaval, Christophe; Ortega-Rodríguez, Inés; Wild, Martin K; Lee, Brendan; Zarbock, Alexander; Hidalgo, Andrés

2013-12-05

Beyond its well-established roles in mediating leukocyte rolling, E-selectin is emerging as a multifunctional receptor capable of inducing integrin activation in neutrophils, and of regulating various biological processes in hematopoietic precursors. Although these effects suggest important homeostatic contributions of this selectin in the immune and hematologic systems, the ligands responsible for transducing these effects in different leukocyte lineages are not well defined. We have characterized mice deficient in E-selectin ligand-1 (ESL-1), or in both P-selectin glycoprotein-1 (PSGL-1) and ESL-1, to explore and compare the contributions of these glycoproteins in immune and hematopoietic cell trafficking. In the steady state, ESL-1 deficiency resulted in a moderate myeloid expansion that became more prominent when both glycoproteins were eliminated. During inflammation, PSGL-1 dominated E-selectin binding, rolling, integrin activation, and extravasation of mature neutrophils, but only the combined deficiency in PSGL-1 and ESL-1 completely abrogated leukocyte recruitment. Surprisingly, we find that the levels of ESL-1 were strongly elevated in hematopoietic progenitor cells. These elevations correlated with a prominent function of ESL-1 for E-selectin binding and for migration of hematopoietic progenitor cells into the bone marrow. Our results uncover dominant roles for ESL-1 in the immature compartment, and a functional shift toward PSGL-1 dependence in mature neutrophils.

Evolutionary conservation of P-selectin glycoprotein ligand-1 primary structure and function

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Schapira Marc

2007-09-01

Full Text Available Abstract Background P-selectin glycoprotein ligand-1 (PSGL-1 plays a critical role in recruiting leukocytes in inflammatory lesions by mediating leukocyte rolling on selectins. Core-2 O-glycosylation of a N-terminal threonine and sulfation of at least one tyrosine residue of PSGL-1 are required for L- and P-selectin binding. Little information is available on the intra- and inter-species evolution of PSGL-1 primary structure. In addition, the evolutionary conservation of selectin binding site on PSGL-1 has not been previously examined in detail. Therefore, we performed multiple sequence alignment of PSGL-1 amino acid sequences of 14 mammals (human, chimpanzee, rhesus monkey, bovine, pig, rat, tree-shrew, bushbaby, mouse, bat, horse, cat, sheep and dog and examined mammalian PSGL-1 interactions with human selectins. Results A signal peptide was predicted in each sequence and a propeptide cleavage site was found in 9/14 species. PSGL-1 N-terminus is poorly conserved. However, each species exhibits at least one tyrosine sulfation site and, except in horse and dog, a T [D/E]PP [D/E] motif associated to the core-2 O-glycosylation of a N-terminal threonine. A mucin-like domain of 250–280 amino acids long was disclosed in all studied species. It lies between the conserved N-terminal O-glycosylated threonine (Thr-57 in human and the transmembrane domain, and contains a central region exhibiting a variable number of decameric repeats (DR. Interspecies and intraspecies polymorphisms were observed. Transmembrane and cytoplasmic domain sequences are well conserved. The moesin binding residues that serve as adaptor between PSGL-1 and Syk, and are involved in regulating PSGL-1-dependent rolling on P-selectin are perfectly conserved in all analyzed mammalian sequences. Despite a poor conservation of PSGL-1 N-terminal sequence, CHO cells co-expressing human glycosyltransferases and human, bovine, pig or rat PSGL-1 efficiently rolled on human L- or P-selectin

Expression of E-selectin ligand-1 (CFR/ESL-1) on hepatic stellate cells: implications for leukocyte extravasation and liver metastasis.

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Antoine, Marianne; Tag, Carmen G; Gressner, Axel M; Hellerbrand, Claus; Kiefer, Paul

2009-02-01

Leukocytes and tumor cells use E-selectin binding ligands to attach to activated endothelial cells expressing E-selectin during inflammation or metastasis. The cysteine-rich fibroblast growth factor receptor (CFR) represents the main E-selectin ligand (ESL-1) on granulocytes and its expression is exclusively modified by alpha(1,3)-fucosyltransferases IV or VII (FucT4 and FucT7). Hepatic stellate cells (HSC) are pericytes of liver sinusoidal endothelial cells. The activation of HSC and transdifferentiation into a myofibroblastic phenotype is involved in the repair of liver tissue injury, liver regeneration and angiogenesis of liver metastases. In the present study, we demonstrated that HSC expressed CFR together with FucT7 and exhibited a functional E-selectin binding activity on their cell surface. Since HSC appear to be oxygen-sensing cells, the expression of E-selectin binding activity was analyzed in HSC under a hypoxic atmosphere. While the expression of the glycoprotein CFR was unaffected by hypoxia, the cell-associated E-selectin binding activity decreased. However, under the same conditions, mRNA expression of the modifying enzyme FucT7 increased. The loss of E-selectin binding activity, therefore, appears to be neither the result of a reduced expression of the modifying transferase nor the expression of the backbone glycoprotein. After the transient transfection of HSC with CFR cDNA, the E-selectin binding activity (ESL-1) was efficiently released into the supernatant. Therefore, we hypothesize that under hypoxia, ESL-1 is shed from activated HSC. Our findings provide a novel perspective on the function of HSC in liver metastasis and inflammatory liver diseases.

A Panel of Recombinant Mucins Carrying a Repertoire of Sialylated O-Glycans Based on Different Core Chains for Studies of Glycan Binding Proteins

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Reeja Maria Cherian

2015-08-01

Full Text Available Sialylated glycans serve as key elements of receptors for many viruses, bacteria, and bacterial toxins. The microbial recognition and their binding specificity can be affected by the linkage of the terminal sugar residue, types of underlying sugar chains, and the nature of the entire glycoconjugate. Owing to the pathobiological significance of sialylated glycans, we have engineered Chinese hamster ovary (CHO cells to secrete mucin-type immunoglobulin-fused proteins carrying terminal α2,3- or α2,6-linked sialic acid on defined O-glycan core saccharide chains. Besides stably expressing P-selectin glycoprotein ligand-1/mouse immunoglobulin G2b cDNA (PSGL-1/mIgG2b, CHO cells were stably transfected with plasmids encoding glycosyltransferases to synthesize core 2 (GCNT1, core 3 (B3GNT6, core 4 (GCNT1 and B3GNT6, or extended core 1 (B3GNT3 chains with or without the type 1 chain-encoding enzyme B3GALT5 and ST6GAL1. Western blot and liquid chromatography-mass spectrometry analysis confirmed the presence of core 1, 2, 3, 4, and extended core 1 chains carrying either type 1 (Galb3GlcNAc or type 2 (Galb4GlcNAc outer chains with or without α2,6-linked sialic acids. This panel of recombinant mucins carrying a repertoire of sialylated O-glycans will be important tools in studies aiming at determining the fine O-glycan binding specificity of sialic acid-specific microbial adhesins and mammalian lectins.

Short consensus repeat domains extend the E-selectin structure in order to grab cells out of flow

KAUST Repository

Aleisa, Fajr A

2017-01-08

Selectins are key adhesion molecules responsible for initiating a multistep process that leads a cell out of the blood circulation and into a tissue or organ. They are composed of an N-terminal extracellular C-type lectin like domain, followed by an Endothelial Growth Factor like domain (EGF), a defined number of short consensus repeats SCR (also called “sushi” domains), a transmembrane domain and a C-terminal cytoplasmic tail. The adhesion of cells (expressing ligands) to the endothelium (expressing the selection i.e., E-selectin) occurs through the interaction between the lectin domain of selectins and sLeX presenting ligands. Structural/function studies to date have mainly focused on investigating the influence of the lectin domain of E-selectin on its ability to bind its ligands while other domains received less atention. We prepared a number of different recombinant E-selectin proteins with changes in the SCR units. Specifically we generated wild-type E-selectin proteins as monomeric or dimeric structures, mutant proteins with varied numbers of SCRs as well as proteins where strategic residues were mutated to change the conformation of the selectin. Using a novel real time immunoprecipitation surface plasmon resonance (SPR)-based in vitro binding study developed in our lab, the interaction of recombinant E-selectin proteins with immunoprecipitated endogenous ligands (i.e. CD44) captured on a CM-5 chip was assessed. These studies provided quantitative binding kinetics with on and off rates of selectin-ligand interactions and suggested that robust binding is dependent on the presence of the SCRs and oligomerization. These results provide significant implications on the functional mechanism of E-selectin binding to its ligands.

Short consensus repeat domains extend the E-selectin structure in order to grab cells out of flow

KAUST Repository

Aleisa, Fajr A; Sakashita, Kosuke; Lee, Jaeman; Abu Samra, Dina Bashir Kamil; Habuchi, Satoshi; Kusakabe, Takahiro; Merzaban, Jasmeen

2017-01-01

Selectins are key adhesion molecules responsible for initiating a multistep process that leads a cell out of the blood circulation and into a tissue or organ. They are composed of an N-terminal extracellular C-type lectin like domain, followed by an Endothelial Growth Factor like domain (EGF), a defined number of short consensus repeats SCR (also called “sushi” domains), a transmembrane domain and a C-terminal cytoplasmic tail. The adhesion of cells (expressing ligands) to the endothelium (expressing the selection i.e., E-selectin) occurs through the interaction between the lectin domain of selectins and sLeX presenting ligands. Structural/function studies to date have mainly focused on investigating the influence of the lectin domain of E-selectin on its ability to bind its ligands while other domains received less atention. We prepared a number of different recombinant E-selectin proteins with changes in the SCR units. Specifically we generated wild-type E-selectin proteins as monomeric or dimeric structures, mutant proteins with varied numbers of SCRs as well as proteins where strategic residues were mutated to change the conformation of the selectin. Using a novel real time immunoprecipitation surface plasmon resonance (SPR)-based in vitro binding study developed in our lab, the interaction of recombinant E-selectin proteins with immunoprecipitated endogenous ligands (i.e. CD44) captured on a CM-5 chip was assessed. These studies provided quantitative binding kinetics with on and off rates of selectin-ligand interactions and suggested that robust binding is dependent on the presence of the SCRs and oligomerization. These results provide significant implications on the functional mechanism of E-selectin binding to its ligands.

E-selectin ligand-1 (ESL-1) is a novel adiponectin binding protein on cell adhesion

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Yamamoto, Hiroyasu; Kuroda, Nana; Uekita, Hiromi; Kochi, Ikoi; Matsumoto, Akane; Niinaga, Ryu [Department of Biomedical Informatics, Division of Health Sciences, Osaka University Graduate School of Medicine, Osaka (Japan); Funahashi, Tohru; Shimomura, Iichiro [Department of Metabolic Medicine, Osaka University Graduate School of Medicine, Osaka (Japan); Kihara, Shinji, E-mail: skihara@sahs.med.osaka-u.ac.jp [Department of Biomedical Informatics, Division of Health Sciences, Osaka University Graduate School of Medicine, Osaka (Japan)

2016-02-05

Background: Adiponectin (APN) is an adipocyte-derived bioactive molecule with anti-diabetic and anti-atherogenic properties. Although anti-diabetic effects are mostly mediated by the adiponectin receptors AdipoR1 and AdipoR2, the anti-atherogenic mechanisms have not been fully elucidated. Methods and Results: In this study, we identified E-selectin ligand (ESL)-1 as a novel APN-binding protein by mass spectrometry analysis of HepG2 cell-derived immunoprecipitant with an anti-APN antibody. Cell adhesion assays using fluorescence-labelled monocyte cell line THP-1 cells and human umbilical vein endothelial cells (HUVECs) revealed that APN-pre-treated THP-1 cells had reduced binding ability to HUVECs. This APN-mediated suppressive effect on monocyte binding to endothelial cells was partially abrogated by targeting ESL-1 with shRNA in THP-1 cells. In addition, serial mutagenesis analysis disclosed that five extracellular amino acids close to the N-terminus of ESL-1 were essential for binding with APN. Conclusion: Our results highlight the fact that interaction between APN and ESL-1 could provide a fundamental mechanism underlying the anti-atherogenic properties of APN. - Highlights: • E-selectin ligand (ESL)-1 was identified as an adiponectin (APN)-binding protein. • ESL-1 bound to APN at its N-terminal 6th-10th amino acids. • shESL-1 reduced the suppressive effect of APN on adhesion of THP-1 cells to HUVECs. • Interaction with ESL may be involved in the anti-atherogenic effects of APN.

E-selectin ligand-1 (ESL-1) is a novel adiponectin binding protein on cell adhesion

International Nuclear Information System (INIS)

Yamamoto, Hiroyasu; Kuroda, Nana; Uekita, Hiromi; Kochi, Ikoi; Matsumoto, Akane; Niinaga, Ryu; Funahashi, Tohru; Shimomura, Iichiro; Kihara, Shinji

2016-01-01

Background: Adiponectin (APN) is an adipocyte-derived bioactive molecule with anti-diabetic and anti-atherogenic properties. Although anti-diabetic effects are mostly mediated by the adiponectin receptors AdipoR1 and AdipoR2, the anti-atherogenic mechanisms have not been fully elucidated. Methods and Results: In this study, we identified E-selectin ligand (ESL)-1 as a novel APN-binding protein by mass spectrometry analysis of HepG2 cell-derived immunoprecipitant with an anti-APN antibody. Cell adhesion assays using fluorescence-labelled monocyte cell line THP-1 cells and human umbilical vein endothelial cells (HUVECs) revealed that APN-pre-treated THP-1 cells had reduced binding ability to HUVECs. This APN-mediated suppressive effect on monocyte binding to endothelial cells was partially abrogated by targeting ESL-1 with shRNA in THP-1 cells. In addition, serial mutagenesis analysis disclosed that five extracellular amino acids close to the N-terminus of ESL-1 were essential for binding with APN. Conclusion: Our results highlight the fact that interaction between APN and ESL-1 could provide a fundamental mechanism underlying the anti-atherogenic properties of APN. - Highlights: • E-selectin ligand (ESL)-1 was identified as an adiponectin (APN)-binding protein. • ESL-1 bound to APN at its N-terminal 6th-10th amino acids. • shESL-1 reduced the suppressive effect of APN on adhesion of THP-1 cells to HUVECs. • Interaction with ESL may be involved in the anti-atherogenic effects of APN.

Comparison of printed glycan array, suspension array and ELISA in the detection of human anti-glycan antibodies.

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Pochechueva, Tatiana; Jacob, Francis; Goldstein, Darlene R; Huflejt, Margaret E; Chinarev, Alexander; Caduff, Rosemarie; Fink, Daniel; Hacker, Neville; Bovin, Nicolai V; Heinzelmann-Schwarz, Viola

2011-12-01

Anti-glycan antibodies represent a vast and yet insufficiently investigated subpopulation of naturally occurring and adaptive antibodies in humans. Recently, a variety of glycan-based microarrays emerged, allowing high-throughput profiling of a large repertoire of antibodies. As there are no direct approaches for comparison and evaluation of multi-glycan assays we compared three glycan-based immunoassays, namely printed glycan array (PGA), fluorescent microsphere-based suspension array (SA) and ELISA for their efficacy and selectivity in profiling anti-glycan antibodies in a cohort of 48 patients with and without ovarian cancer. The ABO blood group glycan antigens were selected as well recognized ligands for sensitivity and specificity assessments. As another ligand we selected P(1), a member of the P blood group system recently identified by PGA as a potential ovarian cancer biomarker. All three glyco-immunoassays reflected the known ABO blood groups with high performance. In contrast, anti-P(1) antibody binding profiles displayed much lower concordance. Whilst anti-P(1) antibody levels between benign controls and ovarian cancer patients were significantly discriminated using PGA (p=0.004), we got only similar results using SA (p=0.03) but not for ELISA. Our findings demonstrate that whilst assays were largely positively correlated, each presents unique characteristic features and should be validated by an independent patient cohort rather than another array technique. The variety between methods presumably reflects the differences in glycan presentation and the antigen/antibody ratio, assay conditions and detection technique. This indicates that the glycan-antibody interaction of interest has to guide the assay selection. © The Author(s) 2011. This article is published with open access at Springerlink.com

Minimal sulfated carbohydrates for recognition by L-selectin and the MECA-79 antibody.

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Bruehl, R E; Bertozzi, C R; Rosen, S D

2000-10-20

Sulfated forms of sialyl-Le(X) containing Gal-6-SO(4) or GlcNAc-6-SO(4) have been implicated as potential recognition determinants on high endothelial venule ligands for L-selectin. The optimal configuration of sulfate esters on the N-acetyllactosamine (Galbeta1-->4GlcNAc) core of sulfosialyl-Le(X), however, remains unsettled. Using a panel of sulfated lactose (Galbeta1-->4Glc) neoglycolipids as substrates in direct binding assays, we found that 6',6-disulfolactose was the preferred structure for L-selectin, although significant binding to 6'- and 6-sulfolactose was observed as well. Binding was EDTA-sensitive and blocked by L-selectin-specific monoclonal antibodies. Surprisingly, 6', 6-disulfolactose was poorly recognized by MECA-79, a carbohydrate- and sulfate-dependent monoclonal antibody that binds competitively to L-selectin ligands. Instead, MECA-79 bound preferentially to 6-sulfolactose. The difference in preferred substrates between L-selectin and MECA-79 may explain the variable activity of MECA-79 as an inhibitor of lymphocyte adhesion to high endothelial venules in lymphoid organs. Our results suggest that both Gal-6-SO(4) and GlcNAc-6-SO(4) may contribute to L-selectin recognition, either as components of sulfosialyl-Le(X) capping groups or in internal structures. By contrast, only GlcNAc-6-SO(4) appears to contribute to MECA-79 binding.

Cancer cell–derived microparticles bearing P-selectin glycoprotein ligand 1 accelerate thrombus formation in vivo

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Thomas, Grace M.; Panicot-Dubois, Laurence; Lacroix, Romaric; Dignat-George, Françoise; Lombardo, Dominique

2009-01-01

Recent publications have demonstrated the presence of tissue factor (TF)–bearing microparticles (MPs) in the blood of patients suffering from cancer. However, whether these MPs are involved in thrombosis remains unknown. We show that pancreatic and lung cancer cells produce MPs that express active TF and P-selectin glycoprotein ligand 1 (PSGL-1). Cancer cell–derived MPs aggregate platelets via a TF-dependent pathway. In vivo, cancer cell–derived MPs, but not their parent cells, infused into a living mouse accumulate at the site of injury and reduce tail bleeding time and the time to occlusion of venules and arterioles. This thrombotic state is also observed in mice developing tumors. In such mice, the amount of circulating platelet-, endothelial cell–, and cancer cell–derived MPs is increased. Endogenous cancer cell–derived MPs shed from the growing tumor are able to accumulate at the site of injury. Infusion of a blocking P-selectin antibody abolishes the thrombotic state observed after injection of MPs or in mice developing a tumor. Collectively, our results indicate that cancer cell–derived MPs bearing PSGL-1 and TF play a key role in thrombus formation in vivo. Targeting these MPs could be of clinical interest in the prevention of thrombosis and to limit formation of metastasis in cancer patients. PMID:19667060

E-selectin: sialyl Lewis, a dependent adhesion of colon cancer cells, is inhibited differently by antibodies against E-selectin ligands.

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Srinivas, U; Påhlsson, P; Lundblad, A

1996-09-01

Recent studies have demonstrated that selectins, a new family of cell-adhesion molecules with similar domain structures, mediate the adhesion of peripheral blood cells to interleukin-1 (IL-1)-activated endothelium. In the present study the authors evaluated the role of E-selectin-Sialyl Lewis x (SLe(x))/ Sialyl Lewis a (SLe(a)) interaction in mediating in vitro adhesion of two colon cancer cell lines, HT-29 and COLO 201, to human umbilical cord endothelial cells (HUVEC). Colon cancer cell lines had a strong expression of blood group-related carbohydrate epitopes as evaluated by fluorescence-activated cell sorter (FACS) analysis. It was established that adhesion of HT-29 and COLO 201 cells to IL-1 stimulated HUVEC was calcium dependent and could be inhibited by a monoclonal antibody directed against E-selectin. Prior incubation of cells with two different antibodies directed against SLe(x) and antibodies directed against related Lewis epitopes, Le(x) and Le(a), had no significant effect on adhesion. Three antibodies directed against SLe(a) differed in their capacity to inhibit the adhesion of HT-29 and COLO 201 cells to HUVEC. Only one antibody directed against the SLe(a) structure was effective in inhibiting adhesion of both COLO 201 and HT-29 cells. The difference could not be attributed to titre, the type or number of glycoproteins, or to a difference in the amount of SLe(a) present on individual proteins, suggesting that presence and right presentation of SLe(a) epitope might be important for adhesion of colon cancer cells. Finally, in the in vitro system used, adhesion of HT-29 and COLO 201 cells to activated HUVEC is mediated predominantly by E-selectin/SLe(a) interaction. SLe(x) and related epitopes, Le(x) and Le(a), seem to have limited relevance for colon cancer cell recognition of E-selectin.

TIM-1 glycoprotein binds the adhesion receptor P-selectin and mediates T cell trafficking during inflammation and autoimmunity

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Angiari, Stefano; Donnarumma, Tiziano; Rossi, Barbara; Dusi, Silvia; Pietronigro, Enrica; Zenaro, Elena; Della Bianca, Vittorina; Toffali, Lara; Piacentino, Gennj; Budui, Simona; Rennert, Paul; Xiao, Sheng; Laudanna, Carlo; Casasnovas, Jose M.; Kuchroo, Vijay K.; Constantin, Gabriela

2014-01-01

SUMMARY Selectins play a central role in leukocyte trafficking by mediating tethering and rolling on vascular surfaces. Here we have reported that T cell immunoglobulin and mucin domain 1 (TIM-1) is a P-selectin ligand. We have shown that human and murine TIM-1 binds to P-selectin, and that TIM-1 mediates tethering and rolling of T helper-1 (Th1) and Th17, but not Th2 and regulatory T cells on P-selectin. Th1 and Th17 cells lacking the TIM-1 mucin domain showed reduced rolling in thrombin-activated mesenteric venules and inflamed brain microcirculation. Inhibition of TIM-1 had no effect on naive T cell homing, but reduced T cell recruitment in a skin hypersensitivity model and blocked experimental autoimmune encephalomyelitis. Uniquely, the TIM-1 IgV domain was also required for P-selectin binding. Our data demonstrate that TIM-1 is a major P-selectin ligand with a specialized role in T cell trafficking during inflammatory responses and the induction of autoimmune disease. PMID:24703780

An automatic braking system that stabilizes leukocyte rolling by an increase in selectin bond number with shear.

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Chen, S; Springer, T A

1999-01-11

Wall shear stress in postcapillary venules varies widely within and between tissues and in response to inflammation and exercise. However, the speed at which leukocytes roll in vivo has been shown to be almost constant within a wide range of wall shear stress, i.e., force on the cell. Similarly, rolling velocities on purified selectins and their ligands in vitro tend to plateau. This may be important to enable rolling leukocytes to be exposed uniformly to activating stimuli on endothelium, independent of local hemodynamic conditions. Wall shear stress increases the rate of dissociation of individual selectin-ligand tether bonds exponentially (, ) thereby destabilizing rolling. We find that this is compensated by a shear-dependent increase in the number of bonds per rolling step. We also find an increase in the number of microvillous tethers to the substrate. This explains (a) the lack of firm adhesion through selectins at low shear stress or high ligand density, and (b) the stability of rolling on selectins to wide variation in wall shear stress and ligand density, in contrast to rolling on antibodies (). Furthermore, our data successfully predict the threshold wall shear stress below which rolling does not occur. This is a special case of the more general regulation by shear of the number of bonds, in which the number of bonds falls below one.

L-selectin and skin damage in systemic sclerosis.

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James V Dunne

Full Text Available L-selectin ligands are induced on the endothelium of inflammatory sites. L-selectin expression on neutrophils and monocytes may mediate the primary adhesion of these cells at sites of inflammation by mediating the leukocyte-leukocyte interactions that facilitate their recruitment. L-selectin retains functional activity in its soluble form. Levels of soluble L-selectin have been reported as both elevated and lowered in patients with systemic sclerosis (SSc. This preliminary study seeks to discern amongst these disparate results and to discover whether there is an association between L-selectin concentrations in plasma and skin damage in SSc patients.Nineteen cases with limited systemic sclerosis (lSSc and 11 cases with diffuse systemic sclerosis (dSSc were compared on a pairwise basis to age- and sex-matched controls. Criteria of the American College of Rheumatology were used to diagnose SSc. Skin involvement was assessed using the modified Rodnan skin score (mRSS. We find no association between mRSS and plasma L-selectin concentration in lSSc cases (p = 0.9944 but a statistically significant negative correlation in dSSc cases (R(2 = 73.11 per cent, p = 0.0008. The interpretation of the slope for dSSc cases is that for each increase of 100 ng/ml in soluble L-selectin concentration, the mRSS drops 4.22 (95 per cent CI: 2.29, 6.16. There was also a highly statistically significant negative correlation between sL-selectin and disease activity (p = 0.0007 and severity (p = 0.0007 in dSSc cases but not in lSSc cases (p = 0.2596, p = 0.7575, respectively.No effective treatments exist for skin damage in SSc patients. Nor is there a laboratory alternative to the modified Rodnan skin score as is the case for other organs within the body. Modulation of circulating L-selectin is a promising target for reducing skin damage in dSSc patients. Plasma levels of soluble L-selectin could serve as an outcome measure for dSSc patients in

Not just a marker: CD34 on human hematopoietic stem/progenitor cells dominates vascular selectin binding along with CD44

KAUST Repository

Abu Samra, Dina Bashir Kamil; Aleisa, Fajr A; Al-Amoodi, Asma S.; Jalal Ahmed, Heba M.; Chin, Chee Jia; AbuElela, Ayman; Bergam, Ptissam; Sougrat, Rachid; Merzaban, Jasmeen

2017-01-01

CD34 is routinely used to identify and isolate human hematopoietic stem/progenitor cells (HSPCs) for use clinically in bone marrow transplantation, but its function on these cells remains elusive. Glycoprotein ligands on HSPCs help guide their migration to specialized microvascular beds in the bone marrow that express vascular selectins (E- and P-selectin). Here, we show that HSPC-enriched fractions from human hematopoietic tissue expressing CD34 (CD34pos) bound selectins, whereas those lacking CD34 (CD34neg) did not. An unbiased proteomics screen identified potential glycoprotein ligands on CD34pos cells revealing CD34 itself as a major vascular selectin ligand. Biochemical and CD34 knockdown analyses highlight a key role for CD34 in the first prerequisite step of cell migration, suggesting that it is not just a marker on these cells. Our results also entice future potential strategies to investigate the glycoforms of CD34 that discriminate normal HSPCs from leukemic cells and to manipulate CD34neg HSPC-enriched bone marrow or cord blood populations as a source of stem cells for clinical use.

Not just a marker: CD34 on human hematopoietic stem/progenitor cells dominates vascular selectin binding along with CD44

KAUST Repository

Abu Samra, Dina Bashir Kamil

2017-12-27

CD34 is routinely used to identify and isolate human hematopoietic stem/progenitor cells (HSPCs) for use clinically in bone marrow transplantation, but its function on these cells remains elusive. Glycoprotein ligands on HSPCs help guide their migration to specialized microvascular beds in the bone marrow that express vascular selectins (E- and P-selectin). Here, we show that HSPC-enriched fractions from human hematopoietic tissue expressing CD34 (CD34pos) bound selectins, whereas those lacking CD34 (CD34neg) did not. An unbiased proteomics screen identified potential glycoprotein ligands on CD34pos cells revealing CD34 itself as a major vascular selectin ligand. Biochemical and CD34 knockdown analyses highlight a key role for CD34 in the first prerequisite step of cell migration, suggesting that it is not just a marker on these cells. Our results also entice future potential strategies to investigate the glycoforms of CD34 that discriminate normal HSPCs from leukemic cells and to manipulate CD34neg HSPC-enriched bone marrow or cord blood populations as a source of stem cells for clinical use.

Expression and Purification of Glycosyltransferases in Pichia Pastoris: Towards Improving the Migration of Stem Cells by Enhancing Surface Expression of Sialyl Lewis X

KAUST Repository

Al-Amoodi, Asma S.

2017-05-01

Recruitment of circulating cells towards target sites is primarily dependent on E-selectin receptor/ligand adhesive interactions. Glycosyltransferase (GTs) are involved in the creation of E-selectin ligands. A sialofucosylated terminal tetrasaccharide like glycan structure known as sialyl Lewis x (sLex), is the most recognized ligand by selectins. This structure is found on the surface of cancer cells and leukocytes but is often absent on the surface of many adult stem cell populations. In order to synthesize sLex, GTs must be endogenously expressed and remain active within the cells. Generally, these stem cells express terminal sialylated lactosamine structures on their glycoproteins which require the addition of alpha-(1,3)-fucose to be converted into an E-selectin ligand. There are a number of fucosyltransferases (FUTs) that are able to modify terminal lactosamine structures to create sLex such as FUT6. In this work we focused on expressing and purifying active recombinant FUTs as a tool to help create sLex structures on the surface of adult stem cells in order to enhance their migration.

Selectins mediate small cell lung cancer systemic metastasis.

Directory of Open Access Journals (Sweden)

Franziska Heidemann

Full Text Available Metastasis formation is the major reason for the extremely poor prognosis in small cell lung cancer (SCLC patients. The molecular interaction partners regulating metastasis formation in SCLC are largely unidentified, however, from other tumor entities it is known that tumor cells use the adhesion molecules of the leukocyte adhesion cascade to attach to the endothelium at the site of the future metastasis. Using the human OH-1 SCLC line as a model, we found that these cells expressed E- and P-selectin binding sites, which could be in part attributed to the selectin binding carbohydrate motif sialyl Lewis A. In addition, protein backbones known to carry these glycotopes in other cell lines including PSGL-1, CD44 and CEA could be detected in in vitro and in vivo grown OH1 SCLC cells. By intravital microscopy of murine mesenterial vasculature we could capture SCLC cells while rolling along vessel walls demonstrating that SCLC cells mimic leukocyte rolling behavior in terms of selectin and selectin ligand interaction in vivo indicating that this mechanism might indeed be important for SCLC cells to seed distant metastases. Accordingly, formation of spontaneous distant metastases was reduced by 50% when OH-1 cells were xenografted into E-/P-selectin-deficient mice compared with wild type mice (p = 0.0181. However, as metastasis formation was not completely abrogated in selectin deficient mice, we concluded that this adhesion cascade is redundant and that other molecules of this cascade mediate metastasis formation as well. Using several of these adhesion molecules as interaction partners presumably make SCLC cells so highly metastatic.

L-selectin-carbohydrate interactions: relevant modifications of the Lewis x trisaccharide.

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Sanders, W J; Katsumoto, T R; Bertozzi, C R; Rosen, S D; Kiessling, L L

1996-11-26

Protein-carbohydrate interactions are known to mediate cell-cell recognition and adhesion events. Specifically, three carbohydrate binding proteins termed selectins (E-, P-, and L-selectin) have been shown to be essential for leukocyte rolling along the vascular endothelium, the first step in the recruitment of leukocytes from the blood into inflammatory sites or into secondary lymphoid organs. Although this phenomenon is well-established, little is known about the molecular-level interactions on which it depends. All three selectins recognize sulfated and sialylated derivatives of the Lewis x [Le(x):Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc] and Lewis a [Le(a): Gal beta 1-->3(Fuc alpha 1-->4)GlcNAc] trisaccharide cores with affinities in the millimolar range, and it is believed that variants of these structures are the carbohydrate determinants of selectin recognition. Recently it was shown that the mucin GlyCAM-1, a secreted physiological ligand for L-selectin, is capped with sulfated derivatives of sialyl Lewis x [sLe(x): Sia alpha 2-->3Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc] and that sulfation is required for the high-affinity interaction between GlyCAM-1 and L-selectin. To elucidate the important sites of sulfation on Le(x) with respect to L-selectin recognition, we have synthesized six sulfated Le(x) analogs and determined their abilities to block binding of a recombinant L-selectin-Ig chimera to immobilized GlyCAM-1. Our results suggest that 6-sulfo sLe(x) binds to L-selectin with higher affinity than does sLe(x) or 6'-sulfo sLe(x) and that sulfation of sLe(x) capping groups on GlyCAM-1 at the 6-position is important for L-selectin recognition.

Identification of the homing molecules that escort pluripotent stem cells-derived hematopoietic stem cells to their niches and human activated T-cells to inflammatory sites.

KAUST Repository

Ali, Amal

2017-12-01

Hematopoietic cells exploit the multistep paradigm of cell migration to ultimately enable them to perform their function. This process is dictated by the ability of adhesion molecules on the circulating hematopoietic cells to find their counter-receptors on endothelial cells. Of those molecules, the selectin family and their respective ligands induce the initial transient interactions between circulating cells and the opposing endothelium. In this thesis, I focused on studying E-selectin mediated cellular migration in two hematopoietic cell types, namely human hematopoietic stem and progenitor cells (HSPCs) and human T-lymphocytes. HSPCs derived from pluripotent sources theoretically offers a novel, unlimited source for hematopoietic stem cell transplantation therapy. In vitro pluripotent stem cell derived- hematopoietic stem/progenitor cells (ES/iPS-HSPCs) behave much like somatic HSPCs in that they exhibit clonal expansion and multilineage hematopoietic capacity. However, unlike somatic sources, ES/iPS-HSPCs do not give rise to effective hematopoietic repopulation, which may be due to insufficient HSPCs homing to the bone marrow. HSPCs exploit E- and P-selectin to home and engraft into bone marrow niches. Thus, one of my objectives in this thesis was to study the expression of E-selectin ligands associated with ES/iPS-HSPCs. I showed that ES/iPS-HSPCs lack functional E-selectin ligand(s). In an effort to enhance the interaction between Eselectin and ES/iPS-HSPCs, we decorated the cell surface with sialyl-Lewis x (sLex) using the ex-vivo glycan engineering technology. However, this decoration did not improve the engraftment capacity of ES/iPS-HSPCs, in vivo. Induction of E-selectin expression during inflammation is key to recruitment of immune cells and therefore I also focused on analyzing the expression of E-selectin ligands on activated human T-cells. I identified several novel glycoproteins that may function as E-selectin ligands. Specifically, I compared the

A single midcycle dose of levonorgestrel similar to emergency contraceptive does not alter the expression of the L-selectin ligand or molecular markers of endometrial receptivity.

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Palomino, Wilder Alberto; Kohen, Paulina; Devoto, Luigi

2010-10-01

To examine the effects of a single-dose of 1.5 mg of levonorgestrel (commonly used as emergency contraceptive) on endometrial receptivity biomarkers through the oral or vaginal route. Prospective randomized single-blinded trial. Affiliated Hospital and University Research Center. Fertile normal women previously sterilized by tubal ligation. Levonorgestrel (1.5 mg) was administered on the day of LH surge either orally (n = 14) or vaginally (n = 13). Molecular assessment of endometrial progesterone receptors, L-selectin ligand, glicodelin-A and αvβ3 integrin by Immunohistochemistry and reverse transcriptase-polymerase chain reaction. Plasma progesterone concentration and endometrial dating were not different. The pattern of progesterone receptors and glycodelin-A expression was not affected during the early and midsecretory phase. Some endometrial biopsies from the group in which levonorgetrel was orally administered showed areas of glandular atrophy and stromal decidualization. However, the expression of the progesterone receptor, L-selectin ligand, αvβ3 integrin, and glycodelin-A were not different between the groups. Levonorgestrel, given as emergency contraceptive on the day of LH surge, does not disrupt either ovulation or progesterone production by the corpus luteum. The contraceptive mechanism of levonorgestrel at the time of LH surge does not include changes in the progesterone receptors or the endometrial receptivity biomarkers. Copyright © 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Direct Enzymatic Branch-End Extension of Glycocluster-Presented Glycans: An Effective Strategy for Programming Glycan Bioactivity.

