Inducible colitis-associated glycome capable of stimulating the proliferation of memory CD4+ T cells.

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Nishida, Atsushi; Nagahama, Kiyotaka; Imaeda, Hirotsugu; Ogawa, Atsuhiro; Lau, Cindy W; Kobayashi, Taku; Hisamatsu, Tadakazu; Preffer, Frederic I; Mizoguchi, Emiko; Ikeuchi, Hiroki; Hibi, Toshifumi; Fukuda, Minoru; Andoh, Akira; Blumberg, Richard S; Mizoguchi, Atsushi

2012-12-17

Immune responses are modified by a diverse and abundant repertoire of carbohydrate structures on the cell surface, which is known as the glycome. In this study, we propose that a unique glycome that can be identified through the binding of galectin-4 is created on local, but not systemic, memory CD4+ T cells under diverse intestinal inflammatory conditions, but not in the healthy state. The colitis-associated glycome (CAG) represents an immature core 1-expressing O-glycan. Development of CAG may be mediated by down-regulation of the expression of core-2 β1,6-N-acetylglucosaminyltransferase (C2GnT) 1, a key enzyme responsible for the production of core-2 O-glycan branch through addition of N-acetylglucosamine (GlcNAc) to a core-1 O-glycan structure. Mechanistically, the CAG seems to contribute to super raft formation associated with the immunological synapse on colonic memory CD4+ T cells and to the consequent stabilization of protein kinase C θ activation, resulting in the stimulation of memory CD4+ T cell expansion in the inflamed intestine. Functionally, CAG-mediated CD4+ T cell expansion contributes to the exacerbation of T cell-mediated experimental intestinal inflammations. Therefore, the CAG may be an attractive therapeutic target to specifically suppress the expansion of effector memory CD4+ T cells in intestinal inflammation such as that seen in inflammatory bowel disease.

Reliable LC-MS quantitative glycomics using iGlycoMab stable isotope labeled glycans as internal standards.

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Zhou, Shiyue; Tello, Nadia; Harvey, Alex; Boyes, Barry; Orlando, Ron; Mechref, Yehia

2016-06-01

Glycans have numerous functions in various biological processes and participate in the progress of diseases. Reliable quantitative glycomic profiling techniques could contribute to the understanding of the biological functions of glycans, and lead to the discovery of potential glycan biomarkers for diseases. Although LC-MS is a powerful analytical tool for quantitative glycomics, the variation of ionization efficiency and MS intensity bias are influencing quantitation reliability. Internal standards can be utilized for glycomic quantitation by MS-based methods to reduce variability. In this study, we used stable isotope labeled IgG2b monoclonal antibody, iGlycoMab, as an internal standard to reduce potential for errors and to reduce variabililty due to sample digestion, derivatization, and fluctuation of nanoESI efficiency in the LC-MS analysis of permethylated N-glycans released from model glycoproteins, human blood serum, and breast cancer cell line. We observed an unanticipated degradation of isotope labeled glycans, tracked a source of such degradation, and optimized a sample preparation protocol to minimize degradation of the internal standard glycans. All results indicated the effectiveness of using iGlycoMab to minimize errors originating from sample handling and instruments. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Glycomic Expression in Esophageal Disease

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Sanjay Mohanty

2012-11-01

Full Text Available Glycosylation is among the most common post translation modifications of proteins in humans. Decades of research have demonstrated that aberrant glycosylation can lead to malignant degeneration. Glycoproteomic studies in the past several years have identified techniques that can successfully characterize a glycan or glycan profile associated with a high-grade dysplastic or malignant state. This review summarizes the current glycomic and glycoproteomic literature with specific reference to esophageal cancer. Esophageal adenocarcinoma represents a highly morbid and mortal cancer with a defined progression from metaplasia (Barrett's esophagus to dysplasia to neoplasia. This disease is highlighted because (1 differences in glycan profiles between the stages of disease progression have been described in the glycoproteomic literature; (2 a glycan biomarker that identifies a given stage may be used as a predictor of disease progression and thus may have significant influence over clinical management; and (3 the differences in glycan profiles between disease and disease-free states in esophageal cancer are more dramatic than in other cancers.

Studying the Effects of Reproductive Hormones and Bacterial Vaginosis on the Glycome of Lavage Samples from the Cervicovaginal Cavity

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Wang, Linlin; Koppolu, Sujeethraj; Chappell, Catherine; Moncla, Bernard J.; Hillier, Sharon L.; Mahal, Lara K.

2015-01-01

The cervicovaginal fluid (CVF) coating the vaginal epithelium is an important immunological mediator, providing a barrier to infection. Glycosylation of CVF proteins, such as mucins, IgG and S-IgA, plays a critical role in their immunological functions. Although multiple factors, such as hormones and microflora, may influence glycosylation of the CVF, few studies have examined their impact on this important immunological fluid. Herein we analyzed the glycosylation of cervicovaginal lavage (CVL) samples collected from 165 women under different hormonal conditions including: (1) no contraceptive, post-menopausal, (2) no contraceptive, days 1-14 of the menstrual cycle, (3) no contraceptive, days 15-28 of the menstrual cycle, (4) combined-oral contraceptive pills for at least 6 months, (5) depo-medroxyprogesterone acetate (Depo-Provera) injections for at least 6 months, (6) levonorgestrel IUD for at least 1 month. Glycomic profiling was obtained using our lectin microarray system, a rapid method to analyze carbohydrate composition. Although some small effects were observed due to hormone levels, the major influence on the glycome was the presence of an altered bacterial cohort due to bacterial vaginosis (BV). Compared to normal women, samples from women with BV contained lower levels of sialic acid and high-mannose glycans in their CVL. The change in high mannose levels was unexpected and may be related to the increased risk of HIV-infection observed in women with BV, as high mannose receptors are a viral entry pathway. Changes in the glycome were also observed with hormonal contraceptive use, in a contraceptive-dependent manner. Overall, microflora had a greater impact on the glycome than hormonal levels, and both of these effects should be more closely examined in future studies given the importance of glycans in the innate immune system. PMID:25993513

Studying the effects of reproductive hormones and bacterial vaginosis on the glycome of lavage samples from the cervicovaginal cavity.

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Wang, Linlin; Koppolu, Sujeethraj; Chappell, Catherine; Moncla, Bernard J; Hillier, Sharon L; Mahal, Lara K

2015-01-01

The cervicovaginal fluid (CVF) coating the vaginal epithelium is an important immunological mediator, providing a barrier to infection. Glycosylation of CVF proteins, such as mucins, IgG and S-IgA, plays a critical role in their immunological functions. Although multiple factors, such as hormones and microflora, may influence glycosylation of the CVF, few studies have examined their impact on this important immunological fluid. Herein we analyzed the glycosylation of cervicovaginal lavage (CVL) samples collected from 165 women under different hormonal conditions including: (1) no contraceptive, post-menopausal, (2) no contraceptive, days 1-14 of the menstrual cycle, (3) no contraceptive, days 15-28 of the menstrual cycle, (4) combined-oral contraceptive pills for at least 6 months, (5) depo-medroxyprogesterone acetate (Depo-Provera) injections for at least 6 months, (6) levonorgestrel IUD for at least 1 month. Glycomic profiling was obtained using our lectin microarray system, a rapid method to analyze carbohydrate composition. Although some small effects were observed due to hormone levels, the major influence on the glycome was the presence of an altered bacterial cohort due to bacterial vaginosis (BV). Compared to normal women, samples from women with BV contained lower levels of sialic acid and high-mannose glycans in their CVL. The change in high mannose levels was unexpected and may be related to the increased risk of HIV-infection observed in women with BV, as high mannose receptors are a viral entry pathway. Changes in the glycome were also observed with hormonal contraceptive use, in a contraceptive-dependent manner. Overall, microflora had a greater impact on the glycome than hormonal levels, and both of these effects should be more closely examined in future studies given the importance of glycans in the innate immune system.

Studying the effects of reproductive hormones and bacterial vaginosis on the glycome of lavage samples from the cervicovaginal cavity.

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Linlin Wang

Full Text Available The cervicovaginal fluid (CVF coating the vaginal epithelium is an important immunological mediator, providing a barrier to infection. Glycosylation of CVF proteins, such as mucins, IgG and S-IgA, plays a critical role in their immunological functions. Although multiple factors, such as hormones and microflora, may influence glycosylation of the CVF, few studies have examined their impact on this important immunological fluid. Herein we analyzed the glycosylation of cervicovaginal lavage (CVL samples collected from 165 women under different hormonal conditions including: (1 no contraceptive, post-menopausal, (2 no contraceptive, days 1-14 of the menstrual cycle, (3 no contraceptive, days 15-28 of the menstrual cycle, (4 combined-oral contraceptive pills for at least 6 months, (5 depo-medroxyprogesterone acetate (Depo-Provera injections for at least 6 months, (6 levonorgestrel IUD for at least 1 month. Glycomic profiling was obtained using our lectin microarray system, a rapid method to analyze carbohydrate composition. Although some small effects were observed due to hormone levels, the major influence on the glycome was the presence of an altered bacterial cohort due to bacterial vaginosis (BV. Compared to normal women, samples from women with BV contained lower levels of sialic acid and high-mannose glycans in their CVL. The change in high mannose levels was unexpected and may be related to the increased risk of HIV-infection observed in women with BV, as high mannose receptors are a viral entry pathway. Changes in the glycome were also observed with hormonal contraceptive use, in a contraceptive-dependent manner. Overall, microflora had a greater impact on the glycome than hormonal levels, and both of these effects should be more closely examined in future studies given the importance of glycans in the innate immune system.

Glycomic analysis of gastric carcinoma cells discloses glycans as modulators of RON receptor tyrosine kinase activation in cancer

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Mereiter, Stefan; Magalhães, Ana; Adamczyk, Barbara

2016-01-01

gastric carcinoma cells transfected with the sialyltransferase ST3GAL4 were established as a model overexpressing sialylated terminal glycans. We have evaluated at the structural level the glycome and the sialoproteome of this gastric cancer cell line applying liquid chromatography and mass spectrometry...... known to be key players in malignancy. Further analysis of RON confirmed its modification with SLe(X) and the concomitant activation. SLe(X) and RON co-expression was validated in gastric tumors. CONCLUSION: The overexpression of ST3GAL4 interferes with the overall glycophenotype of cancer cells...... affecting a multitude of key proteins involved in malignancy. Aberrant glycosylation of the RON receptor was shown as an alternative mechanism of oncogenic activation. GENERAL SIGNIFICANCE: This study provides novel targets and points to an integrative tumor glycomic/proteomic-profiling for gastric cancer...

Quantitative glycomics monitoring of induced pluripotent- and embryonic stem cells during neuronal differentiation

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Michiyo Terashima

2014-11-01

Full Text Available Alterations in the structure of cell surface glycoforms occurring during the stages of stem cell differentiation remain unclear. We describe a rapid glycoblotting-based cellular glycomics method for quantitatively evaluating changes in glycoform expression and structure during neuronal differentiation of murine induced pluripotent stem cells (iPSCs and embryonic stem cells (ESCs. Our results show that changes in the expression of cellular N-glycans are comparable during the differentiation of iPSCs and ESCs. The expression of bisect-type N-glycans was significantly up-regulated in neurons that differentiated from both iPSCs and ESCs. From a glycobiological standpoint, iPSCs are an alternative neural cell source in addition to ESCs.

On the analysis of glycomics mass spectrometry data via the regularized area under the ROC curve

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Lebrilla Carlito B

2007-12-01

Full Text Available Abstract Background Novel molecular and statistical methods are in rising demand for disease diagnosis and prognosis with the help of recent advanced biotechnology. High-resolution mass spectrometry (MS is one of those biotechnologies that are highly promising to improve health outcome. Previous literatures have identified some proteomics biomarkers that can distinguish healthy patients from cancer patients using MS data. In this paper, an MS study is demonstrated which uses glycomics to identify ovarian cancer. Glycomics is the study of glycans and glycoproteins. The glycans on the proteins may deviate between a cancer cell and a normal cell and may be visible in the blood. High-resolution MS has been applied to measure relative abundances of potential glycan biomarkers in human serum. Multiple potential glycan biomarkers are measured in MS spectra. With the objection of maximizing the empirical area under the ROC curve (AUC, an analysis method was considered which combines potential glycan biomarkers for the diagnosis of cancer. Results Maximizing the empirical AUC of glycomics MS data is a large-dimensional optimization problem. The technical difficulty is that the empirical AUC function is not continuous. Instead, it is in fact an empirical 0–1 loss function with a large number of linear predictors. An approach was investigated that regularizes the area under the ROC curve while replacing the 0–1 loss function with a smooth surrogate function. The constrained threshold gradient descent regularization algorithm was applied, where the regularization parameters were chosen by the cross-validation method, and the confidence intervals of the regression parameters were estimated by the bootstrap method. The method is called TGDR-AUC algorithm. The properties of the approach were studied through a numerical simulation study, which incorporates the positive values of mass spectrometry data with the correlations between measurements within person

Chemical shift-based identification of monosaccharide spin-systems with NMR spectroscopy to complement untargeted glycomics.

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Klukowski, Piotr; Schubert, Mario

2018-06-15

A better understanding of oligosaccharides and their wide-ranging functions in almost every aspect of biology and medicine promises to uncover hidden layers of biology and will support the development of better therapies. Elucidating the chemical structure of an unknown oligosaccharide is still a challenge. Efficient tools are required for non-targeted glycomics. Chemical shifts are a rich source of information about the topology and configuration of biomolecules, whose potential is however not fully explored for oligosaccharides. We hypothesize that the chemical shifts of each monosaccharide are unique for each saccharide type with a certain linkage pattern, so that correlated data measured by NMR spectroscopy can be used to identify the chemical nature of a carbohydrate. We present here an efficient search algorithm, GlycoNMRSearch, that matches either a subset or the entire set of chemical shifts of an unidentified monosaccharide spin system to all spin systems in an NMR database. The search output is much more precise than earlier search functions and highly similar matches suggest the chemical structure of the spin system within the oligosaccharide. Thus searching for connected chemical shift correlations within all electronically available NMR data of oligosaccharides is a very efficient way of identifying the chemical structure of unknown oligosaccharides. With an improved database in the future, GlycoNMRSearch will be even more efficient deducing chemical structures of oligosaccharides and there is a high chance that it becomes an indispensable technique for glycomics. The search algorithm presented here, together with a graphical user interface, is available at http://glyconmrsearch.santos.pwr.edu.pl. Supplementary data are available at Bioinformatics online.

Solid-phase glycan isolation for glycomics analysis.

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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.

Nanotechnology in Glycomics: Applications in Diagnostics, Therapy, Imaging, and Separation Processes.

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Dosekova, Erika; Filip, Jaroslav; Bertok, Tomas; Both, Peter; Kasak, Peter; Tkac, Jan

2017-05-01

This review comprehensively covers the most recent achievements (from 2013) in the successful integration of nanomaterials in the field of glycomics. The first part of the paper addresses the beneficial properties of nanomaterials for the construction of biosensors, bioanalytical devices, and protocols for the detection of various analytes, including viruses and whole cells, together with their key characteristics. The second part of the review focuses on the application of nanomaterials integrated with glycans for various biomedical applications, that is, vaccines against viral and bacterial infections and cancer cells, as therapeutic agents, for in vivo imaging and nuclear magnetic resonance imaging, and for selective drug delivery. The final part of the review describes various ways in which glycan enrichment can be effectively done using nanomaterials, molecularly imprinted polymers with polymer thickness controlled at the nanoscale, with a subsequent analysis of glycans by mass spectrometry. A short section describing an active glycoprofiling by microengines (microrockets) is covered as well. © 2016 Wiley Periodicals, Inc.

Genomics meets glycomics-the first GWAS study of human N-Glycome identifies HNF1α as a master regulator of plasma protein fucosylation.

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Gordan Lauc

2010-12-01

Full Text Available Over half of all proteins are glycosylated, and alterations in glycosylation have been observed in numerous physiological and pathological processes. Attached glycans significantly affect protein function; but, contrary to polypeptides, they are not directly encoded by genes, and the complex processes that regulate their assembly are poorly understood. A novel approach combining genome-wide association and high-throughput glycomics analysis of 2,705 individuals in three population cohorts showed that common variants in the Hepatocyte Nuclear Factor 1α (HNF1α and fucosyltransferase genes FUT6 and FUT8 influence N-glycan levels in human plasma. We show that HNF1α and its downstream target HNF4α regulate the expression of key fucosyltransferase and fucose biosynthesis genes. Moreover, we show that HNF1α is both necessary and sufficient to drive the expression of these genes in hepatic cells. These results reveal a new role for HNF1α as a master transcriptional regulator of multiple stages in the fucosylation process. This mechanism has implications for the regulation of immunity, embryonic development, and protein folding, as well as for our understanding of the molecular mechanisms underlying cancer, coronary heart disease, and metabolic and inflammatory disorders.

Glycomic profiling of tissue sections by LC-MS.

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Hu, Yunli; Zhou, Shiyue; Khalil, Sarah I; Renteria, Calvin L; Mechref, Yehia

2013-04-16

Because routine preparation of glycan samples involves multiple reaction and cleaning steps at which sample loss occurs, glycan analysis is typically performed using large tissue samples. This type of analysis yields no detailed molecular spatial information and requires special care to maintain proper storage and shipping conditions. We describe here a new glycan sample preparation protocol using minimized sample preparation steps and optimized procedures. Tissue sections and spotted samples first undergo on-surface enzymatic digestion to release N-glycans. The released glycans are then reduced and permethylated prior to online purification and LC-electrospray ionization (ESI)-MS analysis. The efficiency of this protocol was initially evaluated using model glycoproteins and human blood serum (HBS) spotted on glass or Teflon slides. The new protocol permitted the detection of permethylated N-glycans derived from 10 ng RNase B. On the other hand, 66 N-glycans were identified when injecting the equivalent of permethylated glycans derived from a 0.1-μL aliquot of HBS. On-tissue enzymatic digestion of nude mouse brain tissue permitted the detection of 43 N-glycans. The relative peak areas of these 43 glycans were comparable to those from a C57BL/6 mouse reported by the Consortium for Functional Glycomics (CFG). However, the sample size analyzed in the protocol described here was substantially smaller than for the routine method (submicrogram vs mg). The on-tissue N-glycan profiling method permits high sensitivity and reproducibility and can be widely applied to assess the spatial distribution of glycans associated with tissue sections, and may be correlated with immunoflourescence imaging when adjacent tissue sections are analyzed.

Shotgun glycomics of pig lung identifies natural endogenous receptors for influenza viruses.

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Byrd-Leotis, Lauren; Liu, Renpeng; Bradley, Konrad C; Lasanajak, Yi; Cummings, Sandra F; Song, Xuezheng; Heimburg-Molinaro, Jamie; Galloway, Summer E; Culhane, Marie R; Smith, David F; Steinhauer, David A; Cummings, Richard D

2014-06-03

Influenza viruses bind to host cell surface glycans containing terminal sialic acids, but as studies on influenza binding become more sophisticated, it is becoming evident that although sialic acid may be necessary, it is not sufficient for productive binding. To better define endogenous glycans that serve as viral receptors, we have explored glycan recognition in the pig lung, because influenza is broadly disseminated in swine, and swine have been postulated as an intermediary host for the emergence of pandemic strains. For these studies, we used the technology of "shotgun glycomics" to identify natural receptor glycans. The total released N- and O-glycans from pig lung glycoproteins and glycolipid-derived glycans were fluorescently tagged and separated by multidimensional HPLC, and individual glycans were covalently printed to generate pig lung shotgun glycan microarrays. All viruses tested interacted with one or more sialylated N-glycans but not O-glycans or glycolipid-derived glycans, and each virus demonstrated novel and unexpected differences in endogenous N-glycan recognition. The results illustrate the repertoire of specific, endogenous N-glycans of pig lung glycoproteins for virus recognition and offer a new direction for studying endogenous glycan functions in viral pathogenesis.

The glycomic effect of N-acetylglucosaminyltransferase III overexpression in metastatic melanoma cells. GnT-III modifies highly branched N-glycans.

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Link-Lenczowski, Paweł; Bubka, Monika; Balog, Crina I A; Koeleman, Carolien A M; Butters, Terry D; Wuhrer, Manfred; Lityńska, Anna

2018-04-01

N-acetylglucosaminyltransferase III (GnT-III) is known to catalyze N-glycan "bisection" and thereby modulate the formation of highly branched complex structures within the Golgi apparatus. While active, it inhibits the action of other GlcNAc transferases such as GnT-IV and GnT-V. Moreover, GnT-III is considered as an inhibitor of the metastatic potential of cancer cells both in vitro and in vivo. However, the effects of GnT-III may be more diverse and depend on the cellular context. We describe the detailed glycomic analysis of the effect of GnT-III overexpression in WM266-4-GnT-III metastatic melanoma cells. We used MALDI-TOF and ESI-ion-trap-MS/MS together with HILIC-HPLC of 2-AA labeled N-glycans to study the N-glycome of membrane-attached and secreted proteins. We found that the overexpression of GnT-III in melanoma leads to the modification of a broad range of N-glycan types by the introduction of the "bisecting" GlcNAc residue with highly branched complex structures among them. The presence of these unusual complex N-glycans resulted in stronger interactions of cellular glycoproteins with the PHA-L. Based on the data presented here we conclude that elevated activity of GnT-III in cancer cells does not necessarily lead to a total abrogation of the formation of highly branched glycans. In addition, the modification of pre-existing N-glycans by the introduction of "bisecting" GlcNAc can modulate their capacity to interact with carbohydrate-binding proteins such as plant lectins. Our results suggest further studies on the biological function of "bisected" oligosaccharides in cancer cell biology and their interactions with carbohydrate-binding proteins.

Glycomic analysis of human respiratory tract tissues and correlation with influenza virus infection.

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Trevenan Walther

2013-03-01

Full Text Available The first step in influenza infection of the human respiratory tract is binding of the virus to sialic (Sia acid terminated receptors. The binding of different strains of virus for the receptor is determined by the α linkage of the sialic acid to galactose and the adjacent glycan structure. In this study the N- and O-glycan composition of the human lung, bronchus and nasopharynx was characterized by mass spectrometry. Analysis showed that there was a wide spectrum of both Sia α2-3 and α2-6 glycans in the lung and bronchus. This glycan structural data was then utilized in combination with binding data from 4 of the published glycan arrays to assess whether these current glycan arrays were able to predict replication of human, avian and swine viruses in human ex vivo respiratory tract tissues. The most comprehensive array from the Consortium for Functional Glycomics contained the greatest diversity of sialylated glycans, but was not predictive of productive replication in the bronchus and lung. Our findings indicate that more comprehensive but focused arrays need to be developed to investigate influenza virus binding in an assessment of newly emerging influenza viruses.

Glycomic analyses of mouse models of congenital muscular dystrophy.