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Bayón, Carlos; He, Ning; Deir-Kaspar, Mario; Blasco, Pilar; André, Sabine; Gabius, Hans-Joachim; Rumbero, Ángel; Jiménez-Barbero, Jesús; Fessner, Wolf-Dieter; Hernáiz, María J

2017-01-31

The sequence of a glycan and its topology of presentation team up to determine the specificity and selectivity of recognition by saccharide receptors (lectins). Structure-activity analysis would be furthered if the glycan part of a glycocluster could be efficiently elaborated in situ while keeping all other parameters constant. By using a bacterial α2,6-sialyltransferase and a small library of bi- to tetravalent glycoclusters, we illustrate the complete conversion of scaffold-presented lactoside units into two different sialylated ligands based on N-acetyl/glycolyl-neuraminic acid incorporation. We assess the ensuing effect on their bioactivity for a plant toxin, and present an analysis of the noncovalent substrate binding contacts that the added sialic acid moiety makes to the lectin. Enzymatic diversification of a scaffold-presented glycan can thus be brought to completion in situ, offering a versatile perspective for rational glycocluster engineering. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Role of siglecs and related glycan-binding proteins in immune responses and immunoregulation.

Science.gov (United States)

Bochner, Bruce S; Zimmermann, Nives

2015-03-01

Virtually all cells and extracellular material are heavily decorated by various glycans, yet our understanding of the structure and function of these moieties lags behind the understanding of nucleic acids, lipids, and proteins. Recent years have seen a tremendous acceleration of knowledge in the field of glycobiology, revealing many intricacies and functional contributions that were previously poorly appreciated or even unrecognized. This review highlights several topics relevant to glycoimmunology in which mammalian and pathogen-derived glycans displayed on glycoproteins and other scaffolds are recognized by specific glycan-binding proteins (GBPs), leading to a variety of proinflammatory and anti-inflammatory cellular responses. The focus for this review is mainly on 2 families of GBPs, sialic acid-binding immunoglobulin-like lectins (siglecs) and selectins, that are involved in multiple steps of the immune response, including distinguishing pathogens from self, cell trafficking to sites of inflammation, fine-tuning of immune responses leading to activation or tolerance, and regulation of cell survival. Importantly for the clinician, accelerated rates of discovery in the field of glycoimmunology are being translated into innovative medical approaches that harness the interaction of glycans and GBPs to the benefit of the host and might soon lead to novel diagnostics and therapeutics. Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

E-selectin ligand-1 (ESL-1) is a novel adiponectin binding protein on cell adhesion.

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Yamamoto, Hiroyasu; Kuroda, Nana; Uekita, Hiromi; Kochi, Ikoi; Matsumoto, Akane; Niinaga, Ryu; Funahashi, Tohru; Shimomura, Iichiro; Kihara, Shinji

2016-02-05

Adiponectin (APN) is an adipocyte-derived bioactive molecule with anti-diabetic and anti-atherogenic properties. Although anti-diabetic effects are mostly mediated by the adiponectin receptors AdipoR1 and AdipoR2, the anti-atherogenic mechanisms have not been fully elucidated. In this study, we identified E-selectin ligand (ESL)-1 as a novel APN-binding protein by mass spectrometry analysis of HepG2 cell-derived immunoprecipitant with an anti-APN antibody. Cell adhesion assays using fluorescence-labelled monocyte cell line THP-1 cells and human umbilical vein endothelial cells (HUVECs) revealed that APN-pre-treated THP-1 cells had reduced binding ability to HUVECs. This APN-mediated suppressive effect on monocyte binding to endothelial cells was partially abrogated by targeting ESL-1 with shRNA in THP-1 cells. In addition, serial mutagenesis analysis disclosed that five extracellular amino acids close to the N-terminus of ESL-1 were essential for binding with APN. Our results highlight the fact that interaction between APN and ESL-1 could provide a fundamental mechanism underlying the anti-atherogenic properties of APN. Copyright © 2016 Elsevier Inc. All rights reserved.

Biomechanics of P-selectin PSGL-1 bonds: Shear threshold and integrin-independent cell adhesion

Energy Technology Data Exchange (ETDEWEB)

Xiao, Zhihua; Goldsmith, Harry L.; MacIntosh, Fiona A.; Shankaran, Harish; Neelamegham, Sriram

2006-03-01

Platelet-leukocyte adhesion may contribute to thrombosis and inflammation. We examined the heterotypic interaction between unactivated neutrophils and either thrombin receptor activating peptide (TRAP) stimulated platelets or P-selectin bearing beads (Ps-beads) in suspension. Cone-plate viscometers were used to apply controlled shear rates from 14-3000/s. Platelet-neutrophil and bead-neutrophil adhesion analysis was performed using both flow cytometry and high-speed videomicroscopy. We observed that while blocking antibodies against either P-selectin or P-selectin glycoprotein ligand-1 (PSGL-1) alone inhibited platelet-neutrophil adhesion by ~60% at 140/s, these reagents completely blocked adhesion at 3000/s. Anti-Mac-1 alone did not alter platelet-neutrophil adhesion rates at any shear rate, though in synergy with selectin antagonists it abrogated cell binding. Unstimulated neutrophils avidly bound Ps-beads and activated platelets in an integrin-independent manner, suggesting that purely selectin-dependent cell adhesion is possible. In support of this, antagonists against P-selectin or PSGL-1 dissociated previously formed platelet-neutrophil and Ps-bead neutrophil aggregates under shear in a variety of experimental systems, including in assays performed with whole blood. In studies where medium viscosity and shear rate were varied, a subtle shear threshold for P-selectin PSGL-1 binding was also noted at shear rates<100/s and at force loading rates of ~300pN/sec. Results are discussed in light of biophysical computations that characterize the collision between unequal size particles in linear shear flow. Overall, our studies reveal an integrin-independent regime for cell adhesion that may be physiologically relevant.

Using affinity capillary electrophoresis and computational models for binding studies of heparinoids with p-selectin and other proteins.

Science.gov (United States)

Mozafari, Mona; Balasupramaniam, Shantheya; Preu, Lutz; El Deeb, Sami; Reiter, Christian G; Wätzig, Hermann

2017-06-01

A fast and precise affinity capillary electrophoresis (ACE) method has been developed and applied for the investigation of the binding interactions between P-selectin and heparinoids as potential P-selectin inhibitors in the presence and absence of calcium ions. Furthermore, model proteins and vitronectin were used to appraise the binding behavior of P-selectin. The normalized mobility ratios (∆R/R f ), which provided information about the binding strength and the overall charge of the protein-ligand complex, were used to evaluate the binding affinities. It was found that P-selectin interacts more strongly with heparinoids in the presence of calcium ions. P-selectin was affected by heparinoids at the concentration of 3 mg/L. In addition, the results of the ACE experiments showed that among other investigated proteins, albumins and vitronectin exhibited strong interactions with heparinoids. Especially with P-selectin and vitronectin, the interaction may additionally induce conformational changes. Subsequently, computational models were applied to interpret the ACE experiments. Docking experiments explained that the binding of heparinoids on P-selectin is promoted by calcium ions. These docking models proved to be particularly well suited to investigate the interaction of charged compounds, and are therefore complementary to ACE experiments. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Glycans: bioactive signals decoded by lectins.

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Gabius, Hans-Joachim

2008-12-01

The glycan part of cellular glycoconjugates affords a versatile means to build biochemical signals. These oligosaccharides have an exceptional talent in this respect. They surpass any other class of biomolecule in coding capacity within an oligomer (code word). Four structural factors account for this property: the potential for variability of linkage points, anomeric position and ring size as well as the aptitude for branching (first and second dimensions of the sugar code). Specific intermolecular recognition is favoured by abundant potential for hydrogen/co-ordination bonds and for C-H/pi-interactions. Fittingly, an array of protein folds has developed in evolution with the ability to select certain glycans from the natural diversity. The thermodynamics of this reaction profits from the occurrence of these ligands in only a few energetically favoured conformers, comparing favourably with highly flexible peptides (third dimension of the sugar code). Sequence, shape and local aspects of glycan presentation (e.g. multivalency) are key factors to regulate the avidity of lectin binding. At the level of cells, distinct glycan determinants, a result of enzymatic synthesis and dynamic remodelling, are being defined as biomarkers. Their presence gains a functional perspective by co-regulation of the cognate lectin as effector, for example in growth regulation. The way to tie sugar signal and lectin together is illustrated herein for two tumour model systems. In this sense, orchestration of glycan and lectin expression is an efficient means, with far-reaching relevance, to exploit the coding potential of oligosaccharides physiologically and medically.

E-selectin mediates stem cell adhesion and formation of blood vessels in a murine model of infantile hemangioma.

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Smadja, David M; Mulliken, John B; Bischoff, Joyce

2012-12-01

Hemangioma stem cells (HemSCs) are multipotent cells isolated from infantile hemangioma (IH), which form hemangioma-like lesions when injected subcutaneously into immune-deficient mice. In this murine model, HemSCs are the primary target of corticosteroid, a mainstay therapy for problematic IH. The relationship between HemSCs and endothelial cells that reside in IH is not clearly understood. Adhesive interactions might be critical for the preferential accumulation of HemSCs and/or endothelial cells in the tumor. Therefore, we studied the interactions between HemSCs and endothelial cells (HemECs) isolated from IH surgical specimens. We found that HemECs isolated from proliferating phase IH, but not involuting phase, constitutively express E-selectin, a cell adhesion molecule not present in quiescent endothelial cells. E-selectin was further increased when HemECs were exposed to vascular endothelial growth factor-A or tumor necrosis factor-α. In vitro, HemSC migration and adhesion was enhanced by recombinant E-selectin but not P-selectin; both processes were neutralized by E-selectin-blocking antibodies. E-selectin-positive HemECs also stimulated migration and adhesion of HemSCs. In vivo, neutralizing antibodies to E-selectin strongly inhibited formation of blood vessels when HemSCs and HemECs were co-implanted in Matrigel. These data suggest that endothelial E-selectin could be a major ligand for HemSCs and thereby promote cellular interactions and vasculogenesis in IH. We propose that constitutively expressed E-selectin on endothelial cells in the proliferating phase is one mediator of the stem cell tropism in IH. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Assignment by Negative-Ion Electrospray Tandem Mass Spectrometry of the Tetrasaccharide Backbones of Monosialylated Glycans Released from Bovine Brain Gangliosides

Science.gov (United States)

Chai, Wengang; Zhang, Yibing; Mauri, Laura; Ciampa, Maria G.; Mulloy, Barbara; Sonnino, Sandro; Feizi, Ten

2018-05-01

Gangliosides, as plasma membrane-associated sialylated glycolipids, are antigenic structures and they serve as ligands for adhesion proteins of pathogens, for toxins of bacteria, and for endogenous proteins of the host. The detectability by carbohydrate-binding proteins of glycan antigens and ligands on glycolipids can be influenced by the differing lipid moieties. To investigate glycan sequences of gangliosides as recognition structures, we have underway a program of work to develop a "gangliome" microarray consisting of isolated natural gangliosides and neoglycolipids (NGLs) derived from glycans released from them, and each linked to the same lipid molecule for arraying and comparative microarray binding analyses. Here, in the first phase of our studies, we describe a strategy for high-sensitivity assignment of the tetrasaccharide backbones and application to identification of eight of monosialylated glycans released from bovine brain gangliosides. This approach is based on negative-ion electrospray mass spectrometry with collision-induced dissociation (ESI-CID-MS/MS) of the desialylated glycans. Using this strategy, we have the data on backbone regions of four minor components among the monosialo-ganglioside-derived glycans; these are of the ganglio-, lacto-, and neolacto-series.

Spatio-temporal Characterization of Ligand-Receptor Interactions in Blood Stem-Cell Rolling

KAUST Repository

Al Alwan, Bader

2017-08-16

One of the most important issues in the research on hematopoietic stem/progenitor cells (HSPCs) is to understand the mechanism of the homing process of these cells to the bone marrow after being transplanted into patients and establish the production of various blood cell types. The HSPCs first come in contact with the endothelial cells. This contact is known as adhesion and occurs through a multi-step paradigm ending with transmigration to the bone marrow niche. The initial step of the homing, tethering and rolling of HSPCs is mediated by P- and E-Selectins expressed on the endothelial cell surface through their interactions with the ligands expressed by HSPCs. Here we developed a novel experimental method to unravel the molecular mechanisms of the selectin-ligands interactions in vitro at the single molecule level by combining microfluidics and single-molecule fluorescence imaging. Our method enables direct visualization of the nanoscale spatiotemporal dynamics of the E-selectin-ligand (PSGL-1) interactions under conditions of shear stress acting on the cells at the molecular level in real time.

Regulation of cytokine receptors by Golgi N-glycan processing and endocytosis.

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Partridge, Emily A; Le Roy, Christine; Di Guglielmo, Gianni M; Pawling, Judy; Cheung, Pam; Granovsky, Maria; Nabi, Ivan R; Wrana, Jeffrey L; Dennis, James W

2004-10-01

The Golgi enzyme beta1,6 N-acetylglucosaminyltransferase V (Mgat5) is up-regulated in carcinomas and promotes the substitution of N-glycan with poly N-acetyllactosamine, the preferred ligand for galectin-3 (Gal-3). Here, we report that expression of Mgat5 sensitized mouse cells to multiple cytokines. Gal-3 cross-linked Mgat5-modified N-glycans on epidermal growth factor and transforming growth factor-beta receptors at the cell surface and delayed their removal by constitutive endocytosis. Mgat5 expression in mammary carcinoma was rate limiting for cytokine signaling and consequently for epithelial-mesenchymal transition, cell motility, and tumor metastasis. Mgat5 also promoted cytokine-mediated leukocyte signaling, phagocytosis, and extravasation in vivo. Thus, conditional regulation of N-glycan processing drives synchronous modification of cytokine receptors, which balances their surface retention against loss via endocytosis.

Synthesis and Functional Characterization of Novel Sialyl LewisX Mimic-Decorated Liposomes for E-selectin-Mediated Targeting to Inflamed Endothelial Cells.

Science.gov (United States)

Chantarasrivong, Chanikarn; Ueki, Akiharu; Ohyama, Ryutaro; Unga, Johan; Nakamura, Shinya; Nakanishi, Isao; Higuchi, Yuriko; Kawakami, Shigeru; Ando, Hiromune; Imamura, Akihiro; Ishida, Hideharu; Yamashita, Fumiyoshi; Kiso, Makoto; Hashida, Mitsuru

2017-05-01

Sialyl LewisX (sLeX) is a natural ligand of E-selectin that is overexpressed by inflamed and tumor endothelium. Although sLeX is a potential ligand for drug targeting, synthesis of the tetrasaccharide is complicated with many reaction steps. In this study, structurally simplified novel sLeX analogues were designed and linked with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-2000 (DSPE-PEG) for E-selectin-mediated liposomal delivery. The sLeX structural simplification strategies include (1) replacement of the Gal-GlcNAc disaccharide unit with lactose to reduce many initial steps and (2) substitution of neuraminic acid with a negatively charged group, i.e., 3'-sulfo, 3'-carboxymethyl (3'-CM), or 3'-(1-carboxy)ethyl (3'-CE). While all the liposomes developed were similar in particle size and charge, the 3'-CE sLeX mimic liposome demonstrated the highest uptake in inflammatory cytokine-treated human umbilical vein endothelial cells (HUVECs), being even more potent than native sLeX-decorated liposomes. Inhibition studies using antiselectin antibodies revealed that their uptake was mediated primarily by overexpressed E-selectin on inflamed HUVECs. Molecular dynamics simulations were performed to gain mechanistic insight into the E-selectin binding differences among native and mimic sLeX. The terminally branched methyl group of the 3'-CE sLeX mimic oriented and faced the bulk hydrophilic solution during E-selectin binding. Since this state is entropically unfavorable, the 3'-CE sLeX mimic molecule might be pushed toward the binding pocket of E-selectin by a hydrophobic effect, leading to a higher probability of hydrogen-bond formation than native sLeX and the 3'-CM sLeX mimic. This corresponded with the fact that the 3'-CE sLeX mimic liposome exhibited much greater uptake than the 3'-CM sLeX mimic liposome.

Glycan arrays and other tools produced by automated glycan assembly

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Peter H. Seeberger

2017-01-01

Full Text Available Carbohydrates are the dominant biopolymer on earth and play important roles ranging from building material for plants to function in many biological systems. Glycans remain poorly studied due to a lack of synthetic tools. The goal of my laboratory has been to develop a general method for the automated assembly of glycans. The general protocols we developed resulted in the commercialisation of the Glyconeer 2.1™ synthesizer as well as the building blocks and all reagents. Oligosaccharides as long as 50-mers are now accessible within days. Rapid access to defined oligosaccharides has been the foundation to many applications including synthetic tools such as glycan microarrays, glycan nanoparticles and anti-glycan antibodies. The platform technology is helping to address real-life problems by the creation of new vaccines and diagnostics. After addressing mainly mammalian glycobiology earlier, material science and plant biology are benefitting increasingly from synthetic glycans.

Soluble L-selectin levels predict survival in sepsis

DEFF Research Database (Denmark)

Seidelin, Jakob B; Nielsen, Ole H; Strøm, Jens

2002-01-01

OBJECTIVE: To evaluate serum soluble L-selectin as a prognostic factor for survival in patients with sepsis. DESIGN: A prospective study of mortality in patients with sepsis whose serum levels of sL-selectin were measured on admission to an intensive care unit (ICU) and 4 days later. Follow-up data......, and 3 and 12 months after admission. Serum sL-selectin levels were significantly lower in the patients than in the controls. Sepsis nonsurvivors had significantly lower levels than survivors. Efficiency analysis and receiver operation characteristics showed that the ideal cutoff point for s......L-selectin as a test for sepsis survival was 470 ng/ml. The accumulated mortality in patients with subnormal sL-selectin levels on admission was significantly increased. No correlation was found between clinical or paraclinical markers, including SAPS II and sL-selectin, and no relationship to the microbial diagnosis...

MCAW-DB: A glycan profile database capturing the ambiguity of glycan recognition patterns.

Science.gov (United States)

Hosoda, Masae; Takahashi, Yushi; Shiota, Masaaki; Shinmachi, Daisuke; Inomoto, Renji; Higashimoto, Shinichi; Aoki-Kinoshita, Kiyoko F

2018-05-11

Glycan-binding protein (GBP) interaction experiments, such as glycan microarrays, are often used to understand glycan recognition patterns. However, oftentimes the interpretation of glycan array experimental data makes it difficult to identify discrete GBP binding patterns due to their ambiguity. It is known that lectins, for example, are non-specific in their binding affinities; the same lectin can bind to different monosaccharides or even different glycan structures. In bioinformatics, several tools to mine the data generated from these sorts of experiments have been developed. These tools take a library of predefined motifs, which are commonly-found glycan patterns such as sialyl-Lewis X, and attempt to identify the motif(s) that are specific to the GBP being analyzed. In our previous work, as opposed to using predefined motifs, we developed the Multiple Carbohydrate Alignment with Weights (MCAW) tool to visualize the state of the glycans being recognized by the GBP under analysis. We previously reported on the effectiveness of our tool and algorithm by analyzing several glycan array datasets from the Consortium of Functional Glycomics (CFG). In this work, we report on our analysis of 1081 data sets which we collected from the CFG, the results of which we have made publicly and freely available as a database called MCAW-DB. We introduce this database, its usage and describe several analysis results. We show how MCAW-DB can be used to analyze glycan-binding patterns of GBPs amidst their ambiguity. For example, the visualization of glycan-binding patterns in MCAW-DB show how they correlate with the concentrations of the samples used in the array experiments. Using MCAW-DB, the patterns of glycans found to bind to various GBP-glycan binding proteins are visualized, indicating the binding "environment" of the glycans. Thus, the ambiguity of glycan recognition is numerically represented, along with the patterns of monosaccharides surrounding the binding region. The

Biotinylated N-Acetyllactosamine- and N,N-Diacetyllactosamine-Based Oligosaccharides as Novel Ligands for Human Galectin-3

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Sophia Böcker

2017-04-01

Full Text Available Galectin inhibitor design is an emerging research field due to the involvement of galectins in cancer. Galectin-3, in particular, plays an important role in tumor progression. To generate inhibitors, modifications of the glycan structure can be introduced. Conjugation of hydrophobic compounds to saccharides has proven to be promising as increased binding of galectin-3 can be observed. In the present study, we report on neo-glycans carrying hydrophobic biotin as novel ligands for human galectin-3. We modified N-acetyllactosamine- and N,N-diacetyllactosamine-based tetrasaccharides at the C6-position of the terminal saccharide unit using selective enzymatic oxidation and subsequent chemical conjugation of biotinamidohexanoic acid hydrazide. These neo-glycans were much better bound by galectin-3 than the unmodified counterparts. High selectivity for galectin-3 over galectin-1 was also proven. We generated multivalent neo-glycoproteins by conjugation of neo-glycans to bovine serum albumin showing high affinity for galectin-3. Compared to non-biotinylated neo-glycoproteins, we achieved high binding levels of galectin-3 with a lesser amount of conjugated neo-glycans. Multivalent ligand presentation of neo-glycoproteins significantly increased the inhibitory potency towards galectin-3 binding to asialofetuin when compared to free monovalent glycans. Our findings show the positive impact of 6-biotinylation of tetrasaccharides on galectin-3 binding, which broadens the recent design approaches for producing high-affinity ligands.

The alpha(1,3)fucosyltransferases FucT-IV and FucT-VII exert collaborative control over selectin-dependent leukocyte recruitment and lymphocyte homing

Czech Academy of Sciences Publication Activity Database

Homeister, J. W.; Thall, A. D.; Petryniak, B.; Malý, Petr; Rogers, C. E.; Smith, P. L.; Kelly, R. J.; Gersten, K. M.; Askari, S. W.; Cheng, G.

2001-01-01

Roč. 15, č. 1 (2001), s. 115-126 ISSN 1074-7613 Institutional research plan: CEZ:AV0Z5052915 Keywords : fucosyltransferase * selectin ligand * gene knock-out Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 18.866, year: 2001

Improved method for drawing of a glycan map, and the first page of glycan atlas, which is a compilation of glycan maps for a whole organism.

Directory of Open Access Journals (Sweden)

Shunji Natsuka

Full Text Available Glycan Atlas is a set of glycan maps over the whole body of an organism. The glycan map that includes data of glycan structure and quantity displays micro-heterogeneity of the glycans in a tissue, an organ, or cells. The two-dimensional glycan mapping is widely used for structure analysis of N-linked oligosaccharides on glycoproteins. In this study we developed a comprehensive method for the mapping of both N- and O-glycans with and without sialic acid. The mapping data of 150 standard pyridylaminated glycans were collected. The empirical additivity rule which was proposed in former reports was able to adapt for this extended glycan map. The adapted rule is that the elution time of pyridylamino glycans on high performance liquid chromatography (HPLC is expected to be the simple sum of the partial elution times assigned to each monosaccharide residue. The comprehensive mapping method developed in this study is a powerful tool for describing the micro-heterogeneity of the glycans. Furthermore, we prepared 42 pyridylamino (PA- glycans from human serum and were able to draw the map of human serum N- and O-glycans as an initial step of Glycan Atlas editing.

Restricted N-glycan conformational space in the PDB and its implication in glycan structure modeling.

Science.gov (United States)

Jo, Sunhwan; Lee, Hui Sun; Skolnick, Jeffrey; Im, Wonpil

2013-01-01

Understanding glycan structure and dynamics is central to understanding protein-carbohydrate recognition and its role in protein-protein interactions. Given the difficulties in obtaining the glycan's crystal structure in glycoconjugates due to its flexibility and heterogeneity, computational modeling could play an important role in providing glycosylated protein structure models. To address if glycan structures available in the PDB can be used as templates or fragments for glycan modeling, we present a survey of the N-glycan structures of 35 different sequences in the PDB. Our statistical analysis shows that the N-glycan structures found on homologous glycoproteins are significantly conserved compared to the random background, suggesting that N-glycan chains can be confidently modeled with template glycan structures whose parent glycoproteins share sequence similarity. On the other hand, N-glycan structures found on non-homologous glycoproteins do not show significant global structural similarity. Nonetheless, the internal substructures of these N-glycans, particularly, the substructures that are closer to the protein, show significantly similar structures, suggesting that such substructures can be used as fragments in glycan modeling. Increased interactions with protein might be responsible for the restricted conformational space of N-glycan chains. Our results suggest that structure prediction/modeling of N-glycans of glycoconjugates using structure database could be effective and different modeling approaches would be needed depending on the availability of template structures.

Restricted N-glycan conformational space in the PDB and its implication in glycan structure modeling.

Directory of Open Access Journals (Sweden)

Sunhwan Jo

Full Text Available Understanding glycan structure and dynamics is central to understanding protein-carbohydrate recognition and its role in protein-protein interactions. Given the difficulties in obtaining the glycan's crystal structure in glycoconjugates due to its flexibility and heterogeneity, computational modeling could play an important role in providing glycosylated protein structure models. To address if glycan structures available in the PDB can be used as templates or fragments for glycan modeling, we present a survey of the N-glycan structures of 35 different sequences in the PDB. Our statistical analysis shows that the N-glycan structures found on homologous glycoproteins are significantly conserved compared to the random background, suggesting that N-glycan chains can be confidently modeled with template glycan structures whose parent glycoproteins share sequence similarity. On the other hand, N-glycan structures found on non-homologous glycoproteins do not show significant global structural similarity. Nonetheless, the internal substructures of these N-glycans, particularly, the substructures that are closer to the protein, show significantly similar structures, suggesting that such substructures can be used as fragments in glycan modeling. Increased interactions with protein might be responsible for the restricted conformational space of N-glycan chains. Our results suggest that structure prediction/modeling of N-glycans of glycoconjugates using structure database could be effective and different modeling approaches would be needed depending on the availability of template structures.

Differing patterns of P-selectin expression in lung injury

DEFF Research Database (Denmark)

Bless, N M; Tojo, S J; Kawarai, H

1998-01-01

Using two models of acute lung inflammatory injury in rats (intrapulmonary deposition of immunoglobulin G immune complexes and systemic activation of complement after infusion of purified cobra venom factor), we have analyzed the requirements and patterns for upregulation of lung vascular P......-selectin. In the immune complex model, upregulation of P-selectin was defined by Northern and Western blot analysis of lung homogenates, by immunostaining of lung tissue, and by vascular fixation of 125I-labeled anti-P-selectin. P-selectin protein was detected by 1 hour (long before detection of mRNA) and expression......-selectin was dependent on an intact complement system, and the presence of blood neutrophils was susceptible to the antioxidant dimethyl sulfoxide and required C5a but not tumor necrosis factor alpha. In contrast, in the cobra venom factor model, upregulation of P-selectin, which is C5a dependent, was also dimethyl...

The high affinity selectin glycan ligand C2-O-sLex and mRNA transcripts of the core 2 β-1,6-N-acetylglusaminyltransferase (C2GnT1) gene are highly expressed in human colorectal adenocarcinomas

International Nuclear Information System (INIS)

St Hill, Catherine A; Farooqui, Mariya; Mitcheltree, Gregory; Gulbahce, H Evin; Jessurun, Jose; Cao, Qing; Walcheck, Bruce

2009-01-01

The metastasis of cancer cells and leukocyte extravasation into inflamed tissues share common features. Specialized carbohydrates modified with sialyl Lewis x (sLe x ) antigens on leukocyte membranes are ligands for selectin adhesion molecules on activated vascular endothelial cells at inflammatory sites. The activity of the enzyme core 2 β1,6 N-acetylglucosaminyltransferase (C2GnT1) in leukocytes greatly increases their ability to bind to endothelial selectins. C2GnT1 is essential for the synthesis of core 2-branched O-linked carbohydrates terminated with sLe x (C2-O-sLe x ). Our goal was to determine the expression profiles of C2-O-sLe x in the malignant progression and metastasis of colorectal adenocarcinomas. The well characterized CHO-131 monoclonal antibody (mAb) specifically recognizes C2-O-sLe x present in human leukocytes and carcinoma cells. Using CHO-131 mAb, we investigated whether C2-O-sLe x was present in 113 human primary colorectal adenocarcinomas, 10 colorectal adenomas, 46 metastatic liver tumors, 28 normal colorectal tissues, and 5 normal liver tissues by immunohistochemistry. We also examined mRNA levels of the enzyme core 2 β1,6-N-acetylglucosaminyltransferase (C2GnT1) in 20 well, 15 moderately, and 2 poorly differentiated colorectal adenocarcinomas, and in 5 normal colorectal tissues by using quantitative real-time polymerase chain reactions (RT-PCR). We observed high reactivity with CHO-131 mAb in approximately 70% of colorectal carcinomas and 87% of metastatic liver tumors but a lack of reactivity in colorectal adenomas and normal colonic and liver tissues. Positive reactivity with CHO-131 mAb was very prominent in neoplastic colorectal glands of well to moderately differentiated adenocarcinomas. The most intense staining with CHO-131 mAb was observed at the advancing edge of tumors with the deepest invasive components. Finally, we analyzed C2GnT1 mRNA levels in 37 colorectal adenocarcinomas and 5 normal colorectal tissues by RT

Analysis of O-glycans as 9-fluorenylmethyl derivatives and its application to the studies on glycan array.

Science.gov (United States)

Yamada, Keita; Hirabayashi, Jun; Kakehi, Kazuaki

2013-03-19

A method is proposed for the analysis of O-glycans as 9-fluorenylmethyl (Fmoc) derivatives. After releasing the O-glycans from the protein backbone in the presence of ammonia-based media, the glycosylamines thus formed are conveniently labeled with Fmoc-Cl and analyzed by HPLC and MALDI-TOF MS after easy purification. Fmoc labeled O-glycans showed 3.5 times higher sensitivities than those labeled with 2-aminobenzoic acid in fluorescent detection. Various types of O-glycans having sialic acids, fucose, and/or sulfate residues were successfully labeled with Fmoc and analyzed by HPLC and MALDI-TOF MS. The method was applied to the comprehensive analysis of O-glycans expressed on MKN45 cells (human gastric adenocarcinoma). In addition, Fmoc-derivatized O-glycans were easily converted to free hemiacetal or glycosylamine-form glycans that are available for fabrication of glycan array and neoglycoproteins. To demonstrate the availability of our methods, we fabricate the glycan array with Fmoc labeled glycans derived from mucin samples and cancer cells. The model studies using the glycan array showed clear interactions between immobilized glycans and some lectins.

Raised soluble P-selectin moderately accelerates atherosclerotic plaque progression.

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Kevin J Woollard

Full Text Available Soluble P-selectin (sP-selectin, a biomarker of inflammatory related pathologies including cardiovascular and peripheral vascular diseases, also has pro-atherosclerotic effects including the ability to increase leukocyte recruitment and modulate thrombotic responses in vivo. The current study explores its role in progressing atherosclerotic plaque disease. Apoe-/- mice placed on a high fat diet (HFD were given daily injections of recombinant dimeric murine P-selectin (22.5 µg/kg/day for 8 or 16 weeks. Saline or sE-selectin injections were used as negative controls. In order to assess the role of sP-selectin on atherothrombosis an experimental plaque remodelling murine model, with sm22α-hDTR Apoe-/- mice on a HFD in conjunction with delivery of diphtheria toxin to induce targeted vascular smooth muscle apoptosis, was used. These mice were similarly given daily injections of sP-selectin for 8 or 16 weeks. While plaque mass and aortic lipid content did not change with sP-selectin treatment in Apoe-/- or SM22α-hDTR Apoe-/- mice on HFD, increased plasma MCP-1 and a higher plaque CD45 content in Apoe-/- HFD mice was observed. As well, a significant shift towards a more unstable plaque phenotype in the SM22α-hDTR Apoe-/- HFD mice, with increased macrophage accumulation and lower collagen content, leading to a lower plaque stability index, was observed. These results demonstrate that chronically raised sP-selectin favours progression of an unstable atherosclerotic plaque phenotype.

Soluble L-selectin levels predict survival in sepsis

DEFF Research Database (Denmark)

Seidelin, Jakob B; Nielsen, Ole H; Strøm, Jens

2002-01-01

To evaluate serum soluble L-selectin as a prognostic factor for survival in patients with sepsis.......To evaluate serum soluble L-selectin as a prognostic factor for survival in patients with sepsis....

P-selectin- and heparanase-dependent antimetastatic activity of non-anticoagulant heparins.

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Hostettler, Nina; Naggi, Annamaria; Torri, Giangiacomo; Ishai-Michaeli, Riva; Casu, Benito; Vlodavsky, Israel; Borsig, Lubor

2007-11-01

Vascular cell adhesion molecules, P- and L-selectins, facilitate metastasis of cancer cells in mice by mediating interactions with platelets, endothelium, and leukocytes. Heparanase is an endoglycosidase that degrades heparan sulfate of extracellular matrix, thereby promoting tumor invasion and metastasis. Heparin is known to efficiently attenuate metastasis in different tumor models. Here we identified modified, nonanticoagulant species of heparin that specifically inhibit selectin-mediated cell-cell interactions, heparanase enzymatic activity, or both. We show that selective inhibition of selectin interactions or heparanase with specific heparin derivatives in mouse models of MC-38 colon carcinoma and B16-BL6 melanoma attenuates metastasis. Selectin-specific heparin derivatives attenuated metastasis of MC-38 carcinoma, but heparanase-specific derivatives had no effect, in accordance with the virtual absence of heparanase activity in these cells. Heparin derivatives had no further effect on metastasis in mice deficient in P- and L-selectin, indicating that selectins are the primary targets of heparin antimetastatic activity. Selectin-specific and heparanase-specific derivatives attenuated metastasis of B16-BL6 melanomas to a similar extent. When mice were injected with a derivative containing both heparanase and selectin inhibitory activity, no additional attenuation of metastasis could be observed. Thus, selectin-specific heparin derivatives efficiently attenuated metastasis of both tumor cell types whereas inhibition of heparanase led to reduction of metastasis only in tumor cells producing heparanase.

Improve accuracy and sensibility in glycan structure prediction by matching glycan isotope abundance

International Nuclear Information System (INIS)

Xu Guang; Liu Xin; Liu Qingyan; Zhou Yanhong; Li Jianjun

2012-01-01

Highlights: ► A glycan isotope pattern recognition strategy for glycomics. ► A new data preprocessing procedure to detect ion peaks in a giving MS spectrum. ► A linear soft margin SVM classification for isotope pattern recognition. - Abstract: Mass Spectrometry (MS) is a powerful technique for the determination of glycan structures and is capable of providing qualitative and quantitative information. Recent development in computational method offers an opportunity to use glycan structure databases and de novo algorithms for extracting valuable information from MS or MS/MS data. However, detecting low-intensity peaks that are buried in noisy data sets is still a challenge and an algorithm for accurate prediction and annotation of glycan structures from MS data is highly desirable. The present study describes a novel algorithm for glycan structure prediction by matching glycan isotope abundance (mGIA), which takes isotope masses, abundances, and spacing into account. We constructed a comprehensive database containing 808 glycan compositions and their corresponding isotope abundance. Unlike most previously reported methods, not only did we take into count the m/z values of the peaks but also their corresponding logarithmic Euclidean distance of the calculated and detected isotope vectors. Evaluation against a linear classifier, obtained by training mGIA algorithm with datasets of three different human tissue samples from Consortium for Functional Glycomics (CFG) in association with Support Vector Machine (SVM), was proposed to improve the accuracy of automatic glycan structure annotation. In addition, an effective data preprocessing procedure, including baseline subtraction, smoothing, peak centroiding and composition matching for extracting correct isotope profiles from MS data was incorporated. The algorithm was validated by analyzing the mouse kidney MS data from CFG, resulting in the identification of 6 more glycan compositions than the previous annotation

PANP is a novel O-glycosylated PILR{alpha} ligand expressed in neural tissues

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Kogure, Amane [Department of Immunochemistry, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871 (Japan); Laboratory of Immunochemistry, WPI Immunology Frontier Research Center, Osaka University, Osaka 565-0871 (Japan); Shiratori, Ikuo [Department of Immunochemistry, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871 (Japan); Wang, Jing [Department of Immunochemistry, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871 (Japan); Laboratory of Immunochemistry, WPI Immunology Frontier Research Center, Osaka University, Osaka 565-0871 (Japan); Lanier, Lewis L. [Department of Microbiology and Immunology and the Cancer Research Institute, University of California San Francisco, San Francisco, CA 94143 (United States); Arase, Hisashi, E-mail: arase@biken.osaka-u.ac.jp [Department of Immunochemistry, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871 (Japan); Laboratory of Immunochemistry, WPI Immunology Frontier Research Center, Osaka University, Osaka 565-0871 (Japan); JST CREST, Saitama 332-0012 (Japan)

2011-02-18

Research highlights: {yields} A Novel molecule, PANP, was identified to be a PILR{alpha} ligand. {yields} Sialylated O-glycan structures on PANP were required for PILR{alpha} recognition. {yields} Transcription of PANP was mainly observed in neural tissues. {yields} PANP seems to be involved in immune regulation as a ligand for PILR{alpha}. -- Abstract: PILR{alpha} is an immune inhibitory receptor possessing an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain enabling it to deliver inhibitory signals. Binding of PILR{alpha} to its ligand CD99 is involved in immune regulation; however, whether there are other PILR{alpha} ligands in addition to CD99 is not known. Here, we report that a novel molecule, PILR-associating neural protein (PANP), acts as an additional ligand for PILR{alpha}. Transcription of PANP was mainly observed in neural tissues. PILR{alpha}-Ig fusion protein bound cells transfected with PANP and the transfectants stimulated PILR{alpha} reporter cells. Specific O-glycan structures on PANP were found to be required for PILR recognition of this ligand. These results suggest that PANP is involved in immune regulation as a ligand of the PILR{alpha}.