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Stalnaker, Stephanie H; Aoki, Kazuhiro; Lim, Jae-Min; Porterfield, Mindy; Liu, Mian; Satz, Jakob S; Buskirk, Sean; Xiong, Yufang; Zhang, Peng; Campbell, Kevin P; Hu, Huaiyu; Live, David; Tiemeyer, Michael; Wells, Lance

2011-06-17

Dystroglycanopathies are a subset of congenital muscular dystrophies wherein α-dystroglycan (α-DG) is hypoglycosylated. α-DG is an extensively O-glycosylated extracellular matrix-binding protein and a key component of the dystrophin-glycoprotein complex. Previous studies have shown α-DG to be post-translationally modified by both O-GalNAc- and O-mannose-initiated glycan structures. Mutations in defined or putative glycosyltransferase genes involved in O-mannosylation are associated with a loss of ligand-binding activity of α-DG and are causal for various forms of congenital muscular dystrophy. In this study, we sought to perform glycomic analysis on brain O-linked glycan structures released from proteins of three different knock-out mouse models associated with O-mannosylation (POMGnT1, LARGE (Myd), and DAG1(-/-)). Using mass spectrometry approaches, we were able to identify nine O-mannose-initiated and 25 O-GalNAc-initiated glycan structures in wild-type littermate control mouse brains. Through our analysis, we were able to confirm that POMGnT1 is essential for the extension of all observed O-mannose glycan structures with β1,2-linked GlcNAc. Loss of LARGE expression in the Myd mouse had no observable effect on the O-mannose-initiated glycan structures characterized here. Interestingly, we also determined that similar amounts of O-mannose-initiated glycan structures are present on brain proteins from α-DG-lacking mice (DAG1) compared with wild-type mice, indicating that there must be additional proteins that are O-mannosylated in the mammalian brain. Our findings illustrate that classical β1,2-elongation and β1,6-GlcNAc branching of O-mannose glycan structures are dependent upon the POMGnT1 enzyme and that O-mannosylation is not limited solely to α-DG in the brain.

Stable isotopic labeling-based quantitative targeted glycomics (i-QTaG).

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Kim, Kyoung-Jin; Kim, Yoon-Woo; Kim, Yun-Gon; Park, Hae-Min; Jin, Jang Mi; Hwan Kim, Young; Yang, Yung-Hun; Kyu Lee, Jun; Chung, Junho; Lee, Sun-Gu; Saghatelian, Alan

2015-01-01

Mass spectrometry (MS) analysis combined with stable isotopic labeling is a promising method for the relative quantification of aberrant glycosylation in diseases and disorders. We developed a stable isotopic labeling-based quantitative targeted glycomics (i-QTaG) technique for the comparative and quantitative analysis of total N-glycans using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We established the analytical procedure with the chemical derivatizations (i.e., sialic acid neutralization and stable isotopic labeling) of N-glycans using a model glycoprotein (bovine fetuin). Moreover, the i-QTaG using MALDI-TOF MS was evaluated with various molar ratios (1:1, 1:2, 1:5) of (13) C6 /(12) C6 -2-aminobenzoic acid-labeled glycans from normal human serum. Finally, this method was applied to direct comparison of the total N-glycan profiles between normal human sera (n = 8) and prostate cancer patient sera (n = 17). The intensities of the N-glycan peaks from i-QTaG method showed a good linearity (R(2) > 0.99) with the amount of the bovine fetuin glycoproteins. The ratios of relative intensity between the isotopically 2-AA labeled N-glycans were close to the theoretical molar ratios (1:1, 1:2, 1:5). We also demonstrated that the up-regulation of the Lewis antigen (~82%) in sera from prostate cancer patients. In this proof-of-concept study, we demonstrated that the i-QTaG method, which enables to achieve a reliable comparative quantitation of total N-glycans via MALDI-TOF MS analysis, has the potential to diagnose and monitor alterations in glycosylation associated with disease states or biotherapeutics. © 2015 American Institute of Chemical Engineers.

Glycomic characterization of basal tears and changes with diabetes and diabetic retinopathy.

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

2015-03-01

As a secreted fluid, the state of tear glycosylation is particularly important in the role of immunity of the ocular surface. Tears are a valuable source of non-invasive biomarkers for disease and there are continued efforts to characterize their components thoroughly. In this study, a small volume of basal tears (5 μL) was collected from healthy controls, patients with diabetes without retinopathy and patients with diabetes and retinopathy. The detailed N- and O-linked tear protein glycome was characterized and the relative abundance of each structure determined. Of the 50 N-linked glycans found, 89% were complex with 50% containing a bisecting N-acetylglucosamine, 65% containing a core fucose whilst 33% were sialylated. Of the 8 O-linked glycans detected, 3 were of cores 1 and 5 of core 2 type, with a majority of them being sialylated (90%). Additionally, these glycan structures were profiled across the three diabetic disease groups. Whilst the higher abundant structures did not alter across the three groups, only five low abundance N-linked glycans and 1 O-linked glycan did alter with the onset of diabetes mellitus and diabetic retinopathy (DR). These results suggest the conservation of glycan types on basal tear proteins between individuals and point to only small changes in glycan expression on the proteins in tears with the development of diabetes and DR. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Phosphoneurofilament heavy chain and N-glycomics from the cerebrospinal fluid in amyotrophic lateral sclerosis.

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Gonçalves, Margarida; Tillack, Linda; de Carvalho, Mamede; Pinto, Susana; Conradt, Harald S; Costa, Júlia

2015-01-01

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease of the motor neuron for which no clinically validated biomarkers have been identified. We have quantified by ELISA the biomarker phosphoneurofilament heavy chain (pNFH) in the cerebrospinal fluid (CSF) of ALS patients (n=29) and age-matched control patients with other diseases (n=19) by ELISA. Furthermore, we compared protein N-glycosylation of the CSF in ALS patients and controls, by applying a glycomics approach based on liquid chromatography and mass spectrometry. pNFH levels were significantly higher in ALS patients in comparison with controls (P<0.0001) in particular in fast progressors. The N-glycans found in the CSF were predominantly complex diantennary with sialic acid in α2,3- and α2,6-linkage, and bisecting N-acetylglucosamine-containing structures as well as peripherally fucosylated structures were found. As compared with controls the ALS group had a significant increase of a peak composed of the monosialylated diantennary glycans A2G2S(6)1 and FA2G2S(3)1 (P=0.0348). Our results underscore the value of pNFH as a biomarker in ALS. In addition, we identified a variation of the N-glycosylation pattern in ALS, suggesting that this change should be explored in future studies as potential biomarker. Copyright © 2014 Elsevier B.V. All rights reserved.

Molecular glycopathology by capillary electrophoresis: Analysis of the N-glycome of formalin-fixed paraffin-embedded mouse tissue samples.

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Donczo, Boglarka; Szarka, Mate; Tovari, Jozsef; Ostoros, Gyorgyi; Csanky, Eszter; Guttman, Andras

2017-06-01

Capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection was used to analyze endoglycosidase released and fluorophore-labeled N-glycans from formalin-fixed paraffin-embedded (FFPE) mouse tissue samples of lung, brain, heart, spleen, liver, kidney and intestine. The FFPE samples were first deparaffinized followed by solubilization and glycoprotein retrieval. PNGase F mediated release of the N-linked oligosaccharides was followed by labeling with aminopyrene trisulfonate. After CE-LIF glycoprofiling of the FFPE mouse tissues, the N-glycan pool of the lung specimen was subject to further investigation by exoglycosidase array based carbohydrate sequencing. Structural assignment of the oligosaccharides was accomplished by the help of the GUcal software and the associated database, based on the mobility shifts after treatments with the corresponding exoglycosidase reaction mixtures. Sixteen major N-linked carbohydrate structures were sequenced from the mouse lung FFPE tissue glycome and identified, as high mannose (3) neutral biantennary (3) sialylated monoantennary (1) and sialylated bianennary (9) oligosaccharides. Two of these latter ones also possessed alpha(1-3) linked galactose residues. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Glycomic Analysis of Life Stages of the Human Parasite Schistosoma mansoni Reveals Developmental Expression Profiles of Functional and Antigenic Glycan Motifs.

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Smit, Cornelis H; van Diepen, Angela; Nguyen, D Linh; Wuhrer, Manfred; Hoffmann, Karl F; Deelder, André M; Hokke, Cornelis H

2015-07-01

Glycans present on glycoproteins and glycolipids of the major human parasite Schistosoma mansoni induce innate as well as adaptive immune responses in the host. To be able to study the molecular characteristics of schistosome infections it is therefore required to determine the expression profiles of glycans and antigenic glycan-motifs during a range of critical stages of the complex schistosome lifecycle. We performed a longitudinal profiling study covering schistosome glycosylation throughout worm- and egg-development using a mass spectrometry-based glycomics approach. Our study revealed that during worm development N-glycans with Galβ1-4(Fucα1-3)GlcNAc (LeX) and core-xylose motifs were rapidly lost after cercariae to schistosomula transformation, whereas GalNAcβ1-4GlcNAc (LDN)-motifs gradually became abundant and predominated in adult worms. LeX-motifs were present on glycolipids up to 2 weeks of schistosomula development, whereas glycolipids with mono- and multifucosylated LDN-motifs remained present up to the adult worm stage. In contrast, expression of complex O-glycans diminished to undetectable levels within days after transformation. During egg development, a rich diversity of N-glycans with fucosylated motifs was expressed, but with α3-core fucose and a high degree of multifucosylated antennae only in mature eggs and miracidia. N-glycan antennae were exclusively LDN-based in miracidia. O-glycans in the mature eggs were also diverse and contained LeX- and multifucosylated LDN, but none of these were associated with miracidia in which we detected only the Galβ1-3(Galβ1-6)GalNAc core glycan. Immature eggs also exhibited short O-glycan core structures only, suggesting that complex fucosylated O-glycans of schistosome eggs are derived primarily from glycoproteins produced by the subshell envelope in the developed egg. Lipid glycans with multifucosylated GlcNAc repeats were present throughout egg development, but with the longer highly fucosylated

Oligosaccharide substrate preferences of human extracellular sulfatase Sulf2 using liquid chromatography-mass spectrometry based glycomics approaches.

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Yu Huang

Full Text Available Sulfs are extracellular endosulfatases that selectively remove the 6-O-sulfate groups from cell surface heparan sulfate (HS chain. By altering the sulfation at these particular sites, Sulfs function to remodel HS chains. As a result of the remodeling activity, HSulf2 regulates a multitude of cell-signaling events that depend on interactions between proteins and HS. Previous efforts to characterize the substrate specificity of human Sulfs (HSulfs focused on the analysis of HS disaccharides and synthetic repeating units. In this study, we characterized the substrate preferences of human HSulf2 using HS oligosaccharides with various lengths and sulfation degrees from several naturally occurring HS sources by applying liquid chromatography mass spectrometry based glycomics methods. The results showed that HSulf2 preferentially digests highly sulfated HS oligosaccharides with zero acetyl groups and this preference is length dependent. In terms of length of oligosaccharides, HSulf2 digestion induced more sulfation decrease on DP6 (DP: degree of polymerization compared to DP2, DP4 and DP8. In addition, the HSulf2 preferentially digests the oligosaccharide domain located at the non-reducing end (NRE of the HS and heparin chain. In addition, the HSulf2 digestion products were altered only for specific isomers. HSulf2 treated NRE oligosaccharides also showed greater decrease in cell proliferation than those from internal domains of the HS chain. After further chromatographic separation, we identified the three most preferred unsaturated hexasaccharide for HSulf2.

Glycomics and glycoproteomics focused on aging and age-related diseases--Glycans as a potential biomarker for physiological alterations.

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Miura, Yuri; Endo, Tamao

2016-08-01

Since glycosylation depends on glycosyltransferases, glycosidases, and sugar nucleotide donors, it is susceptible to the changes associated with physiological and pathological conditions. Therefore, alterations in glycan structures may be good targets and biomarkers for monitoring health conditions. Since human aging and longevity are affected by genetic and environmental factors such as diseases, lifestyle, and social factors, a scale that reflects various environmental factors is required in the study of human aging and longevity. We herein focus on glycosylation changes elucidated by glycomic and glycoproteomic studies on aging, longevity, and age-related diseases including cognitive impairment, diabetes mellitus, and frailty. We also consider the potential of glycan structures as biomarkers and/or targets for monitoring physiological and pathophysiological changes. Glycan structures are altered in age-related diseases. These glycans and glycoproteins may be involved in the pathophysiology of these diseases and, thus, be useful diagnostic markers. Age-dependent changes in N-glycans have been reported previously in cohort studies, and characteristic N-glycans in extreme longevity have been proposed. These findings may lead to a deeper understanding of the mechanisms underlying aging as well as the factors influencing longevity. Alterations in glycosylation may be good targets and biomarkers for monitoring health conditions, and be applicable to studies on age-related diseases and healthy aging. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc. Copyright © 2016 Elsevier B.V. All rights reserved.

Evaluation of a novel suture material for closure of intestinal anastomoses in canine cadavers.

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Hansen, Lane A; Monnet, Eric L

2012-11-01

To compare leakage and maximum intraluminal pressures for a novel suture material with pressures for comparable suture material when used in closure of intestinal anastomoses in canine cadavers. Healthy intestines from cadavers of dogs euthanized for reasons unrelated to the study. 18 anastomoses were performed on intestinal sections within 72 hours after dogs were euthanized and intestinal samples collected. Anastomoses were performed with a simple continuous suture pattern. Leakage and maximum intraluminal pressures were measured and recorded for 6 control segments and 18 anastomosed sections. A barbed glycomer 631 suture (size 4-0 United States Pharmacopeia [USP]) was compared with glycomer 631 sutures (sizes 3-0 and 4-0 USP). Results for leakage and maximum intraluminal pressures were compared via an ANOVA. The barbed glycomer 631 suture material leaked at a significantly higher pressure than did the comparable glycomer 631 suture materials. Maximum intraluminal pressures were not significantly different among the suture materials. Barbed glycomer 631 4-0 USP suture material was as effective as glycomer 631 suture materials and may be a safe alternative for use in closure of enterectomies in dogs.

Simultaneous Release and Labeling of O- and N-Glycans Allowing for Rapid Glycomic Analysis by Online LC-UV-ESI-MS/MS.

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Wang, Chengjian; Lu, Yu; Han, Jianli; Jin, Wanjun; Li, Lingmei; Zhang, Ying; Song, Xuezheng; Huang, Linjuan; Wang, Zhongfu

2018-05-24

Most glycoproteins and biological protein samples undergo both O- and N-glycosylation, making characterization of their structures very complicated and time-consuming. Nevertheless, to fully understand the biological functions of glycosylation, both the glycosylation forms need to be analyzed. Herein we report a versatile, convenient one-pot method in which O- and N-glycans are simultaneously released from glycoproteins and chromogenically labeled in situ and thus available for further characterization. In this procedure, glycoproteins are incubated with 1-phenyl-3-methyl-5-pyrazolone (PMP) in aqueous ammonium hydroxide, making O-glycans released from protein backbones by β-elimination and N-glycans liberated by alkaline hydrolysis. The released glycans are promptly derivatized with PMP in situ by Knoevenagel condensation and Michael addition, with peeling degradation almost completely prevented. The recovered mixture of O- and N-glycans as bis-PMP derivatives features strong ultraviolet (UV) absorbing ability and hydrophobicity, allowing for high-resolution chromatographic separation and high-sensitivity spectrometric detection. Using this technique, O- and N-glycans were simultaneously prepared from some model glycoproteins and complex biological samples, without significant peeling, desialylation, deacetylation, desulfation or other side-reactions, and then comprehensively analyzed by online HILIC-UV-ESI-MS/MS and RP-HPLC-UV-ESI-MS/MS, with which some novel O- and N-glycan structures were first found. This method provides a simple, versatile strategy for high-throughput glycomics analysis.

Insights into plant cell wall structure, architecture, and integrity using glycome profiling of native and AFEXTM-pre-treated biomass

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Pattathil, Sivakumar; Hahn, Michael G.; Dale, Bruce E.; Chundawat, Shishir P. S.

2015-01-01

Cell walls, which constitute the bulk of plant biomass, vary considerably in their structure, composition, and architecture. Studies on plant cell walls can be conducted on both native and pre-treated plant biomass samples, allowing an enhanced understanding of these structural and compositional variations. Here glycome profiling was employed to determine the relative abundance of matrix polysaccharides in several phylogenetically distinct native and pre-treated plant biomasses. Eight distinct biomass types belonging to four different subgroups (i.e. monocot grasses, woody dicots, herbaceous dicots, and softwoods) were subjected to various regimes of AFEX™ (ammonia fiber expansion) pre-treatment [AFEX is a trademark of MBI, Lansing (http://www.mbi.org]. This approach allowed detailed analysis of close to 200 cell wall glycan epitopes and their relative extractability using a high-throughput platform. In general, irrespective of the phylogenetic origin, AFEX™ pre-treatment appeared to cause loosening and improved accessibility of various xylan epitope subclasses in most plant biomass materials studied. For most biomass types analysed, such loosening was also evident for other major non-cellulosic components including subclasses of pectin and xyloglucan epitopes. The studies also demonstrate that AFEX™ pre-treatment significantly reduced cell wall recalcitrance among diverse phylogenies (except softwoods) by inducing structural modifications to polysaccharides that were not detectable by conventional gross composition analyses. It was found that monitoring changes in cell wall glycan compositions and their relative extractability for untreated and pre-treated plant biomass can provide an improved understanding of variations in structure and composition of plant cell walls and delineate the role(s) of matrix polysaccharides in cell wall recalcitrance. PMID:25911738

Glycoscience aids in biomarker discovery

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Serenus Hua1,2 & Hyun Joo An1,2,*

2012-06-01

Full Text Available The glycome consists of all glycans (or carbohydrates within abiological system, and modulates a wide range of important biologicalactivities, from protein folding to cellular communications.The mining of the glycome for disease markers representsa new paradigm for biomarker discovery; however, this effortis severely complicated by the vast complexity and structuraldiversity of glycans. This review summarizes recent developmentsin analytical technology and methodology as applied tothe fields of glycomics and glycoproteomics. Mass spectrometricstrategies for glycan compositional profiling are described, as arepotential refinements which allow structure-specific profiling.Analytical methods that can discern protein glycosylation at aspecific site of modification are also discussed in detail.Biomarker discovery applications are shown at each level ofanalysis, highlighting the key role that glycoscience can play inhelping scientists understand disease biology.

Glycoblotting method allows for rapid and efficient glycome profiling of human Alzheimer's disease brain, serum and cerebrospinal fluid towards potential biomarker discovery.

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Gizaw, Solomon T; Ohashi, Tetsu; Tanaka, Masakazu; Hinou, Hiroshi; Nishimura, Shin-Ichiro

2016-08-01

Understanding of the significance of posttranslational glycosylation in Alzheimer's disease (AD) is of growing importance for the investigation of the pathogenesis of AD as well as discovery research of the disease-specific serum biomarkers. We designed a standard protocol for the glycoblotting combined with MALDI-TOFMS to perform rapid and quantitative profiling of the glycan parts of glycoproteins (N-glycans) and glycosphingolipids (GSLs) using human AD's post-mortem samples such as brain tissues (dissected cerebral cortices such as frontal, parietal, occipital, and temporal domains), serum and cerebrospinal fluid (CSF). The structural profiles of the major N-glycans released from glycoproteins and the total expression levels of the glycans were found to be mostly similar between the brain tissues of the AD patients and those of the normal control group. In contrast, the expression levels of the serum and CSF protein N-glycans such as bisect-type and multiply branched glycoforms were increased significantly in AD patient group. In addition, the levels of some gangliosides such as GM1, GM2 and GM3 appeared to alter in the AD patient brain and serum samples when compared with the normal control groups. Alteration of the expression levels of major N- and GSL-glycans in human brain tissues, serum and CSF of AD patients can be monitored quantitatively by means of the glycoblotting-based standard protocols. The changes in the expression levels of the glycans derived from the human post-mortem samples uncovered by the standardized glycoblotting method provides potential serum biomarkers in central nervous system disorders and can contribute to the insight into the molecular mechanisms in the pathogenesis of neurodegenerative diseases and future drug discovery. Most importantly, the present preliminary trials using human post-mortem samples of AD patients suggest that large-scale serum glycomics cohort by means of various-types of human AD patients as well as the normal

The Effects of Hormones and Vaginal Microflora on the Glycome of the Female Genital Tract: Cervical-Vaginal Fluid.

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Moncla, Bernard J; Chappell, Catherine A; Debo, Brian M; Meyn, Leslie A

2016-01-01

In this study, we characterized the glycome of cervical-vaginal fluid, collected with a Catamenial cup. We quantified: glycosidase levels; sialic acid and high mannose specific lectin binding; mucins, MUC1, MUC4, MUC5AC, MUC7; and albumin in the samples collected. These data were analyzed in the context of hormonal status (day of menstrual cycle, hormonal contraception use) and role, if any, of the type of the vaginal microflora present. When the Nugent score was used to stratify the subjects by microflora as normal, intermediate, or bacterial vaginosis, several important differences were observed. The activities of four of six glycosidases in the samples from women with bacterial vaginosis were significantly increased when compared to normal or intermediate women: sialidase, P = <0.001; α-galactosidase, P = 0.006; β-galactosidase, P = 0.005; α-glucosidase, P = 0.056. Sialic acid binding sites as measured by two lectins, Maackia amurensis and Sambucus nigra binding, were significantly lower in women with BV compared to women with normal and intermediate scores (P = <0.0001 and 0.008 respectively). High mannose binding sites, a measure of innate immunity were also significantly lower in women with BV (P = <0.001). Additionally, we observed significant increases in MUC1, MUC4, MUC5AC, and MUC7 concentrations in women with BV (P = <0.001, 0.001, <0.001, 0.02 respectively). Among normal women we found that the membrane bound mucin MUC4 and the secreted MUC5AC were decreased in postmenopausal women (P = 0.02 and 0.07 respectively), while MUC7 (secreted) was decreased in women using levonorgestrel-containing IUDs (P = 0.02). The number of sialic acid binding sites was lower in the postmenopausal group (P = 0.04), but the number of high mannose binding sites, measured with Griffithsin, was not significantly different among the 6 hormonal groups. The glycosidase levels in the cervical-vaginal mucus were rather low in the groups, with exception of α-glucosidase activity

The Effects of Hormones and Vaginal Microflora on the Glycome of the Female Genital Tract: Cervical-Vaginal Fluid.

Directory of Open Access Journals (Sweden)

Bernard J Moncla

Full Text Available In this study, we characterized the glycome of cervical-vaginal fluid, collected with a Catamenial cup. We quantified: glycosidase levels; sialic acid and high mannose specific lectin binding; mucins, MUC1, MUC4, MUC5AC, MUC7; and albumin in the samples collected. These data were analyzed in the context of hormonal status (day of menstrual cycle, hormonal contraception use and role, if any, of the type of the vaginal microflora present. When the Nugent score was used to stratify the subjects by microflora as normal, intermediate, or bacterial vaginosis, several important differences were observed. The activities of four of six glycosidases in the samples from women with bacterial vaginosis were significantly increased when compared to normal or intermediate women: sialidase, P = <0.001; α-galactosidase, P = 0.006; β-galactosidase, P = 0.005; α-glucosidase, P = 0.056. Sialic acid binding sites as measured by two lectins, Maackia amurensis and Sambucus nigra binding, were significantly lower in women with BV compared to women with normal and intermediate scores (P = <0.0001 and 0.008 respectively. High mannose binding sites, a measure of innate immunity were also significantly lower in women with BV (P = <0.001. Additionally, we observed significant increases in MUC1, MUC4, MUC5AC, and MUC7 concentrations in women with BV (P = <0.001, 0.001, <0.001, 0.02 respectively. Among normal women we found that the membrane bound mucin MUC4 and the secreted MUC5AC were decreased in postmenopausal women (P = 0.02 and 0.07 respectively, while MUC7 (secreted was decreased in women using levonorgestrel-containing IUDs (P = 0.02. The number of sialic acid binding sites was lower in the postmenopausal group (P = 0.04, but the number of high mannose binding sites, measured with Griffithsin, was not significantly different among the 6 hormonal groups. The glycosidase levels in the cervical-vaginal mucus were rather low in the groups, with exception of �

The carbohydrate sequence markup language (CabosML): an XML description of carbohydrate structures.