CROSSWORK for Glycans: Glycan Identificatin Through Mass Spectrometry and Bioinformatics

DEFF Research Database (Denmark)

Rasmussen, Morten; Thaysen-Andersen, Morten; Højrup, Peter

  We have developed "GLYCANthrope " - CROSSWORKS for glycans:  a bioinformatics tool, which assists in identifying N-linked glycosylated peptides as well as their glycan moieties from MS2 data of enzymatically digested glycoproteins. The program runs either as a stand-alone application or as a plug...

Intranasal delivery of E-selectin reduces atherosclerosis in ApoE-/- mice.

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Xinhui Li

Full Text Available Mucosal tolerance to E-selectin prevents stroke and protects against ischemic brain damage in experimental models of stroke studying healthy animals or spontaneously hypertensive stroke-prone rats. A reduction in inflammation and neural damage was associated with immunomodulatory or "tolerogenic" responses to E-selectin. The purpose of the current study on ApoE deficient mice is to assess the capacity of this stroke prevention innovation to influence atherosclerosis, a major underlying cause for ischemic strokes; human E-selectin is being translated as a potential clinical prevention strategy for secondary stroke. Female ApoE-/- mice received intranasal delivery of E-selectin prior to (pre-tolerization or simultaneously with initiation of a high-fat diet. After 7 weeks on the high-fat diet, lipid lesions in the aorta, serum triglycerides, and total cholesterol were assessed as markers of atherosclerosis development. We also assessed E-selectin-specific antibodies and cytokine responses, in addition to inflammatory responses that included macrophage infiltration of the aorta and altered gene expression profiles of aortic mRNA. Intranasal delivery of E-selectin prior to initiation of high-fat chow decreased atherosclerosis, serum total cholesterol, and expression of the leucocyte chemoattractant CCL21 that is typically upregulated in atherosclerotic lesions of ApoE-/- mice. This response was associated with the induction of E-selectin specific cells producing the immunomodulatory cytokine IL-10 and immunosuppressive antibody isotypes. Intranasal administration of E-selectin generates E-selectin specific immune responses that are immunosuppressive in nature and can ameliorate atherosclerosis, a major risk factor for ischemic stroke. These results provide additional preclinical support for the potential of induction of mucosal tolerance to E-selectin to prevent stroke.

The GlycanBuilder: a fast, intuitive and flexible software tool for building and displaying glycan structures.

Science.gov (United States)

Ceroni, Alessio; Dell, Anne; Haslam, Stuart M

2007-08-07

Carbohydrates play a critical role in human diseases and their potential utility as biomarkers for pathological conditions is a major driver for characterization of the glycome. However, the additional complexity of glycans compared to proteins and nucleic acids has slowed the advancement of glycomics in comparison to genomics and proteomics. The branched nature of carbohydrates, the great diversity of their constituents and the numerous alternative symbolic notations, make the input and display of glycans not as straightforward as for example the amino-acid sequence of a protein. Every glycoinformatic tool providing a user interface would benefit from a fast, intuitive, appealing mechanism for input and output of glycan structures in a computer readable format. A software tool for building and displaying glycan structures using a chosen symbolic notation is described here. The "GlycanBuilder" uses an automatic rendering algorithm to draw the saccharide symbols and to place them on the drawing board. The information about the symbolic notation is derived from a configurable graphical model as a set of rules governing the aspect and placement of residues and linkages. The algorithm is able to represent a structure using only few traversals of the tree and is inherently fast. The tool uses an XML format for import and export of encoded structures. The rendering algorithm described here is able to produce high-quality representations of glycan structures in a chosen symbolic notation. The automated rendering process enables the "GlycanBuilder" to be used both as a user-independent component for displaying glycans and as an easy-to-use drawing tool. The "GlycanBuilder" can be integrated in web pages as a Java applet for the visual editing of glycans. The same component is available as a web service to render an encoded structure into a graphical format. Finally, the "GlycanBuilder" can be integrated into other applications to create intuitive and appealing user

The GlycanBuilder: a fast, intuitive and flexible software tool for building and displaying glycan structures

Directory of Open Access Journals (Sweden)

Dell Anne

2007-08-01

Full Text Available Abstract Background Carbohydrates play a critical role in human diseases and their potential utility as biomarkers for pathological conditions is a major driver for characterization of the glycome. However, the additional complexity of glycans compared to proteins and nucleic acids has slowed the advancement of glycomics in comparison to genomics and proteomics. The branched nature of carbohydrates, the great diversity of their constituents and the numerous alternative symbolic notations, make the input and display of glycans not as straightforward as for example the amino-acid sequence of a protein. Every glycoinformatic tool providing a user interface would benefit from a fast, intuitive, appealing mechanism for input and output of glycan structures in a computer readable format. Results A software tool for building and displaying glycan structures using a chosen symbolic notation is described here. The "GlycanBuilder" uses an automatic rendering algorithm to draw the saccharide symbols and to place them on the drawing board. The information about the symbolic notation is derived from a configurable graphical model as a set of rules governing the aspect and placement of residues and linkages. The algorithm is able to represent a structure using only few traversals of the tree and is inherently fast. The tool uses an XML format for import and export of encoded structures. Conclusion The rendering algorithm described here is able to produce high-quality representations of glycan structures in a chosen symbolic notation. The automated rendering process enables the "GlycanBuilder" to be used both as a user-independent component for displaying glycans and as an easy-to-use drawing tool. The "GlycanBuilder" can be integrated in web pages as a Java applet for the visual editing of glycans. The same component is available as a web service to render an encoded structure into a graphical format. Finally, the "GlycanBuilder" can be integrated into other

Nano-motion dynamics are determined by surface-tethered selectin mechanokinetics and bond formation.

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Brian J Schmidt

2009-12-01

Full Text Available The interaction of proteins at cellular interfaces is critical for many biological processes, from intercellular signaling to cell adhesion. For example, the selectin family of adhesion receptors plays a critical role in trafficking during inflammation and immunosurveillance. Quantitative measurements of binding rates between surface-constrained proteins elicit insight into how molecular structural details and post-translational modifications contribute to function. However, nano-scale transport effects can obfuscate measurements in experimental assays. We constructed a biophysical simulation of the motion of a rigid microsphere coated with biomolecular adhesion receptors in shearing flow undergoing thermal motion. The simulation enabled in silico investigation of the effects of kinetic force dependence, molecular deformation, grouping adhesion receptors into clusters, surface-constrained bond formation, and nano-scale vertical transport on outputs that directly map to observable motions. Simulations recreated the jerky, discrete stop-and-go motions observed in P-selectin/PSGL-1 microbead assays with physiologic ligand densities. Motion statistics tied detailed simulated motion data to experimentally reported quantities. New deductions about biomolecular function for P-selectin/PSGL-1 interactions were made. Distributing adhesive forces among P-selectin/PSGL-1 molecules closely grouped in clusters was necessary to achieve bond lifetimes observed in microbead assays. Initial, capturing bond formation effectively occurred across the entire molecular contour length. However, subsequent rebinding events were enhanced by the reduced separation distance following the initial capture. The result demonstrates that vertical transport can contribute to an enhancement in the apparent bond formation rate. A detailed analysis of in silico motions prompted the proposition of wobble autocorrelation as an indicator of two-dimensional function. Insight into two

Protein mobilities and P-selectin storage in Weibel-Palade bodies.

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Kiskin, Nikolai I; Hellen, Nicola; Babich, Victor; Hewlett, Lindsay; Knipe, Laura; Hannah, Matthew J; Carter, Tom

2010-09-01

Using fluorescence recovery after photobleaching (FRAP) we measured the mobilities of EGFP-tagged soluble secretory proteins in the endoplasmic reticulum (ER) and in individual Weibel-Palade bodies (WPBs) at early (immature) and late (mature) stages in their biogenesis. Membrane proteins (P-selectin, CD63, Rab27a) were also studied in individual WPBs. In the ER, soluble secretory proteins were mobile; however, following insertion into immature WPBs larger molecules (VWF, Proregion, tPA) and P-selectin became immobilised, whereas small proteins (ssEGFP, eotaxin-3) became less mobile. WPB maturation led to further decreases in mobility of small proteins and CD63. Acute alkalinisation of mature WPBs selectively increased the mobilities of small soluble proteins without affecting larger molecules and the membrane proteins. Disruption of the Proregion-VWF paracrystalline core by prolonged incubation with NH(4)Cl rendered P-selectin mobile while VWF remained immobile. FRAP of P-selectin mutants revealed that immobilisation most probably involves steric entrapment of the P-selectin extracellular domain by the Proregion-VWF paracrystal. Significantly, immobilisation contributed to the enrichment of P-selectin in WPBs; a mutation of P-selectin preventing immobilisation led to a failure of enrichment. Together these data shed new light on the transitions that occur for soluble and membrane proteins following their entry and storage into post-Golgi-regulated secretory organelles.

Novel cleavage of reductively aminated glycan-tags by N-bromosuccinimide to regenerate free, reducing glycans

OpenAIRE

Song, Xuezheng; Johns, Brian A.; Ju, Hong; Lasanajak, Yi; Zhao, Chunmei; Smith, David F.; Cummings, Richard D.

2013-01-01

Glycans that are fluorescently tagged by reductive amination have been useful for functional glycomic studies. However, the existing tags can introduce unwanted properties to the glycans and complicate structural and functional studies. Here we describe a facile method using N-bromosuccinimide (NBS) to remove the tags and efficiently regenerate free reducing glycans. The regenerated free reducing glycans can be easily analyzed by routine mass spectrometry or re-tagged with different tags for ...

Development of a Schistosoma mansoni shotgun O-glycan microarray and application to the discovery of new antigenic schistosome glycan motifs.

Science.gov (United States)

van Diepen, Angela; van der Plas, Arend-Jan; Kozak, Radoslaw P; Royle, Louise; Dunne, David W; Hokke, Cornelis H

2015-06-01

Upon infection with Schistosoma, antibody responses are mounted that are largely directed against glycans. Over the last few years significant progress has been made in characterising the antigenic properties of N-glycans of Schistosoma mansoni. Despite also being abundantly expressed by schistosomes, much less is understood about O-glycans and antibody responses to these have not yet been systematically analysed. Antibody binding to schistosome glycans can be analysed efficiently and quantitatively using glycan microarrays, but O-glycan array construction and exploration is lagging behind because no universal O-glycanase is available, and release of O-glycans has been dependent on chemical methods. Recently, a modified hydrazinolysis method has been developed that allows the release of O-glycans with free reducing termini and limited degradation, and we applied this method to obtain O-glycans from different S. mansoni life stages. Two-dimensional HPLC separation of 2-aminobenzoic acid-labelled O-glycans generated 362 O-glycan-containing fractions that were printed on an epoxide-modified glass slide, thereby generating the first shotgun O-glycan microarray containing naturally occurring schistosome O-glycans. Monoclonal antibodies and mass spectrometry showed that the O-glycan microarray contains well-known antigenic glycan motifs as well as numerous other, potentially novel, antibody targets. Incubations of the microarrays with sera from Schistosoma-infected humans showed substantial antibody responses to O-glycans in addition to those observed to the previously investigated N- and glycosphingolipid glycans. This underlines the importance of the inclusion of these often schistosome-specific O-glycans in glycan antigen studies and indicates that O-glycans contain novel antigenic motifs that have potential for use in diagnostic methods and studies aiming at the discovery of vaccine targets. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights

Solid-phase glycan isolation for glycomics analysis.

Science.gov (United States)

Yang, Shuang; Zhang, Hui

2012-12-01

Glycosylation is one of the most significant protein PTMs. The biological activities of proteins are dramatically changed by the glycans associated with them. Thus, structural analysis of the glycans of glycoproteins in complex biological or clinical samples is critical in correlation with the functions of glycans with diseases. Profiling of glycans by HPLC-MS is a commonly used technique in analyzing glycan structures and quantifying their relative abundance in different biological systems. Methods relied on MS require isolation of glycans from negligible salts and other contaminant ions since salts and ions may interfere with the glycans, resulting in poor glycan ionization. To accomplish those objectives, glycan isolation and clean-up methods including SPE, liquid-phase extraction, chromatography, and electrophoresis have been developed. Traditionally, glycans are isolated from proteins or peptides using a combination of hydrophobic and hydrophilic columns: proteins and peptides remain on hydrophobic absorbent while glycans, salts, and other hydrophilic reagents are collected as flowthrough. The glycans in the flowthrough are then purified through graphite-activated carbon column by hydrophilic interaction LC. Yet, the drawback in these affinity-based approaches is nonspecific binding. As a result, chemical methods by hydrazide or oxime have been developed for solid-phase isolation of glycans with high specificity and yield. Combined with high-resolution MS, specific glycan isolation techniques provide tremendous potentials as useful tools for glycomics analysis. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Neonatal protection by an innate immune system of human milk consisting of oligosaccharides and glycans.

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Newburg, D S

2009-04-01

This review discusses the role of human milk glycans in protecting infants, but the conclusion that the human milk glycans constitute an innate immune system whereby the mother protects her offspring may have general applicability in all mammals, including species of commercial importance. Infants that are not breastfed have a greater incidence of severe diarrhea and respiratory diseases than those who are breastfed. In the past, this had been attributed primarily to human milk secretory antibodies. However, the oligosaccharides are major components of human milk, and milk is also rich in other glycans, including glycoproteins, mucins, glycosaminoglycans, and glycolipids. These milk glycans, especially the oligosaccharides, are composed of thousands of components. The milk factor that promotes gut colonization by Bifidobacterium bifidum was found to be a glycan, and such prebiotic characteristics may contribute to protection against infectious agents. However, the ability of human milk glycans to protect the neonate seems primarily to be due to their inhibition of pathogen binding to their host cell target ligands. Many such examples include specific fucosylated oligosaccharides and glycans that inhibit specific pathogens. Most human milk oligosaccharides are fucosylated, and their production depends on fucosyltransferase enzymes; mutations in these fucosyltransferase genes are common and underlie the various Lewis blood types in humans. Variable expression of specific fucosylated oligosaccharides in milk, also a function of these genes (and maternal Lewis blood type), is significantly associated with the risk of infectious disease in breastfed infants. Human milk also contains major quantities and large numbers of sialylated oligosaccharides, many of which are also present in bovine colostrum. These could similarly inhibit several common viral pathogens. Moreover, human milk oligosaccharides strongly attenuate inflammatory processes in the intestinal mucosa. These

Predominant Expression of Hybrid N-Glycans Has Distinct Cellular Roles Relative to Complex and Oligomannose N-Glycans

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M. Kristen Hall

2016-06-01

Full Text Available Glycosylation modulates growth, maintenance, and stress signaling processes. Consequently, altered N-glycosylation is associated with reduced fitness and disease. Therefore, expanding our understanding of N-glycans in altering biological processes is of utmost interest. Herein, clustered regularly interspaced short palindromic repeats/caspase9 (CRISPR/Cas9 technology was employed to engineer a glycosylation mutant Chinese Hamster Ovary (CHO cell line, K16, which expresses predominantly hybrid type N-glycans. This newly engineered cell line enabled us to compare N-glycan effects on cellular properties of hybrid type N-glycans, to the well-established Pro−5 and Lec1 cell lines, which express complex and oligomannose types of N-glycans, respectively. Lectin binding studies revealed the predominant N-glycan expressed in K16 is hybrid type. Cell dissociation and migration assays demonstrated the greatest strength of cell–cell adhesion and fastest migratory rates for oligomannose N-glycans, and these properties decreased as oligomannose type were converted to hybrid type, and further decreased upon conversion to complex type. Next, we examined the roles of three general types of N-glycans on ectopic expression of E-cadherin, a cell–cell adhesion protein. Microscopy revealed more functional E-cadherin at the cell–cell border when N-glycans were oligomannose and these levels decreased as the oligomannose N-glycans were processed to hybrid and then to complex. Thus, we provide evidence that all three general types of N-glycans impact plasma membrane architecture and cellular properties.

Cytotoxic protein from the mushroom Coprinus comatus possesses a unique mode for glycan binding and specificity.

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Zhang, Peilan; Li, Kunhua; Yang, Guang; Xia, Changqing; Polston, Jane E; Li, Gengnan; Li, Shiwu; Lin, Zhao; Yang, Li-Jun; Bruner, Steven D; Ding, Yousong

2017-08-22

Glycans possess significant chemical diversity; glycan binding proteins (GBPs) recognize specific glycans to translate their structures to functions in various physiological and pathological processes. Therefore, the discovery and characterization of novel GBPs and characterization of glycan-GBP interactions are significant to provide potential targets for therapeutic intervention of many diseases. Here, we report the biochemical, functional, and structural characterization of a 130-amino-acid protein, Y3, from the mushroom Coprinus comatus Biochemical studies of recombinant Y3 from a yeast expression system demonstrated the protein is a unique GBP. Additionally, we show that Y3 exhibits selective and potent cytotoxicity toward human T-cell leukemia Jurkat cells compared with a panel of cancer cell lines via inducing caspase-dependent apoptosis. Screening of a glycan array demonstrated GalNAcβ1-4(Fucα1-3)GlcNAc (LDNF) as a specific Y3-binding ligand. To provide a structural basis for function, the crystal structure was solved to a resolution of 1.2 Å, revealing a single-domain αβα-sandwich motif. Two monomers were dimerized to form a large 10-stranded, antiparallel β-sheet flanked by α-helices on each side, representing a unique oligomerization mode among GBPs. A large glycan binding pocket extends into the dimeric interface, and docking of LDNF identified key residues for glycan interactions. Disruption of residues predicted to be involved in LDNF/Y3 interactions resulted in the significant loss of binding to Jurkat T-cells and severely impaired their cytotoxicity. Collectively, these results demonstrate Y3 to be a GBP with selective cytotoxicity toward human T-cell leukemia cells and indicate its potential use in cancer diagnosis and treatment.

Investigating the feasibility of stem cell enrichment mediated by immobilized selectins.

Science.gov (United States)

Charles, Nichola; Liesveld, Jane L; King, Michael R

2007-01-01

Hematopoietic stem cell therapy is used to treat both malignant and non-malignant diseases, and enrichment of the hematopoietic stem and progenitor cells (HSPCs) has the potential to reduce the likelihood of graft vs host disease or relapse, potentially fatal complications associated with the therapy. Current commercial HSPC isolation technologies rely solely on the CD34 surface marker, and while they have proven to be invaluable, they can be time-consuming with variable recoveries reported. We propose that selectin-mediated enrichment could prove to be a quick and effective method for recovering HSPCs from adult bone marrow (ABM) on the basis of differences in rolling velocities and independently of CD34 expression. Purified CD34+ ABM cells and the unselected CD34- ABM cells were perfused over immobilized P-, E-, and L-selectin-IgG at physiologic wall shear stresses, and rolling velocities and cell retention data were collected. CD34+ ABM cells generally exhibited lower rolling velocities and higher retention than the unselected CD34- ABM cells on all three selectins. For initial CD34+ ABM cell concentrations ranging from 1% to 5%, we predict an increase in purity ranging from 5.2% to 36.1%, depending on the selectin used. Additionally, selectin-mediated cell enrichment is not limited to subsets of cells with inherent differences in rolling velocities. CD34+ KG1a cells and CD34- HL60 cells exhibited nearly identical rolling velocities on immobilized P-selectin-IgG over the entire range of shear stresses studied. However, when anti-CD34 antibody was co-immobilized with the P-selectin-IgG, the rolling velocity of the CD34+ KG1a cells was significantly reduced, making selectin-mediated cell enrichment a feasible option. Optimal cell enrichment in immobilized selectin surfaces can be achieved within 10 min, much faster than most current commercially available systems.

Novel cleavage of reductively aminated glycan-tags by N-bromosuccinimide to regenerate free, reducing glycans.

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Song, Xuezheng; Johns, Brian A; Ju, Hong; Lasanajak, Yi; Zhao, Chunmei; Smith, David F; Cummings, Richard D

2013-11-15

Glycans that are fluorescently tagged by reductive amination have been useful for functional glycomic studies. However, the existing tags can introduce unwanted properties to the glycans and complicate structural and functional studies. Here, we describe a facile method using N-bromosuccinimide (NBS) to remove the tags and efficiently regenerate free reducing glycans. The regenerated free reducing glycans can be easily analyzed by routine mass spectrometry or retagged with different tags for further studies. This new method can be used to efficiently remove a variety of fluorescent tags installed by reductive amination, including 2-aminobenzoic acid and 2-aminopyridine. NBS treatment essentially transforms the commonly used 2-aminobenzoic linkage to a cleavable linkage. It can be used to cleave printed glycans from microarrays and cleave neoglycopeptides containing a 2-aminobenzoic linker.

Solid-phase glycan isolation for glycomics analysis

OpenAIRE

Yang, Shuang; Zhang, Hui

2012-01-01

Glycosylation is one of the most significant protein PTMs. The biological activities of proteins are dramatically changed by the glycans associated with them. Thus, structural analysis of the glycans of glycoproteins in complex biological or clinical samples is critical in correlation with the functions of glycans with diseases. Profiling of glycans by HPLC-MS is a commonly used technique in analyzing glycan structures and quantifying their relative abundance in different biological systems. ...

Substrate recognition and catalysis by GH47 α-mannosidases involved in Asn-linked glycan maturation in the mammalian secretory pathway

Energy Technology Data Exchange (ETDEWEB)

Xiang, Yong; Karaveg, Khanita; Moremen, Kelley W.

2016-11-17

Asn-linked glycosylation of newly synthesized polypeptides occurs in the endoplasmic reticulum of eukaryotic cells. Glycan structures are trimmed and remodeled as they transit the secretory pathway, and processing intermediates play various roles as ligands for folding chaperones and signals for quality control and intracellular transport. Key steps for the generation of these trimmed intermediates are catalyzed by glycoside hydrolase family 47 (GH47) α-mannosidases that selectively cleave α1,2-linked mannose residues. Despite the sequence and structural similarities among the GH47 enzymes, the molecular basis for residue-specific cleavage remains obscure. The present studies reveal enzyme–substrate complex structures for two related GH47 α-mannosidases and provide insights into how these enzymes recognize the same substrates differently and catalyze the complementary glycan trimming reactions necessary for glycan maturation.

Structural analysis of N-glycans by the glycan-labeling method using 3-aminoquinoline-based liquid matrix in negative-ion MALDI-MS.

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Nishikaze, Takashi; Kaneshiro, Kaoru; Kawabata, Shin-ichirou; Tanaka, Koichi

2012-11-06

Negative-ion fragmentation of underivatized N-glycans has been proven to be more informative than positive-ion fragmentation. Fluorescent labeling via reductive amination is often employed for glycan analysis, but little is known about the influence of the labeling group on negative-ion fragmentation. We previously demonstrated that the on-target glycan-labeling method using 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid (3AQ/CHCA) liquid matrix enables highly sensitive, rapid, and quantitative N-glycan profiling analysis. The current study investigates the suitability of 3AQ-labeled N-glycans for structural analysis based on negative-ion collision-induced dissociation (CID) spectra. 3AQ-labeled N-glycans exhibited simple and informative CID spectra similar to those of underivatized N-glycans, with product ions due to cross-ring cleavages of the chitobiose core and ions specific to two antennae (D and E ions). The interpretation of diagnostic fragment ions suggested for underivatized N-glycans could be directly applied to the 3AQ-labeled N-glycans. However, fluorescently labeled N-glycans by conventional reductive amination, such as 2-aminobenzamide (2AB)- and 2-pyrydilamine (2PA)-labeled N-glycans, exhibited complicated CID spectra consisting of numerous signals formed by dehydration and multiple cleavages. The complicated spectra of 2AB- and 2PA-labeled N-glycans was found to be due to their open reducing-terminal N-acetylglucosamine (GlcNAc) ring, rather than structural differences in the labeling group in the N-glycan derivative. Finally, as an example, the on-target 3AQ labeling method followed by negative-ion CID was applied to structurally analyze neutral N-glycans released from human epidermal growth factor receptor type 2 (HER2) protein. The glycan-labeling method using 3AQ-based liquid matrix should facilitate highly sensitive quantitative and qualitative analyses of glycans.

21 CFR 172.898 - Bakers yeast glycan.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Bakers yeast glycan. 172.898 Section 172.898 Food... Multipurpose Additives § 172.898 Bakers yeast glycan. Bakers yeast glycan may be safely used in food in accordance with the following conditions: (a) Bakers yeast glycan is the comminuted, washed, pasteurized, and...

Serum concentrations of soluble (s)L- and (s)P-selectins in women with ovarian cancer.

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Majchrzak-Baczmańska, Dominika B; Głowacka, Ewa; Wilczyński, Miłosz; Malinowski, Andrzej

2018-03-01

The aim of the study was to compare serum concentration of soluble L- and P-selectins in women with ovarian cancer (OC) and healthy controls, and to investigate sL- and sP-selectin levels with regard to clinical and pathological parameters. Correlation analysis was used to measure the following: sL- and sP-selectin concentration and Ca125; sP-selectin and platelet concentrations; and sL-selectin and serum leukocyte levels in women with OC. The study included 29 patients with OC and 23 healthy controls. Serum concentrations of sL- and sP-selectins were measured in all subjects. Routine diagnostic tests: CBC and USG (both groups) and Ca125 (study group) were performed. Significantly higher serum concentrations of sL- and sP-selectins were found in the study group as compared to controls. Lower levels of serum sL-selectin were observed in women with poorly-differentiated OC (G3) and advanced stages of the disease (FIGO III, IV), but the results were statistically insignificant. No statistically significant relationship was detected between sP-selectin serum concentration in women with OC and tumour differentiation, histological type, and stage of the disease. No significant correlation was found between sL- and sP-selectins and Ca125 levels. A weak correlation was found between serum concentration of sP-selectin in women with OC and platelet count. No statistically significant correlation was observed between sL-selectin concentration and serum leukocyte levels in women with OC. The analysis of sL- and sP-selectin concentrations may be a useful tool in the diagnosis of OC. The levels of sL-selectin decrease with disease progression.

Chemoenzymatic assembly of mammalian O-mannose glycans.

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Cao, Hongzhi; Meng, Caicai; Sasmal, Aniruddha; Zhang, Yan; Gao, Tian; Liu, Chang-Cheng; Khan, Naazneen; Varki, Ajit; Wang, Fengshan

2018-05-26

O-Mannose glycans account up to 30% of total O-glycans in brain. Previous synthesis and functional studies only focused on the Core M3 O-mannose glycans of α-dystroglycan which are a causative factor for various muscular diseases. In this study, a highly efficient chemoenzymatic strategy was developed that enabled the first collective synthesis of 63 Core M1 and Core M2 O-mannose glycans. This chemoenzymatic strategy features the gram-scale chemical synthesis of 5 judiciously designed core structures, and the diversity-oriented modification of the core structures with 3 enzyme modules to provide 58 complex O-mannose glycans in a linear sequence that does not exceed 4 steps. The binding profiles of synthetic O-mannose glycans with a panel of lectins, antibodies and brain proteins were also explored using the printed O-mannose glycan array. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Blood Plasma-Derived Anti-Glycan Antibodies to Sialylated and Sulfated Glycans Identify Ovarian Cancer Patients.

Science.gov (United States)

Pochechueva, Tatiana; Chinarev, Alexander; Schoetzau, Andreas; Fedier, André; Bovin, Nicolai V; Hacker, Neville F; Jacob, Francis; Heinzelmann-Schwarz, Viola

2016-01-01

Altered levels of naturally occurring anti-glycan antibodies (AGA) circulating in human blood plasma are found in different pathologies including cancer. Here the levels of AGA directed against 22 negatively charged (sialylated and sulfated) glycans were assessed in high-grade serous ovarian cancer (HGSOC, n = 22) patients and benign controls (n = 31) using our previously developed suspension glycan array (SGA). Specifically, the ability of AGA to differentiate between controls and HGSOC, the most common and aggressive type of ovarian cancer with a poor outcome was determined. Results were compared to CA125, the commonly used ovarian cancer biomarker. AGA to seven glycans that significantly (P<0.05) differentiated between HGSOC and control were identified: AGA to top candidates SiaTn and 6-OSulfo-TF (both IgM) differentiated comparably to CA125. The area under the curve (AUC) of a panel of AGA to 5 glycans (SiaTn, 6-OSulfo-TF, 6-OSulfo-LN, SiaLea, and GM2) (0.878) was comparable to CA125 (0.864), but it markedly increased (0.985) when combined with CA125. AGA to SiaTn and 6-OSulfo-TF were also valuable predictors for HGSOC when CA125 values appeared inconclusive, i.e. were below a certain threshold. AGA-glycan binding was in some cases isotype-dependent and sensitive to glycosidic linkage switch (α2-6 vs. α2-3), to sialylation, and to sulfation of the glycans. In conclusion, plasma-derived AGA to sialylated and sulfated glycans including SiaTn and 6-OSulfo-TF detected by SGA present a valuable alternative to CA125 for differentiating controls from HGSOC patients and for predicting the likelihood of HGSOC, and may be potential HGSOC tumor markers.

Structural Analysis of N- and O-glycans Using ZIC-HILIC/Dialysis Coupled to NMR Detection

Energy Technology Data Exchange (ETDEWEB)

Qu, Yi; Feng, Ju; Deng, Shuang; Cao, Li; Zhang, Qibin; Zhao, Rui; Zhang, Zhaorui; Jiang, Yuxuan; Zink, Erika M.; Baker, Scott E.; Lipton, Mary S.; Pasa-Tolic, Ljiljana; Hu, Jian Z.; Wu, Si

2014-11-19

Protein glycosylation, an important and complex post-translational modification (PTM), is involved in various biological processes including the receptor-ligand and cell-cell interaction, and plays a crucial role in many biological functions. However, little is known about the glycan structures of important biological complex samples, and the conventional glycan enrichment strategy (i.e., size-exclusion column [SEC] separation,) prior to nuclear magnetic resonance (NMR) detection is time-consuming and tedious. In this study, we employed SEC, Zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC), and ZIC-HILIC coupled with dialysis strategies to enrich the glycopeptides from the pronase E digests of RNase B, followed by NMR analysis of the glycoconjugate. Our results suggest that the ZIC-HILIC enrichment coupled with dialysis is the most efficient, which was thus applied to the analysis of biological complex sample, the pronase E digest of the secreted proteins from the fungi Aspergillus niger. The NMR spectra revealed that the secreted proteins from A. niger contain both N-linked glycans with a high-mannose core and O-linked glycans bearing mannose and glucose with 1->3 and 1->6 linkages. In all, our study provides compelling evidence that ZIC-HILIC separation coupled to dialysis is superior to the commonly used SEC separation to prepare glycopeptides for the downstream NMR analysis, which could greatly facilitate the future NMR-based glycoproteomics research.

Mass spectrometric-based stable isotopic 2-aminobenzoic acid glycan mapping for rapid glycan screening of biotherapeutics.

Science.gov (United States)

Prien, Justin M; Prater, Bradley D; Qin, Qiang; Cockrill, Steven L

2010-02-15

Fast, sensitive, robust methods for "high-level" glycan screening are necessary during various stages of a biotherapeutic product's lifecycle, including clone selection, process changes, and quality control for lot release testing. Traditional glycan screening involves chromatographic or electrophoretic separation-based methods, and, although reproducible, these methods can be time-consuming. Even ultrahigh-performance chromatographic and microfluidic integrated LC/MS systems, which work on the tens of minute time scale, become lengthy when hundreds of samples are to be analyzed. Comparatively, a direct infusion mass spectrometry (MS)-based glycan screening method acquires data on a millisecond time scale, exhibits exquisite sensitivity and reproducibility, and is amenable to automated peak annotation. In addition, characterization of glycan species via sequential mass spectrometry can be performed simultaneously. Here, we demonstrate a quantitative high-throughput MS-based mapping approach using stable isotope 2-aminobenzoic acid (2-AA) for rapid "high-level" glycan screening.

Circulating endothelial cells (CECs and E-selectin: Predictors of preeclampsia

Directory of Open Access Journals (Sweden)

Ferdous Mehrabian

2012-01-01

Full Text Available Background: Circulating endothelial cells (CECs and E-selectin are known as sensitive and specific markers of en-dothelial dysfunction. This study investigated whether CECs and E-selectin are surrogate biomarkers of preeclampsia and if measurement of CECs and E-selectin, early in the third trimester, could be a means of predicting preeclampsia. Methods: In this prospective, descriptive-analytic study, rollover test was performed on 523 pregnant women during 28-30 weeks of gestation. CECs were measured by anti-CD 146-driven immunomagnetic isolation in women with posi-tive rollover test. They were followed up prospectively until delivery without any active intervention. Women with and without preeclampsia were determined. The number of CECs and level of E-selectin were compared in the two studied groups. Results: From the 47 pregnant women with positive rollover test who were selected and followed up, 22 individuals were diagnosed with preeclampsia while the remainder were normotensive. Mean CEC numbers was significantly high-er in preeclamptic women than normal pregnancies (24.7 cells/mL vs. 13 cells/mL. The best cut-off point for CEC numbers was 6.5 with a sensitivity of 78.9% and a specificity of 69.1%. The level of E-selectin was significantly higher in mothers with preeclampsia (p < 0.05. Conclusions: Higher levels of CECs and E-selectin in women with positive rollover test who developed preeclampsia prior to onset of the complication were predictive of preeclampsia. However, larger studies are needed to confirm these findings.

Sulfated Hexasaccharides Attenuate Metastasis by Inhibition of P-selectin and Heparanase

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Lubor Borsig

2011-05-01

Full Text Available Development of compounds that target both heparanase and selectins is emerging as a promising approach for cancer therapy. Selectins are vascular cell adhesion molecules that mediate tumor cell interactions with platelets, leukocytes, and the vascular endothelium. Heparanase is an endoglycosidase that degrades heparan sulfate in the tumor microenvironment, cell surfaces, and vessel wall. Acting together, these molecules facilitate tumor cell arrest, extravasation, and metastasis. Here, we report the preparation of novel semisynthetic sulfated tri mannose C-C-linked dimers (STMCs endowed with heparanase and selectin inhibitory activity. The P-selectin specificity of the STMC was defined by the anomeric linkage of the C-C bond. This STMC hexasaccharide is an effective inhibitor of P-selectin in vivo. We show that selective inhibition of heparanase attenuates metastasis in B16-BL6 melanoma cells, expressing high levels of this endoglycosidase, but has no effect on the metastasis of MC-38 carcinoma cells that express little or no heparanase activity. P-selectin-specific STMC attenuated metastasis in both animal models, indicating that inhibition of tumor cell interaction with the vascular endothelium is critical for cancer dissemination. Thus, the small size, the stability of the C-C bond, and the chemically defined structure of the newly generated STMCs make them superior to heparin derivatives and signify STMCs as valuable candidates for further evaluation.

Correlation between serum E-selectin levels and panoramic nailfold capillaroscopy in systemic sclerosis

Directory of Open Access Journals (Sweden)

Valim V.