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Kikuchi, Norihiro; Kameyama, Akihiko; Nakaya, Shuuichi; Ito, Hiromi; Sato, Takashi; Shikanai, Toshihide; Takahashi, Yoriko; Narimatsu, Hisashi

2005-04-15

Bioinformatics resources for glycomics are very poor as compared with those for genomics and proteomics. The complexity of carbohydrate sequences makes it difficult to define a common language to represent them, and the development of bioinformatics tools for glycomics has not progressed. In this study, we developed a carbohydrate sequence markup language (CabosML), an XML description of carbohydrate structures. The language definition (XML Schema) and an experimental database of carbohydrate structures using an XML database management system are available at http://www.phoenix.hydra.mki.co.jp/CabosDemo.html kikuchi@hydra.mki.co.jp.

Quantitative O-glycomics based on improvement of the one-pot method for nonreductive O-glycan release and simultaneous stable isotope labeling with 1-(d0/d5)phenyl-3-methyl-5-pyrazolone followed by mass spectrometric analysis.

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Wang, Chengjian; Zhang, Ping; Jin, Wanjun; Li, Lingmei; Qiang, Shan; Zhang, Ying; Huang, Linjuan; Wang, Zhongfu

2017-01-06

O-glycomic comparison between perch and salmon eggs by ESI-MS, MS/MS and online RP-HPLC-UV-ESI-MS/MS, demonstrating its excellent applicability to various complex biological samples. O-Linked glycoproteins, generated via a widely existing glycosylation modification process on serine (Ser) or threonine (Thr) residues of nascent proteins, play essential roles in a series of biological processes. As a type of informational molecule, the O-glycans of these glycoproteins participate directly in these biological mechanisms. Thus, the characteristic differences or changes of O-glycans in expression level usually relate to pathologies of many diseases and represent an important opportunity to uncover the functional mechanisms of various glycoprotein O-glycans. The novel strategy introduced here provides a simple and versatile analytical method for the precise quantitation of glycoprotein O-glycans by mass spectrometry, enabling rapid evaluation of the differences or changes of O-glycans in expression level. It is attractive for the field of quantitative/comparative O-glycomics, which has great significance for exploring the complex structure-function relationship of O-glycans, as well as for the search of O-glycan biomarkers of some major diseases and O-glycan related targets of some drugs. Copyright © 2016 Elsevier B.V. All rights reserved.

CSF N-glycoproteomics for early diagnosis in Alzheimer's disease.

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Palmigiano, Angelo; Barone, Rita; Sturiale, Luisa; Sanfilippo, Cristina; Bua, Rosaria Ornella; Romeo, Donata Agata; Messina, Angela; Capuana, Maria Luisa; Maci, Tiziana; Le Pira, Francesco; Zappia, Mario; Garozzo, Domenico

2016-01-10

This work aims at exploring the human CSF (Cerebrospinal fluid) N-glycome by MALDI MS techniques, in order to assess specific glycosylation pattern(s) in patients with Alzheimer's disease (n:24) and in subjects with mild cognitive impairment (MCI) (n:11), these last as potential AD patients at a pre-dementia stage. For comparison, 21 healthy controls were studied. We identified a group of AD and MCI subjects (about 40-50% of the studied sample) showing significant alteration of CSF N-glycome profiling, consisting of a decrease in the overall sialylation degree and an increase in species bearing bisecting GlcNAc. Noteworthy, all the MCI patients that converted to AD within the clinical follow-up, had an abnormal CSF glycosylation profile. Based on the studied cohort, CSF glycosylation changes may occur before an AD clinical onset. Previous studies specifically focused on the key role of glycosyltransferase GnT-III on AD-pathogenesis, addressing the patho-mechanism to specific sugar modification of BACE-1 glycoprotein with bisecting GlcNAc. Our patients addressed protein N-glycosylation changes at an early phase of the whole biomolecular misregulation on AD, pointing to CSF N-glycome analyses as promising tool to enhance early detection of AD and also suggesting alternative therapeutics target molecules, such as specific glyco-enzymes. Copyright © 2015 Elsevier B.V. All rights reserved.

Multidimensional fractionation is a requirement for quantitation of Golgi-resident glycosylation enzymes from cultured human cells.

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Lin, Chi-Hung; Chik, Jenny H L; Packer, Nicolle H; Molloy, Mark P

2015-02-06

Glycosylation results from the concerted action of glycosylation enzymes in the secretory pathway. In general, gene expression serves as the primary control mechanism, but post-translational fine-tuning of glycosylation enzyme functions is often necessary for efficient synthesis of specific glycan epitopes. While the field of glycomics has rapidly advanced, there lacks routine proteomic methods to measure expression of specific glycosylation enzymes needed to fill the gap between mRNA expression and the glycomic profile in a "reverse genomics" workflow. Toward developing this workflow we enriched Golgi membranes from two human colon cancer cell lines by sucrose density centrifugation and further mass-based fractionation by SDS-PAGE. We then applied mass spectrometry to demonstrate a doubling in the number of Golgi resident proteins identified, compared to the unenriched, low speed centrifuged supernatant of lysed cells. A total of 35 Golgi-resident glycosylation enzymes, of which 23 were glycosyltransferases, were identified making this the largest protein database so far of Golgi resident glycosylation enzymes experimentally identified in cultured human cells. We developed targeted mass spectrometry assays for specific quantitation of many of these glycosylation enzymes. Our results show that alterations in abundance of glycosylation enzymes at the protein level were generally consistent with the resultant glycomic profiles, but not necessarily with the corresponding glycosyltransferase mRNA expression as exemplified by the case of O-glycan core 1 T synthase.

Highly specific purification of N-glycans using phosphate-based derivatization as an affinity tag in combination with Ti{sup 4+}-SPE enrichment for mass spectrometric analysis

Energy Technology Data Exchange (ETDEWEB)

Zhang, Ying [Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University, Shanghai 200032 (China); Key Laboratory of Glycoconjugates Research Ministry of Public Health, Fudan University, Shanghai 200032 (China); Peng, Ye; Bin, Zhichao [Department of Chemistry, Fudan University, Shanghai 200032 (China); Wang, Huijie [Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University, Shanghai 200032 (China); Lu, Haojie, E-mail: luhaojie@fudan.edu.cn [Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University, Shanghai 200032 (China); Department of Chemistry, Fudan University, Shanghai 200032 (China); Key Laboratory of Glycoconjugates Research Ministry of Public Health, Fudan University, Shanghai 200032 (China)

2016-08-31

N-linked protein glycosylation is involved in regulation of a wide variety of cellular processes and associated with numerous diseases. Highly specific identification of N-glycome remains a challenge while its biological significance is acknowledged. The relatively low abundance of glycan in complex biological mixtures, lack of basic sites for protonation, and suppression by other highly abundant proteins/peptides lead to the particularly poor detection sensitivity of N-glycans in the MS analysis. Therefore, the highly specific purification procedure becomes a crucial step prior to MS analysis of the N-glycome. Herein, a novel N-glycans enrichment approach based on phosphate derivatization combined with Ti{sup 4+}-SPE (solid phase extraction) was developed. Briefly, in this strategy, N-glycans were chemically labeled with a phospho-group at their reducing ends, such that the Ti{sup 4+}-SPE microspheres were able to capture the phospho-containing glycans. The enrichment method was developed and optimized using model oligosaccharides (maltoheptaose DP7 and sialylated glycan A1) and also glycans from a standard glycoprotein (asialofetuin, ASF). This method experimentally showed high derivatization efficiency (almost 100%), excellent selectivity (analyzing DP7 in the digests of bovine serum albumin at a mass ratio of 1:100), high enriching recovery (90%), good reproducibility (CV<15%) as well as high sensitivity (LOD at fmol level). At last, the proposed method was successfully applied in the profiling of N-glycome in human serum, in which a total of 31 N-glycan masses were identified. - Graphical abstract: A selective enrichment method for the N-glycome is reported. N-glycans were chemically labeled with a phosphate derivatization reagent (AMS), then the phospho-containing glycans were enriched using Ti{sup 4+}-microspheres. - Highlights: • A highly specific N-glycans purification method based on phosphate derivatization combined with Ti{sup 4+}-SPE was developed

Glycomic Analysis of Prostate Cancer

Science.gov (United States)

2012-07-01

A, Mucci LA, Ekbom A, et al. The risk of liver and bile duct cancer in patients with chronic viral hepatitis, alcoholism, or cirrhosis. Hepatology...the AA and EA men in both GSE6956 and GSE17356 datasets. Table 5. Glycogenes selected from Table 2. N-glycans in liver -secreted and immunoglogulin...January 2012 Available online 3 February 2012 N-glycosylation of proteins provides a rich source of information on liver disease progression

Applications of Azide-Based Bioorthogonal Click Chemistry in Glycobiology

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Xiu Zhang

2013-06-01

Full Text Available Click chemistry is a powerful chemical reaction with excellent bioorthogonality features: biocompatible, rapid and highly specific in biological environments. For glycobiology, bioorthogonal click chemistry has created a new method for glycan non-invasive imaging in living systems, selective metabolic engineering, and offered an elite chemical handle for biological manipulation and glycomics studies. Especially the [3 + 2] dipolar cycloadditions of azides with strained alkynes and the Staudinger ligation of azides and triarylphosphines have been widely used among the extant click reactions. This review focuses on the azide-based bioorthogonal click chemistry, describing the characteristics and development of these reactions, introducing some recent applications in glycobiology research, especially in glycan metabolic engineering, including glycan non-invasive imaging, glycomics studies and viral surface manipulation for drug discovery as well as other applications like activity-based protein profiling and carbohydrate microarrays.

Systems glycobiology for glycoengineering

DEFF Research Database (Denmark)

Spahn, Philipp N.; Lewis, Nathan

2014-01-01

, systems-level analyses of glycan diversity are elucidating deeper insights into the mechanisms underlying glycosylation. As computational models of glycosylation continue to be expanded, refined, and leveraged for detailed analysis of glycomics data, they will become invaluable resources for cell line...

High-throughput antibody development and retrospective epitope mapping

DEFF Research Database (Denmark)

Rydahl, Maja Gro

the binding profile - in more or less high resolution - of two small molecular probes, 11 carbohydrate binding modules and 24 monoclonal antibodies. This was made possible by combining the HTP multiplexing capacity of carbohydrate microarrays with diverse glycomic tools, to downstream characterize...

Variation of Human Salivary O-Glycome.

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Radoslaw P Kozak

Full Text Available The study of saliva O-glycosylation is receiving increasing attention due to the potential of glycans for disease biomarkers, but also due to easy access and non-invasive collection of saliva as biological fluid. Saliva is rich in glycoproteins which are secreted from the bloodstream or produced by salivary glands. Mucins, which are highly O-glycosylated proteins, are particularly abundant in human saliva. Their glycosylation is associated with blood group and secretor status, and represents a reservoir of potential disease biomarkers. This study aims to analyse and compare O-glycans released from whole human mouth saliva collected 3 times a day from a healthy individual over a 5 days period. O-linked glycans were released by hydrazinolysis, labelled with procainamide and analysed by ultra-high performance liquid chromatography with fluorescence detection (UHPLC-FLR coupled to electrospray ionization mass spectrometry (ESI-MS/MS. The sample preparation method showed excellent reproducibility and can therefore be used for biomarker discovery. Our data demonstrates that the O-glycosylation in human saliva changes significantly during the day. These changes may be related to changes in the salivary concentrations of specific proteins.

The S-Layer Glycome—Adding to the Sugar Coat of Bacteria

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Robin Ristl

2011-01-01

Full Text Available The amazing repertoire of glycoconjugates present on bacterial cell surfaces includes lipopolysaccharides, capsular polysaccharides, lipooligosaccharides, exopolysaccharides, and glycoproteins. While the former are constituents of Gram-negative cells, we review here the cell surface S-layer glycoproteins of Gram-positive bacteria. S-layer glycoproteins have the unique feature of self-assembling into 2D lattices providing a display matrix for glycans with periodicity at the nanometer scale. Typically, bacterial S-layer glycans are O-glycosidically linked to serine, threonine, or tyrosine residues, and they rely on a much wider variety of constituents, glycosidic linkage types, and structures than their eukaryotic counterparts. As the S-layer glycome of several bacteria is unravelling, a picture of how S-layer glycoproteins are biosynthesized is evolving. X-ray crystallography experiments allowed first insights into the catalysis mechanism of selected enzymes. In the future, it will be exciting to fully exploit the S-layer glycome for glycoengineering purposes and to link it to the bacterial interactome.

Protein-carbohydrate complex reveals circulating metastatic cells in a microfluidic assay

KAUST Repository

Simone, Giuseppina

2013-02-11

Advances in carbohydrate sequencing technologies reveal the tremendous complexity of the glycome and the role that glycomics might have to bring insight into the biological functions. Carbohydrate-protein interactions, in particular, are known to be crucial to most mammalian physiological processes as mediators of cell adhesion and metastasis, signal transducers, and organizers of protein interactions. An assay is developed here to mimic the multivalency of biological complexes that selectively and sensitively detect carbohydrate-protein interactions. The binding of β-galactosides and galectin-3 - a protein that is correlated to the progress of tumor and metastasis - is examined. The efficiency of the assay is related to the expression of the receptor while anchoring to the interaction\\'s strength. Comparative binding experiments reveal molecular binding preferences. This study establishes that the assay is robust to isolate metastatic cells from colon affected patients and paves the way to personalized medicine. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Xylan hydrolysis in Populus trichocarpa × P. deltoides and model substrates during hydrothermal pretreatment.

Science.gov (United States)

Trajano, Heather L; Pattathil, Sivakumar; Tomkins, Bruce A; Tschaplinski, Timothy J; Hahn, Michael G; Van Berkel, Gary J; Wyman, Charles E

2015-03-01

Previous studies defined easy and difficult to hydrolyze fractions of hemicellulose that may result from bonds among cellulose, hemicellulose, and lignin. To understand how such bonds affect hydrolysis, Populus trichocarpa × Populus deltoides, holocellulose isolated from P. trichocarpa × P. deltoides and birchwood xylan were subjected to hydrothermal flow-through pretreatment. Samples were characterized by glycome profiling, HPLC, and UPLC-MS. Glycome profiling revealed steady fragmentation and removal of glycans from solids during hydrolysis. The extent of polysaccharide fragmentation, hydrolysis rate, and total xylose yield were lowest for P. trichocarpa × P. deltoides and greatest for birchwood xylan. Comparison of results from P. trichocarpa × P. deltoides and holocellulose suggested that lignin-carbohydrate complexes reduce hydrolysis rates and limit release of large xylooligomers. Smaller differences between results with holocellulose and birchwood xylan suggest xylan-cellulose hydrogen bonds limited hydrolysis, but to a lesser extent. These findings imply cell wall structure strongly influences hydrolysis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Protein-carbohydrate complex reveals circulating metastatic cells in a microfluidic assay

KAUST Repository

Simone, Giuseppina; Malara, Natalia Maria; Trunzo, Valentina; Perozziello, Gerardo; Neužil, Pavel; Francardi, Marco; Roveda, Laura; Renne, Maria; Prati, Ubaldo; Mollace, Vincenzo; Manz, Andreas; Di Fabrizio, Enzo M.

2013-01-01

Advances in carbohydrate sequencing technologies reveal the tremendous complexity of the glycome and the role that glycomics might have to bring insight into the biological functions. Carbohydrate-protein interactions, in particular, are known to be crucial to most mammalian physiological processes as mediators of cell adhesion and metastasis, signal transducers, and organizers of protein interactions. An assay is developed here to mimic the multivalency of biological complexes that selectively and sensitively detect carbohydrate-protein interactions. The binding of β-galactosides and galectin-3 - a protein that is correlated to the progress of tumor and metastasis - is examined. The efficiency of the assay is related to the expression of the receptor while anchoring to the interaction's strength. Comparative binding experiments reveal molecular binding preferences. This study establishes that the assay is robust to isolate metastatic cells from colon affected patients and paves the way to personalized medicine. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Changes in the expression of N- and O-glycopeptides in patients with colorectal cancer and hepatocellular carcinoma quantified by full-MS scan FT-ICR and multiple reaction monitoring

Czech Academy of Sciences Publication Activity Database

Darebná, P.; Novák, Petr; Kučera, R.; Topolčan, O.; Sanda, M.; Goldman, R.; Pompach, Petr

2017-01-01

Roč. 153, SI (2017), s. 44-52 ISSN 1874-3919 Grant - others:Ministerstvo pro místní rozvoj(CZ) CZ2.16./3.1.00/24023 Institutional support: RVO:61388971 Keywords : Mass spectrometry * Glycomics * FT-ICR Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 3.914, year: 2016

Chemomics-based marker compounds mining and mimetic processing for exploring chemical mechanisms in traditional processing of herbal medicines, a continuous study on Rehmanniae Radix.

Science.gov (United States)

Zhou, Li; Xu, Jin-Di; Zhou, Shan-Shan; Shen, Hong; Mao, Qian; Kong, Ming; Zou, Ye-Ting; Xu, Ya-Yun; Xu, Jun; Li, Song-Lin

2017-12-29

Exploring processing chemistry, in particular the chemical transformation mechanisms involved, is a key step to elucidate the scientific basis in traditional processing of herbal medicines. Previously, taking Rehmanniae Radix (RR) as a case study, the holistic chemome (secondary metabolome and glycome) difference between raw and processed RR was revealed by integrating hyphenated chromatographic techniques-based targeted glycomics and untargeted metabolomics. Nevertheless, the complex chemical transformation mechanisms underpinning the holistic chemome variation in RR processing remain to be extensively clarified. As a continuous study, here a novel strategy by combining chemomics-based marker compounds mining and mimetic processing is proposed for further exploring the chemical mechanisms involved in herbal processing. First, the differential marker compounds between raw and processed herbs were rapidly discovered by untargeted chemomics-based mining approach through multivariate statistical analysis of the chemome data obtained by integrated metabolomics and glycomics analysis. Second, the marker compounds were mimetically processed under the simulated physicochemical conditions as in the herb processing, and the final reaction products were chemically characterized by targeted chemomics-based mining approach. Third, the main chemical transformation mechanisms involved were clarified by linking up the original marker compounds and their mimetic processing products. Using this strategy, a set of differential marker compounds including saccharides, glycosides and furfurals in raw and processed RR was rapidly found, and the major chemical mechanisms involved in RR processing were elucidated as stepwise transformations of saccharides (polysaccharides, oligosaccharides and monosaccharides) and glycosides (iridoid glycosides and phenethylalcohol glycosides) into furfurals (glycosylated/non-glycosylated hydroxymethylfurfurals) by deglycosylation and/or dehydration. The

Determination of N-glycans by high performance liquid chromatography using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate as the glycosylamine labeling reagent.

Science.gov (United States)

Wu, Yike; Sha, Qiuyue; Du, Juan; Wang, Chang; Zhang, Liang; Liu, Bi-Feng; Lin, Yawei; Liu, Xin

2018-02-02

Robust, efficient identification and accurate quantification of N-glycans are of great significance in N-glycomics analysis. Here, a simple and rapid derivatization method, based on the combination of microwave-assisted deglycosylation and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) labeling, was developed for the analysis of N-glycan by high performance liquid chromatography with fluorescence detection (HPLC-FLD). After optimizing various parameters affecting deglycosylation and derivatization by RNase B, the time for N-glycan labeling was shortened to 50 min with ∼10-fold enhancement in detection sensitivity comparing to conventional 2-aminobenzoic acid (2-AA) labeling method. Additionally, the method showed good linearity (correlation coefficients > 0.991) and reproducibility (RSD < 8.7%). These advantages of the proposed method were further validated by the analysis of complex samples, including fetuin and human serum. Investigation of serum N-glycome for preliminary diagnosis of human lung cancer was conducted, where significant changes of several N-glycans corresponding to core-fucosylated, mono- and disialylated glycans have been evidenced by a series of statistical analysis. Copyright © 2018 Elsevier B.V. All rights reserved.

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. ...

Total plasma N-glycome changes during pregnancy.

NARCIS (Netherlands)

Ruhaak, L.R.; Uh, H.W.; Deelder, A.M.; Dolhain, R.E.; Wuhrer, M.

2014-01-01

During pregnancy, the mother faces a major immunological challenge. Most of the major plasma proteins have important immunological functions, and altered levels of these major proteins have been reported during pregnancy, potentially providing immunosuppression. A large number of the high abundance

A Multi-Omic View of Host-Pathogen-Commensal Interplay in Salmonella-Mediated Intestinal Infection

Energy Technology Data Exchange (ETDEWEB)

Kaiser, Brooke LD; Li, Jie; Sanford, James A.; Kim, Young-Mo; Kronewitter, Scott R.; Jones, Marcus B.; Peterson, Christine; Peterson, Scott N.; Frank, Bryan C.; Purvine, Samuel O.; Brown, Joseph N.; Metz, Thomas O.; Smith, Richard D.; Heffron, Fred; Adkins, Joshua N.

2013-06-26

The potential for commensal microorganisms indigenous to a host (the ‘microbiome’ or ‘microbiota’) to alter infection outcome by influencing host-pathogen interplay is largely unknown. We used a multi-omics “systems” approach, incorporating proteomics, metabolomics, glycomics, and metagenomics, to explore the molecular interplay between the murine host, the pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), and commensal gut microorganisms during intestinal infection with S. Typhimurium. We find proteomic evidence that S. Typhimurium thrives within the infected 129/SvJ mouse gut without antibiotic pre-treatment, inducing inflammation and disrupting the intestinal microbiome (e.g., suppressing Bacteroidetes and Firmicutes while promoting growth of Salmonella and Enterococcus). Alteration of the host microbiome population structure was highly correlated with gut environmental changes, including the accumulation of metabolites normally consumed by commensal microbiota. Finally, the less characterized phase of S. Typhimurium’s lifecycle was investigated, and both proteomic and glycomic evidence suggests S. Typhimurium may take advantage of increased fucose moieties to metabolize fucose while growing in the gut. The application of multiple omics measurements to Salmonella-induced intestinal inflammation provides insights into complex molecular strategies employed during pathogenesis between host, pathogen, and the microbiome.

Human Plasma N-glycosylation as Analyzed by Matrix-Assisted Laser Desorption/Ionization-Fourier Transform Ion Cyclotron Resonance-MS Associates with Markers of Inflammation and Metabolic Health*

Science.gov (United States)

Reiding, Karli R.; Ruhaak, L. Renee; Uh, Hae-Won; el Bouhaddani, Said; van den Akker, Erik B.; Plomp, Rosina; McDonnell, Liam A.; Houwing-Duistermaat, Jeanine J.; Slagboom, P. Eline; Beekman, Marian; Wuhrer, Manfred

2017-01-01

Glycosylation is an abundant co- and post-translational protein modification of importance to protein processing and activity. Although not template-defined, glycosylation does reflect the biological state of an organism and is a high-potential biomarker for disease and patient stratification. However, to interpret a complex but informative sample like the total plasma N-glycome, it is important to establish its baseline association with plasma protein levels and systemic processes. Thus far, large-scale studies (n >200) of the total plasma N-glycome have been performed with methods of chromatographic and electrophoretic separation, which, although being informative, are limited in resolving the structural complexity of plasma N-glycans. MS has the opportunity to contribute additional information on, among others, antennarity, sialylation, and the identity of high-mannose type species. Here, we have used matrix-assisted laser desorption/ionization (MALDI)-Fourier transform ion cyclotron resonance (FTICR)-MS to study the total plasma N-glycome of 2144 healthy middle-aged individuals from the Leiden Longevity Study, to allow association analysis with markers of metabolic health and inflammation. To achieve this, N-glycans were enzymatically released from their protein backbones, labeled at the reducing end with 2-aminobenzoic acid, and following purification analyzed by negative ion mode intermediate pressure MALDI-FTICR-MS. In doing so, we achieved the relative quantification of 61 glycan compositions, ranging from Hex4HexNAc2 to Hex7HexNAc6dHex1Neu5Ac4, as well as that of 39 glycosylation traits derived thereof. Next to confirming known associations of glycosylation with age and sex by MALDI-FTICR-MS, we report novel associations with C-reactive protein (CRP), interleukin 6 (IL-6), body mass index (BMI), leptin, adiponectin, HDL cholesterol, triglycerides (TG), insulin, gamma-glutamyl transferase (GGT), alanine aminotransferase (ALT), and smoking. Overall, the

Systematic Comparison of Reverse Phase and Hydrophilic Interaction Liquid Chromatography Platforms for the Analysis of N-linked Glycans

Science.gov (United States)

Walker, S. Hunter; Carlisle, Brandon C.; Muddiman, David C.