2004-01-01

Full Text Available E-selectin is expressed by the activated endothelium and its plasma levels are increased in patients with systemic sclerosis. Eighteen patients fulfilling the American Rheumatism Association criteria for systemic sclerosis, 15 females and 3 males, 42-70 years old, 9 with diffuse and 9 with limited forms, were sequentially recruited for this study. Serum E-selectin levels were determined by commercially available ELISA and their association with nailfold capillaroscopic abnormalities was investigated. Nailfold capillaries were analyzed by 16X magnification wide-field capillaroscopy. Two parameters on capillaroscopy were used to correlate to serum E-selectin: deletion and ectasia. Data were analyzed statistically by the Student t-test and Spearman correlation. Two-tailed P values below 0.05 were considered significant. E-selectin range was 38 to 200 ng/ml (80 ± 39.94. There was a correlation between serum E-selectin levels and the deletion capillaroscopic score (r = 0.50, P < 0.035. This correlation was even stronger within the first 48 months of diagnosis (r = 0.63, P < 0.048. On the other hand, no association was observed between selectin and ectasia. Patients with diffuse disease presented higher serum E-selectin levels than patients with limited disease, although the difference was not statistically significant (96.44 ± 48.04 vs 63.56 ± 21.77 ng/dl; P = 0.08. The present study is the first showing a correlation between soluble serum E-selectin levels and alterations in capillaroscopy. The stronger correlation of deletion score in capillaroscopy in early disease suggests that serum E-selectin levels might be a useful biochemical marker of disease activity in systemic sclerosis.

Correlation between serum E-selectin levels and panoramic nailfold capillaroscopy in systemic sclerosis

Directory of Open Access Journals (Sweden)

V. Valim

2004-09-01

Full Text Available E-selectin is expressed by the activated endothelium and its plasma levels are increased in patients with systemic sclerosis. Eighteen patients fulfilling the American Rheumatism Association criteria for systemic sclerosis, 15 females and 3 males, 42-70 years old, 9 with diffuse and 9 with limited forms, were sequentially recruited for this study. Serum E-selectin levels were determined by commercially available ELISA and their association with nailfold capillaroscopic abnormalities was investigated. Nailfold capillaries were analyzed by 16X magnification wide-field capillaroscopy. Two parameters on capillaroscopy were used to correlate to serum E-selectin: deletion and ectasia. Data were analyzed statistically by the Student t-test and Spearman correlation. Two-tailed P values below 0.05 were considered significant. E-selectin range was 38 to 200 ng/ml (80 ± 39.94. There was a correlation between serum E-selectin levels and the deletion capillaroscopic score (r = 0.50, P < 0.035. This correlation was even stronger within the first 48 months of diagnosis (r = 0.63, P < 0.048. On the other hand, no association was observed between selectin and ectasia. Patients with diffuse disease presented higher serum E-selectin levels than patients with limited disease, although the difference was not statistically significant (96.44 ± 48.04 vs 63.56 ± 21.77 ng/dl; P = 0.08. The present study is the first showing a correlation between soluble serum E-selectin levels and alterations in capillaroscopy. The stronger correlation of deletion score in capillaroscopy in early disease suggests that serum E-selectin levels might be a useful biochemical marker of disease activity in systemic sclerosis.

Differential N-glycan patterns identified in lung adenocarcinoma by N-glycan profiling of formalin-fixed paraffin-embedded (FFPE) tissue sections.

Science.gov (United States)

Wang, Xiaoning; Deng, Zaian; Huang, Chuncui; Zhu, Tong; Lou, Jiatao; Wang, Lin; Li, Yan

2018-02-10

N-glycan profiling is a powerful approach for analyzing the functional relationship between N-glycosylation and cancer. Current methods rely on either serum or fresh tissue samples; however, N-glycan patterns may differ between serum and tissue, as the proteins of serum originate from a variety of tissues. Furthermore, fresh tissue samples are difficult to ship and store. Here, we used a profiling method based on formalin-fixed paraffin-embedded (FFPE) tissue sections from lung adenocarcinoma patients. We found that our method was highly reproducible. We identified 58 N-glycan compositions from lung adenocarcinoma FFPE samples, 51 of which were further used for MS n -based structure prediction. We show that high mannose type N-glycans are upregulated, while sialylated N-glycans are downregulated in our FFPE lung adenocarcinoma samples, compared to the control samples. Our receiver operating characteristic (ROC) curve analysis shows that high mannose type and sialylated N-glycans are useful discriminators to distinguish between lung adenocarcinoma and control tissue. Together, our results indicate that expression levels of specific N-glycans correlate well with lung adenocarcinoma, and strongly suggest that our FFPE-based method will be useful for N-glycan profiling of cancer tissues. Glycosylation is one of the most important post-translational protein modifications, and is associated with several physiopathological processes, including carcinogenesis. In this study, we tested the feasibility of using formalin-fixed paraffin-embedded (FFPE) tissue sections to identify changes in N-glycan patterns and identified the differentially expressed N-glycans of lung adenocarcinoma. Our study shows that the FFPE-based N-glycan profiling method is useful for clinical diagnosis as well as identification of potential biomarkers, and our data expand current knowledge of differential N-glycan patterns of lung adenocarcinoma. Copyright © 2017 Elsevier B.V. All rights reserved.

Cytotoxic protein from the mushroom Coprinus comatus possesses a unique mode for glycan binding and specificity

Science.gov (United States)

Zhang, Peilan; Yang, Guang; Xia, Changqing; Polston, Jane E.; Li, Gengnan; Li, Shiwu; Lin, Zhao; Yang, Li-jun; Bruner, Steven D.

2017-01-01

Glycans possess significant chemical diversity; glycan binding proteins (GBPs) recognize specific glycans to translate their structures to functions in various physiological and pathological processes. Therefore, the discovery and characterization of novel GBPs and characterization of glycan–GBP interactions are significant to provide potential targets for therapeutic intervention of many diseases. Here, we report the biochemical, functional, and structural characterization of a 130-amino-acid protein, Y3, from the mushroom Coprinus comatus. Biochemical studies of recombinant Y3 from a yeast expression system demonstrated the protein is a unique GBP. Additionally, we show that Y3 exhibits selective and potent cytotoxicity toward human T-cell leukemia Jurkat cells compared with a panel of cancer cell lines via inducing caspase-dependent apoptosis. Screening of a glycan array demonstrated GalNAcβ1–4(Fucα1–3)GlcNAc (LDNF) as a specific Y3-binding ligand. To provide a structural basis for function, the crystal structure was solved to a resolution of 1.2 Å, revealing a single-domain αβα-sandwich motif. Two monomers were dimerized to form a large 10-stranded, antiparallel β-sheet flanked by α-helices on each side, representing a unique oligomerization mode among GBPs. A large glycan binding pocket extends into the dimeric interface, and docking of LDNF identified key residues for glycan interactions. Disruption of residues predicted to be involved in LDNF/Y3 interactions resulted in the significant loss of binding to Jurkat T-cells and severely impaired their cytotoxicity. Collectively, these results demonstrate Y3 to be a GBP with selective cytotoxicity toward human T-cell leukemia cells and indicate its potential use in cancer diagnosis and treatment. PMID:28784797

Correlation between serum E-selectin levels and panoramic nailfold capillaroscopy in systemic sclerosis.

Science.gov (United States)

Valim, V; Assis, L S S; Simões, M F J; Trevisani, V F M; Pucinelli, M L C; Andrade, L E C

2004-09-01

E-selectin is expressed by the activated endothelium and its plasma levels are increased in patients with systemic sclerosis. Eighteen patients fulfilling the American Rheumatism Association criteria for systemic sclerosis, 15 females and 3 males, 42-70 years old, 9 with diffuse and 9 with limited forms, were sequentially recruited for this study. Serum E-selectin levels were determined by commercially available ELISA and their association with nailfold capillaroscopic abnormalities was investigated. Nailfold capillaries were analyzed by 16X magnification wide-field capillaroscopy. Two parameters on capillaroscopy were used to correlate to serum E-selectin: deletion and ectasia. Data were analyzed statistically by the Student t-test and Spearman correlation. Two-tailed P values below 0.05 were considered significant. E-selectin range was 38 to 200 ng/ml (80 +/- 39.94). There was a correlation between serum E-selectin levels and the deletion capillaroscopic score (r = 0.50, P capillaroscopy. The stronger correlation of deletion score in capillaroscopy in early disease suggests that serum E-selectin levels might be a useful biochemical marker of disease activity in systemic sclerosis.

Cimetidine attenuates vinorelbine-induced phlebitis in mice by militating E-selectin expression.

Science.gov (United States)

Wang, Zhuo; Ma, Lijuan; Wang, Xuebin; Cai, Heping; Huang, Jin; Liu, Jiyong; Hu, Jinhong; Su, Dingfeng

2014-08-01

We investigated E-selectin expression in mice and rabbits with vinorelbine-induced phlebitis and the effect of cimetidine. To find the relationship between E-selectin expression and vinorelbine-induced phlebitis. Mouse and rabbit model of vinorelbine-induced phlebitis was established by intravenous infusion of vinorelbine. Pathological observation, molecular-biological determination of E-selectin and protein function of it was evaluated. Grossly, we observed swelling, edema and cord-like vessel changes in mice receiving vinorelbine but only mild edema in mice pretreated with cimetidine. Pathological scoring yielded a total score of 37 for vinorelbine-treated mice and 17 for mice pretreated with cimetidine (P phlebitis in mice probably by suppressing increased expression of E-selectin.

Qrator: A web-based curation tool for glycan structures

Science.gov (United States)

Eavenson, Matthew; Kochut, Krys J; Miller, John A; Ranzinger, René; Tiemeyer, Michael; Aoki, Kazuhiro; York, William S

2015-01-01

Most currently available glycan structure databases use their own proprietary structure representation schema and contain numerous annotation errors. These cause problems when glycan databases are used for the annotation or mining of data generated in the laboratory. Due to the complexity of glycan structures, curating these databases is often a tedious and labor-intensive process. However, rigorously validating glycan structures can be made easier with a curation workflow that incorporates a structure-matching algorithm that compares candidate glycans to a canonical tree that embodies structural features consistent with established mechanisms for the biosynthesis of a particular class of glycans. To this end, we have implemented Qrator, a web-based application that uses a combination of external literature and database references, user annotations and canonical trees to assist and guide researchers in making informed decisions while curating glycans. Using this application, we have started the curation of large numbers of N-glycans, O-glycans and glycosphingolipids. Our curation workflow allows creating and extending canonical trees for these classes of glycans, which have subsequently been used to improve the curation workflow. PMID:25165068

Glycan array data management at Consortium for Functional Glycomics.

Science.gov (United States)

Venkataraman, Maha; Sasisekharan, Ram; Raman, Rahul

2015-01-01

Glycomics or the study of structure-function relationships of complex glycans has reshaped post-genomics biology. Glycans mediate fundamental biological functions via their specific interactions with a variety of proteins. Recognizing the importance of glycomics, large-scale research initiatives such as the Consortium for Functional Glycomics (CFG) were established to address these challenges. Over the past decade, the Consortium for Functional Glycomics (CFG) has generated novel reagents and technologies for glycomics analyses, which in turn have led to generation of diverse datasets. These datasets have contributed to understanding glycan diversity and structure-function relationships at molecular (glycan-protein interactions), cellular (gene expression and glycan analysis), and whole organism (mouse phenotyping) levels. Among these analyses and datasets, screening of glycan-protein interactions on glycan array platforms has gained much prominence and has contributed to cross-disciplinary realization of the importance of glycomics in areas such as immunology, infectious diseases, cancer biomarkers, etc. This manuscript outlines methodologies for capturing data from glycan array experiments and online tools to access and visualize glycan array data implemented at the CFG.

Sulfated Hexasaccharides Attenuate Metastasis by Inhibition of P-selectin and Heparanase1

Science.gov (United States)

Borsig, Lubor; Vlodavsky, Israel; Ishai-Michaeli, Rivka; Torri, Giangiacomo; Vismara, Elena

2011-01-01

Development of compounds that target both heparanase and selectins is emerging as a promising approach for cancer therapy. Selectins are vascular cell adhesion molecules that mediate tumor cell interactions with platelets, leukocytes, and the vascular endothelium. Heparanase is an endoglycosidase that degrades heparan sulfate in the tumor microenvironment, cell surfaces, and vessel wall. Acting together, these molecules facilitate tumor cell arrest, extravasation, and metastasis. Here, we report the preparation of novel semisynthetic sulfated tri mannose C-C-linked dimers (STMCs) endowed with heparanase and selectin inhibitory activity. The P-selectin specificity of the STMC was defined by the anomeric linkage of the C-C bond. This STMC hexasaccharide is an effective inhibitor of P-selectin in vivo. We show that selective inhibition of heparanase attenuates metastasis in B16-BL6 melanoma cells, expressing high levels of this endoglycosidase, but has no effect on the metastasis of MC-38 carcinoma cells that express little or no heparanase activity. P-selectin-specific STMC attenuated metastasis in both animal models, indicating that inhibition of tumor cell interaction with the vascular endothelium is critical for cancer dissemination. Thus, the small size, the stability of the C-C bond, and the chemically defined structure of the newly generated STMCs make them superior to heparin derivatives and signify STMCs as valuable candidates for further evaluation. PMID:21532885

A time-resolved luminescent competitive assay to detect L-selectin using aptamers as recognition elements

International Nuclear Information System (INIS)

Cywiński, Piotr J.; Olejko, Lydia; Löhmannsröben, Hans-Gerd

2015-01-01

L-selectin is a protein with potential importance for numerous diseases and clinical disorders. In this paper, we present a new aptamer-based luminescent assay developed to detect L-selectin. The sensing system working principle is based on Förster Resonance Energy Transfer (FRET) from a donor terbium complex (TbC) to an acceptor cyanine dye (Cy5). In the present approach, the biotinylated aptamer is combined with Cy5-labelled streptavidin (Cy5-Strep) to yield an aptamer-based acceptor construct (Apta-Cy5-Strep), while L-selectin is conjugated using luminescent TbC. Upon aptamer binding to the TbC-labelled L-selectin (L-selectin-TbC), permanent donor-acceptor proximity is established which allows for radiationless energy transfer to occur. However, when unlabelled L-selectin is added, it competes with the L-selectin-TbC and the FRET signal decreases as the L-selectin concentration increases. FRET from the TbC to Cy5 was observed with time-gated time-resolved luminescence spectroscopy. A significant change in the corrected luminescence signal was observed in the dynamic range of 10–500 ng/mL L-selectin, the concentration range relevant for accelerated cognitive decline of Alzheimer's disease, with a limit of detection (LOD) equal to 10 ng/mL. The aptasensor-based assay is homogeneous and can be realized within one hour. Therefore, this method has the potential to become an alternative to tedious heterogeneous analytical methods, e.g. based on enzyme-linked immunosorbent assay (ELISA). - Highlights: • Tb-based FRET assay with aptamers toward a protein is presented for the first time. • L-selectin can be detected in concentrations relevant for the Alzheimer's Disease. • The assay can be realized in one hour with the LOD equal to 10 ng/ml

A time-resolved luminescent competitive assay to detect L-selectin using aptamers as recognition elements

Energy Technology Data Exchange (ETDEWEB)

Cywiński, Piotr J., E-mail: piotr.cywinski@iap.fraunhofer.de [Functional Materials and Devices, Fraunhofer Institute for Applied Polymer Research, Geiselberstr.69, 14476 Potsdam-Golm (Germany); Department of Physical Chemistry, Institute of Chemistry, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam-Golm (Germany); Olejko, Lydia; Löhmannsröben, Hans-Gerd [Department of Physical Chemistry, Institute of Chemistry, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam-Golm (Germany)

2015-08-05

L-selectin is a protein with potential importance for numerous diseases and clinical disorders. In this paper, we present a new aptamer-based luminescent assay developed to detect L-selectin. The sensing system working principle is based on Förster Resonance Energy Transfer (FRET) from a donor terbium complex (TbC) to an acceptor cyanine dye (Cy5). In the present approach, the biotinylated aptamer is combined with Cy5-labelled streptavidin (Cy5-Strep) to yield an aptamer-based acceptor construct (Apta-Cy5-Strep), while L-selectin is conjugated using luminescent TbC. Upon aptamer binding to the TbC-labelled L-selectin (L-selectin-TbC), permanent donor-acceptor proximity is established which allows for radiationless energy transfer to occur. However, when unlabelled L-selectin is added, it competes with the L-selectin-TbC and the FRET signal decreases as the L-selectin concentration increases. FRET from the TbC to Cy5 was observed with time-gated time-resolved luminescence spectroscopy. A significant change in the corrected luminescence signal was observed in the dynamic range of 10–500 ng/mL L-selectin, the concentration range relevant for accelerated cognitive decline of Alzheimer's disease, with a limit of detection (LOD) equal to 10 ng/mL. The aptasensor-based assay is homogeneous and can be realized within one hour. Therefore, this method has the potential to become an alternative to tedious heterogeneous analytical methods, e.g. based on enzyme-linked immunosorbent assay (ELISA). - Highlights: • Tb-based FRET assay with aptamers toward a protein is presented for the first time. • L-selectin can be detected in concentrations relevant for the Alzheimer's Disease. • The assay can be realized in one hour with the LOD equal to 10 ng/ml.

P-Selectin: An Unpredicted Factor for Deep Vein Thrombosis after Total Hip Arthroplasty

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Dongquan Shi

2014-01-01

Full Text Available Introduction. Deep vein thrombosis (DVT is a severe complication after total hip arthroplasty (THA. It leads to acute pulmonary embolism, a life-threatening disease. P-selectin is a 140-kDa transmembrane glycoprotein. Elevated P-selectin was associated with 1.7-fold increase in the risk of venous thrombosis. Materials and Methods. To confirm the association, a total of 91 subjects who received primary total hip arthroplasty using lateral approach performed by one skilled orthopedic surgeon were studied. All the patients were consecutively enrolled at the Center of Diagnosis and Treatment for Joint Diseases, Drum Tower Hospital affiliated to the Medical School of Nanjing University from 2010 to 2012. All the subjects received venography 3–5 days after operation. We measured P-selectin by means of a highly sensitive sandwich ELISA technique and a commercially available test reagent set. Results. No significant association was detected between P-selectin and DVT (all P  values>0.05. ΔsP-selectin was correlated with weight, APTT after operation, history of DVT, and diagnosis of primary disease ( P values were 0.03, 0.03, 0.04, and 0.02, resp.. Conclusion. P-selectin may not be a predicted factor for deep vein thrombosis after total hip arthroplasty.

Plasma substance P and soluble P-selectin as biomarkers of β ...

African Journals Online (AJOL)

Samia A. Ebeid

2013-09-19

Sep 19, 2013 ... logic disorder that causes hemolytic anemia because of the de- creased or absent .... AMs), sE-selectin, sP-selectin in sickle cell patients compared to healthy individuals. .... with childhood sickle cell · vasoocclusive crises.

Notable Aspects of Glycan-Protein Interactions

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Miriam Cohen

2015-09-01

Full Text Available This mini review highlights several interesting aspects of glycan-mediated interactions that are common between cells, bacteria, and viruses. Glycans are ubiquitously found on all living cells, and in the extracellular milieu of multicellular organisms. They are known to mediate initial binding and recognition events of both immune cells and pathogens with their target cells or tissues. The host target tissues are hidden under a layer of secreted glycosylated decoy targets. In addition, pathogens can utilize and display host glycans to prevent identification as foreign by the host’s immune system (molecular mimicry. Both the host and pathogens continually evolve. The host evolves to prevent infection and the pathogens evolve to evade host defenses. Many pathogens express both glycan-binding proteins and glycosidases. Interestingly, these proteins are often located at the tip of elongated protrusions in bacteria, or in the leading edge of the cell. Glycan-protein interactions have low affinity and, as a result, multivalent interactions are often required to achieve biologically relevant binding. These enable dynamic forms of adhesion mechanisms, reviewed here, and include rolling (cells, stick and roll (bacteria or surfacing (viruses.

Lewis antigen mediated adhesion of freshly removed human bladder tumors to E-selectin

DEFF Research Database (Denmark)

Skorsteensgaard, Karna; Vestergaard, Else Marie; Langkilde, Niels

1999-01-01

PURPOSE: Twenty fresh surgical specimens of human bladder tumors were tested for their ability to adhere to recombinant P and E-selectin. The adhesion was correlated to immunological detection of carbohydrate structures. MATERIALS AND METHODS: A static titertray assay with immobilized selectins.......003), whereas no correlation was found to secretor and Lewis genotypes. CONCLUSIONS: These data on clinical specimens indicate that Lewis antigen mediated E-selectin adhesion may play a role in the human bladder cancer disease....

Stem Cell Enrichment with Selectin Receptors: Mimicking the pH Environment of Trauma

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Michael R. King

2013-09-01

Full Text Available The isolation of hematopoietic stem and progenitor cells (HSPCs is critical for transplantation therapy and HSPC research, however current isolation techniques can be prohibitively expensive, time-consuming, and produce variable results. Selectin-coated microtubes have shown promise in rapidly isolating HSPCs from human bone marrow, but further purification of HSPCs remains a challenge. Herein, a biomimetic device for HSPC isolation is presented to mimic the acidic vascular microenvironment during trauma, which can enhance the binding frequency between L-selectin and its counter-receptor PSGL-1 and HSPCs. Under acidic pH conditions, L-selectin coated microtubes enhanced CD34+ HSPC adhesion, as evidenced by decreased cell rolling velocity and increased rolling flux. Dynamic light scattering was utilized as a novel sensor to confirm an L-selectin conformational change under acidic conditions, as previously predicted by molecular dynamics. These results suggest that mimicking the acidic conditions of trauma can induce a conformational extension of L-selectin, which can be utilized for flow-based, clinical isolation of HSPCs.

Comprehensive analysis of the N-glycan biosynthetic pathway using bioinformatics to generate UniCorn: A theoretical N-glycan structure database.

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Akune, Yukie; Lin, Chi-Hung; Abrahams, Jodie L; Zhang, Jingyu; Packer, Nicolle H; Aoki-Kinoshita, Kiyoko F; Campbell, Matthew P

2016-08-05

Glycan structures attached to proteins are comprised of diverse monosaccharide sequences and linkages that are produced from precursor nucleotide-sugars by a series of glycosyltransferases. Databases of these structures are an essential resource for the interpretation of analytical data and the development of bioinformatics tools. However, with no template to predict what structures are possible the human glycan structure databases are incomplete and rely heavily on the curation of published, experimentally determined, glycan structure data. In this work, a library of 45 human glycosyltransferases was used to generate a theoretical database of N-glycan structures comprised of 15 or less monosaccharide residues. Enzyme specificities were sourced from major online databases including Kyoto Encyclopedia of Genes and Genomes (KEGG) Glycan, Consortium for Functional Glycomics (CFG), Carbohydrate-Active enZymes (CAZy), GlycoGene DataBase (GGDB) and BRENDA. Based on the known activities, more than 1.1 million theoretical structures and 4.7 million synthetic reactions were generated and stored in our database called UniCorn. Furthermore, we analyzed the differences between the predicted glycan structures in UniCorn and those contained in UniCarbKB (www.unicarbkb.org), a database which stores experimentally described glycan structures reported in the literature, and demonstrate that UniCorn can be used to aid in the assignment of ambiguous structures whilst also serving as a discovery database. Copyright © 2016 Elsevier Ltd. All rights reserved.

Selected immunological changes in patients with Goeckerman's therapy TNF-alpha, sE-selectin, sP-selectin, sICAM-1 and IL-8

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Borska, L.; Fiala, Z.; Krejsek, J.; Andrys, C.; Vokurkova, D.; Hamakova, K.; Kremlacek, J.; Ettler, K. [Charles University, Hradec Kralove (Czech Republic). Faculty of Medicine

2006-07-01

Psoriasis is one of the most frequent inflammatory skin diseases in which abnormal individual immune reactivity plays an important role. The aim of the present study was to describe selected immunological changes, concerning pro-inflammatory cytokines (TNF-alpha, IL-8) and adhesion molecules (sE-selectin, sP-selectin, sICAM-1), in 56 patients cured by Goeckerman's therapy (GT). GT includes dermal application of crude coal tar (containing polycyclic aromatic hydrocarbons) and exposure to UV radiation.

Comparison of L-selectin blood level and gene polymorphism in tuberculosis patients with healthy individuals.

Science.gov (United States)

Eini, Peyman; Shirvani, Maria; Hajilooi, Mehrdad; Esna-Ashari, Farzaneh

2018-02-12

The inflammatory response to Mycobacterium tuberculosis bacilli influences tuberculosis (TB) progression. In this study, we aimed to identify the Phe206Leu polymorphism and serum L-selectin level in TB patients, compared to healthy individuals. Ninety patients with a diagnosis of TB and 90 healthy controls were selected in this study. The serum L-selectin level was determined, using ELISA. L-selectin polymorphism was also evaluated using PCR. For data analysis, SPSS was used at a significance level of 0.05. According to the findings, the mean±SD age of the participants was 57.5 ± 18.4 and 56.5 ± 17.5 years in the TB and healthy groups, respectively. The TB group showed a significantly higher serum L-selectin level (1721.1 ± 330.9) versus the healthy controls (1624 ± 279). The L-selectin Phe allele frequencies were higher than the Leu allele frequencies in the main population, whereas the patients and controls were not significantly different. Eight (0.04%) subjects had Leu/Leu genotypes, 84 (46.6%) carried Phe/Leu genotypes, and 88 (48.8%) had Phe/Phe genotypes. Our results showed that the groups were not significantly different regarding L-selectin genotypes. TB patients had a significantly higher serum L-selectin level, compared to the controls. Based on the findings, the incidence of TB and L-selectin polymorphism in the Phe206Leu gene had no significant association. © 2018 Wiley Periodicals, Inc.

A spin column-free approach to sodium hydroxide-based glycan permethylation.

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Hu, Yueming; Borges, Chad R

2017-07-24

Glycan permethylation was introduced as a tool to facilitate the study of glycans in 1903. Since that time, permethylation procedures have been continually modified to improve permethylation efficiency and qualitative applicability. Typically, however, either laborious preparation steps or cumbersome and uneconomical spin columns have been needed to obtain decent permethylation yields on small glycan samples. Here we describe a spin column-free (SCF) glycan permethylation procedure that is applicable to both O- and N-linked glycans and can be employed upstream to intact glycan analysis by MALDI-MS, ESI-MS, or glycan linkage analysis by GC-MS. The SCF procedure involves neutralization of NaOH beads by acidified phosphate buffer, which eliminates the risk of glycan oxidative degradation and avoids the use of spin columns. Optimization of the new permethylation procedure provided high permethylation efficiency for both hexose (>98%) and HexNAc (>99%) residues-yields which were comparable to (or better than) those of some widely-used spin column-based procedures. A light vs. heavy labelling approach was employed to compare intact glycan yields from a popular spin-column based approach to the SCF approach. Recovery of intact N-glycans was significantly better with the SCF procedure (p 0.75; p < 0.01). In summary, the SCF permethylation procedure expedites and economizes both intact glycan analysis and linkage analysis of glycans from whole biospecimens.

A spin column-free approach to sodium hydroxide-based glycan permethylation†

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Hu, Yueming; Borges, Chad R.

2018-01-01

Glycan permethylation was introduced as a tool to facilitate the study of glycans in 1903. Since that time, permethylation procedures have been continually modified to improve permethylation efficiency and qualitative applicability. Typically, however, either laborious preparation steps or cumbersome and uneconomical spin columns have been needed to obtain decent permethylation yields on small glycan samples. Here we describe a spin column-free (SCF) glycan permethylation procedure that is applicable to both O- and N-linked glycans and can be employed upstream to intact glycan analysis by MALDI-MS, ESI-MS, or glycan linkage analysis by GC-MS. The SCF procedure involves neutralization of NaOH beads by acidified phosphate buffer, which eliminates the risk of glycan oxidative degradation and avoids the use of spin columns. Optimization of the new permethylation procedure provided high permethylation efficiency for both hexose (>98%) and HexNAc (>99%) residues—yields which were comparable to (or better than) those of some widely-used spin column-based procedures. A light vs. heavy labelling approach was employed to compare intact glycan yields from a popular spin-column based approach to the SCF approach. Recovery of intact N-glycans was significantly better with the SCF procedure (p 0.75; p < 0.01). In summary, the SCF permethylation procedure expedites and economizes both intact glycan analysis and linkage analysis of glycans from whole biospecimens. PMID:28635997

Glycotherapy: new advances inspire a reemergence of glycans in medicine.

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Hudak, Jason E; Bertozzi, Carolyn R

2014-01-16

The beginning of the 20(th) century marked the dawn of modern medicine with glycan-based therapies at the forefront. However, glycans quickly became overshadowed as DNA- and protein-focused treatments became readily accessible. The recent development of new tools and techniques to study and produce structurally defined carbohydrates has spurred renewed interest in the therapeutic applications of glycans. This review focuses on advances within the past decade that are bringing glycan-based treatments back to the forefront of medicine and the technologies that are driving these efforts. These include the use of glycans themselves as therapeutic molecules as well as engineering protein and cell surface glycans to suit clinical applications. Glycan therapeutics offer a rich and promising frontier for developments in the academic, biopharmaceutical, and medical fields. Copyright © 2014 Elsevier Ltd. All rights reserved.

Structural basis of glycan specificity of P[19] VP8*: Implications for rotavirus zoonosis and evolution.

Science.gov (United States)

Liu, Yang; Xu, Shenyuan; Woodruff, Andrew L; Xia, Ming; Tan, Ming; Kennedy, Michael A; Jiang, Xi

2017-11-01

Recognition of specific cell surface glycans, mediated by the VP8* domain of the spike protein VP4, is the essential first step in rotavirus (RV) infection. Due to lack of direct structural information of virus-ligand interactions, the molecular basis of ligand-controlled host ranges of the major human RVs (P[8] and P[4]) in P[II] genogroup remains unknown. Here, through characterization of a minor P[II] RV (P[19]) that can infect both animals (pigs) and humans, we made an important advance to fill this knowledge gap by solving the crystal structures of the P[19] VP8* in complex with its ligands. Our data showed that P[19] RVs use a novel binding site that differs from the known ones of other genotypes/genogroups. This binding site is capable of interacting with two types of glycans, the mucin core and type 1 histo-blood group antigens (HBGAs) with a common GlcNAc as the central binding saccharide. The binding site is apparently shared by other P[II] RVs and possibly two genotypes (P[10] and P[12]) in P[I] as shown by their highly conserved GlcNAc-interacting residues. These data provide strong evidence of evolutionary connections among these human and animal RVs, pointing to a common ancestor in P[I] with a possible animal host origin. While the binding properties to GlcNAc-containing saccharides are maintained, changes in binding to additional residues, such as those in the polymorphic type 1 HBGAs may occur in the course of RV evolution, explaining the complex P[II] genogroup that mainly causes diseases in humans but also in some animals.

Ion Mobility Mass Spectrometry for Extracting Spectra of N-Glycans Directly from Incubation Mixtures Following Glycan Release: Application to Glycans from Engineered Glycoforms of Intact, Folded HIV gp120

Science.gov (United States)

Harvey, David J.; Sobott, Frank; Crispin, Max; Wrobel, Antoni; Bonomelli, Camille; Vasiljevic, Snezana; Scanlan, Christopher N.; Scarff, Charlotte A.; Thalassinos, Konstantinos; Scrivens, James H.

2011-03-01

The analysis of glycosylation from native biological sources is often frustrated by the low abundances of available material. Here, ion mobility combined with electrospray ionization mass spectrometry have been used to extract the spectra of N-glycans released with PNGase F from a serial titration of recombinantly expressed envelope glycoprotein, gp120, from the human immunodeficiency virus (HIV). Analysis was also performed on gp120 expressed in the α-mannosidase inhibitor, and in a matched mammalian cell line deficient in GlcNAc transferase I. Without ion mobility separation, ESI spectra frequently contained no observable ions from the glycans whereas ions from other compounds such as detergents and residual buffer salts were abundant. After ion mobility separation on a Waters T-wave ion mobility mass spectrometer, the N-glycans fell into a unique region of the ion mobility/ m/z plot allowing their profiles to be extracted with good signal:noise ratios. This method allowed N-glycan profiles to be extracted from crude incubation mixtures with no clean-up even in the presence of surfactants such as NP40. Furthermore, this technique allowed clear profiles to be obtained from sub-microgram amounts of glycoprotein. Glycan profiles were similar to those generated by MALDI-TOF MS although they were more susceptible to double charging and fragmentation. Structural analysis could be accomplished by MS/MS experiments in either positive or negative ion mode but negative ion mode gave the most informative spectra and provided a reliable approach to the analysis of glycans from small amounts of glycoprotein.

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.

N-glycan sialylation in a silkworm-baculovirus expression system.

Science.gov (United States)

Suganuma, Masatoshi; Nomura, Tsuyoshi; Higa, Yukiko; Kataoka, Yukiko; Funaguma, Shunsuke; Okazaki, Hironobu; Suzuki, Takeo; Fujiyama, Kazuhito; Sezutsu, Hideki; Tatematsu, Ken-Ichiro; Tamura, Toshiki

2018-02-09

A silkworm-baculovirus system is particularly effective for producing recombinant proteins, including glycoproteins. However, N-glycan structures in silkworm differ from those in mammals. Glycoproteins in silkworm are secreted as pauci-mannose type N-glycans without sialic acid or galactose residues. Sialic acid on N-glycans plays important roles in protein functions. Therefore, we developed pathways for galactosylation and sialylation in silkworm. Sialylated N-glycans on proteins were successfully produced in silkworm by co-expressing galactosyltransferase and sialyltransferase and providing an external supply of a sialylation-related substrate. α2,3/α2,6 Sialylation to N-glycans was controlled by changing the type of sialyltransferase expressed in silkworm. Furthermore, the co-expression of N-acetylglucosaminyltransferase II facilitated the formation of additional di-sialylated N-glycan structures. Our results provide new information on the control of N-glycosylation in silkworm. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Imaging specific cellular glycan structures using glycosyltransferases via click chemistry.

Science.gov (United States)

Wu, Zhengliang L; Person, Anthony D; Anderson, Matthew; Burroughs, Barbara; Tatge, Timothy; Khatri, Kshitij; Zou, Yonglong; Wang, Lianchun; Geders, Todd; Zaia, Joseph; Sackstein, Robert

2018-02-01

Heparan sulfate (HS) is a polysaccharide fundamentally important for biologically activities. T/Tn antigens are universal carbohydrate cancer markers. Here, we report the specific imaging of these carbohydrates using a mesenchymal stem cell line and human umbilical vein endothelial cells (HUVEC). The staining specificities were demonstrated by comparing imaging of different glycans and validated by either removal of target glycans, which results in loss of signal, or installation of target glycans, which results in gain of signal. As controls, representative key glycans including O-GlcNAc, lactosaminyl glycans and hyaluronan were also imaged. HS staining revealed novel architectural features of the extracellular matrix (ECM) of HUVEC cells. Results from T/Tn antigen staining suggest that O-GalNAcylation is a rate-limiting step for O-glycan synthesis. Overall, these highly specific approaches for HS and T/Tn antigen imaging should greatly facilitate the detection and functional characterization of these biologically important glycans. © The Author(s) 2017. Published by Oxford University Press.

Simple Sugars to Complex Disease—Mucin-Type O-Glycans in Cancer

Science.gov (United States)

Kudelka, Matthew R.; Ju, Tongzhong; Heimburg-Molinaro, Jamie; Cummings, Richard D.

2017-01-01

Mucin-type O-glycans are a class of glycans initiated with N-acetylgalactosamine (GalNAc) α-linked primarily to Ser/Thr residues within glycoproteins and often extended or branched by sugars or saccharides. Most secretory and membrane-bound proteins receive this modification, which is important in regulating many biological processes. Alterations in mucin-type O-glycans have been described across tumor types and include expression of relatively small-sized, truncated O-glycans and altered terminal structures, both of which are associated with patient prognosis. New discoveries in the identity and expression of tumor-associated O-glycans are providing new avenues for tumor detection and treatment. This chapter describes mucin-type O-glycan biosynthesis, altered mucin-type O-glycans in primary tumors, including mechanisms for structural changes and contributions to the tumor phenotype, and clinical approaches to detect and target altered O-glycans for cancer treatment and management. PMID:25727146

Glycan-deficient PrP stimulates VEGFR2 signaling via glycosaminoglycan.

Science.gov (United States)

Gao, Zhenxing; Zhang, Huixia; Hu, Fei; Yang, Liheng; Yang, Xiaowen; Zhu, Ying; Sy, Man-Sun; Li, Chaoyang

2016-06-01

Whether the two N-linked glycans are important in prion, PrP, biology is unresolved. In Chinese hamster ovary (CHO) cells, the two glycans are clearly not important in the cell surface expression of transfected human PrP. Compared to fully-glycosylated PrP, glycan-deficient PrP preferentially partitions to lipid raft. In CHO cells glycan-deficient PrP also interacts with glycosaminoglycan (GAG) and vascular endothelial growth factor receptor 2 (VEGFR2), resulting in VEGFR2 activation and enhanced Akt phosphorylation. Accordingly, CHO cells expressing glycan-deficient PrP lacking the GAG binding motif or cells treated with heparinase to remove GAG show diminished Akt signaling. Being in lipid raft is critical, chimeric glycan-deficient PrP with CD4 transmembrane and cytoplasmic domains is absent in lipid raft and does not activate Akt signaling. CHO cells bearing glycan-deficient PrP also exhibit enhanced cellular adhesion and migration. Based on these findings, we propose a model in which glycan-deficient PrP, GAG, and VEGFR2 interact, activating VEGFR2 and resulting in changes in cellular behavior. Copyright © 2016 Elsevier Inc. All rights reserved.

The Analysis of Sialylation, N-Glycan Branching, and Expression of O-Glycans in Seminal Plasma of Infertile Men

Directory of Open Access Journals (Sweden)

Ewa M. Kratz

2015-01-01

Full Text Available Carbohydrates are known to mediate some events involved in successful fertilization. Although some studies on the glycosylation of seminal plasma proteins are available, the total glycan profile was rarely analyzed as a feature influencing fertilization potential. In this work we aimed to compare some glycosylation traits in seminal plasma glycoproteins of fertile and infertile men. The following findings emerge from our studies: (1 in human seminal plasma the presence and alterations of O-linked glycans were observed; (2 the expression of SNA-reactive sialic acid significantly differs between asthenozoospermia and both normozoospermic (fertile and infertile groups; (3 the expression of PHA-L-reactive highly branched N-glycans was significantly lower in oligozoospermic patients than in both normozoospermic groups. Indication of the appropriate lectins that would enable the possibly precise determination of the glycan profile seems to be a good supplement to mass spectrum analysis. Extension of the lectin panel is useful for the further research.

Kinetics of N-Glycan Release from Human Immunoglobulin G (IgG) by PNGase F: All Glycans Are Not Created Equal.

Science.gov (United States)

Huang, Yining; Orlando, Ron

2017-12-01

The biologic activity of IgG molecules is modulated by its crystallizable fragment N-glycosylation, and thus, the analysis of IgG glycosylation is critical. A standard approach to analyze glycosylation of IgGs involves the release of the N-glycans by the enzyme peptide N-glycosidase F, which cleaves the linkage between the asparagine residue and innermost N-acetylglucosamine (GlcNAc) of all N-glycans except those containing a 3-linked fucose attached to the reducing terminal GlcNAc residue. The importance of obtaining complete glycan release for accurate quantitation led us to investigate the kinetics of this de-glycosylation reaction for IgG glycopeptides and to determine the effect of glycan structure and amino acid sequence on the rate of glycan release from glycopeptides of IgGs. This study revealed that the slight differences in amino acid sequences did not lead to a statistically different deglycosylation rate. However, statistically significant differences in the deglycosylation rate constants were observed between glycopeptides differing only in glycan structure ( i.e. , nonfucosylated, fucosylated, bisecting-GlcNAc, sialylated, etc .). For example, a single sialic acid residue was found to decrease the rate by a factor of 3. Similar reductions in rate were associated with the presence of a bisecting-GlcNAc. We predict the differences in release kinetics can lead to significant quantitative variations of the glycosylation study of IgGs.

Genetically engineered tissue to screen for glycan function in tissue formation

DEFF Research Database (Denmark)

M., Adamopoulou; E.M., Pallesen; A., Levann

2017-01-01

engineered GlycoSkin tissue models can be used to study biological interactions involving glycan structure on lipids, or glycosaminoglycans. This engineering approach will allow us to investigate the functions of glycans in homeostasis and elucidate the role of glycans in normal epithelial formation....... We use genetic engineering with CRISPR/Cas9 combined with 3D organotypic skin models to examine how distinct glycans influence epithelial formation. We have performed knockout and knockin of more than 100 select genes in the genome of human immortalized human keratinocytes, enabling a systematic...... analysis of the impact of specific glycans in the formation and transformation of the human skin. The genetic engineered human skin models (GlycoSkin) was designed with and without all major biosynthetic pathways in mammalian glycan biosynthesis, including GalNAc-O-glycans, O-fucosylation, O...

Plasma E-selectin levels can play a role in the development of diabetic retinopathy.

Science.gov (United States)

Kasza, Márta; Meleg, J; Vardai, J; Nagy, B; Szalai, E; Damjanovich, J; Csutak, A; Ujhelyi, B; Nagy, V

2017-01-01

Diabetic retinopathy is one of the leading causes of blindness. There are several risk factors, such as the duration of diabetes or glycemic control of the patient; however, several biochemical factors also alter the process. Our aim was to investigate the role of soluble E-selectin in the formation of diabetic retinopathy. Fifty-seven patients (37 female and 20 male, aged 61.71 ± 12.31 years) and 14 healthy control subjects (ten female and four male, aged 63.06 ± 10.46 years) were enrolled in the study. We measured the soluble E-selectin level in the plasma of patients by ELISA. All patients underwent careful ophthalmological examination, including ophthalmoscopy and color fundus photography, while diabetic retinopathy grading was performed in line with the 2012 classification of the American Academy of Ophthalmology (AAO). The soluble E-selectin level was significantly higher in patients with diabetes compared to controls (32.95 ng/ml vs. 26.55 ng/ml, p = 0.03). Dividing patients into groups by the presence of retinopathy, the E-selectin level was also significantly higher in the retinopathy group (p diabetic patients by the severity of retinopathy (groups A, B, and C, by the guidelines of the AAO), however, we did not find any significant difference in soluble E-selectin levels, although it tended to be higher in group B. An elevated E-selectin level can play a role in the development of diabetic retinopathy, but it does not seem to alter disease severity. However, glycemic control and the reduction of cardiovascular risk factors may also alter the level of E-selectin that might play a role in the prevention of diabetic retinopathy.

Architecture effects on multivalent interactions by polypeptide-based multivalent ligands

Science.gov (United States)

Liu, Shuang

protein materials, including structural as well as functional proteins. Therefore, polypeptide-based multivalent scaffolds are used to display ligands to assess the contribution of different architectural parameters to the multivalent binding events. In this work, a family of alanine-rich alpha-helical glycopolypeptides was designed and synthesized by a combination of protein engineering and chemical coupling, to display two types of saccharide ligands for two different multivalent binding systems. The valencies, chain length and spacing between adjacent ligands of these multivalent ligands were designed in order to study architecture effects on multivalent interactions. The polypeptides and their glycoconjugates were characterized via various methods, including SDS-PAGE, NMR, HPLC, amino acid analysis (AAA), MALDI, circular dichroism (CD) and GPC. In the first multivalent binding system, cholera toxin B pentamer (CT B5) was chosen to be the protein receptor due to its well-characterized structure, lack of significant steric interference of binding to multiple binding sites, and requirement of only simple monosaccharide as ligands. Galactopyranoside was incorporated into polypeptide scaffolds through amine-carboxylic acid coupling to the side chains of glutamic acid residues. The inhibition and binding to CT B5 of these glycopolypeptide ligands were evaluated by direct enzyme-linked assay (DELA). As a complement method, weak affinity chromatography (WAC) was also used to evaluate glycopolypeptides binding to a CT B5 immobilized column. The architecture effects on CT B 5 inhibition are discussed. In the second system, cell surface receptor L-selectin was targeted by polypeptide-based multivalent ligands containing disulfated galactopyranoside ligands, due to its important roles in various immunological activities. The effects of glycopolypeptide architectural variables L-selectin shedding were evaluated via ELISA-based assays. These polypeptide-based multivalent ligands

The hemostatic agent ethamsylate enhances P-selectin membrane expression in human platelets and cultured endothelial cells.

Science.gov (United States)

Alvarez-Guerra, Miriam; Hernandez, Maria Rosa; Escolar, Ginés; Chiavaroli, Carlo; Garay, Ricardo P; Hannaert, Patrick

2002-09-15

Ethamsylate possesses antihemorrhagic properties, but whether or not it directly activates blood platelets is unclear. Here we investigated the platelet activation potential of ethamsylate, by measuring membrane P-selectin expression with flow cytometry in human whole blood and also by immunofluorescence imaging of isolated human platelets. Moreover, we measured membrane P-selectin expression in the SV40-transformed aortic rat endothelial cell line (SVAREC) and 14C-ethamsylate membrane binding and/or uptake in platelets and endothelial cells. Whole blood flow cytometry showed a modest, but statistically significant increase by ethamsylate in the percentage of platelets expressing P-selectin (from 2% to 4-5%, p ethamsylate tested (1 microM), with maximal enhancement of P-selectin expression (75-90%) at 10 microM ethamsylate. Similar results were obtained in SVAREC endothelial cells. 14C-ethamsylate specifically bound to platelets and endothelial cell membranes, without significant uptake into the cell interior. In conclusion, ethamsylate enhances membrane P-selectin expression in human platelets and in cultured endothelial cells. Ethamsylate specifically binds to some protein receptor in platelet and endothelial cell membranes, receptor which can signal for membrane P-selectin expression. These results support the view that ethamsylate acts on the first step of hemostasis, by improving platelet adhesiveness and restoring capillary resistance. Copyright 2002 Elsevier Science Ltd.

P-selectin targeting to secretory lysosomes of Rbl-2H3 cells

OpenAIRE

Kaur, J.; Cutler, D. F.

2002-01-01

The biogenesis of secretory lysosomes, which combine characteristics of both lysosomes and secretory granules, is currently of high interest. In particular, it is not clear whether delivery of membrane proteins to the secretory lysosome requires lysosomal, secretory granule, or some novel targeting determinants. Heterologous expression of P-selectin has established that this membrane protein contains targeting signals for both secretory granules and lysosomes. P-selectin is therefore an ideal...

Glycosaminoglycans analogues from marine invertebrates: structure, biological effects and potential as new therapeutics.

Directory of Open Access Journals (Sweden)

Mauro Sergio Pavao

2014-09-01

Full Text Available In this review, several glycosaminoglycan analogs obtained from different marine invertebrate are reported. The structure, biological activity and mechanism of action of these unique molecules are detailed reviewed and exemplified by experiments in vitro and in vivo. Among the glycans studied are low-sulfated heparin-like polymers from ascidians, containing significant anticoagulant activity and no bleeding effect; dermatan sulfates with significant neurite outgrowth promoting activity and anti-P-selectin from ascidians, and a unique fucosylated chondroitin sulfate from sea cucumbers, possessing anticoagulant activity after oral administration and high anti P- and L-selectin activities. The therapeutic value and safety of these invertebrate glycans have been extensively proved by several experimental animal models of diseases, including thrombosis, inflammation and metastasis. These invertebrate glycans can be obtained in high concentrations from marine organisms that have been used as a food source for decades, and usually obtained from marine farms in sufficient quantities to be used as starting material for new therapeutics.

CROSSWORK for Glycans: Glycan Identificatin Through Mass Spectrometry and Bioinformatics. / Rasmussen, Morten ; Thaysen-Andersen, Morten ; Højrup, Peter. 2009

DEFF Research Database (Denmark)

Rasmussen, Morten

, and observed that the glyco-peptides were identified correctly in 11/11 cases and the glycan moieties were annotated in 11/11 cases. Finally, glycan structures were proposed in 10/11 cases, all of which were in agreement with previously reported structures.    As a stand-alone program, GLYCANthrope is a useful...

Neutral glycans from sandfish skin can reduce friction of polymers

Science.gov (United States)

Vihar, Boštjan; Hanisch, Franz Georg; Baumgartner, Werner

2016-01-01

The lizard Scincus scincus, also known as sandfish, can move through aeolian desert sand in a swimming-like manner. A prerequisite for this ability is a special integument, i.e. scales with a very low friction for sand and a high abrasion resistance. Glycans in the scales are causally related to the low friction. Here, we analysed the glycans and found that neutral glycans with five to nine mannose residues are important. If these glycans were covalently bound to acrylic polymers like poly(methyl methacrylate) or acrylic car coatings at a density of approximately one molecule per 4 nm², friction for and adhesion of sand particles could be reduced to levels close to those observed with sandfish scales. This was also found true, if the glycans were isolated from sources other than sandfish scales like plants such as almonds or mistletoe. We speculate that these neutral glycans act as low density spacers separating sand particles from the dense scales thereby reducing van der Waals forces. PMID:27030038

Reductive Alkaline Release of N-Glycans Generates a Variety of Unexpected, Useful Products.

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Figl, Rudolf; Altmann, Friedrich

2018-02-01

Release of O-glycans by reductive β-elimination has become routine in many glyco-analytical laboratories and concomitant release of N-glycans has repeatedly been observed. Revisiting this somewhat forgotten mode of N-glycan release revealed that all kinds of N-glycans including oligomannosidic and complex-type N-glycans from plants with 3-linked fucose and from mammals with or without 6-linked fucose and with sialic acid could be recovered. However, the mass spectra of the obtained products revealed very surprising facts. Even after 16 h incubation in 1 M sodium borohydride, a large part of the glycans occurred in reducing form. Moreover, about one third emerged in the form of the stable amino-functionalized 1-amino-1-deoxy-glycitol. When avoiding acidic conditions, considerable amounts of glycosylamine were observed. In addition, a compound with a reduced asparagine and de-N-acetylation products, in particular of sialylated glycans, was seen. The relative yields of the products reducing glycosylamine, reducing N-glycan, 1-amino-1-deoxy-glycitol or glycitol could be controlled by the release conditions, foremost by temperature and borohydride concentration. Thus, chemical release of N-glycans constitutes a cost-saving alternative to enzymatic hydrolysis for the preparation of precursors for the production of reference compounds for various formats of N-glycan analysis. Moreover, it allows to obtain a stable amino-functionalized glycan derivative, which can be employed to construct glycan arrays or affinity matrices. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

P-selectin in preterm infants suffering necrotizing enterocolitis ...

African Journals Online (AJOL)

All neonates were subjected to perinatal history, clinical examination, routine investigations (CBC, plain X-ray and abdominal ultrasonography (US), arterial blood gases and serum bicarbonate, serum sodium, CRP and blood culture), and measurement of blood P-selectin by direct immunofluorescent staining. Results: ...

Human Milk Contains Novel Glycans That Are Potential Decoy Receptors for Neonatal Rotaviruses*

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Yu, Ying; Lasanajak, Yi; Song, Xuezheng; Hu, Liya; Ramani, Sasirekha; Mickum, Megan L.; Ashline, David J.; Prasad, B. V. Venkataram; Estes, Mary K.; Reinhold, Vernon N.; Cummings, Richard D.; Smith, David F.

2014-01-01

Human milk contains a rich set of soluble, reducing glycans whose functions and bioactivities are not well understood. Because human milk glycans (HMGs) have been implicated as receptors for various pathogens, we explored the functional glycome of human milk using shotgun glycomics. The free glycans from pooled milk samples of donors with mixed Lewis and Secretor phenotypes were labeled with a fluorescent tag and separated via multidimensional HPLC to generate a tagged glycan library containing 247 HMG targets that were printed to generate the HMG shotgun glycan microarray (SGM). To investigate the potential role of HMGs as decoy receptors for rotavirus (RV), a leading cause of severe gastroenteritis in children, we interrogated the HMG SGM with recombinant forms of VP8* domains of the RV outer capsid spike protein VP4 from human neonatal strains N155(G10P[11]) and RV3(G3P[6]) and a bovine strain, B223(G10P[11]). Glycans that were bound by RV attachment proteins were selected for detailed structural analyses using metadata-assisted glycan sequencing, which compiles data on each glycan based on its binding by antibodies and lectins before and after exo- and endo-glycosidase digestion of the SGM, coupled with independent MSn analyses. These complementary structural approaches resulted in the identification of 32 glycans based on RV VP8* binding, many of which are novel HMGs, whose detailed structural assignments by MSn are described in a companion report. Although sialic acid has been thought to be important as a surface receptor for RVs, our studies indicated that sialic acid is not required for binding of glycans to individual VP8* domains. Remarkably, each VP8* recognized specific glycan determinants within a unique subset of related glycan structures where specificity differences arise from subtle differences in glycan structures. PMID:25048705

Clinical significance of changes of serum of P-selectin, CEA and TSGF levels after operation in patients with rectal cancer

International Nuclear Information System (INIS)

Wang Zhizhong; Huang Jin

2007-01-01

Objective: To study the clinical significance of postoperative changes of serum P-selectin, CEA and TSGF levels in patients with rectal cancer. Methods: Serum CEA (with RIA), P-selectin (with ELISA), and TSGF (with biochemistry levels were determined) in 32 patients with rectal cancer both before and after operation as well as in 30 controls. Results: Before operation, the serum P-selectin, CEA and TSGF levels were significantly higher than those in controls (P < 0.01), Twenty -two of the 30 patients underwent operative therapy showed no sign of recurrence at one year and their serum P-selectin, CEA and TSGF levels dropped to within normal range. Hower in the 8 patients with recurrence, the serum levels of P-selectin, CEA and TSGF remained abnormally high. Conclusion: Serum P-selectin, CEA and TSGF levels were closely related to the diseases process of rectal cancer and were of prognostic values. (authors)

Improving N-Glycan Coverage using HPLC-MS with Electrospray Ionization at Subambient Pressure

Energy Technology Data Exchange (ETDEWEB)

Marginean, Ioan; Kronewitter, Scott R.; Moore, Ronald J.; Slysz, Gordon W.; Monroe, Matthew E.; Anderson, Gordon A.; Tang, Keqi; Smith, Richard D.

2012-10-01

Human serum glycan profiling with mass spectrometry (MS) has been employed to study several disease conditions and is demonstrating promise for e.g. clinical biomarker discovery. However, the poor glycan ionization efficiency and the large dynamic range of glycan concentrations in human sera hinder comprehensive profiling. In particular, large glycans are problematic because they are present at low concentrations and prone to fragmentation. Here we show that the sub-ambient pressure ionization with nanoelectrospray (SPIN)-MS can expand the serum glycome profile when compared with the conventional atmospheric pressure electrospray ionization (ESI)-MS with a heated capillary inlet. Notably, the ions generated by the SPIN interface were observed at higher charge states for 50% of the annotated glycans. Out of a total of 130 detected glycans, 34 were only detected with the SPIN-MS, resulting in improved coverage of glycan families as well as of glycans with larger numbers of labile monosaccharides.

Glycan gimmickry by parasitic helminths: a strategy for modulating the host immune response?

Science.gov (United States)

van Die, Irma; Cummings, Richard D

2010-01-01

Parasitic helminths (worms) co-evolved with vertebrate immune systems to enable long-term survival of worms in infected hosts. Among their survival strategies, worms use their glycans within glycoproteins and glycolipids, which are abundant on helminth surfaces and in their excretory/ secretory products, to regulate and suppress host immune responses. Many helminths express unusual and antigenic (nonhost-like) glycans, including those containing polyfucose, tyvelose, terminal GalNAc, phosphorylcholine, methyl groups, and sugars in unusual linkages. In addition, some glycan antigens are expressed that share structural features with those in their intermediate and vertebrate hosts (host-like glycans), including Le(X) (Galbeta1-4[Fucalpha1-3]GlcNAc-), LDNF (GalNAcbeta1-4[Fucalpha1-3]GlcNAc-), LDN (GalNAcbeta1-4GlcNAc-), and Tn (GalNAcalpha1-O-Thr/Ser) antigens. The expression of host-like glycan determinants is remarkable and suggests that helminths may gain advantages by synthesizing such glycans. The expression of host-like glycans by parasites previously led to the concept of "molecular mimicry," in which molecules are either derived from the pathogen or acquired from the host to evade recognition by the host immune system. However, recent discoveries into the potential of host glycan-binding proteins (GBPs), such as C-type lectin receptors and galectins, to functionally interact with various host-like helminth glycans provide new insights. Host GBPs through their interactions with worm-derived glycans participate in shaping innate and adaptive immune responses upon infection. We thus propose an alternative concept termed "glycan gimmickry," which is defined as an active strategy of parasites to use their glycans to target GBPs within the host to promote their survival.

Hydrazinonicotinic acid derivatization for selective ionization and improved glycan structure characterization by MALDI-MS.

Science.gov (United States)

Jiao, Jing; Yang, Lijun; Zhang, Ying; Lu, Haojie

2015-08-21

The analysis of glycan is important for understanding cell biology and disease processes because the glycans play a key role in many important biological behaviors, such as cell division, cellular localization, tumor immunology and inflammation. Nevertheless, it is still hard work to analyze glycans by MALDI-MS, which generally stems from the inherent low abundance and the low ionization efficiency of glycans. Moreover, the difficulty in generating informative fragmentations further hinders glycans structure characterization. In this work, hydrazinonicotinic acid (HYNIC) was used as a novel derivatized reagent for improved and selective detection of glycans. Through tagging the reducing terminus of glycans with the diazanyl group of HYNIC, significant enhancement of the ionization efficiency of glycans was achieved. After derivatization, the signal to noise ratio (S/N) of the maltoheptaose was improved by more than one order of magnitude in positive mode. HYNIC derivatization also allowed the sensitive detection of sialylated glycan in negative mode, with a 15 fold enhancement of S/N. Interestingly, it is noteworthy that the HYNIC reagent not only effectively labeled the reducing end of glycans in the presence of tryptic peptides, but also suppressed the ionization of peptides, enabling the direct detection of glycans from glycoprotein without separation. Therefore, analysis of glycans became easier due to the omission of a pre-separation step. Importantly, by using different acid reagents as the catalyst, derivatized product signals corresponding to [M + Na](+) or [M + H](+) were obtained respectively, which yield complementary fragmentation patterns for the structure elucidation of glycans. Finally, more than 40 N-glycans were successfully detected in 10 μL human serum using this method.

Determination of site-specific glycan heterogeneity on glycoproteins

DEFF Research Database (Denmark)

Kolarich, Daniel; Jensen, Pia Hønnerup; Altmann, Friedrich

2012-01-01

and the determination of site-specific glycan heterogeneity. The described workflow takes approximately 3-5 d, including sample preparation and data analysis. The data obtained from analyzing released glycans of rHuEPO and IgG, described in the second protocol of this series (10.1038/nprot.2012.063), provide...

The hydroxyl-functionalized magnetic particles for purification of glycan-binding proteins.

Science.gov (United States)

Sun, Xiuxuan; Yang, Ganglong; Sun, Shisheng; Quan, Rui; Dai, Weiwei; Li, Bin; Chen, Chao; Li, Zheng

2009-12-01

Glycan-protein interactions play important biological roles in biological processes. Although there are some methods such as glycan arrays that may elucidate recognition events between carbohydrates and protein as well as screen the important glycan-binding proteins, there is a lack of simple effectively separate method to purify them from complex samples. In proteomics studies, fractionation of samples can help to reduce their complexity and to enrich specific classes of proteins for subsequent downstream analyses. Herein, a rapid simple method for purification of glycan-binding proteins from proteomic samples was developed using hydroxyl-coated magnetic particles coupled with underivatized carbohydrate. Firstly, the epoxy-coated magnetic particles were further hydroxyl functionalized with 4-hydroxybenzhydrazide, then the carbohydrates were efficiently immobilized on hydroxyl functionalized surface of magnetic particles by formation of glycosidic bond with the hemiacetal group at the reducing end of the suitable carbohydrates via condensation. All conditions of this method were optimized. The magnetic particle-carbohydrate conjugates were used to purify the glycan-binding proteins from human serum. The fractionated glycan-binding protein population was displayed by SDS-PAGE. The result showed that the amount of 1 mg magnetic particles coupled with mannose in acetate buffer (pH 5.4) was 10 micromol. The fractionated glycan-binding protein population in human serum could be eluted from the magnetic particle-mannose conjugates by 0.1% SDS. The methodology could work together with the glycan microarrays for screening and purification of the important GBPs from complex protein samples.

Infection's Sweet Tooth: How Glycans Mediate Infection and Disease Susceptibility.

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Taylor, Steven L; McGuckin, Michael A; Wesselingh, Steve; Rogers, Geraint B

2018-02-01

Glycans form a highly variable constituent of our mucosal surfaces and profoundly affect our susceptibility to infection and disease. The diversity and importance of these surface glycans can be seen in individuals who lack a functional copy of the fucosyltransferase gene, FUT2. Representing around one-fifth of the population, these individuals have an altered susceptibility to many bacterial and viral infections and diseases. The mediation of host-pathogen interactions by mucosal glycans, such as those added by FUT2, is poorly understood. We highlight, with specific examples, important mechanisms by which host glycans influence infection dynamics, including by: acting as pathogen receptors (or receptor-decoys), promoting microbial stability, altering the physical characteristics of mucus, and acting as immunological markers. We argue that the effect glycans have on infection dynamics has profound implications for many aspects of healthcare and policy, including clinical management, outbreak control, and vaccination policy. Copyright © 2017 Elsevier Ltd. All rights reserved.

ANALYSIS OF GLYCANS DERIVED FROM GLYCOCONJUGATES BY CAPILLARY ELECTROPHORESIS-MASS SPECTROMETRY

Science.gov (United States)

Mechref, Yehia

2012-01-01

The high structural variation of glycan derived from glycoconjugates, which substantially increases with the molecular size of a protein, contributes to the complexity of glycosylation patterns commonly associated with glycoconjugates. In the case of glycoproteins, such variation originates from the multiple glycosylation sites of proteins and the number of glycan structures associated with each site (microheterogeneity). The ability to comprehensively characterize highly complex mixture of glycans has been analytically stimulating and challenging. Although the most powerful mass spectrometric (MS) and tandem MS techniques are capable of providing a wealth of structural information, they are still not able to readily identify isomeric glycan structures without high order tandem MS (MSn). The analysis of isomeric glycan structures has been attained using several separation methods, including high-pH anion exchange chromatography (HPAEC), hydrophilic interaction chromatography (HILIC) and gas chromatography (GC). However, capillary electrophoresis (CE) and microfluidics capillary electrophoresis (MCE) offer high separation efficiency and resolutions, allowing the separation of closely related glycan structures. Therefore, interfacing CE and MCE to MS is a powerful analytical approach, allowing potentially comprehensive and sensitive analysis of complex glycan samples. This review describes and discusses the utility of different CE and MCE approaches in the structural characterization of glycoproteins and the feasibility of interfacing these approaches to mass spectrometry. PMID:22180203

MALDI Mass Spectrometry Imaging of N-Linked Glycans in Cancer Tissues.

Science.gov (United States)

Drake, R R; Powers, T W; Jones, E E; Bruner, E; Mehta, A S; Angel, P M

2017-01-01

Glycosylated proteins account for a majority of the posttranslation modifications of cell surface, secreted, and circulating proteins. Within the tumor microenvironment, the presence of immune cells, extracellular matrix proteins, cell surface receptors, and interactions between stroma and tumor cells are all processes mediated by glycan binding and recognition reactions. Changes in glycosylation during tumorigenesis are well documented to occur and affect all of these associated adhesion and regulatory functions. A MALDI imaging mass spectrometry (MALDI-IMS) workflow for profiling N-linked glycan distributions in fresh/frozen tissues and formalin-fixed paraffin-embedded tissues has recently been developed. The key to the approach is the application of a molecular coating of peptide-N-glycosidase to tissues, an enzyme that cleaves asparagine-linked glycans from their protein carrier. The released N-linked glycans can then be analyzed by MALDI-IMS directly on tissue. Generally 40 or more individual glycan structures are routinely detected, and when combined with histopathology localizations, tumor-specific glycans are readily grouped relative to nontumor regions and other structural features. This technique is a recent development and new approach in glycobiology and mass spectrometry imaging research methodology; thus, potential uses such as tumor-specific glycan biomarker panels and other applications are discussed. © 2017 Elsevier Inc. All rights reserved.

Efficient adhesion-based plasma membrane isolation for cell surface N-glycan analysis.

Science.gov (United States)

Mun, Ji-Young; Lee, Kyung Jin; Seo, Hoon; Sung, Min-Sun; Cho, Yee Sook; Lee, Seung-Goo; Kwon, Ohsuk; Oh, Doo-Byoung

2013-08-06

Glycans, which decorate cell surfaces, play crucial roles in various physiological events involving cell surface recognition. Despite the importance of surface glycans, most analyses have been performed using total cells or whole membranes rather than plasma membranes due to difficulties related to isolation. In the present study, we employed an adhesion-based method for plasma membrane isolation to analyze N-glycans on cell surfaces. Cells were attached to polylysine-coated glass plates and then ruptured by hypotonic pressure. After washing to remove intracellular organelles, only a plasma membrane fraction remained attached to the plates, as confirmed by fluorescence imaging using organelle-specific probes. The plate was directly treated with trypsin to digest and detach the glycoproteins from the plasma membrane. From the resulting glycopeptides, N-glycans were released and analyzed using MALDI-TOF mass spectrometry and HPLC. When N-glycan profiles obtained by this method were compared to those by other methods, the amount of high-mannose type glycans mainly contaminated from the endoplasmic reticulum was dramatically reduced, which enabled the efficient detection of complex type glycans present on the cell surface. Moreover, this method was successfully used to analyze the increase of high-mannose glycans on the surface as induced by a mannosidase inhibitor treatment.

Glycan-mediated modification of the immune response

DEFF Research Database (Denmark)

Madsen, Caroline B; Pedersen, Anders E; Wandall, Hans H

2013-01-01

Aberrantly glycosylated tumor antigens represent promising targets for the development of anti-cancer vaccines, yet how glycans influence immune responses is poorly understood. Recent studies have demonstrated that GalNAc-glycosylation enhances antigen uptake by dendritic cells as well as CD4(+) T......-cell and humoral responses, but prevents CD8(+) T-cell activation. Here, we briefly discuss the relevance of glycans as candidate targets for anti-cancer vaccines....

Development of Rare Bacterial Monosaccharide Analogs for Metabolic Glycan Labeling in Pathogenic Bacteria.

Science.gov (United States)

Clark, Emily L; Emmadi, Madhu; Krupp, Katharine L; Podilapu, Ananda R; Helble, Jennifer D; Kulkarni, Suvarn S; Dube, Danielle H

2016-12-16

Bacterial glycans contain rare, exclusively bacterial monosaccharides that are frequently linked to pathogenesis and essentially absent from human cells. Therefore, bacterial glycans are intriguing molecular targets. However, systematic discovery of bacterial glycoproteins is hampered by the presence of rare deoxy amino sugars, which are refractory to traditional glycan-binding reagents. Thus, the development of chemical tools that label bacterial glycans is a crucial step toward discovering and targeting these biomolecules. Here, we explore the extent to which metabolic glycan labeling facilitates the studying and targeting of glycoproteins in a range of pathogenic and symbiotic bacterial strains. We began with an azide-containing analog of the naturally abundant monosaccharide N-acetylglucosamine and discovered that it is not broadly incorporated into bacterial glycans, thus revealing a need for additional azidosugar substrates to broaden the utility of metabolic glycan labeling in bacteria. Therefore, we designed and synthesized analogs of the rare deoxy amino d-sugars N-acetylfucosamine, bacillosamine, and 2,4-diacetamido-2,4,6-trideoxygalactose and established that these analogs are differentially incorporated into glycan-containing structures in a range of pathogenic and symbiotic bacterial species. Further application of these analogs will refine our knowledge of the glycan repertoire in diverse bacteria and may find utility in treating a variety of infectious diseases with selectivity.

High-throughput profiling of anti-glycan humoral responses to SIV vaccination and challenge.

Directory of Open Access Journals (Sweden)

Christopher T Campbell

Full Text Available Recent progress toward an HIV vaccine highlights both the potential of vaccines to end the AIDS pandemic and the need to boost efficacy by incorporating additional vaccine strategies. Although many aspects of the immune response can contribute to vaccine efficacy, the key factors have not been defined fully yet. A particular area that may yield new insights is anti-glycan immune responses, such as those against the glycan shield that HIV uses to evade the immune system. In this study, we used glycan microarray technology to evaluate anti-glycan antibody responses induced by SIV vaccination and infection in a non-human primate model of HIV infection. This comprehensive profiling of circulating anti-glycan antibodies found changes in anti-glycan antibody levels after both vaccination with the Ad5hr-SIV vaccine and SIV infection. Notably, SIV infection produced generalized declines in anti-glycan IgM antibodies in a number of animals. Additionally, some infected animals generated antibodies to the Tn antigen, which is a cryptic tumor-associated antigen exposed by premature termination of O-linked glycans; however, the Ad5hr-SIV vaccine did not induce anti-Tn IgG antibodies. Overall, this study demonstrates the potential contributions that glycan microarrays can make for HIV vaccine development.

Composition and Antigenic Effects of Individual Glycan Sites of a Trimeric HIV-1 Envelope Glycoprotein

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Anna-Janina Behrens

2016-03-01

Full Text Available The HIV-1 envelope glycoprotein trimer is covered by an array of N-linked glycans that shield it from immune surveillance. The high density of glycans on the trimer surface imposes steric constraints limiting the actions of glycan-processing enzymes, so that multiple under-processed structures remain on specific areas. These oligomannose glycans are recognized by broadly neutralizing antibodies (bNAbs that are not thwarted by the glycan shield but, paradoxically, target it. Our site-specific glycosylation analysis of a soluble, recombinant trimer (BG505 SOSIP.664 maps the extremes of simplicity and diversity of glycan processing at individual sites and reveals a mosaic of dense clusters of oligomannose glycans on the outer domain. Although individual sites usually minimally affect the global integrity of the glycan shield, we identify examples of how deleting some glycans can subtly influence neutralization by bNAbs that bind at distant sites. The network of bNAb-targeted glycans should be preserved on vaccine antigens.

Comprehensive functional analysis of N-linked glycans on Ebola virus GP1.

Science.gov (United States)

Lennemann, Nicholas J; Rhein, Bethany A; Ndungo, Esther; Chandran, Kartik; Qiu, Xiangguo; Maury, Wendy

2014-01-28

Ebola virus (EBOV) entry requires the virion surface-associated glycoprotein (GP) that is composed of a trimer of heterodimers (GP1/GP2). The GP1 subunit contains two heavily glycosylated domains, the glycan cap and the mucin-like domain (MLD). The glycan cap contains only N-linked glycans, whereas the MLD contains both N- and O-linked glycans. Site-directed mutagenesis was performed on EBOV GP1 to systematically disrupt N-linked glycan sites to gain an understanding of their role in GP structure and function. All 15 N-glycosylation sites of EBOV GP1 could be removed without compromising the expression of GP. The loss of these 15 glycosylation sites significantly enhanced pseudovirion transduction in Vero cells, which correlated with an increase in protease sensitivity. Interestingly, exposing the receptor-binding domain (RBD) by removing the glycan shield did not allow interaction with the endosomal receptor, NPC1, indicating that the glycan cap/MLD domains mask RBD residues required for binding. The effects of the loss of GP1 N-linked glycans on Ca(2+)-dependent (C-type) lectin (CLEC)-dependent transduction were complex, and the effect was unique for each of the CLECs tested. Surprisingly, EBOV entry into murine peritoneal macrophages was independent of GP1 N-glycans, suggesting that CLEC-GP1 N-glycan interactions are not required for entry into this important primary cell. Finally, the removal of all GP1 N-glycans outside the MLD enhanced antiserum and antibody sensitivity. In total, our results provide evidence that the conserved N-linked glycans on the EBOV GP1 core protect GP from antibody neutralization despite the negative impact the glycans have on viral entry efficiency. Filovirus outbreaks occur sporadically throughout central Africa, causing high fatality rates among the general public and health care workers. These unpredictable hemorrhagic fever outbreaks are caused by multiple species of Ebola viruses, as well as Marburg virus. While filovirus

Increased levels of anti-glycan antibodies in patients with cystic fibrosis

Directory of Open Access Journals (Sweden)

Hirche TO

2011-09-01

Full Text Available Abstract Background The prevalence of Crohn's disease (CD is increased in patients with cystic fibrosis (CF. Anti-Saccharomyces cerevisiae antibodies (ASCA have been suggested as a screening tool to detect CD in CF. Recently, several new anti-glycan antibodies have been reported in CD. Materials and methods The sera of 119 CF patients of various age groups were prospectively screened for ASCA type IgG (gASCA, anti-laminaribioside carbohydrate IgG antibodies (ALCA, anti-chitobioside carbohydrate IgA antibodies (ACCA, and anti-mannobioside carbohydrate IgG antibodies (AMCA. The frequency of these anti-glycan antibodies was then compared in patients with CD, ulcerative colitis, rheumatoid arthritis and healthy volunteers. Results A significant number of CF patients were positive for gASCA (51.3% [41.6-60.6] and up to three other anti-glycan antibodies concurrently. Serum levels of anti-glycan antibodies in CF and CD were not related to parameters of inflammation. Despite the well-documented difference in clinical course between male and female CF patients no gender difference of anti-glycan antibodies was found. In contrast, there was a significant positive correlation between anti-glycan markers and age in CF patients. Conclusions Our findings demonstrate for the first time the increased frequency of a panel of anti-glycan antibodies in CF and provide a link between the presence of these serological biomarkers and patient's age. Anti-glycan antibody profiling may therefore become a valuable tool in the care of patients with CF.