2013-01-01

Due to the hydrophilic nature of glycans, reverse phase chromatography has not been widely used as a glycomic separation technique coupled to mass spectrometry. Other approaches such as hydrophilic interaction chromatography and porous graphitized carbon chromatography are often employed, though these strategies frequently suffer from decreased chromatographic resolution, long equilibration times, indefinite retention, and column bleed. Herein, it is shown that through an efficient hydrazone formation derivatization of N-linked glycans (∼4 hr of additional sample preparation time which is carried out in parallel), numerous experimental and practical advantages are gained when analyzing the glycans by online reverse phase chromatography. These benefits include an increased number of glycans detected, increased peak capacity of the separation, and the ability to analyze glycans on the identical liquid chromatography-mass spectrometry platform commonly used for proteomic analyses. The data presented show that separation of derivatized N-linked glycans by reverse phase chromatography significantly out-performs traditional separation of native or derivatized glycans by hydrophilic interaction chromatography. Furthermore, the movement to a more ubiquitous separation technique will afford numerous research groups the opportunity to analyze both proteomic and glycomic samples on the same platform with minimal time and physical change between experiments, increasing the efficiency of ‘multi-omic’ biological approaches. PMID:22954204

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.

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 ...

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

Human Plasma N-glycosylation as Analyzed by Matrix-Assisted Laser Desorption/Ionization-Fourier Transform Ion Cyclotron Resonance-MS Associates with Markers of Inflammation and Metabolic Health.

Science.gov (United States)

Reiding, Karli R; Ruhaak, L Renee; Uh, Hae-Won; El Bouhaddani, Said; van den Akker, Erik B; Plomp, Rosina; McDonnell, Liam A; Houwing-Duistermaat, Jeanine J; Slagboom, P Eline; Beekman, Marian; Wuhrer, Manfred

2017-02-01

Glycosylation is an abundant co- and post-translational protein modification of importance to protein processing and activity. Although not template-defined, glycosylation does reflect the biological state of an organism and is a high-potential biomarker for disease and patient stratification. However, to interpret a complex but informative sample like the total plasma N-glycome, it is important to establish its baseline association with plasma protein levels and systemic processes. Thus far, large-scale studies (n >200) of the total plasma N-glycome have been performed with methods of chromatographic and electrophoretic separation, which, although being informative, are limited in resolving the structural complexity of plasma N-glycans. MS has the opportunity to contribute additional information on, among others, antennarity, sialylation, and the identity of high-mannose type species.Here, we have used matrix-assisted laser desorption/ionization (MALDI)-Fourier transform ion cyclotron resonance (FTICR)-MS to study the total plasma N-glycome of 2144 healthy middle-aged individuals from the Leiden Longevity Study, to allow association analysis with markers of metabolic health and inflammation. To achieve this, N-glycans were enzymatically released from their protein backbones, labeled at the reducing end with 2-aminobenzoic acid, and following purification analyzed by negative ion mode intermediate pressure MALDI-FTICR-MS. In doing so, we achieved the relative quantification of 61 glycan compositions, ranging from Hex 4 HexNAc 2 to Hex 7 HexNAc 6 dHex 1 Neu5Ac 4 , as well as that of 39 glycosylation traits derived thereof. Next to confirming known associations of glycosylation with age and sex by MALDI-FTICR-MS, we report novel associations with C-reactive protein (CRP), interleukin 6 (IL-6), body mass index (BMI), leptin, adiponectin, HDL cholesterol, triglycerides (TG), insulin, gamma-glutamyl transferase (GGT), alanine aminotransferase (ALT), and smoking. Overall

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

One-pot multienzyme (OPME) systems for chemoenzymatic synthesis of carbohydrates.

Science.gov (United States)

Yu, Hai; Chen, Xi

2016-03-14

Glycosyltransferase-catalyzed enzymatic and chemoenzymatic syntheses are powerful approaches for the production of oligosaccharides, polysaccharides, glycoconjugates, and their derivatives. Enzymes involved in the biosynthesis of sugar nucleotide donors can be combined with glycosyltransferases in one pot for efficient production of the target glycans from simple monosaccharides and acceptors. The identification of enzymes involved in the salvage pathway of sugar nucleotide generation has greatly facilitated the development of simplified and efficient one-pot multienzyme (OPME) systems for synthesizing major glycan epitopes in mammalian glycomes. The applications of OPME methods are steadily gaining popularity mainly due to the increasing availability of wild-type and engineered enzymes. Substrate promiscuity of these enzymes and their mutants allows OPME synthesis of carbohydrates with naturally occurring post-glycosylational modifications (PGMs) and their non-natural derivatives using modified monosaccharides as precursors. The OPME systems can be applied in sequence for synthesizing complex carbohydrates. The sequence of the sequential OPME processes, the glycosyltransferase used, and the substrate specificities of the glycosyltransferases define the structures of the products. The OPME and sequential OPME strategies can be extended to diverse glycans in other glycomes when suitable enzymes with substrate promiscuity become available. This Perspective summarizes the work of the authors and collaborators on the development of glycosyltransferase-based OPME systems for carbohydrate synthesis. Future directions are also discussed.

Chemoselective Reactions for the Synthesis of Glycoconjugates from Unprotected Carbohydrates

DEFF Research Database (Denmark)

Villadsen, Klaus; Martos Maldonado, Manuel Cristo; Jensen, Knud Jørgen

2017-01-01

Glycobiology is the comprehensive biological investigation of carbohydrates. The study of the role and function of complex carbohydrates often requires the attachment of carbohydrates to surfaces, their tagging with fluorophores, or their conversion into natural or non-natural glycoconjugates......, such as glycopeptides or glycolipids. Glycobiology and its “omics”, glycomics, require easy and robust chemical methods for the construction of these glycoconjugates. This review gives an overview of the rapidly expanding field of chemical reactions that selectively convert unprotected carbohydrates...

Glycomics: revealing the dynamic ecology and evolution of sugar molecules.

Science.gov (United States)

Springer, Stevan A; Gagneux, Pascal

2016-03-01

Sugars are the most functionally and structurally diverse molecules in the biological world. Glycan structures range from tiny single monosaccharide units to giant chains thousands of units long. Some glycans are branched, their monosaccharides linked together in many different combinations and orientations. Some exist as solitary molecules; others are conjugated to proteins and lipids and alter their collective functional properties. In addition to structural and storage roles, glycan molecules participate in and actively regulate physiological and developmental processes. Glycans also mediate cellular interactions within and between individuals. Their roles in ecology and evolution are pivotal, but not well studied because glycan biochemistry requires different methods than standard molecular biology practice. The properties of glycans are in some ways convenient, and in others challenging. Glycans vary on organismal timescales, and in direct response to physiological and ecological conditions. Their mature structures are physical records of both genetic and environmental influences during maturation. We describe the scope of natural glycan variation and discuss how studying glycans will allow researchers to further integrate the fields of ecology and evolution. Copyright © 2015 Elsevier B.V. All rights reserved.

Comparative transcriptomics indicate changes in cell wall organization and stress response in seedlings during spaceflight.

Science.gov (United States)

Johnson, Christina M; Subramanian, Aswati; Pattathil, Sivakumar; Correll, Melanie J; Kiss, John Z

2017-08-21

Plants will play an important role in the future of space exploration as part of bioregenerative life support. Thus, it is important to understand the effects of microgravity and spaceflight on gene expression in plant development. We analyzed the transcriptome of Arabidopsis thaliana using the Biological Research in Canisters (BRIC) hardware during Space Shuttle mission STS-131. The bioinformatics methods used included RMA (robust multi-array average), MAS5 (Microarray Suite 5.0), and PLIER (probe logarithmic intensity error estimation). Glycome profiling was used to analyze cell wall composition in the samples. In addition, our results were compared to those of two other groups using the same hardware on the same mission (BRIC-16). In our BRIC-16 experiments, we noted expression changes in genes involved in hypoxia and heat shock responses, DNA repair, and cell wall structure between spaceflight samples compared to the ground controls. In addition, glycome profiling supported our expression analyses in that there was a difference in cell wall components between ground control and spaceflight-grown plants. Comparing our studies to those of the other BRIC-16 experiments demonstrated that, even with the same hardware and similar biological materials, differences in results in gene expression were found among these spaceflight experiments. A common theme from our BRIC-16 space experiments and those of the other two groups was the downregulation of water stress response genes in spaceflight. In addition, all three studies found differential regulation of genes associated with cell wall remodeling and stress responses between spaceflight-grown and ground control plants. © 2017 Botanical Society of America.

Validation of standard operating procedures in a multicenter retrospective study to identify -omics biomarkers for chronic low back pain.

Directory of Open Access Journals (Sweden)

Concetta Dagostino

Full Text Available Chronic low back pain (CLBP is one of the most common medical conditions, ranking as the greatest contributor to global disability and accounting for huge societal costs based on the Global Burden of Disease 2010 study. Large genetic and -omics studies provide a promising avenue for the screening, development and validation of biomarkers useful for personalized diagnosis and treatment (precision medicine. Multicentre studies are needed for such an effort, and a standardized and homogeneous approach is vital for recruitment of large numbers of participants among different centres (clinical and laboratories to obtain robust and reproducible results. To date, no validated standard operating procedures (SOPs for genetic/-omics studies in chronic pain have been developed. In this study, we validated an SOP model that will be used in the multicentre (5 centres retrospective "PainOmics" study, funded by the European Community in the 7th Framework Programme, which aims to develop new biomarkers for CLBP through three different -omics approaches: genomics, glycomics and activomics. The SOPs describe the specific procedures for (1 blood collection, (2 sample processing and storage, (3 shipping details and (4 cross-check testing and validation before assays that all the centres involved in the study have to follow. Multivariate analysis revealed the absolute specificity and homogeneity of the samples collected by the five centres for all genetics, glycomics and activomics analyses. The SOPs used in our multicenter study have been validated. Hence, they could represent an innovative tool for the correct management and collection of reliable samples in other large-omics-based multicenter studies.

Prebiotics: why definitions matter

Science.gov (United States)

Hutkins, Robert W; Krumbeck, Janina A; Bindels, Laure B; Cani, Patrice D; Fahey, George; Goh, Yong Jun; Hamaker, Bruce; Martens, Eric C; Mills, David A; Rastal, Robert A; Vaughan, Elaine; Sanders, Mary Ellen

2015-01-01

The prebiotic concept was introduced twenty years ago, and despite several revisions to the original definition, the scientific community has continued to debate what it means to be a prebiotic. How prebiotics are defined is important not only for the scientific community, but also for regulatory agencies, the food industry, consumers and healthcare professionals. Recent developments in community-wide sequencing and glycomics have revealed that more complex interactions occur between putative prebiotic substrates and the gut microbiota than previously considered. A consensus among scientists on the most appropriate definition of a prebiotic is necessary to enable continued use of the term. PMID:26431716

Studies and Applications of Metals for the Synthesis of Carbinols, Amides and Carbohydrates

DEFF Research Database (Denmark)

Osztrovszky, Gyorgyi

for the amidation. These two systems do not show any significant differences in reactivity indicating that the same catalytically active species is operating. Project 3: Synthesis of a trisaccharide probe as a putative dengue virus receptor At the Institute for Glycomics major research has been devoted to identify...... putative receptors for dengue virus (DENV). Based on previous studies the GlcNAcß1-3Galß1-4GlcNAc trisaccharide was considered as a putative virus receptor. The synthesis of the trisaccharide probe has been achieved by the coupling of the corresponding D-glucosamine donor and the lactosamine acceptor...