Parallel analysis and orthogonal identification of N-glycans with different capillary electrophoresis mechanisms

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Feng, Hua-tao [Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543 (Singapore); NUS Environmental Research Institute, 5A Engineering Drive 1, T-Lab Building, Singapore 117411 (Singapore); Su, Min; Rifai, Farida Nur [Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543 (Singapore); Li, Pingjing [NUS Environmental Research Institute, 5A Engineering Drive 1, T-Lab Building, Singapore 117411 (Singapore); Li, Sam F.Y., E-mail: chmlifys@nus.edu.sg [Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543 (Singapore); NUS Environmental Research Institute, 5A Engineering Drive 1, T-Lab Building, Singapore 117411 (Singapore)

2017-02-08

The deep involvement of glycans or carbohydrate moieties in biological processes makes glycan patterns an important direction for the clinical and medicine researches. A multiplexing CE mapping method for glycan analysis was developed in this study. By applying different CE separation mechanisms, the potential of combined parallel applications of capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC) and capillary gel electrophoresis (CGE) for rapid and accurate identification of glycan was investigated. The combination of CZE and MEKC demonstrated enhancing chromatography separation capacity without the compromises of sample pre-treatment and glycan concentration. The separation mechanisms for multiplexing platform were selected based on the orthogonalities of the separation of glycan standards. MEKC method exhibited promising ability for the analysis of small GU value glycans and thus complementing the unavailability of CZE. The method established required only small amount of samples, simple instrument and single fluorescent labelling for sensitive detection. This integrated method can be used to search important glycan patterns appearing in biopharmaceutical products and other glycoproteins with clinical importance. - Highlights: • Cross-validation of analytes in complex samples was done with different CE separation mechanisms. • A simple strategy is used to confirm peak identification and extend capacity of CE separation. • The method uses small amount of sample, simple instrument and single fluorescent labeling. • Selection of mechanisms is based on orthogonalities of GU values of glycan standards. • Micellar electrokinetic chromatography was suitable for analysis of small or highly sialylated glycans.

Parallel analysis and orthogonal identification of N-glycans with different capillary electrophoresis mechanisms

International Nuclear Information System (INIS)

Feng, Hua-tao; Su, Min; Rifai, Farida Nur; Li, Pingjing; Li, Sam F.Y.

2017-01-01

The deep involvement of glycans or carbohydrate moieties in biological processes makes glycan patterns an important direction for the clinical and medicine researches. A multiplexing CE mapping method for glycan analysis was developed in this study. By applying different CE separation mechanisms, the potential of combined parallel applications of capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC) and capillary gel electrophoresis (CGE) for rapid and accurate identification of glycan was investigated. The combination of CZE and MEKC demonstrated enhancing chromatography separation capacity without the compromises of sample pre-treatment and glycan concentration. The separation mechanisms for multiplexing platform were selected based on the orthogonalities of the separation of glycan standards. MEKC method exhibited promising ability for the analysis of small GU value glycans and thus complementing the unavailability of CZE. The method established required only small amount of samples, simple instrument and single fluorescent labelling for sensitive detection. This integrated method can be used to search important glycan patterns appearing in biopharmaceutical products and other glycoproteins with clinical importance. - Highlights: • Cross-validation of analytes in complex samples was done with different CE separation mechanisms. • A simple strategy is used to confirm peak identification and extend capacity of CE separation. • The method uses small amount of sample, simple instrument and single fluorescent labeling. • Selection of mechanisms is based on orthogonalities of GU values of glycan standards. • Micellar electrokinetic chromatography was suitable for analysis of small or highly sialylated glycans.

Glycans in Medicinal Chemistry: An Underexploited Resource.

Science.gov (United States)

Fernández-Tejada, Alberto; Cañada, F Javier; Jiménez-Barbero, Jesús

2015-08-01

The biological relevance of glycans as mediators of key physiological processes, including disease-related mechanisms, makes them attractive targets for a wide range of medical applications. Despite their important biological roles, especially as molecular recognition elements, carbohydrates have not been fully exploited as therapeutics mainly due to the scarcity of structure-activity correlations and their non-drug-like properties. A more detailed understanding of the complex carbohydrate structures and their associated functions should contribute to the development of new glycan-based pharmaceuticals. Recent significant progress in oligosaccharide synthesis and chemical glycobiology has renewed the interest of the medicinal chemistry community in carbohydrates. This promises to increase our possibilities to harness them in drug discovery efforts for the development of new and more effective, synthetic glycan-based therapeutics and vaccines. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A computational framework for the automated construction of glycosylation reaction networks.

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Liu, Gang; Neelamegham, Sriram

2014-01-01

Glycosylation is among the most common and complex post-translational modifications identified to date. It proceeds through the catalytic action of multiple enzyme families that include the glycosyltransferases that add monosaccharides to growing glycans, and glycosidases which remove sugar residues to trim glycans. The expression level and specificity of these enzymes, in part, regulate the glycan distribution or glycome of specific cell/tissue systems. Currently, there is no systematic method to describe the enzymes and cellular reaction networks that catalyze glycosylation. To address this limitation, we present a streamlined machine-readable definition for the glycosylating enzymes and additional methodologies to construct and analyze glycosylation reaction networks. In this computational framework, the enzyme class is systematically designed to store detailed specificity data such as enzymatic functional group, linkage and substrate specificity. The new classes and their associated functions enable both single-reaction inference and automated full network reconstruction, when given a list of reactants and/or products along with the enzymes present in the system. In addition, graph theory is used to support functions that map the connectivity between two or more species in a network, and that generate subset models to identify rate-limiting steps regulating glycan biosynthesis. Finally, this framework allows the synthesis of biochemical reaction networks using mass spectrometry (MS) data. The features described above are illustrated using three case studies that examine: i) O-linked glycan biosynthesis during the construction of functional selectin-ligands; ii) automated N-linked glycosylation pathway construction; and iii) the handling and analysis of glycomics based MS data. Overall, the new computational framework enables automated glycosylation network model construction and analysis by integrating knowledge of glycan structure and enzyme biochemistry. All

A computational framework for the automated construction of glycosylation reaction networks.

Directory of Open Access Journals (Sweden)

Gang Liu

Full Text Available Glycosylation is among the most common and complex post-translational modifications identified to date. It proceeds through the catalytic action of multiple enzyme families that include the glycosyltransferases that add monosaccharides to growing glycans, and glycosidases which remove sugar residues to trim glycans. The expression level and specificity of these enzymes, in part, regulate the glycan distribution or glycome of specific cell/tissue systems. Currently, there is no systematic method to describe the enzymes and cellular reaction networks that catalyze glycosylation. To address this limitation, we present a streamlined machine-readable definition for the glycosylating enzymes and additional methodologies to construct and analyze glycosylation reaction networks. In this computational framework, the enzyme class is systematically designed to store detailed specificity data such as enzymatic functional group, linkage and substrate specificity. The new classes and their associated functions enable both single-reaction inference and automated full network reconstruction, when given a list of reactants and/or products along with the enzymes present in the system. In addition, graph theory is used to support functions that map the connectivity between two or more species in a network, and that generate subset models to identify rate-limiting steps regulating glycan biosynthesis. Finally, this framework allows the synthesis of biochemical reaction networks using mass spectrometry (MS data. The features described above are illustrated using three case studies that examine: i O-linked glycan biosynthesis during the construction of functional selectin-ligands; ii automated N-linked glycosylation pathway construction; and iii the handling and analysis of glycomics based MS data. Overall, the new computational framework enables automated glycosylation network model construction and analysis by integrating knowledge of glycan structure and enzyme

Expression and Purification of Glycosyltransferases in Pichia Pastoris: Towards Improving the Migration of Stem Cells by Enhancing Surface Expression of Sialyl Lewis X

KAUST Repository

Al-Amoodi, Asma S.

2017-01-01

Recruitment of circulating cells towards target sites is primarily dependent on E-selectin receptor/ligand adhesive interactions. Glycosyltransferase (GTs) are involved in the creation of E-selectin ligands. A sialofucosylated terminal

Improved sample preparation for CE-LIF analysis of plant N-glycans.

Science.gov (United States)

Nagels, Bieke; Santens, Francis; Weterings, Koen; Van Damme, Els J M; Callewaert, Nico

2011-12-01

In view of glycomics studies in plants, it is important to have sensitive tools that allow one to analyze and characterize the N-glycans present on plant proteins in different species. Earlier methods combined plant-based sample preparations with CE-LIF N-glycan analysis but suffered from background contaminations, often resulting in non-reproducible results. This publication describes a reproducible and sensitive protocol for the preparation and analysis of plant N-glycans, based on a combination of the 'in-gel release method' and N-glycan analysis on a multicapillary DNA sequencer. Our protocol makes it possible to analyze plant N-glycans starting from low amounts of plant material with highly reproducible results. The developed protocol was validated for different plant species and plant cells. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Saikosaponin D Isolated from Bupleurum falcatum Inhibits Selectin-Mediated Cell Adhesion

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Myoung-Jun Jang

2014-12-01

Full Text Available Three saikosaponins were isolated from the MeOH extract of the roots of Bupleurum falcatum L.: saikosaponins B3 (1; B4 (2; and D (3. Of the three, compound 3 inhibited the interaction of selectins (E, L, and P and THP-1 cells with IC50 values of 1.8, 3.0 and 4.3 µM, respectively. Also, the aglycone structure 4 of compound 3 showed moderate inhibitory activity on L-selectin-mediated cell adhesion. From these results, we suspect that compound 3 isolated from Bupleurum falcatum roots would be a good candidate for therapeutic strategies to treat inflammation.

Glycan Reader: Automated Sugar Identification and Simulation Preparation for Carbohydrates and Glycoproteins

Science.gov (United States)

Jo, Sunhwan; Song, Kevin C.; Desaire, Heather; MacKerell, Alexander D.; Im, Wonpil

2011-01-01

Understanding how glycosylation affects protein structure, dynamics, and function is an emerging and challenging problem in biology. As a first step toward glycan modeling in the context of structural glycobiology, we have developed Glycan Reader and integrated it into the CHARMM-GUI, http://www.charmm-gui.org/input/glycan. Glycan Reader greatly simplifies the reading of PDB structure files containing glycans through (i) detection of carbohydrate molecules, (ii) automatic annotation of carbohydrates based on their three-dimensional structures, (iii) recognition of glycosidic linkages between carbohydrates as well as N-/O-glycosidic linkages to proteins, and (iv) generation of inputs for the biomolecular simulation program CHARMM with the proper glycosidic linkage setup. In addition, Glycan Reader is linked to other functional modules in CHARMM-GUI, allowing users to easily generate carbohydrate or glycoprotein molecular simulation systems in solution or membrane environments and visualize the electrostatic potential on glycoprotein surfaces. These tools are useful for studying the impact of glycosylation on protein structure and dynamics. PMID:21815173

Data for analysis of mannose-6-phosphate glycans labeled with fluorescent tags.

Science.gov (United States)

Kang, Ji-Yeon; Kwon, Ohsuk; Gil, Jin Young; Oh, Doo-Byoung

2016-06-01

Mannose-6-phosphate (M-6-P) glycan plays an important role in lysosomal targeting of most therapeutic enzymes for treatment of lysosomal storage diseases. This article provides data for the analysis of M-6-P glycans by high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The identities of M-6-P glycan peaks in HPLC profile were confirmed by measuring the masses of the collected peak eluates. The performances of three fluorescent tags (2-aminobenzoic acid [2-AA], 2-aminobenzamide [2-AB], and 3-(acetyl-amino)-6-aminoacridine [AA-Ac]) were compared focusing on the analysis of bi-phosphorylated glycan (containing two M-6-Ps). The bi-phosphorylated glycan analysis is highly affected by the attached fluorescent tag and the hydrophilicity of elution solvent used in HPLC. The data in this article is associated with the research article published in "Comparison of fluorescent tags for analysis of mannose-6-phosphate glycans" (Kang et al., 2016 [1]).

Structural characterization of complex O-linked glycans from insect-derived material.

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Garenaux, Estelle; Maes, Emmanuel; Levêque, S; Brassart, Colette; Guerardel, Yann

2011-07-01

Although insects are among the most diverse groups of the animal kingdom and may be found in nearly all environments, one can observe an obvious lack of structural data on their glycosylation ability. Hymenoptera is the second largest of all insect orders with more than 110,000 identified species and includes the most famous examples of social insects' species such as wasps, bees and ants. In this report, the structural variety of O-glycans has been studied in two Hymenoptera species. In a previous study, we showed that major O-glycans from common wasp (Vespula germanica) salivary mucins correspond to T and Tn antigen, eventually substituted by phosphoethanolamine or phosphate groups. More detailed structural analysis performed by mass spectrometry revealed numerous minor O-glycan structures bearing Gal, GlcNAc, GalNAc and Fuc residues. Thus, in order to investigate glycosylation diversity in insects, we used common wasp nest (V. germanica) and hornet nest (Vespa cabro) as starting materials. These materials were submitted to reductive β-elimination and the released oligosaccharide-alditols further fractionated by multidimensional HPLC. Tandem mass spectrometry analyses combined with NMR data revealed the presence of various families of complex O-glycans differing accordingly to both core structures and external motifs. Glycans from wasp were characterized by the presence of core types 1 and 2, Lewis X and internal Gal-Gal motifs. We also observed unusual O-glycans containing a reducing GalNAc unit directly substituted by a fucose residue. In contrast, hornet O-glycans appeared as a rather homogeneous family of core 1 type O-glycans extended by galactose oligomers. Copyright © 2011 Elsevier Ltd. All rights reserved.

Glycan involvement in the adhesion of Pseudomonas aeruginosa to tears.

Science.gov (United States)

Kautto, Liisa; Nguyen-Khuong, Terry; Everest-Dass, Arun; Leong, Andrea; Zhao, Zhenjun; Willcox, Mark D P; Packer, Nicolle H; Peterson, Robyn

2016-04-01

The human eye is constantly bathed by tears, which protect the ocular surface via a variety of mechanisms. The O-linked glycans of tear mucins have long been considered to play a role in binding to pathogens and facilitating their removal in the tear flow. Other conjugated glycans in tears could similarly contribute to pathogen binding and removal but have received less attention. In the work presented here we assessed the contribution of glycan moieties, in particular the protein attached N-glycans, presented by the broad complement of tear proteins to the adhesion of the opportunistic pathogen Pseudomonas aeruginosa, a leading cause of microbial keratitis and ulceration of the cornea. Our adhesion assay involved immobilising the macromolecular components of tears into the wells of a polyvinyl difluoride (PVDF) microtitre filter plate and probing the binding of fluorescently labelled bacteria. Three P. aeruginosa strains were studied: a cytotoxic strain (6206) and an invasive strain (6294) from eye infections, and an invasive strain (320) from a urinary tract infection (UTI). The ocular isolates adhered two to three times more to human tears than to human saliva or porcine gastric mucin, suggesting ocular niche-specific adaptation. Support for the role of the N-glycans carried by human tear proteins in the binding and removal of P. aeruginosa from the eye was shown by: 1) pre-incubation of the bacteria with free component sugars, galactose, mannose, fucose and sialyl lactose (or combination thereof) inhibiting adhesion of all the P. aeruginosa strains to the immobilised tear proteins, with the greatest inhibition of binding of the ocular cytotoxic 6206 and least for the invasive 6294 strain; 2) pre-incubation of the bacteria with N-glycans released from the commercially available human milk lactoferrin, an abundant protein that carries N-linked glycans in tears, inhibiting the adhesion to tears of the ocular bacteria by up to 70%, which was significantly more

MicroRNA-1185 Promotes Arterial Stiffness though Modulating VCAM-1 and E-Selectin Expression

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Haoyuan Deng

2017-04-01

Full Text Available Background/Aims: Atherosclerosis is the primary cause of cardiovascular ischaemic events; arterial stiffness is a characteristic of the atherosclerotic process. MicroRNAs (miRNAs have been revealed as crucial modulators of atherosclerosis. However, the role of arterial stiffness-related miRNAs in the atherosclerotic process is still unclear. Methods: Four hundred six participants from Northern China were enrolled in this study. Circulating miR-1185 and adhesion molecule levels were measured. Multiple linear regression models were used to evaluate the association of miR-1185 levels with brachial-ankle pulse wave velocity (baPWV and adhesion molecule levels. A mediation analysis was also performed to examine the mediating effect. Cell adhesion molecule levels were measured in primary human umbilical vein endothelial cells (pHUVECs and human umbilical vein smooth cells (HUVSMCs transfected with miR-1185 or co-transfected with a miR-1185 inhibitor. Results: miR-1185 was independently correlated with arterial stiffness. A positive relationship between miR-1185 and vascular cell adhesion molecule-1 (VCAM-1 and E-selectin levels was observed. VCAM- 1 and E-selectin partially mediated the correlation between miR-1185 and arterial stiffness. miR-1185 induced a significant increase in the VCAM-1 and E-selectin levels in pHUVECs and HUVSMCs in vitro. According to our mechanistic analysis, VCAM-1 and E-selectin mediated miR-1185-induced arterial stiffening. Conclusions: miR-1185 modulated the expression of VCAM-1 and E-selectin to promote arterial stiffening, suggesting that miR-1185 plays a crucial role in the development of atherosclerosis and may serve as a novel therapeutic target for atherosclerosis.

A synthetic glycan microarray enables epitope mapping of plant cell wall glycan-directed antibodies

DEFF Research Database (Denmark)

Ruprecht, Colin; Bartetzko, Max P; Senf, Deborah

2017-01-01

In the last three decades, more than 200 monoclonal antibodies have been raised against most classes of plant cell wall polysaccharides by different laboratories world-wide. These antibodies are widely used to identify differences in plant cell wall components in mutants, organ and tissue types......, and developmental stages. Despite their importance and broad use, the precise binding epitope for only a few of these antibodies has been determined. Here, we use a plant glycan microarray equipped with 88 synthetic oligosaccharides to comprehensively map the epitopes of plant cell wall glycan-directed antibodies....... Our results reveal the binding epitopes for 78 arabinogalactan-, rhamnogalacturonan-, xylan-, and xyloglucan-directed antibodies. We demonstrate that, with knowledge of the exact epitopes recognized by individual antibodies, specific glycosyl hydrolases can be implemented into immunological cell wall...

Detection of early stage atherosclerotic plaques using PET and CT fusion imaging targeting P-selectin in low density lipoprotein receptor-deficient mice

Energy Technology Data Exchange (ETDEWEB)

Nakamura, Ikuko, E-mail: nakamuri@riken.jp [RIKEN Center for Molecular Imaging Science, Kobe (Japan); Department of Cardiovascular Medicine, Saga University, Saga (Japan); Hasegawa, Koki [RIKEN Center for Molecular Imaging Science, Kobe (Japan); Department of Pathology and Experimental Medicine, Kumamoto University, Kumamoto (Japan); Wada, Yasuhiro [RIKEN Center for Molecular Imaging Science, Kobe (Japan); Hirase, Tetsuaki; Node, Koichi [Department of Cardiovascular Medicine, Saga University, Saga (Japan); Watanabe, Yasuyoshi, E-mail: yywata@riken.jp [RIKEN Center for Molecular Imaging Science, Kobe (Japan)

2013-03-29

Highlights: ► P-selectin regulates leukocyte recruitment as an early stage event of atherogenesis. ► We developed an antibody-based molecular imaging probe targeting P-selectin for PET. ► This is the first report on successful PET imaging for delineation of P-selectin. ► P-selectin is a candidate target for atherosclerotic plaque imaging by clinical PET. -- Abstract: Background: Sensitive detection and qualitative analysis of atherosclerotic plaques are in high demand in cardiovascular clinical settings. The leukocyte–endothelial interaction mediated by an adhesion molecule P-selectin participates in arterial wall inflammation and atherosclerosis. Methods and results: A {sup 64}Cu-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid conjugated anti-P-selectin monoclonal antibody ({sup 64}Cu-DOTA-anti-P-selectin mAb) probe was prepared by conjugating an anti-P-selectin monoclonal antibody with DOTA followed by {sup 64}Cu labeling. Thirty-six hours prior to PET and CT fusion imaging, 3 MBq of {sup 64}Cu-DOTA-anti-P-selectin mAb was intravenously injected into low density lipoprotein receptor-deficient Ldlr-/- mice. After a 180 min PET scan, autoradiography and biodistribution of {sup 64}Cu-DOTA-anti-P-selectin monoclonal antibody was examined using excised aortas. In Ldlr-/- mice fed with a high cholesterol diet for promotion of atherosclerotic plaque development, PET and CT fusion imaging revealed selective and prominent accumulation of the probe in the aortic root. Autoradiography of aortas that demonstrated probe uptake into atherosclerotic plaques was confirmed by Oil red O staining for lipid droplets. In Ldlr-/- mice fed with a chow diet to develop mild atherosclerotic plaques, probe accumulation was barely detectable in the aortic root on PET and CT fusion imaging. Probe biodistribution in aortas was 6.6-fold higher in Ldlr-/- mice fed with a high cholesterol diet than in those fed with a normal chow diet. {sup 64}Cu-DOTA-anti-P-selectin m

Mucin glycan foraging in the human gut microbiome

Science.gov (United States)

Tailford, Louise E.; Crost, Emmanuelle H.; Kavanaugh, Devon; Juge, Nathalie

2015-01-01

The availability of host and dietary carbohydrates in the gastrointestinal (GI) tract plays a key role in shaping the structure-function of the microbiota. In particular, some gut bacteria have the ability to forage on glycans provided by the mucus layer covering the GI tract. The O-glycan structures present in mucin are diverse and complex, consisting predominantly of core 1-4 mucin-type O-glycans containing α- and β- linked N-acetyl-galactosamine, galactose and N-acetyl-glucosamine. These core structures are further elongated and frequently modified by fucose and sialic acid sugar residues via α1,2/3/4 and α2,3/6 linkages, respectively. The ability to metabolize these mucin O-linked oligosaccharides is likely to be a key factor in determining which bacterial species colonize the mucosal surface. Due to their proximity to the immune system, mucin-degrading bacteria are in a prime location to influence the host response. However, despite the growing number of bacterial genome sequences available from mucin degraders, our knowledge on the structural requirements for mucin degradation by gut bacteria remains fragmented. This is largely due to the limited number of functionally characterized enzymes and the lack of studies correlating the specificity of these enzymes with the ability of the strain to degrade and utilize mucin and mucin glycans. This review focuses on recent findings unraveling the molecular strategies used by mucin-degrading bacteria to utilize host glycans, adapt to the mucosal environment, and influence human health. PMID:25852737

Dual modifications strategy to quantify neutral and sialylated N-glycans simultaneously by MALDI-MS.

Science.gov (United States)

Zhou, Hui; Warren, Peter G; Froehlich, John W; Lee, Richard S

2014-07-01

Differences in ionization efficiency among neutral and sialylated glycans prevent direct quantitative comparison by their respective mass spectrometric signals. To overcome this challenge, we developed an integrated chemical strategy, Dual Reactions for Analytical Glycomics (DRAG), to quantitatively compare neutral and sialylated glycans simultaneously by MALDI-MS. Initially, two glycan samples to be compared undergo reductive amination with 2-aminobenzoic acid and 2-(13)[C6]-aminobenzoic acid, respectively. The different isotope-incorporated glycans are then combined and subjected to the methylamidation of the sialic acid residues in one mixture, homogenizing the ionization responses for all neutral and sialylated glycans. By this approach, the expression change of relevant glycans between two samples is proportional to the ratios of doublet signals with a static 6 Da mass difference in MALDI-MS and the change in relative abundance of any glycan within samples can also be determined. The strategy was chemically validated using well-characterized N-glycans from bovine fetuin and IgG from human serum. By comparing the N-glycomes from a first morning (AM) versus an afternoon (PM) urine sample obtained from a single donor, we further demonstrated the ability of DRAG strategy to measure subtle quantitative differences in numerous urinary N-glycans.

Assessment of the E-Selectin rs5361 (561A>C Polymorphism and Soluble Protein Concentration in Acute Coronary Syndrome: Association with Circulating Levels

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Elena Sandoval-Pinto

2014-01-01

Full Text Available Introduction. The acute coronary syndrome (ACS is a complex disease where genetic and environmental factors are involved. E-selectin gene is a candidate for ACS progression due to its contribution in the inflammatory process and endothelial function. The rs5361 (561A>C polymorphism in the E-selectin gene has been linked to changes in gene expression, affinity for its receptor, and plasmatic levels; therefore it is associated with an increased risk of cardiovascular disease. The aim of this study was to determine the association of the rs5361 polymorphism with ACS and to measure serum levels of soluble E-selectin (sE-selectin. Materials and Methods. 283 ACS patients and 205 healthy subjects (HS from Western Mexico were included. The polymerase chain reaction-restriction fragment length polymorphism was used to determine the rs5361 polymorphism. The sE-selectin levels were measured by enzyme-linked immunosorbent assay. Results. Neither genotype nor allele frequencies of the rs5361 polymorphism showed statistical differences between groups. The sE-selectin levels were significantly higher in ACS patients compared to HS (54.58 versus 40.41 ng/ml, P=0.02. The C allele had no effect on sE-selectin levels. Conclusions. The rs5361 E-selectin gene polymorphism is not a susceptibility marker for ACS in Western Mexico population. However, sE-selectin may be a biological marker of ACS.

Differential Fragmentation of Mobility-Selected Glycans via Ultraviolet Photodissociation and Ion Mobility-Mass Spectrometry

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Morrison, Kelsey A.; Clowers, Brian H.

2017-06-01

The alternative dissociation pathways initiated by ultraviolet photodissociation (UVPD) compared with collision-induced dissociation (CID) may provide useful diagnostic fragments for biomolecule identification, including glycans. However, underivatized glycans do not commonly demonstrate strong UV absorbance, resulting in low fragmentation yields for UVPD spectra. In contrast to UVPD experiments that leverage covalent modification of glycans, we detail the capacity of metal adduction to yield comparatively rich UVPD fragmentation patterns and enhance separation factors for an isomeric glycan set in a drift tube ion mobility system. Ion mobility and UVPD-MS spectra for two N-acetyl glycan isomers were examined, each adducted with sodium or cobalt cations, with the latter providing fragment yield gains of an order of magnitude versus sodium adducts. Furthermore, our glycan analysis incorporated front-end ion mobility separation such that the structural glycan isomers could still be identified even as a mixture and not simply composite spectra of isomeric standards. Cobalt adduction proved influential in the glycan separation by yielding an isomer resolution of 0.78 when analyzed simultaneously versus no discernable separation obtained with the sodium adducts. It is the combined enhancement of both isomeric drift time separation and isomer distinction with improved UVPD fragment ion yields that further bolster multivalent metal adduction for advancing glycan IM-MS experiments. [Figure not available: see fulltext.

Comparison of fluorescent tags for analysis of mannose-6-phosphate glycans.

Science.gov (United States)

Kang, Ji-Yeon; Kwon, Ohsuk; Gil, Jin Young; Oh, Doo-Byoung

2016-05-15

Mannose-6-phosphate (M-6-P) glycan analysis is important for quality control of therapeutic enzymes for lysosomal storage diseases. Here, we found that the analysis of glycans containing two M-6-Ps was highly affected by the hydrophilicity of the elution solvent used in high-performance liquid chromatography (HPLC). In addition, the performances of three fluorescent tags--2-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), and 3-(acetyl-amino)-6-aminoacridine (AA-Ac)--were compared with each other for M-6-P glycan analysis using HPLC and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The best performance for analyzing M-6-P glycans was shown by 2-AA labeling in both analyses. Copyright © 2016 Elsevier Inc. All rights reserved.

Rapid chemical de-N-glycosylation and derivatization for liquid chromatography of immunoglobulin N-linked glycans.

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Akihiko Kameyama

Full Text Available Glycan analysis may result in exploitation of glycan biomarkers and evaluation of heterogeneity of glycosylation of biopharmaceuticals. For N-linked glycan analysis, we investigated alkaline hydrolysis of the asparagine glycosyl carboxamide of glycoproteins as a deglycosylation reaction. By adding hydroxylamine into alkaline de-N-glycosylation, we suppressed the degradation of released glycans and obtained a mixture of oximes, free glycans, and glycosylamines. The reaction was completed within 1 h, and the mixture containing oximes was easily tagged with 2-aminobenzamide by reductive amination. Here, we demonstrated N-linked glycan analysis using this method for a monoclonal antibody, and examined whether this method could liberate glycans without degradation from apo-transferrin containing NeuAc and NeuGc and horseradish peroxidase containing Fuc α1-3 GlcNAc at the reducing end. Furthermore, we compared glycan recoveries between conventional enzymatic glycan release and this method. Increasing the reaction temperature and reaction duration led to degradation, whereas decreasing these parameters resulted in lower release. Considering this balance, we proposed to carry out the reaction at 80°C for 1 h for asialo glycoproteins from mammals and at 50°C for 1 h for sialoglycoproteins.

Quantitative glycan profiling of normal human plasma derived immunoglobulin and its fragments Fab and Fc.

Science.gov (United States)

Anumula, Kalyan Rao

2012-08-31

Typical clinical grade human IgG (intravenous immunoglobulin, IVIG), used for carbohydrate analysis, is derived from thousands of healthy donors. Quantitative high-resolution glycan profiles of IgG and its Fc-Fab fragments are presented here. Glycan profiles were established following digestions with Fc specific endoglycosidase S and generic PNGase F under denaturing and non-denaturing (native) conditions. The native PNGase F glycan profile of IgG was similar (but not identical) to that of Endo S. Endo S profiles did not contain the glycans with bisecting GlcNAc. PNGase F glycan profiles were the same for Fc fragments that were isolated from pepsin and Ide S protease digests. Both isolated Fab fragments and the previously deglycosylated IVIG (native conditions) yielded the same glycan profile. Glycan profiles were established using high resolution HPLC with 2-aminobenzoic acid (2AA) labeling. An accurate determination of sialylation levels can be made by this method. Carbohydrate content in Fc and Fab was determined using an internal standard and corrected for both protein and glycan recoveries. Fab portion contained about 14% of the total carbohydrate which translates to 2.3 sugar chains per mol in IVIG where 2 chains are located in the CH2 domain of the Fc. Fc glycans consisted of neutral (N) 84.5%; mono-sialylated (S1) 15% and di-sialylated (S2) 0.5%. In contrast, Fab contained N, 21%; S1, 43% and S2, 36%. The distribution of bisecting N-acetylglucosamine and fucose was found to be very different in various glycans (N, S1 and S2) found in Fab and Fc. Total IgG glycan profile (Fab plus Fc) contained N, 78.5%; S1, 17% and S2, 4.5%. Percent distribution of glycans G0, G1 and G2 (with 0, 1 and 2 two galactoses) was 26, 49 and 25 respectively within the 78% of the neutral glycans. Glycan profiles were nearly the same for various clinical grade IVIG preparations from various manufacturers. A fast HPLC profiling method was developed for the separation and quantitation

NF-kB activity-dependent P-selectin involved in ox-LDL-induced foam cell formation in U937 cell

International Nuclear Information System (INIS)

Wang, Yi; Wang, Xiang; Sun, Minghui; Zhang, Zhenyu; Cao, Heng; Chen, Xiaoqing

2011-01-01

Highlights: → Ox-LDL induced foam cell formation in the human U937 promonocytic cell line in a dose- and time-dependent manner. → Ox-LDL induced expression of P-selectin through degradation of IkBa and augment of NF-kB activity and protein level during macrophage-derived foam cell formation. → P-selectin and NF-kB may be identified as pivotal regulators of ox-LDL-induced foam cell formation. → Therapy based on the inhibition of P-selectin and NF-kB may complement conventional treatments to prevent atherosclerosis. -- Abstract: Oxidized low-density lipoprotein (ox-LDL) plays a critical role in regulation of atherosclerosis. However, little is known about the role of Nuclear factor kB (NF-kB) activity-dependent P-selectin in ox-LDL-induced foam cell formation during atherosclerosis. In this study, we first investigated ox-LDL induced foam cell formation in the human U937 promonocytic cell line in a dose- and time-dependent manner. Treatment of U937 cells with ox-LDL increased lipid accumulation as well as intracellular cholesterol content. Next, a comparative analysis of gene expression profiling using cDNA microarray and Real-time-PCR indicated that ox-LDL exposure induced, in three treated groups, an extremely marked increase in the mRNA level of P-selectin. Protein levels of P-selectin and its upstream regulators IkBa and NF-kB showed that NF-kB pathway is involved in the ox-LDL-induced foam cell formation. Finally, overexpression of NF-kB significantly accelerated, whereas, inhibition of NF-kB with siRNA remarkably attenuated ox-LDL-induced macrophage-derived foam cell formation. It was concluded that the activity of NF-kB is augmented during macrophage-derived foam cell formation. Activation of NF-kB increased, whereas, inhibition of NF-kB decreased ox-LDL-induced P-selectin expression and lipid accumulation in macrophages, suggesting ox-LDL induced expression of P-selectin through degradation of IkBa and activation of NF-kB in the regulation of foam

NF-kB activity-dependent P-selectin involved in ox-LDL-induced foam cell formation in U937 cell

Energy Technology Data Exchange (ETDEWEB)

Wang, Yi, E-mail: wangyi2004a@126.com [Department of Cardiology, Shanghai First People' s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200080 (China); Wang, Xiang; Sun, Minghui; Zhang, Zhenyu; Cao, Heng; Chen, Xiaoqing [Department of Cardiology, Shanghai First People' s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200080 (China)

2011-08-05

Highlights: {yields} Ox-LDL induced foam cell formation in the human U937 promonocytic cell line in a dose- and time-dependent manner. {yields} Ox-LDL induced expression of P-selectin through degradation of IkBa and augment of NF-kB activity and protein level during macrophage-derived foam cell formation. {yields} P-selectin and NF-kB may be identified as pivotal regulators of ox-LDL-induced foam cell formation. {yields} Therapy based on the inhibition of P-selectin and NF-kB may complement conventional treatments to prevent atherosclerosis. -- Abstract: Oxidized low-density lipoprotein (ox-LDL) plays a critical role in regulation of atherosclerosis. However, little is known about the role of Nuclear factor kB (NF-kB) activity-dependent P-selectin in ox-LDL-induced foam cell formation during atherosclerosis. In this study, we first investigated ox-LDL induced foam cell formation in the human U937 promonocytic cell line in a dose- and time-dependent manner. Treatment of U937 cells with ox-LDL increased lipid accumulation as well as intracellular cholesterol content. Next, a comparative analysis of gene expression profiling using cDNA microarray and Real-time-PCR indicated that ox-LDL exposure induced, in three treated groups, an extremely marked increase in the mRNA level of P-selectin. Protein levels of P-selectin and its upstream regulators IkBa and NF-kB showed that NF-kB pathway is involved in the ox-LDL-induced foam cell formation. Finally, overexpression of NF-kB significantly accelerated, whereas, inhibition of NF-kB with siRNA remarkably attenuated ox-LDL-induced macrophage-derived foam cell formation. It was concluded that the activity of NF-kB is augmented during macrophage-derived foam cell formation. Activation of NF-kB increased, whereas, inhibition of NF-kB decreased ox-LDL-induced P-selectin expression and lipid accumulation in macrophages, suggesting ox-LDL induced expression of P-selectin through degradation of IkBa and activation of NF-kB in the

Hexose rearrangements upon fragmentation of N-glycopeptides and reductively aminated N-glycans.

Science.gov (United States)

Wuhrer, Manfred; Koeleman, Carolien A M; Deelder, André M

2009-06-01

Tandem mass spectrometry of glycans and glycoconjugates in protonated form is known to result in rearrangement reactions leading to internal residue loss. Here we studied the occurrence of hexose rearrangements in tandem mass spectrometry of N-glycopeptides and reductively aminated N-glycans by MALDI-TOF/TOF-MS/MS and ESI-ion trap-MS/MS. Fragmentation of proton adducts of oligomannosidic N-glycans of ribonuclease B that were labeled with 2-aminobenzamide and 2-aminobenzoic acid resulted in transfer of one to five hexose residues to the fluorescently tagged innermost N-acetylglucosamine. Glycopeptides from various biological sources with oligomannosidic glycans were likewise shown to undergo hexose rearrangement reactions, resulting in chitobiose cleavage products that have acquired one or two hexose moieties. Tryptic immunoglobulin G Fc-glycopeptides with biantennary N-glycans likewise showed hexose rearrangements resulting in hexose transfer to the peptide moiety retaining the innermost N-acetylglucosamine. Thus, as a general phenomenon, tandem mass spectrometry of reductively aminated glycans as well as glycopeptides may result in hexose rearrangements. This characteristic of glycopeptide MS/MS has to be considered when developing tools for de novo glycopeptide structural analysis.

Reciprocal Prioritization to Dietary Glycans by Gut Bacteria in a Competitive Environment Promotes Stable Coexistence

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Yunus E. Tuncil

2017-10-01

Full Text Available When presented with nutrient mixtures, several human gut Bacteroides species exhibit hierarchical utilization of glycans through a phenomenon that resembles catabolite repression. However, it is unclear how closely these observed physiological changes, often measured by altered transcription of glycan utilization genes, mirror actual glycan depletion. To understand the glycan prioritization strategies of two closely related human gut symbionts, Bacteroides ovatus and Bacteroides thetaiotaomicron, we performed a series of time course assays in which both species were individually grown in a medium with six different glycans that both species can degrade. Disappearance of the substrates and transcription of the corresponding polysaccharide utilization loci (PULs were measured. Each species utilized some glycans before others, but with different priorities per species, providing insight into species-specific hierarchical preferences. In general, the presence of highly prioritized glycans repressed transcription of genes involved in utilizing lower-priority nutrients. However, transcriptional sensitivity to some glycans varied relative to the residual concentration in the medium, with some PULs that target high-priority substrates remaining highly expressed even after their target glycan had been mostly depleted. Coculturing of these organisms in the same mixture showed that the hierarchical orders generally remained the same, promoting stable coexistence. Polymer length was found to be a contributing factor for glycan utilization, thereby affecting its place in the hierarchy. Our findings not only elucidate how B. ovatus and B. thetaiotaomicron strategically access glycans to maintain coexistence but also support the prioritization of carbohydrate utilization based on carbohydrate structure, advancing our understanding of the relationships between diet and the gut microbiome.

Glycosylation with ribitol-phosphate in mammals: New insights into the O-mannosyl glycan.

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Manya, Hiroshi; Endo, Tamao

2017-10-01

O-mannosyl glycans have been found in a limited number of glycoproteins of the brain, nerves, and skeletal muscles, particularly in α-dystroglycan (α-DG). Defects in O-mannosyl glycan on α-DG are the primary cause of a group of congenital muscular dystrophies, which are collectively termed α-dystroglycanopathy. Recent studies have revealed various O-mannosyl glycan structures, which can be classified as core M1, core M2, and core M3 glycans. Although many dystroglycanopathy genes are involved in core M3 processing, the structure and biosynthesis of core M3 glycan remains only partially understood. This review presents recent findings about the structure, biosynthesis, and pathology of O-mannosyl glycans. Recent studies have revealed that the entire structure of core M3 glycan, including ribitol-5-phosphate, is a novel structure in mammals; its unique biosynthetic pathway has been elucidated by the identification of new causative genes for α-dystroglycanopathies and their functions. O-mannosyl glycan has a novel, unique structure that is important for the maintenance of brain and muscle functions. These findings have opened up a new field in glycoscience. These studies will further contribute to the understanding of the pathomechanism of α-dystroglycanopathy and the development of glycotherapeutics. 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.

Angiogenesis mediated by soluble forms of E-selectin and vascular cell adhesion molecule-1

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Koch, Alisa E.; Halloran, Margaret M.; Haskell, Catherine J.; Shah, Manisha R.; Polverini, Peter J.

1995-08-01

ENDOTHELIAL adhesion molecules facilitate the entry of leukocytes into inflamed tissues. This in turn promotes neovascularization, a process central to the progression of rheumatoid arthritis, tumour growth and wound repair1. Here we test the hypothesis that soluble endothelial adhesion molecules promote angiogenesis2á¤-4. Human recombinant soluble E-selectin and soluble vascular cell adhesion molecule-1 induced chemotaxis of human endothelial cells in vitro and were angiogenic in rat cornea. Soluble E-selectin acted on endothelial cells in part through a sialyl Lewis-X-dependent mechanism, while soluble vascular cell adhesion molecule-1 acted on endothelial cells in part through a very late antigen (VLA)-4 dependent mechanism. The chemotactic activity of rheumatoid synovial fluid for endothelial cells, and also its angiogenic activity, were blocked by antibodies to either soluble E-selectin or soluble vascular cell adhesion molecule-1. These results suggest a novel function for soluble endothelial adhesion molecules as mediators of angiogenesis.

Immunogenicity of glycans on biotherapeutic drugs produced in plant expression systems—The taliglucerase alfa story

Science.gov (United States)

Rup, Bonita; Alon, Sari; Amit-Cohen, Bat-Chen; Brill Almon, Einat; Chertkoff, Raul; Rudd, Pauline M.

2017-01-01

Plants are a promising alternative for the production of biotherapeutics. Manufacturing in-planta adds plant specific glycans. To understand immunogenic potential of these glycans, we developed a validated method to detect plant specific glycan antibodies in human serum. Using this assay, low prevalence of pre-existing anti-plant glycan antibodies was found in healthy humans (13.5%) and in glucocerebrosidase-deficient Gaucher disease (GD) patients (5%). A low incidence (9% in naïve patient and none in treatment experienced patients) of induced anti-plant glycan antibodies was observed in GD patients after up to 30 months replacement therapy treatment with taliglucerase alfa, a version of human glucocerebrosidase produced in plant cells. Detailed evaluation of clinical safety and efficacy endpoints indicated that anti-plant glycan antibodies did not affect the safety or efficacy of taliglucerase alfa in patients. This study shows the benefit of using large scale human trials to evaluate the immunogenicity risk of plant derived glycans, and indicates no apparent risk related to anti-plant glycan antibodies. PMID:29088235

A nonself sugar mimic of the HIV glycan shield shows enhanced antigenicity

Energy Technology Data Exchange (ETDEWEB)

Doores, Katie J.; Fulton, Zara; Hong, Vu; Patel, Mitul K.; Scanlan, Christopher N.; Wormald, Mark R.; Finn, M.G.; Burton, Dennis R.; Wilson, Ian A.; Davis, Benjamin G. (Scripps); (Oxford)

2011-08-24

Antibody 2G12 uniquely neutralizes a broad range of HIV-1 isolates by binding the high-mannose glycans on the HIV-1 surface glycoprotein, gp120. Antigens that resemble these natural epitopes of 2G12 would be highly desirable components for an HIV-1 vaccine. However, host-produced (self)-carbohydrate motifs have been unsuccessful so far at eliciting 2G12-like antibodies that cross-react with gp120. Based on the surprising observation that 2G12 binds nonproteinaceous monosaccharide D-fructose with higher affinity than D-mannose, we show here that a designed set of nonself, synthetic monosaccharides are potent antigens. When introduced to the terminus of the D1 arm of protein glycans recognized by 2G12, their antigenicity is significantly enhanced. Logical variation of these unnatural sugars pinpointed key modifications, and the molecular basis of this increased antigenicity was elucidated using high-resolution crystallographic analyses. Virus-like particle protein conjugates containing such nonself glycans are bound more tightly by 2G12. As immunogens they elicit higher titers of antibodies than those immunogenic conjugates containing the self D1 glycan motif. These antibodies generated from nonself immunogens also cross-react with this self motif, which is found in the glycan shield, when it is presented in a range of different conjugates and glycans. However, these antibodies did not bind this glycan motif when present on gp120.

GLYCAN-DIRECTED CAR-T CELLS.

Science.gov (United States)

Steentoft, Catharina; Migliorini, Denis; King, Tiffany R; Mandel, Ulla; June, Carl H; Posey, Avery D

2018-01-23

Cancer immunotherapy is rapidly advancing in the treatment of a variety of hematopoietic cancers, including pediatric acute lymphoblastic leukemia and diffuse large B cell lymphoma, with chimeric antigen receptor (CAR)-T cells. CARs are genetically encoded artificial T cell receptors that combine the antigen specificity of an antibody with the machinery of T cell activation. However, implementation of CAR technology in the treatment of solid tumors has been progressing much slower. Solid tumors are characterized by a number of challenges that need to be overcome, including cellular heterogeneity, immunosuppressive tumor microenvironment (TME), and, in particular, few known cancer-specific targets. Post-translational modifications that differentially occur in malignant cells generate valid cell surface, cancer-specific targets for CAR-T cells. We previously demonstrated that CAR-T cells targeting an aberrant O-glycosylation of MUC1, a common cancer marker associated with changes in cell adhesion, tumor growth, and poor prognosis, could control malignant growth in mouse models. Here, we discuss the field of glycan-directed CAR-T cells and review the different classes of antibodies specific for glycan-targeting, including the generation of high affinity O-glycopeptide antibodies. Finally, we discuss historic and recently investigated glycan targets for CAR-T cells and provide our perspective on how targeting the tumor glycoproteome and/or glycome will improve CAR-T immunotherapy. © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Direct imaging of glycans in Arabidopsis roots via click labeling of metabolically incorporated azido-monosaccharides.

Science.gov (United States)

Hoogenboom, Jorin; Berghuis, Nathalja; Cramer, Dario; Geurts, Rene; Zuilhof, Han; Wennekes, Tom

2016-10-10

Carbohydrates, also called glycans, play a crucial but not fully understood role in plant health and development. The non-template driven formation of glycans makes it impossible to image them in vivo with genetically encoded fluorescent tags and related molecular biology approaches. A solution to this problem is the use of tailor-made glycan analogs that are metabolically incorporated by the plant into its glycans. These metabolically incorporated probes can be visualized, but techniques documented so far use toxic copper-catalyzed labeling. To further expand our knowledge of plant glycobiology by direct imaging of its glycans via this method, there is need for novel click-compatible glycan analogs for plants that can be bioorthogonally labelled via copper-free techniques. Arabidopsis seedlings were incubated with azido-containing monosaccharide analogs of N-acetylglucosamine, N-acetylgalactosamine, L-fucose, and L-arabinofuranose. These azido-monosaccharides were metabolically incorporated in plant cell wall glycans of Arabidopsis seedlings. Control experiments indicated active metabolic incorporation of the azido-monosaccharide analogs into glycans rather than through non-specific absorption of the glycan analogs onto the plant cell wall. Successful copper-free labeling reactions were performed, namely an inverse-electron demand Diels-Alder cycloaddition reaction using an incorporated N-acetylglucosamine analog, and a strain-promoted azide-alkyne click reaction. All evaluated azido-monosaccharide analogs were observed to be non-toxic at the used concentrations under normal growth conditions. Our results for the metabolic incorporation and fluorescent labeling of these azido-monosaccharide analogs expand the possibilities for studying plant glycans by direct imaging. Overall we successfully evaluated five azido-monosaccharide analogs for their ability to be metabolically incorporated in Arabidopsis roots and their imaging after fluorescent labeling. This expands

The development of retrosynthetic glycan libraries to profile and classify the human serum N-linked glycome.

Science.gov (United States)

Kronewitter, Scott R; An, Hyun Joo; de Leoz, Maria Lorna; Lebrilla, Carlito B; Miyamoto, Suzanne; Leiserowitz, Gary S

2009-06-01

Annotation of the human serum N-linked glycome is a formidable challenge but is necessary for disease marker discovery. A new theoretical glycan library was constructed and proposed to provide all possible glycan compositions in serum. It was developed based on established glycobiology and retrosynthetic state-transition networks. We find that at least 331 compositions are possible in the serum N-linked glycome. By pairing the theoretical glycan mass library with a high mass accuracy and high-resolution MS, human serum glycans were effectively profiled. Correct isotopic envelope deconvolution to monoisotopic masses and the high mass accuracy instruments drastically reduced the amount of false composition assignments. The high throughput capacity enabled by this library permitted the rapid glycan profiling of large control populations. With the use of the library, a human serum glycan mass profile was developed from 46 healthy individuals. This paper presents a theoretical N-linked glycan mass library that was used for accurate high-throughput human serum glycan profiling. Rapid methods for evaluating a patient's glycome are instrumental for studying glycan-based markers.

Self-recognition of high-mannose type glycans mediating adhesion of embryonal fibroblasts.

Science.gov (United States)

Yoon, Seon-Joo; Utkina, Natalia; Sadilek, Martin; Yagi, Hirokazu; Kato, Koichi; Hakomori, Sen-itiroh

2013-07-01

High-mannose type N-linked glycan with 6 mannosyl residues, termed "M6Gn2", displayed clear binding to the same M6Gn2, conjugated with ceramide mimetic (cer-m) and incorporated in liposome, or coated on polystyrene plates. However, the conjugate of M6Gn2-cer-m did not interact with complex-type N-linked glycan with various structures having multiple GlcNAc termini, conjugated with cer-m. The following observations indicate that hamster embryonic fibroblast NIL-2 K cells display homotypic autoadhesion, mediated through the self-recognition capability of high-mannose type glycans expressed on these cells: (i) NIL-2 K cells display clear binding to lectins capable of binding to high-mannose type glycans (e.g., ConA), but not to other lectins capable of binding to other carbohydrates (e.g. GS-II). (ii) NIL-2 K cells adhere strongly to plates coated with M6Gn2-cer-m, but not to plates coated with complex-type N-linked glycans having multiple GlcNAc termini, conjugated with cer-m; (iii) degree of NIL-2 K cell adhesion to plates coated with M6Gn2-cer-m showed a clear dose-dependence on the amount of M6Gn2-cer-m; and (iv) the degree of NIL-2 K adhesion to plates coated with M6Gn2-cer-m was inhibited in a dose-dependent manner by α1,4-L-mannonolactone, the specific inhibitor in high-mannose type glycans addition. These data indicate that adhesion of NIL-2 K is mediated by self-aggregation of high mannose type glycan. Further studies are to be addressed on auto-adhesion of other types of cells based on self interaction of high mannose type glycans.

Clinical significance of determination of serum hypersensitive C-reactive protein (hs-CRP) and E-selectin levels in patients with coronary heart diseases

International Nuclear Information System (INIS)

Yang Chunxiu

2007-01-01

Objective: To investigate the significance of determination of serum contents of hs-CRP and E-Selectin in patients with coronary heart diseases (CHD). Methods: Serum hs-CRP Contents were determined with immuno-turbidity and E-Selectin contents were determined with ELISA in 58 patients with CHD (35SAP, 20UAP, 13AMI) and 35 controls. Results: Serum levels of hs-CRP and E-Selectin in CHD patients were significantly higher than those in controls (P 0.05). Conclusion: The serum levels of hs-CRP and E-Selectin were correlated to the development of CHD, but not to the coronary artery calibers. (authors)

Glycan elongation beyond the mucin associated Tn antigen protects tumor cells from immune-mediated killing

DEFF Research Database (Denmark)

Madsen, Caroline B; Lavrsen, Kirstine; Steentoft, Catharina

2013-01-01

are recognized as cancer associated truncated glycans, and are expressed in many adenocarcinomas, e.g. breast- and pancreatic cancer cells. To investigate the role of the cancer associated glycan truncations in immune-mediated killing we created glyco-engineered breast- and pancreatic cancer cells expressing...... only the shortest possible mucin-like glycans (Tn and STn). Glyco-engineering was performed by zinc finger nuclease (ZFN) knockout (KO) of the Core 1 enzyme chaperone COSMC, thereby preventing glycan elongation beyond the initial GalNAc residue in O-linked glycans. We find that COSMC KO in the breast...

Quantification of the Impact of the HIV-1-Glycan Shield on Antibody Elicitation

Directory of Open Access Journals (Sweden)

Tongqing Zhou

2017-04-01

Full Text Available While the HIV-1-glycan shield is known to shelter Env from the humoral immune response, its quantitative impact on antibody elicitation has been unclear. Here, we use targeted deglycosylation to measure the impact of the glycan shield on elicitation of antibodies against the CD4 supersite. We engineered diverse Env trimers with select glycans removed proximal to the CD4 supersite, characterized their structures and glycosylation, and immunized guinea pigs and rhesus macaques. Immunizations yielded little neutralization against wild-type viruses but potent CD4-supersite neutralization (titers 1: >1,000,000 against four-glycan-deleted autologous viruses with over 90% breadth against four-glycan-deleted heterologous strains exhibiting tier 2 neutralization character. To a first approximation, the immunogenicity of the glycan-shielded protein surface was negligible, with Env-elicited neutralization (ID50 proportional to the exponential of the protein-surface area accessible to antibody. Based on these high titers and exponential relationship, we propose site-selective deglycosylated trimers as priming immunogens to increase the frequency of site-targeting antibodies.

Quantification of the Impact of the HIV-1-Glycan Shield on Antibody Elicitation

Energy Technology Data Exchange (ETDEWEB)

Zhou, Tongqing; Doria-Rose, Nicole A.; Cheng, Cheng; Stewart-Jones, Guillaume B. E.; Chuang, Gwo-Yu; Chambers, Michael; Druz, Aliaksandr; Geng, Hui; McKee, Krisha; Kwon, Young Do; O’Dell, Sijy; Sastry, Mallika; Schmidt, Stephen D.; Xu, Kai; Chen, Lei; Chen, Rita E.; Louder, Mark K.; Pancera, Marie; Wanninger, Timothy G.; Zhang, Baoshan; Zheng, Anqi; Farney, S. Katie; Foulds, Kathryn E.; Georgiev, Ivelin S.; Joyce, M. Gordon; Lemmin, Thomas; Narpala, Sandeep; Rawi, Reda; Soto, Cinque; Todd, John-Paul; Shen, Chen-Hsiang; Tsybovsky, Yaroslav; Yang, Yongping; Zhao, Peng; Haynes, Barton F.; Stamatatos, Leonidas; Tiemeyer, Michael; Wells, Lance; Scorpio, Diana G.; Shapiro, Lawrence; McDermott, Adrian B.; Mascola, John R.; Kwong, Peter D.

2017-04-01

While the HIV-1-glycan shield is known to shelter Env from the humoral immune response, its quantitative impact on antibody elicitation has been unclear. Here, we use targeted deglycosylation to measure the impact of the glycan shield on elicitation of antibodies against the CD4 supersite. We engineered diverse Env trimers with select glycans removed proximal to the CD4 supersite, characterized their structures and glycosylation, and immunized guinea pigs and rhesus macaques. Immunizations yielded little neutralization against wild-type viruses but potent CD4-supersite neutralization (titers 1: >1,000,000 against four-glycan-deleted autologous viruses with over 90% breadth against four-glycan-deleted heterologous strains exhibiting tier 2 neutralization character). To a first approximation, the immunogenicity of the glycan-shielded protein surface was negligible, with Env-elicited neutralization (ID50) proportional to the exponential of the protein-surface area accessible to antibody. Based on these high titers and exponential relationship, we propose site-selective deglycosylated trimers as priming immunogens to increase the frequency of site-targeting antibodies.

Phase 1 Study of the E-Selectin Inhibitor GMI 1070 in Patients with Sickle Cell Anemia

Science.gov (United States)

Wun, Ted; Styles, Lori; DeCastro, Laura; Telen, Marilyn J.; Kuypers, Frans; Cheung, Anthony; Kramer, William; Flanner, Henry; Rhee, Seungshin; Magnani, John L.; Thackray, Helen

2014-01-01

Background Sickle cell anemia is an inherited disorder of hemoglobin that leads to a variety of acute and chronic complications. Abnormal cellular adhesion, mediated in part by selectins, has been implicated in the pathophysiology of the vaso-occlusion seen in sickle cell anemia, and selectin inhibition was able to restore blood flow in a mouse model of sickle cell disease. Methods We performed a Phase 1 study of the selectin inhibitor GMI 1070 in patients with sickle cell anemia. Fifteen patients who were clinically stable received GMI 1070 in two infusions. Results The drug was well tolerated without significant adverse events. There was a modest increase in total peripheral white blood cell count without clinical symptoms. Plasma concentrations were well-described by a two-compartment model with an elimination T1/2 of 7.7 hours and CLr of 19.6 mL/hour/kg. Computer-assisted intravital microscopy showed transient increases in red blood cell velocity in 3 of the 4 patients studied. Conclusions GMI 1070 was safe in stable patients with sickle cell anemia, and there was suggestion of increased blood flow in a subset of patients. At some time points between 4 and 48 hours after treatment with GMI 1070, there were significant decreases in biomarkers of endothelial activation (sE-selectin, sP-selectin, sICAM), leukocyte activation (MAC-1, LFA-1, PM aggregates) and the coagulation cascade (tissue factor, thrombin-antithrombin complexes). Development of GMI 1070 for the treatment of acute vaso-occlusive crisis is ongoing. Trial Registration ClinicalTrials.gov NCT00911495 PMID:24988449

Phase 1 study of the E-selectin inhibitor GMI 1070 in patients with sickle cell anemia.

Directory of Open Access Journals (Sweden)

Ted Wun

Full Text Available Sickle cell anemia is an inherited disorder of hemoglobin that leads to a variety of acute and chronic complications. Abnormal cellular adhesion, mediated in part by selectins, has been implicated in the pathophysiology of the vaso-occlusion seen in sickle cell anemia, and selectin inhibition was able to restore blood flow in a mouse model of sickle cell disease.We performed a Phase 1 study of the selectin inhibitor GMI 1070 in patients with sickle cell anemia. Fifteen patients who were clinically stable received GMI 1070 in two infusions.The drug was well tolerated without significant adverse events. There was a modest increase in total peripheral white blood cell count without clinical symptoms. Plasma concentrations were well-described by a two-compartment model with an elimination T1/2 of 7.7 hours and CLr of 19.6 mL/hour/kg. Computer-assisted intravital microscopy showed transient increases in red blood cell velocity in 3 of the 4 patients studied.GMI 1070 was safe in stable patients with sickle cell anemia, and there was suggestion of increased blood flow in a subset of patients. At some time points between 4 and 48 hours after treatment with GMI 1070, there were significant decreases in biomarkers of endothelial activation (sE-selectin, sP-selectin, sICAM, leukocyte activation (MAC-1, LFA-1, PM aggregates and the coagulation cascade (tissue factor, thrombin-antithrombin complexes. Development of GMI 1070 for the treatment of acute vaso-occlusive crisis is ongoing.ClinicalTrials.gov NCT00911495.

Phase 1 study of the E-selectin inhibitor GMI 1070 in patients with sickle cell anemia.

Science.gov (United States)

Wun, Ted; Styles, Lori; DeCastro, Laura; Telen, Marilyn J; Kuypers, Frans; Cheung, Anthony; Kramer, William; Flanner, Henry; Rhee, Seungshin; Magnani, John L; Thackray, Helen

2014-01-01

Sickle cell anemia is an inherited disorder of hemoglobin that leads to a variety of acute and chronic complications. Abnormal cellular adhesion, mediated in part by selectins, has been implicated in the pathophysiology of the vaso-occlusion seen in sickle cell anemia, and selectin inhibition was able to restore blood flow in a mouse model of sickle cell disease. We performed a Phase 1 study of the selectin inhibitor GMI 1070 in patients with sickle cell anemia. Fifteen patients who were clinically stable received GMI 1070 in two infusions. The drug was well tolerated without significant adverse events. There was a modest increase in total peripheral white blood cell count without clinical symptoms. Plasma concentrations were well-described by a two-compartment model with an elimination T1/2 of 7.7 hours and CLr of 19.6 mL/hour/kg. Computer-assisted intravital microscopy showed transient increases in red blood cell velocity in 3 of the 4 patients studied. GMI 1070 was safe in stable patients with sickle cell anemia, and there was suggestion of increased blood flow in a subset of patients. At some time points between 4 and 48 hours after treatment with GMI 1070, there were significant decreases in biomarkers of endothelial activation (sE-selectin, sP-selectin, sICAM), leukocyte activation (MAC-1, LFA-1, PM aggregates) and the coagulation cascade (tissue factor, thrombin-antithrombin complexes). Development of GMI 1070 for the treatment of acute vaso-occlusive crisis is ongoing. ClinicalTrials.gov NCT00911495.

Mutations in HNF1A Result in Marked Alterations of Plasma Glycan Profile

Science.gov (United States)

Thanabalasingham, Gaya; Huffman, Jennifer E.; Kattla, Jayesh J.; Novokmet, Mislav; Rudan, Igor; Gloyn, Anna L.; Hayward, Caroline; Adamczyk, Barbara; Reynolds, Rebecca M.; Muzinic, Ana; Hassanali, Neelam; Pucic, Maja; Bennett, Amanda J.; Essafi, Abdelkader; Polasek, Ozren; Mughal, Saima A.; Redzic, Irma; Primorac, Dragan; Zgaga, Lina; Kolcic, Ivana; Hansen, Torben; Gasperikova, Daniela; Tjora, Erling; Strachan, Mark W.J.; Nielsen, Trine; Stanik, Juraj; Klimes, Iwar; Pedersen, Oluf B.; Njølstad, Pål R.; Wild, Sarah H.; Gyllensten, Ulf; Gornik, Olga; Wilson, James F.; Hastie, Nicholas D.; Campbell, Harry; McCarthy, Mark I.; Rudd, Pauline M.; Owen, Katharine R.; Lauc, Gordan; Wright, Alan F.

2013-01-01

A recent genome-wide association study identified hepatocyte nuclear factor 1-α (HNF1A) as a key regulator of fucosylation. We hypothesized that loss-of-function HNF1A mutations causal for maturity-onset diabetes of the young (MODY) would display altered fucosylation of N-linked glycans on plasma proteins and that glycan biomarkers could improve the efficiency of a diagnosis of HNF1A-MODY. In a pilot comparison of 33 subjects with HNF1A-MODY and 41 subjects with type 2 diabetes, 15 of 29 glycan measurements differed between the two groups. The DG9-glycan index, which is the ratio of fucosylated to nonfucosylated triantennary glycans, provided optimum discrimination in the pilot study and was examined further among additional subjects with HNF1A-MODY (n = 188), glucokinase (GCK)-MODY (n = 118), hepatocyte nuclear factor 4-α (HNF4A)-MODY (n = 40), type 1 diabetes (n = 98), type 2 diabetes (n = 167), and nondiabetic controls (n = 98). The DG9-glycan index was markedly lower in HNF1A-MODY than in controls or other diabetes subtypes, offered good discrimination between HNF1A-MODY and both type 1 and type 2 diabetes (C statistic ≥0.90), and enabled us to detect three previously undetected HNF1A mutations in patients with diabetes. In conclusion, glycan profiles are altered substantially in HNF1A-MODY, and the DG9-glycan index has potential clinical value as a diagnostic biomarker of HNF1A dysfunction. PMID:23274891

The influence of propofol on P-selectin expression and nitric oxide production in re-oxygenated human umbilical vein endothelial cells.

LENUS (Irish Health Repository)

Corcoran, T B

2012-02-03

BACKGROUND: Reperfusion injury is characterized by free radical production and endothelial inflammation. Neutrophils mediate much of the end-organ injury that occurs, requiring P-selectin-mediated neutrophil-endothelial adhesion, and this is associated with decreased endothelial nitric oxide production. Propofol has antioxidant properties in vitro which might abrogate this inflammation. METHODS: Cultured human umbilical vein endothelial cells were exposed to 20 h of hypoxia and then returned to normoxic conditions. Cells were treated with saline, Diprivan 5 microg\\/l or propofol 5 microg\\/l for 4 h after re-oxygenation and were then examined for P-selectin expression and supernatant nitric oxide concentrations for 24 h. P-selectin was determined by flow cytometry, and culture supernatant nitric oxide was measured as nitrite. RESULTS: In saline-treated cells, a biphasic increase in P-selectin expression was demonstrated at 30 min (P = 0.01) and 4 h (P = 0.023) after re-oxygenation. Propofol and Diprivan prevented these increases in P-selectin expression (P < 0.05). Four hours after re-oxygenation, propofol decreased endothelial nitric oxide production (P = 0.035). CONCLUSION: This is the first study to demonstrate an effect of propofol upon endothelial P-selectin expression. Such an effect may be important in situations of reperfusion injury such as cardiac transplantation and coronary artery bypass surgery. We conclude that propofol attenuates re-oxygenation-induced endothelial inflammation in vitro.

Global site-specific analysis of glycoprotein N-glycan processing.

Science.gov (United States)

Cao, Liwei; Diedrich, Jolene K; Ma, Yuanhui; Wang, Nianshuang; Pauthner, Matthias; Park, Sung-Kyu Robin; Delahunty, Claire M; McLellan, Jason S; Burton, Dennis R; Yates, John R; Paulson, James C

2018-06-01

N-glycans contribute to the folding, stability and functions of the proteins they decorate. They are produced by transfer of the glycan precursor to the sequon Asn-X-Thr/Ser, followed by enzymatic trimming to a high-mannose-type core and sequential addition of monosaccharides to generate complex-type and hybrid glycans. This process, mediated by the concerted action of multiple enzymes, produces a mixture of related glycoforms at each glycosite, making analysis of glycosylation difficult. To address this analytical challenge, we developed a robust semiquantitative mass spectrometry (MS)-based method that determines the degree of glycan occupancy at each glycosite and the proportion of N-glycans processed from high-mannose type to complex type. It is applicable to virtually any glycoprotein, and a complete analysis can be conducted with 30 μg of protein. Here, we provide a detailed description of the method that includes procedures for (i) proteolytic digestion of glycoprotein(s) with specific and nonspecific proteases; (ii) denaturation of proteases by heating; (iii) sequential treatment of the glycopeptide mixture with two endoglycosidases, Endo H and PNGase F, to create unique mass signatures for the three glycosylation states; (iv) LC-MS/MS analysis; and (v) data analysis for identification and quantitation of peptides for the three glycosylation states. Full coverage of site-specific glycosylation of glycoproteins is achieved, with up to thousands of high-confidence spectra hits for each glycosite. The protocol can be performed by an experienced technician or student/postdoc with basic skills for proteomics experiments and takes ∼7 d to complete.

Glycan structures contain information for the spatial arrangement of glycoproteins in the plasma membrane.

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M Kristen Hall

Full Text Available Glycoconjugates at the cell surface are crucial for cells to communicate with each other and the extracellular microenvironment. While it is generally accepted that glycans are vectorial biopolymers, their information content is unclear. This report provides evidence that distinct N-glycan structures influence the spatial arrangement of two integral membrane glycoproteins, Kv3.1 and E-cadherin, at the adherent membrane which in turn alter cellular properties. Distinct N-glycan structures were generated by heterologous expression of these glycoproteins in parental and glycosylation mutant Chinese hamster ovary cell lines. Unlike the N-linked glycans, the O-linked glycans of the mutant cell lines are similar to those of the parental cell line. Western and lectin blots of total membranes and GFP immunopurified samples, combined with glycosidase digestion reactions, were employed to verify the glycoproteins had predominantly complex, oligomannose, and bisecting type N-glycans from Pro(-5, Lec1, and Lec10B cell lines, respectively. Based on total internal reflection fluorescence and differential interference contrast microscopy techniques, and cellular assays of live parental and glycosylation mutant CHO cells, we propose that glycoproteins with complex, oligomannose or bisecting type N-glycans relay information for localization of glycoproteins to various regions of the plasma membrane in both a glycan-specific and protein-specific manner, and furthermore cell-cell interactions are required for deciphering much of this information. These distinct spatial arrangements also impact cell adhesion and migration. Our findings provide direct evidence that N-glycan structures of glycoproteins contribute significantly to the information content of cells.

Serum Levels of Soluble P-Selectin Are Increased and Associated With Disease Activity in Patients With Behçet's Syndrome

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Yusuf Turkoz

2005-01-01

Full Text Available Behçet's syndrome (BS is a relapsing, chronic, inflammatory disease characterized by endothelial dysfunction, atherothromboembogenesis, and leukocytoclastic vasculitis with complex immunologic molecular interactions. Generalized derangements of the lymphocyte and neutrophil populations, activated monocytes, and increased PMNLs motility with upregulated cell surface molecules such as ICAM-1, VCAM-1, and E-selectin, which are found on the endothelial cells, leukocytes, and platelets, have all been demonstrated during the course of BS. Our aim is to investigate the association of serum concentrations of soluble P-selectin in patients with BS, and to evaluate whether disease activity has an effect on their blood levels. This multicenter study included 31 patients with BS (15 men and 16 women and 20 age- and sex-matched healthy control volunteers (11 men and nine women. Neutrophil count, erythrocyte sedimentation rate, and acute-phase reactants as well as soluble P-selectin levels were determined. The mean age and sex distributions were similar (P>.05 between BS patients (35 years and control volunteers (36 years. Serum levels of soluble P-selectin in patients with BS (399 ± 72 ng/mL were significantly (P<.001 higher when compared with control subjects (164±40   ng/mL. In addition, active BS patients (453±37 ng/mL had significantly (P<.001 elevated levels of soluble P-selectin than those in inactive period (341±52 ng/mL. This study clearly demonstrated that serum soluble P-selectin levels are increased in BS patients when compared with control subjects, suggesting a modulator role for soluble P-selectin during the course of platelet activation and therefore, atherothrombogenesis formation in BS, especially in active disease.

In silico determination of intracellular glycosylation and phosphorylation sites in human selectins: Implications for biological function

DEFF Research Database (Denmark)

Ahmad, I.; Hoessli, D.C.; Gupta, Ramneek

2007-01-01

Post-translational modifications provide the proteins with the possibility to perform functions in addition to those determined by their primary sequence. However, analysis of multifunctional protein structures in the environment of cells and body fluids is made especially difficult by the presence...... both modifications are likely to occur can also be predicted (YinYang sites), to suggest further functional versatility. Structural modifications of hydroxyl groups of P-, E-, and L-selectins have been predicted and possible functions resulting from such modifications are proposed. Functional changes...... of the three selectins are based on the assumption that transitory and reversible protein modifications by phosphate and O-GlcNAc cause specific conformational changes and generate binding sites for other proteins. The computer-assisted prediction of glycosylation and phosphorylation sites in selectins should...

Significant sE-Selectin levels reduction after 6 months of anti-TNF-α therapy in non-diabetic patients with moderate-to-severe psoriasis.

Science.gov (United States)

Genre, Fernanda; Armesto, Susana; Corrales, Alfonso; López-Mejías, Raquel; Remuzgo-Martínez, Sara; Pina, Trinitario; Ubilla, Begoña; Mijares, Verónica; Martín-Varillas, José Luis; Rueda-Gotor, Javier; Portilla, Virginia; Dierssen-Sotos, Trinidad; González-López, Marcos Antonio; González-Vela, María Del Carmen; Blanco, Ricardo; Llorca, Javier; Hernández, José Luis; González-Gay, Miguel Ángel

2017-12-01

Psoriasis patients have high risk of atherosclerosis, characterized by endothelial dysfunction. We aimed to study the association of the endothelial activation biomarkers monocyte chemoattractant protein 1 (MCP-1), soluble (s) E-selectin and P-selectin with disease activity and severity in psoriasis patients treated with anti-TNF-α therapy. Also, to evaluate the relationship of metabolic syndrome features with these biomarkers and the effect of anti-TNF-α therapy on these molecules. Twenty-nine consecutive non-diabetic patients with moderate-to-severe psoriasis who underwent 6 months of anti-TNF-α-adalimumab therapy were studied. Metabolic and clinical evaluation was performed prior to anti-TNF-α treatment (time 0) and 6 months later. MCP-1, sE-selectin and sP-selectin serum levels were determined by ELISA. Dyslipidemic and obese patients showed higher MCP-1 levels at month 6 from the onset of anti-TNF-α therapy (p = .05 and .01, respectively). sE-selectin positively correlated with pro-inflammatory molecules such as asymmetric dimethylarginine, sP-selectin and resistin at baseline and month 6 (p psoriasis. Adalimumab therapy led to a reduction in sE-selectin levels, supporting the beneficial effect of anti-TNF-α therapy on mechanisms associated with the development of atherosclerosis in psoriasis.

Automated Glycan Assembly of Complex Oligosaccharides Related to Blood Group Determinants.

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Hahm, Heung Sik; Liang, Chien-Fu; Lai, Chian-Hui; Fair, Richard J; Schuhmacher, Frank; Seeberger, Peter H

2016-07-15

Lactotetraosyl (Lc4) and neo-lactotetraosyl (nLc4) are backbones that are common to many glycans. Using automated glycan assembly, these common core structures were constructed and elaborated to access synthetically challenging glycans of biological relevance. The incorporation of α-fucoses is demonstrated for H-type I and II; α(1,3)-galactose epitopes were prepared, and the pentasaccharide HNK-1 required incorporation of a 3-O-sulfate. In addition to preparing the target structures, essential insights were gained regarding the relationships of glycosylating agents and nucleophiles as well as the linker stability.

Biological significance of complex N-glycans in plants and their impact on plant physiology.

Science.gov (United States)

Strasser, Richard

2014-01-01

Asparagine (N)-linked protein glycosylation is a ubiquitous co- and post-translational modification which can alter the biological function of proteins and consequently affects the development, growth, and physiology of organisms. Despite an increasing knowledge of N-glycan biosynthesis and processing, we still understand very little about the biological function of individual N-glycan structures in plants. In particular, the N-glycan-processing steps mediated by Golgi-resident enzymes create a structurally diverse set of protein-linked carbohydrate structures. Some of these complex N-glycan modifications like the presence of β1,2-xylose, core α1,3-fucose or the Lewis a-epitope are characteristic for plants and are evolutionary highly conserved. In mammals, complex N-glycans are involved in different cellular processes including molecular recognition and signaling events. In contrast, the complex N-glycan function is still largely unknown in plants. Here, in this short review, I focus on important recent developments and discuss their implications for future research in plant glycobiology and plant biotechnology.

N-glycans released from glycoproteins using a commercial kit and comprehensively analyzed with a hypothetical database

Directory of Open Access Journals (Sweden)

Xue Sun

2017-04-01

Full Text Available The glycosylation of proteins is responsible for their structural and functional roles in many cellular activities. This work describes a strategy that combines an efficient release, labeling and liquid chromatography-mass spectral analysis with the use of a comprehensive database to analyze N-glycans. The analytical method described relies on a recently commercialized kit in which quick deglycosylation is followed by rapid labeling and cleanup of labeled glycans. This greatly improves the separation, mass spectrometry (MS analysis and fluorescence detection of N-glycans. A hypothetical database, constructed using GlycResoft, provides all compositional possibilities of N-glycans based on the common sugar residues found in N-glycans. In the initial version this database contains >8,700 N-glycans, and is compatible with MS instrument software and expandable. N-glycans from four different well-studied glycoproteins were analyzed by this strategy. The results provided much more accurate and comprehensive data than had been previously reported. This strategy was then used to analyze the N-glycans present on the membrane glycoproteins of gastric carcinoma cells with different degrees of differentiation. Accurate and comprehensive N-glycan data from those cells was obtained efficiently and their differences compared corresponding to their differentiation states. Thus, the novel strategy developed greatly improves accuracy, efficiency and comprehensiveness of N-glycan analysis.

Variable Domain N-Linked Glycans Acquired During Antigen-Specific Immune Responses Can Contribute to Immunoglobulin G Antibody Stability

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Fleur S. van de Bovenkamp

2018-04-01

Full Text Available Immunoglobulin G (IgG can contain N-linked glycans in the variable domains, the so-called Fab glycans, in addition to the Fc glycans in the CH2 domains. These Fab glycans are acquired following introduction of N-glycosylation sites during somatic hypermutation and contribute to antibody diversification. We investigated whether Fab glycans may—in addition to affecting antigen binding—contribute to antibody stability. By analyzing thermal unfolding profiles of antibodies with or without Fab glycans, we demonstrate that introduction of Fab glycans can improve antibody stability. Strikingly, removal of Fab glycans naturally acquired during antigen-specific immune responses can deteriorate antibody stability, suggesting in vivo selection of stable, glycosylated antibodies. Collectively, our data show that variable domain N-linked glycans acquired during somatic hypermutation can contribute to IgG antibody stability. These findings indicate that introducing Fab glycans may represent a mechanism to improve therapeutic/diagnostic antibody stability.

E-selectin gene polymorphisms and essential hypertension in Asian population: an updated meta-analysis.

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Gaojun Cai

Full Text Available Epidemiological studies have shown that E-selectin gene polymorphisms (A561C and C1839T may be associated with essential hypertension (EH, but the results are conflicting in different ethnic populations. Thus, we performed this meta-analysis to investigate a more authentic association between E-selectin gene polymorphisms and the risk of EH.We searched the relevant studies for the present meta-analysis from the following electronic databases: PubMed, Embase, Cochrane Library, Google Scholar, Web of Science, Wanfang Data, and China National Knowledge Infrastructure (CNKI. Odds ratios (OR with 95% confidence interval (CI were used to evaluate the strength of the association between E-selectin gene polymorphisms and EH susceptibility. The pooled ORs were performed for dominant model, allelic model and recessive model. The publication bias was examined by Begg's funnel plots and Egger's test.A total of eleven studies met the inclusion criteria. All studies came from Asians. Ten studies (12 cohorts evaluated the A561C polymorphism and EH risk, including 2,813 cases and 2,817 controls. The pooled OR was 2.280 (95%CI: 1.893-2.748, P<0.001 in dominant model, 5.284 (95%CI: 2.679-10.420, P<0.001 in recessive model and 2.359 (95%CI: 1.981-2.808, P = 0.001 in allelic model. Four studies (six cohorts evaluated C1839T polymorphism and EH risk, including 1,700 cases and 1,681 controls. The pooled OR was 0.785 (95%CI: 0.627-0.983, P = 0.035 in dominant model, 1.250 (95%CI: 0.336-4.652, P = 0.739 in recessive model and 0.805 (95%CI: 0.649-0.999, P = 0.049 in allelic model.The current meta-analysis concludes that the C allele of E-selectin A561C gene polymorphism might increase the EH risk in Asian population, whereas the T allele of E-selectin C1839T gene polymorphism might decrease the EH risk.

The Impact of O-Glycan Chemistry on the Stability of Intrinsically Disordered Proteins

Energy Technology Data Exchange (ETDEWEB)

Beckham, Gregg T [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Prates, Erica T [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Crowley, Michael F [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Guan, Xiaoyang [University of Colorado; Li, Yaohao [University of Colorado; Wang, Xinfeng [University of Colorado; Chaffey, Patrick K. [University of Colorado; Skaf, Munir S. [University of Campinas; Tan, Zhongping [University of Colorado

2018-03-02

Protein glycosylation is a diverse post-translational modification that serves myriad biological functions. O-linked glycans in particular vary widely in extent and chemistry in eukaryotes, with secreted proteins from fungi and yeast commonly exhibiting O-mannosylation in intrinsically disordered regions of proteins, likely for proteolysis protection, among other functions. However, it is not well understood why mannose is often the preferred glycan, and more generally, if the neighboring protein sequence and glycan have coevolved to protect against proteolysis in glycosylated intrinsically disordered proteins (IDPs). Here, we synthesized variants of a model IDP, specifically a natively O-mannosylated linker from a fungal enzyme, with a-O-linked mannose, glucose, and galactose moieties, along with a non-glycosylated linker. Upon exposure to thermolysin, O-mannosylation, by far, provides the highest extent of proteolysis protection. To explain this observation, extensive molecular dynamics simulations were conducted, revealing that the axial configuration of the C2-hydroxyl group (2-OH) of a-mannose adjacent to the glycan-peptide bond strongly influences the conformational features of the linker. Specifically, a-mannose restricts the torsions of the IDP main chain more than other glycans whose equatorial 2-OH groups exhibit interactions that favor perpendicular glycan-protein backbone orientation. We suggest that IDP stiffening due to O-mannosylation impairs protease action, with contributions from protein-glycan interactions, protein flexibility, and protein stability. Our results further imply that resistance to proteolysis is an important driving force for evolutionary selection of a-mannose in eukaryotic IDPs, and more broadly, that glycan motifs for proteolysis protection likely coevolve with the protein sequence to which they attach.

Glycan Reader is improved to recognize most sugar types and chemical modifications in the Protein Data Bank.

Science.gov (United States)

Park, Sang-Jun; Lee, Jumin; Patel, Dhilon S; Ma, Hongjing; Lee, Hui Sun; Jo, Sunhwan; Im, Wonpil

2017-10-01

Glycans play a central role in many essential biological processes. Glycan Reader was originally developed to simplify the reading of Protein Data Bank (PDB) files containing glycans through the automatic detection and annotation of sugars and glycosidic linkages between sugar units and to proteins, all based on atomic coordinates and connectivity information. Carbohydrates can have various chemical modifications at different positions, making their chemical space much diverse. Unfortunately, current PDB files do not provide exact annotations for most carbohydrate derivatives and more than 50% of PDB glycan chains have at least one carbohydrate derivative that could not be correctly recognized by the original Glycan Reader. Glycan Reader has been improved and now identifies most sugar types and chemical modifications (including various glycolipids) in the PDB, and both PDB and PDBx/mmCIF formats are supported. CHARMM-GUI Glycan Reader is updated to generate the simulation system and input of various glycoconjugates with most sugar types and chemical modifications. It also offers a new functionality to edit the glycan structures through addition/deletion/modification of glycosylation types, sugar types, chemical modifications, glycosidic linkages, and anomeric states. The simulation system and input files can be used for CHARMM, NAMD, GROMACS, AMBER, GENESIS, LAMMPS, Desmond, OpenMM, and CHARMM/OpenMM. Glycan Fragment Database in GlycanStructure.Org is also updated to provide an intuitive glycan sequence search tool for complex glycan structures with various chemical modifications in the PDB. http://www.charmm-gui.org/input/glycan and http://www.glycanstructure.org. wonpil@lehigh.edu. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Physiological significance of Fuc and Sialic acid containing glycans in the body

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Muhammad Ramzan Manwar Hussain

2016-09-01

Full Text Available Complex biomolecular machinery carrying diverse glycan chains are involved in a wide range of physiological activities including blood group determination, cancer recognition protein stabilization and sperm–egg interaction. Diversity of glycan chains, linked to lipids and proteins is due to isomeric and conformational modifications of various sugar residues, giving rise to unique carbohydrate structures with a wide range of anomeric linkages. This unique and significant structural diversity of naturally occurring oligosaccharide structures make them the best recognition markers for countless physiological activities. This is a challenging task to explore the relationship between biological processes and stereochemical behavior of sugar residues. Current review article is related with the physiological significance of glycans carrying fucose and/or sialic residues in complex biomolecular assemblies. Both the sugar units have a diverse range of anomery and linkages with the penultimate sugars. The existing literature and databases did not contain comprehensive information regarding structure–function relationship of glycans. Therefore, the current study is scheduled to debate on the structure–function relationship of glycans carrying Fuc and sialic acid in their backbone structures.

Data for analysis of mannose-6-phosphate glycans labeled with fluorescent tags

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Ji-Yeon Kang

2016-06-01

Full Text Available Mannose-6-phosphate (M-6-P glycan plays an important role in lysosomal targeting of most therapeutic enzymes for treatment of lysosomal storage diseases. This article provides data for the analysis of M-6-P glycans by high-performance liquid chromatography (HPLC and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF mass spectrometry. The identities of M-6-P glycan peaks in HPLC profile were confirmed by measuring the masses of the collected peak eluates. The performances of three fluorescent tags (2-aminobenzoic acid [2-AA], 2-aminobenzamide [2-AB], and 3-(acetyl-amino-6-aminoacridine [AA-Ac] were compared focusing on the analysis of bi-phosphorylated glycan (containing two M-6-Ps. The bi-phosphorylated glycan analysis is highly affected by the attached fluorescent tag and the hydrophilicity of elution solvent used in HPLC. The data in this article is associated with the research article published in “Comparison of fluorescent tags for analysis of mannose-6-phosphate glycans” (Kang et al., 2016 [1].

Glycan structure of Gc Protein-derived Macrophage Activating Factor as revealed by mass spectrometry.

Science.gov (United States)

Borges, Chad R; Rehder, Douglas S

2016-09-15

Disagreement exists regarding the O-glycan structure attached to human vitamin D binding protein (DBP). Previously reported evidence indicated that the O-glycan of the Gc1S allele product is the linear core 1 NeuNAc-Gal-GalNAc-Thr trisaccharide. Here, glycan structural evidence is provided from glycan linkage analysis and over 30 serial glycosidase-digestion experiments which were followed by analysis of the intact protein by electrospray ionization mass spectrometry (ESI-MS). Results demonstrate that the O-glycan from the Gc1F protein is the same linear trisaccharide found on the Gc1S protein and that the hexose residue is galactose. In addition, the putative anti-cancer derivative of DBP known as Gc Protein-derived Macrophage Activating Factor (GcMAF, which is formed by the combined action of β-galactosidase and neuraminidase upon DBP) was analyzed intact by ESI-MS, revealing that the activating E. coli β-galactosidase cleaves nothing from the protein-leaving the glycan structure of active GcMAF as a Gal-GalNAc-Thr disaccharide, regardless of the order in which β-galactosidase and neuraminidase are applied. Moreover, glycosidase digestion results show that α-N-Acetylgalactosamindase (nagalase) lacks endoglycosidic function and only cleaves the DBP O-glycan once it has been trimmed down to a GalNAc-Thr monosaccharide-precluding the possibility of this enzyme removing the O-glycan trisaccharide from cancer-patient DBP in vivo. Copyright © 2016 Elsevier Inc. All rights reserved.

Mutations in HNF1A result in marked alterations of plasma glycan profile

DEFF Research Database (Denmark)

Thanabalasingham, Gaya; Huffman, Jennifer E; Kattla, Jayesh J

2013-01-01

A recent genome-wide association study identified hepatocyte nuclear factor 1-α (HNF1A) as a key regulator of fucosylation. We hypothesized that loss-of-function HNF1A mutations causal for maturity-onset diabetes of the young (MODY) would display altered fucosylation of N-linked glycans on plasma...... proteins and that glycan biomarkers could improve the efficiency of a diagnosis of HNF1A-MODY. In a pilot comparison of 33 subjects with HNF1A-MODY and 41 subjects with type 2 diabetes, 15 of 29 glycan measurements differed between the two groups. The DG9-glycan index, which is the ratio of fucosylated...... to nonfucosylated triantennary glycans, provided optimum discrimination in the pilot study and was examined further among additional subjects with HNF1A-MODY (n = 188), glucokinase (GCK)-MODY (n = 118), hepatocyte nuclear factor 4-α (HNF4A)-MODY (n = 40), type 1 diabetes (n = 98), type 2 diabetes (n = 167...

Simple and Robust N-Glycan Analysis Based on Improved 2-Aminobenzoic Acid Labeling for Recombinant Therapeutic Glycoproteins.

Science.gov (United States)

Jeong, Yeong Ran; Kim, Sun Young; Park, Young Sam; Lee, Gyun Min

2018-03-21

N-glycans of therapeutic glycoproteins are critical quality attributes that should be monitored throughout all stages of biopharmaceutical development. To reduce both the time for sample preparation and the variations in analytical results, we have developed an N-glycan analysis method that includes improved 2-aminobenzoic acid (2-AA) labeling to easily remove deglycosylated proteins. Using this analytical method, 15 major 2-AA-labeled N-glycans of Enbrel ® were separated into single peaks in hydrophilic interaction chromatography mode and therefore could be quantitated. 2-AA-labeled N-glycans were also highly compatible with in-line quadrupole time-of-flight mass spectrometry (MS) for structural identification. The structures of 15 major and 18 minor N-glycans were identified from their mass values determined by quadrupole time-of-flight MS. Furthermore, the structures of 14 major N-glycans were confirmed by interpreting the MS/MS data of each N-glycan. This analytical method was also successfully applied to neutral N-glycans of Humira ® and highly sialylated N-glycans of NESP ® . Furthermore, the analysis data of Enbrel ® that were accumulated for 2.5 years demonstrated the high-level consistency of this analytical method. Taken together, the results show that a wide repertoire of N-glycans of therapeutic glycoproteins can be analyzed with high efficiency and consistency using the improved 2-AA labeling-based N-glycan analysis method. Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Endothelial galectin-1 binds to specific glycans on nipah virus fusion protein and inhibits maturation, mobility, and function to block syncytia formation.

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Omai B Garner

2010-07-01

Full Text Available Nipah virus targets human endothelial cells via NiV-F and NiV-G envelope glycoproteins, resulting in endothelial syncytia formation and vascular compromise. Endothelial cells respond to viral infection by releasing innate immune effectors, including galectins, which are secreted proteins that bind to specific glycan ligands on cell surface glycoproteins. We demonstrate that galectin-1 reduces NiV-F mediated fusion of endothelial cells, and that endogenous galectin-1 in endothelial cells is sufficient to inhibit syncytia formation. Galectin-1 regulates NiV-F mediated cell fusion at three distinct points, including retarding maturation of nascent NiV-F, reducing NiV-F lateral mobility on the plasma membrane, and directly inhibiting the conformational change in NiV-F required for triggering fusion. Characterization of the NiV-F N-glycome showed that the critical site for galectin-1 inhibition is rich in glycan structures known to bind galectin-1. These studies identify a unique set of mechanisms for regulating pathophysiology of NiV infection at the level of the target cell.

N-linked glycans are required on epithelial Na+ channel subunits for maturation and surface expression.

Science.gov (United States)

Kashlan, Ossama B; Kinlough, Carol L; Myerburg, Michael M; Shi, Shujie; Chen, Jingxin; Blobner, Brandon M; Buck, Teresa M; Brodsky, Jeffrey L; Hughey, Rebecca P; Kleyman, Thomas R

2018-03-01

Epithelial Na + channel (ENaC) subunits undergo N-linked glycosylation in the endoplasmic reticulum where they assemble into an αβγ complex. Six, 13, and 5 consensus sites (Asn-X-Ser/Thr) for N-glycosylation reside in the extracellular domains of the mouse α-, β-, and γ-subunits, respectively. Because the importance of ENaC N-linked glycans has not been fully addressed, we examined the effect of preventing N-glycosylation of specific subunits on channel function, expression, maturation, and folding. Heterologous expression in Xenopus oocytes or Fischer rat thyroid cells with αβγ-ENaC lacking N-linked glycans on a single subunit reduced ENaC activity as well as the inhibitory response to extracellular Na + . The lack of N-linked glycans on the β-subunit also precluded channel activation by trypsin. However, channel activation by shear stress was N-linked glycan independent, regardless of which subunit was modified. We also discovered that the lack of N-linked glycans on any one subunit reduced the total and surface levels of cognate subunits. The lack of N-linked glycans on the β-subunit had the largest effect on total levels, with the lack of N-linked glycans on the γ- and α-subunits having intermediate and modest effects, respectively. Finally, channels with wild-type β-subunits were more sensitive to limited trypsin proteolysis than channels lacking N-linked glycans on the β-subunit. Our results indicate that N-linked glycans on each subunit are required for proper folding, maturation, surface expression, and function of the channel.

Top-Down Chemoenzymatic Approach to Synthesizing Diverse High-Mannose N-Glycans and Related Neoglycoproteins for Carbohydrate Microarray Analysis.

Science.gov (United States)

Toonstra, Christian; Wu, Lisa; Li, Chao; Wang, Denong; Wang, Lai-Xi

2018-05-22

High-mannose-type N-glycans are an important component of neutralizing epitopes on HIV-1 envelope glycoprotein gp120. They also serve as signals for protein folding, trafficking, and degradation in protein quality control. A number of lectins and antibodies recognize high-mannose-type N-glycans, and glycan array technology has provided an avenue to probe these oligomannose-specific proteins. We describe in this paper a top-down chemoenzymatic approach to synthesize a library of high-mannose N-glycans and related neoglycoproteins for glycan microarray analysis. The method involves the sequential enzymatic trimming of two readily available natural N-glycans, the Man 9 GlcNAc 2 Asn prepared from soybean flour and the sialoglycopeptide (SGP) isolated from chicken egg yolks, coupled with chromatographic separation to obtain a collection of a full range of natural high-mannose N-glycans. The Asn-linked N-glycans were conjugated to bovine serum albumin (BSA) to provide neoglycoproteins containing the oligomannose moieties. The glycoepitopes displayed were characterized using an array of glycan-binding proteins, including the broadly virus-neutralizing agents, glycan-specific antibody 2G12, Galanthus nivalis lectin (GNA), and Narcissus pseudonarcissus lectin (NPA).

Using Proteomics to 1) Identify the Bone Marrow Homing Receptors Expressed on Human Hematopoietic Stem Cells and 2) Elucidate Critical Signaling Pathways Responsible for the Blockage of Hematopoietic Differentiation in Leukemia

KAUST Repository

Chin, Chee J.

2011-05-22

Successful hematopoiesis requires the trafficking of hematopoietic stem/progenitor cells (HSPCs) to their bone marrow (BM) niche, where they can differentiate to produce all blood lineages. Leukemia arises when there is a blockage of differentiation and uncontrolled proliferation in the hematopoietic cells during their development. To refine therapies for leukemia, this study sought to improve the homing of healthy donor HSPCs for better transplantation and to find new candidates for differentiating and blocking proliferation in leukemic cells. Characterizing the molecular effectors mediating cell migration forms the basis for improving clinical transplantation of HSPCs. E-selectin/ligand interactions play a critical role in the homing of HSPCs to the BM, however, the identity of E-selectin ligands remains elusive. We aimed to use mass spectrometry (MS) to fully analyze the E-selectin ligands expressed on HSPCs. Immunoprecipitation studies coupled with MS confirmed the expression of three known E-selectin ligands, the hematopoietic cell E-/L-selectin ligand (HCELL), P-selectin glycoprotein ligand-1 (PSGL-1) and CD43, and revealed the presence of many interesting candidates on HSPCs-like cell line and on primary human BM CD34+ cells. The MS dataset represents a rich resource for further characterization of E-selectin ligands, which will lead to improvement of HSPCs transplantation. 4 Understanding the critical pathways underlying the initiation and maintenance of leukemia plays a key role in treating acute myeloid leukemia (AML). Ligation of the glycoprotein, CD44, using monoclonal antibodies or its natural ligand, hyaluronic acid, drives the differentiation of immature leukemic cells towards mature terminally differentiated cells, inhibits their proliferation and in some case induces their apoptosis. The aim of this study is to characterize the phosphoproteome of AML cells in response to CD44-induced differentiation. This will afford novel insights into the

Glycan Markers as Potential Immunological Targets in Circulating Tumor Cells.

Science.gov (United States)

Wang, Denong; Wu, Lisa; Liu, Xiaohe

2017-01-01

We present here an experimental approach for exploring a new class of tumor biomarkers that are overexpressed by circulating tumor cells (CTCs) and are likely targetable in immunotherapy against tumor metastasis. Using carbohydrate microarrays, anti-tumor monoclonal antibodies (mAbs) were scanned against a large panel of carbohydrate antigens to identify potential tumor glycan markers. Subsequently, flow cytometry and fiber-optic array scanning technology (FAST) were applied to determine whether the identified targets are tumor-specific cell-surface markers and are, therefore, likely suitable for targeted immunotherapy. Finally, the tumor glycan-specific antibodies identified were validated using cancer patients' blood samples for their performance in CTC-detection and immunotyping analysis. In this article, identifying breast CTC-specific glycan markers and targeting mAbs serve as examples to illustrate this tumor biomarker discovery strategy.

Glycan bioengineering in immunogen design for tumor T antigen immunotargeting

DEFF Research Database (Denmark)

Sendra, Victor G; Zlocowski, Natacha; Ditamo, Yanina

2009-01-01

MM2 energy function showed that pentalysine (Lys5) linker and benzyl (Bzl) residue enhance TFD rigidity of the glycosidic bond. Antibodies raised against BzlalphaTFD-Lys5 immunogen recognize tumor T antigen. Competitive assays confirm that TFD-related structures are the main glycan epitope...... to the bioengineered glycoconjugate inhibited CT26 tumor cell proliferation and reduced tumor growth in an in vivo mouse model. These results show that TFD bioengineering is a useful immunogenic strategy with potential application in cancer therapy. The same approach can be extended to other glycan immunogens......Bioengineering of Galbeta3GalNAcalpha, known as Thomsen-Friedenreich disaccharide (TFD), is studied to promote glycan immunogenicity and immunotargeting to tumor T antigen (Galbeta3GalNAcalpha-O-Ser/Thr). Theoretical studies on disaccharide conformations by energy minimization of structures using...

Isomer Information from Ion Mobility Separation of High-Mannose Glycan Fragments.

Science.gov (United States)

Harvey, David J; Seabright, Gemma E; Vasiljevic, Snezana; Crispin, Max; Struwe, Weston B

2018-05-01

Extracted arrival time distributions of negative ion CID-derived fragments produced prior to traveling-wave ion mobility separation were evaluated for their ability to provide structural information on N-linked glycans. Fragmentation of high-mannose glycans released from several glycoproteins, including those from viral sources, provided over 50 fragments, many of which gave unique collisional cross-sections and provided additional information used to assign structural isomers. For example, cross-ring fragments arising from cleavage of the reducing terminal GlcNAc residue on Man 8 GlcNAc 2 isomers have unique collision cross-sections enabling isomers to be differentiated in mixtures. Specific fragment collision cross-sections enabled identification of glycans, the antennae of which terminated in the antigenic α-galactose residue, and ions defining the composition of the 6-antenna of several of the glycans were also found to have different cross-sections from isomeric ions produced in the same spectra. Potential mechanisms for the formation of the various ions are discussed and the estimated collisional cross-sections are tabulated. Graphical Abstract ᅟ.

Processing of complex N-glycans in IgG Fc-region is affected by core fucosylation

Science.gov (United States)

Castilho, Alexandra; Gruber, Clemens; Thader, Andreas; Oostenbrink, Chris; Pechlaner, Maria; Steinkellner, Herta; Altmann, Friedrich

2015-01-01

We investigated N-glycan processing of immunoglobulin G1 using the monoclonal antibody cetuximab (CxMab), which has a glycosite in the Fab domain in addition to the conserved Fc glycosylation, as a reporter. Three GlcNAc (Gn) terminating bi-antennary glycoforms of CxMab differing in core fucosylation (α1,3- and α1,6-linkage) were generated in a plant-based expression platform. These GnGn, GnGnF3, and GnGnF6 CxMab variants were subjected in vivo to further processing toward sialylation and GlcNAc diversification (bisected and branching structures). Mass spectrometry-based glycan analyses revealed efficient processing of Fab glycans toward envisaged structures. By contrast, Fc glycan processing largely depend on the presence of core fucose. A particularly strong support of glycan processing in the presence of plant-specific core α1,3-fucose was observed. Consistently, molecular modeling suggests changes in the interactions of the Fc carbohydrate chain depending on the presence of core fucose, possibly changing the accessibility. Here, we provide data that reveal molecular mechanisms of glycan processing of IgG antibodies, which may have implications for the generation of glycan-engineered therapeutic antibodies with improved efficacies. PMID:26067753

Identification of glycan structure alterations on cell membrane proteins in desoxyepothilone B resistant leukemia cells.

Science.gov (United States)

Nakano, Miyako; Saldanha, Rohit; Göbel, Anja; Kavallaris, Maria; Packer, Nicolle H

2011-11-01

Resistance to tubulin-binding agents used in cancer is often multifactorial and can include changes in drug accumulation and modified expression of tubulin isotypes. Glycans on cell membrane proteins play important roles in many cellular processes such as recognition and apoptosis, and this study investigated whether changes to the glycan structures on cell membrane proteins occur when cells become resistant to drugs. Specifically, we investigated the alteration of glycan structures on the cell membrane proteins of human T-cell acute lymphoblastic leukemia (CEM) cells that were selected for resistance to desoxyepothilone B (CEM/dEpoB). The glycan profile of the cell membrane glycoproteins was obtained by sequential release of N- and O-glycans from cell membrane fraction dotted onto polyvinylidene difluoride membrane with PNGase F and β-elimination respectively. The released glycan alditols were analyzed by liquid chromatography (graphitized carbon)-electrospray ionization tandem MS. The major N-glycan on CEM cell was the core fucosylated α2-6 monosialo-biantennary structure. Resistant CEM/dEpoB cells had a significant decrease of α2-6 linked sialic acid on N-glycans. The lower α2-6 sialylation was caused by a decrease in activity of β-galactoside α2-6 sialyltransferase (ST6Gal), and decreased expression of the mRNA. It is clear that the membrane glycosylation of leukemia cells changes during acquired resistance to dEpoB drugs and that this change occurs globally on all cell membrane glycoproteins. This is the first identification of a specific glycan modification on the surface of drug resistant cells and the mechanism of this downstream effect on microtubule targeting drugs may offer a route to new interventions to overcome drug resistance.

A small-molecule switch for Golgi sulfotransferases.

Science.gov (United States)

de Graffenried, Christopher L; Laughlin, Scott T; Kohler, Jennifer J; Bertozzi, Carolyn R

2004-11-30

The study of glycan function is a major frontier in biology that could benefit from small molecules capable of perturbing carbohydrate structures on cells. The widespread role of sulfotransferases in modulating glycan function makes them prime targets for small-molecule modulators. Here, we report a system for conditional activation of Golgi-resident sulfotransferases using a chemical inducer of dimerization. Our approach capitalizes on two features shared by these enzymes: their requirement of Golgi localization for activity on cellular substrates and the modularity of their catalytic and localization domains. Fusion of these domains to the proteins FRB and FKBP enabled their induced assembly by the natural product rapamycin. We applied this strategy to the GlcNAc-6-sulfotransferases GlcNAc6ST-1 and GlcNAc6ST-2, which collaborate in the sulfation of L-selectin ligands. Both the activity and specificity of the inducible enzymes were indistinguishable from their WT counterparts. We further generated rapamycin-inducible chimeric enzymes comprising the localization domain of a sulfotransferase and the catalytic domain of a glycosyltransferase, demonstrating the generality of the system among other Golgi enzymes. The approach provides a means for studying sulfate-dependent processes in cellular systems and, potentially, in vivo.

Glycoengineering of Chinese hamster ovary cells for enhanced erythropoietin N-glycan branching and sialylation

DEFF Research Database (Denmark)

Yin, Bojiao; Gao, Yuan; Chung, Cheng-yu

2015-01-01

Sialic acid, a terminal residue on complex N-glycans, and branching or antennarity can play key roles in both the biological activity and circulatory lifetime of recombinant glycoproteins of therapeutic interest. In order to examine the impact of glycosyltransferase expression on the N-glycosylat......Sialic acid, a terminal residue on complex N-glycans, and branching or antennarity can play key roles in both the biological activity and circulatory lifetime of recombinant glycoproteins of therapeutic interest. In order to examine the impact of glycosyltransferase expression on the N...... increased by 26%. The increase in sialic acid content was further verified by detailed profiling of the N-glycan structures using mass spectra (MS) analysis. In order to enhance antennarity/branching, UDP-N-acetylglucosamine: α-1,3-D-mannoside β1,4-N-acetylglucosaminyltransferase (GnTIV/Mgat4) and UDP...... a mean for enhancing both N-glycan branching complexity and sialylation with opportunities to generate tailored complex N-glycan structures on therapeutic glycoproteins in the future....

Modification of the Campylobacter jejuni N-linked glycan by EptC protein-mediated addition of phosphoethanolamine

DEFF Research Database (Denmark)

Scott, Nichollas E; Nothaft, Harald; Edwards, Alistair V G

2012-01-01

. Interrogation of these data allowed the identification of a phosphoethanolamine (pEtN)-modified variant of the N-glycan that was attached to multiple proteins. The pEtN moiety was attached to the terminal GalNAc of the canonical N-glycan. Deletion of the pEtN transferase eptC removed all evidence of the p......, yet above background levels of pEtN-glycan were also observed in E. coli not expressing eptC, suggesting that endogenous E. coli pEtN transferases can mediate the addition of pEtN to N-glycans. The addition of pEtN must be considered in the context of glycoengineering and may alter C. jejuni glycan...

Organic Electrochemical Transistors for the Detection of Cell Surface Glycans.

Science.gov (United States)

Chen, Lizhen; Fu, Ying; Wang, Naixiang; Yang, Anneng; Li, Yuanzhe; Wu, Jie; Ju, Huangxian; Yan, Feng

2018-05-23

Cell surface glycans play critical roles in diverse biological processes, such as cell-cell communication, immunity, infection, development, and differentiation. Their expressions are closely related to cancer growth and metastasis. This work demonstrates an organic electrochemical transistor (OECT)-based biosensor for the detection of glycan expression on living cancer cells. Herein, mannose on human breast cancer cells (MCF-7) as the target glycan model, poly dimethyl diallyl ammonium chloride-multiwall carbon nanotubes (PDDA-MWCNTs) as the loading interface, concanavalin A (Con A) with active mannose binding sites, aptamer and horseradish peroxidase co-immobilized gold nanoparticles (HRP-aptamer-Au NPs) as specific nanoprobes are used to fabricate the OECT biosensor. In this strategy, PDDA-MWCNT interfaces can enhance the loading of Con A, and the target cells can be captured through Con A via active mannose binding sites. Thus, the expression of cell surface can be reflected by the amount of cells captured on the gate. Specific nanoprobes are introduced to the captured cells to produce an OECT signal because of the reduction of hydrogen peroxide catalyzed by HRP conjugated on Au nanoparticles, while the aptamer on nanoprobes can selectively recognize the MCF-7 cells. It is reasonable that more target cells are captured on the gate electrode, more HRP-nanoprobes are loaded thus a larger signal response. The device shows an obvious response to MCF-7 cells down to 10 cells/μL and can be used to selectively monitor the change of mannose expression on cell surfaces upon a treatment with the N-glycan inhibitor. The OECT-based biosensor is promising for the analysis of glycan expressions on the surfaces of different types of cells.

Microvascular abnormalities in capillaroscopy correlate with higher serum IL-18 and sE-selectin levels in patients with type 1 diabetes complicated by microangiopathy

Directory of Open Access Journals (Sweden)

Maria Górska

2011-04-01

Full Text Available Microvascular abnormalities are one of the most important causes of persistent diabetic complications. The aim of this study was to compare microvascular changes examined by nailfold capillaroscopy with serum concentrations of soluble E-selectin (sE-selectin and IL-18 in type 1 diabetic patients with and without microangiopathy. Serum levels of sE-selectin and IL-18 were determined by an enzyme-linked immunosorbent assay in 106 patients with type 1 diabetes and in 40 healthy controls. All diabetic patients were evaluated by extensive clinical, laboratory and capillaroscopic studies. Morphological changes were observed by nailfold capillaroscopy in 86 out of 106 (81% diabetic patients. Severe capillaroscopic changes were seen in 32 out of 54 (59% patients with microangiopathy, but in only seven out of 52 (13% patients without microangiopathy. Higher serum levels of sE-selectin (p < 0.001 and IL-18 (p < 0.05 were demonstrated in diabetic patients compared to controls. Significant differences of sE-selectin (p < 0.001 and IL-18 (p < 0.01 serum concentrations were observed between diabetic patients with microangiopathy and controls. Moreover, comparison between patients with and without microangiopathic complications showed a significantly higher capillaroscopic score and sE-selectin serum concentration in the group with microangiopathy (p < 0.001. Furthermore, diabetic patients with severe microvascular changes in capillaroscopy showed significantly higher IL-18 (p < 0.001 and sE-selectin (p < 0.05 serum levels than subgroups without changes or with mild abnormalities. Our findings suggest that abnormalities in nailfold capillaroscopy may reflect the extent of microvascular involvement and are associated with higher sE-selectin and IL-18 serum levels, as well as with microangiopathic complications in diabetic patients. (Folia Histochemica et Cytobiologica 2011; Vol. 49, No. 1, pp. 104–110