Cell wall changes during the formation of aerenchyma in sugarcane roots.

Science.gov (United States)

Leite, D C C; Grandis, A; Tavares, E Q P; Piovezani, A R; Pattathil, S; Avci, U; Rossini, A; Cambler, A; De Souza, A P; Hahn, M G; Buckeridge, M S

2017-11-10

Aerenchyma develops in different plant organs and leads to the formation of intercellular spaces that can be used by the plant to transport volatile substances. Little is known about the role of cell walls in this process, although the mechanism of aerenchyma formation is known to involve programmed cell death and some cell wall modifications. We assessed the role that cell wall-related mechanisms might play in the formation of aerenchyma in sugarcane roots. Sections of roots (5 cm) were subjected to microtomography analysis. These roots were divided into 1-cm segments and subjected to cell wall fractionation. We performed analyses of monosaccharides, oligosaccharides and lignin and glycome profiling. Sections were visualized by immunofluorescence and immunogold labelling using selected monoclonal antibodies against polysaccharide epitopes according to the glycome profiles. During aerenchyma formation, gas spaces occupied up to 40 % of the cortex cross-section within the first 5 cm of the root. As some of the cortex cells underwent dissolution of the middle lamellae, leading to cell separation, cell expansion took place along with cell death. Mixed-linkage β-glucan was degraded along with some homogalacturonan and galactan, culminating in the formation of cell wall composites made of xyloglucan, arabinoxylans, cellulose and possibly lignin. The composites formed seem to play a role in the physical-chemical properties of the gas chambers, providing mechanical resistance to forces acting upon the root and at the same time decreasing permeability to gases. © The Author 2017. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com

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

Science.gov (United States)

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

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

Science.gov (United States)

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

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

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

Electrochemistry of Nonconjugated Proteins and Glycoproteins. Toward Sensors for Biomedicine and Glycomics

Czech Academy of Sciences Publication Activity Database

Paleček, Emil; Tkáč, J.; Bartošík, M.; Bertok, T.; Ostatná, Veronika; Paleček, J.

2015-01-01

Roč. 115, č. 5 (2015), s. 2045-2108 ISSN 0009-2665 R&D Projects: GA ČR(CZ) GAP301/11/2055; GA ČR(CZ) GA15-15479S; GA ČR(CZ) GA13-00956S Institutional support: RVO:68081707 Keywords : LABEL-FREE DETECTION * ESCHERICHIA - COLI MUTY * BETA-AMYLOID FIBRILS Subject RIV: BO - Biophysics Impact factor: 37.369, year: 2015

Ion Mobility Separations of Isomers based upon Long Path Length Structures for Lossless Ion Manipulations Combined with Mass Spectrometry

Energy Technology Data Exchange (ETDEWEB)

Deng, Liulin [Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 902 Battelle Blvd Richland, WA 99352 USA; Ibrahim, Yehia M. [Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 902 Battelle Blvd Richland, WA 99352 USA; Baker, Erin S. [Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 902 Battelle Blvd Richland, WA 99352 USA; Aly, Noor A. [Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 902 Battelle Blvd Richland, WA 99352 USA; Hamid, Ahmed M. [Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 902 Battelle Blvd Richland, WA 99352 USA; Zhang, Xing [Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 902 Battelle Blvd Richland, WA 99352 USA; Zheng, Xueyun [Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 902 Battelle Blvd Richland, WA 99352 USA; Garimella, Sandilya V. B. [Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 902 Battelle Blvd Richland, WA 99352 USA; Webb, Ian K. [Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 902 Battelle Blvd Richland, WA 99352 USA; Prost, Spencer A. [Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 902 Battelle Blvd Richland, WA 99352 USA; Sandoval, Jeremy A. [Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 902 Battelle Blvd Richland, WA 99352 USA; Norheim, Randolph V. [Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 902 Battelle Blvd Richland, WA 99352 USA; Anderson, Gordon A. [Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 902 Battelle Blvd Richland, WA 99352 USA; Tolmachev, Aleksey V. [Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 902 Battelle Blvd Richland, WA 99352 USA; Smith, Richard D. [Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 902 Battelle Blvd Richland, WA 99352 USA

2016-07-01

Mass spectrometry (MS)-based multi-omic measurements, including proteomics, metabolomics, lipidomics, and glycomics, are increasingly transforming our ability to characterize and understand biological systems, but, presently have limitations due to the chemical diversity and range of abundances of biomolecules in complex samples. Advances addressing these challenges increasingly are based upon the ability to quickly separate, react and otherwise manipulate sample components for analysis by MS. Here we report on a new approach using Structures for Lossless Ion Manipulations (SLIM) to enable long serpentine path ion mobility spectrometry (IMS) separations followed by MS analyses. This approach provides previously unachieved mobility biomolecule isomer separations for biomolecular species, in conjunction with more effective ion utilization, and producing a basis for the improved characterization of very small samples.

Mapping posttranscriptional regulation of the human glycome uncovers microRNA defining the glycocode

OpenAIRE

Agrawal, Praveen; Kurcon, Tomasz; Pilobello, Kanoelani T.; Rakus, John F.; Koppolu, Sujeethraj; Liu, Zhongyin; Batista, Bianca S.; Eng, William S.; Hsu, Ku-Lung; Liang, Yaxuan; Mahal, Lara K.

2014-01-01

Carbohydrates (glycans) are complex cell surface molecules that control multiple aspects of cell biology, including cell–cell communication, cancer metastasis, and inflammation. Glycan biosynthesis requires the coordination of many enzymes, but how this is regulated is not well understood. Herein we show that microRNA (miRNA), small noncoding RNA, are a major regulator of cell surface glycosylation. We map miRNA expression onto carbohydrate signatures obtained by using lectin microarrays, a g...

'Omics' biomarkers associated with chronic low back pain: protocol of a retrospective longitudinal study.

Science.gov (United States)

Allegri, Massimo; De Gregori, Manuela; Minella, Cristina E; Klersy, Catherine; Wang, Wei; Sim, Moira; Gieger, Christian; Manz, Judith; Pemberton, Iain K; MacDougall, Jane; Williams, Frances Mk; Van Zundert, Jan; Buyse, Klaas; Lauc, Gordan; Gudelj, Ivan; Primorac, Dragan; Skelin, Andrea; Aulchenko, Yurii S; Karssen, Lennart C; Kapural, Leonardo; Rauck, Richard; Fanelli, Guido

2016-10-19

Chronic low back pain (CLBP) produces considerable direct costs as well as indirect burdens for society, industry and health systems. CLBP is characterised by heterogeneity, inclusion of several pain syndromes, different underlying molecular pathologies and interaction with psychosocial factors that leads to a range of clinical manifestations. There is still much to understand in the underlying pathological processes and the non-psychosocial factors which account for differences in outcomes. Biomarkers that may be objectively used for diagnosis and personalised, targeted and cost-effective treatment are still lacking. Therefore, any data that may be obtained at the '-omics' level (glycomics, Activomics and genome-wide association studies-GWAS) may be helpful to use as dynamic biomarkers for elucidating CLBP pathogenesis and may ultimately provide prognostic information too. By means of a retrospective, observational, case-cohort, multicentre study, we aim to investigate new promising biomarkers potentially able to solve some of the issues related to CLBP. The study follows a two-phase, 1:2 case-control model. A total of 12 000 individuals (4000 cases and 8000 controls) will be enrolled; clinical data will be registered, with particular attention to pain characteristics and outcomes of pain treatments. Blood samples will be collected to perform -omics studies. The primary objective is to recognise genetic variants associated with CLBP; secondary objectives are to study glycomics and Activomics profiles associated with CLBP. The study is part of the PainOMICS project funded by European Community in the Seventh Framework Programme. The study has been approved from competent ethical bodies and copies of approvals were provided to the European Commission before starting the study. Results of the study will be reviewed by the Scientific Board and Ethical Committee of the PainOMICS Consortium. The scientific results will be disseminated through peer-reviewed journals

Negligible elongation of mucin glycans with Gal β1-3 units distinguishes the laminated layer of Echinococcus multilocularis from that of Echinococcus granulosus.

Science.gov (United States)

Del Puerto, Lucía; Rovetta, Romina; Navatta, Marco; Fontana, Carolina; Lin, Gerardo; Moyna, Guillermo; Dematteis, Sylvia; Brehm, Klaus; Koziol, Uriel; Ferreira, Fernando; Díaz, Alvaro

2016-05-01

The larval stages of the cestodes Echinococcus multilocularis and Echinococcus granulosus cause the important zoonoses known as larval echinococcoses. These larvae are protected by a unique, massive, mucin-based structure known as the laminated layer. The mucin glycans of the E. granulosus laminated layer are core 1- or core 2-based O-glycans in which the core Galpβ1-3 residue can initiate a chain comprising one to three additional Galpβ1-3 residues, a motif not known in mammalian carbohydrates. This chain can be capped with a Galpα1-4 residue, and can be ramified with GlcNAcpβ1-6 residues. These, as well as the GlcNAcpβ1-6 residue in core 2, can be decorated with the Galpα1-4Galpβ1-4 disaccharide. Here we extend our analysis to the laminated layer of E. multilocularis, showing that the non-decorated cores, together with Galpβ1-3(Galpα1-4Galpβ1-4GlcNAcpβ1-6)GalNAc, comprise over 96% of the glycans in molar terms. This simple laminated layer glycome is exhibited by E. multilocularis grown either in vitro or in vivo. Interestingly, all the differences with the complex laminated layer glycome found in E. granulosus may be explained in terms of strongly reduced activity in E. multilocularis of a putative glycosyltransferase catalysing the elongation with Galpβ1-3. Comparative inter-species analysis of available genomic and transcriptomic data suggested a candidate for this enzyme, amongst more than 20 putative (non-core 1) Gal/GlcNAc β1-3 transferases present in each species as a result of a taeniid-specific gene expansion. The candidate gene was experimentally verified to be transcribed at much higher levels in the larva of E. granulosus than that of E. multilocularis. Copyright © 2016 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Involvement of Aberrant Glycosylation in Thyroid Cancer

Directory of Open Access Journals (Sweden)

Eiji Miyoshi

2010-01-01

Full Text Available Glycosylation is one of the most common posttranslational modification reactions and nearly half of all known proteins in eukaryotes are glycosylated. In fact, changes in oligosaccharides structures are associated with many physiological and pathological events, including cell growth, migration and differentiation, and tumor invasion. Therefore, functional glycomics, which is a comprehensive study of the structures and functions of glycans, is attracting the increasing attention of scientists in various fields of life science. In cases of thyroid cancer, the biological characters and prognosis are completely different in each type of histopathology, and their oligosaccharide structures as well as the expression of glycosyltransferases are also different. In this review, we summarized our previous papers on oligosaccharides and thyroid cancers and discussed a possible function of oligosaccharides in the carcinogenesis in thyroid cancer.

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

Science.gov (United States)

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.

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.

Reversibility of membrane N-glycome of HeLa cells upon treatment with epigenetic inhibitors.

Directory of Open Access Journals (Sweden)

Tomislav Horvat

Full Text Available Glycans are essential regulators of protein function and are now in the focus of research in many physiological and pathophysiological processes. There are numerous modes of regulating their biosynthesis, including epigenetic mechanisms implicated in the expression of glyco-genes. Since N-glycans located at the cell membrane define intercellular communication as well as a cellular response to a given environment, we developed a method to preferentially analyze this fraction of glycans. The method is based on incorporation of living cells into polyacrylamide gels, partial denaturation of membrane proteins with 3 M urea and subsequent release of N-glycans with PNGase F followed by HPLC analysis. Using this newly developed method, we revealed multiple effects of epigenetic inhibitors Trichostatin A, sodium butyrate and zebularine on the composition of N-glycans in human cells. The induced changes were found to be reversible after inhibitor removal. Given that many epigenetic inhibitors are currently explored as a therapeutic strategy in treatment of cancer, wherein surface glycans play an important role, the presented work contributes to our understanding of their efficiency in altering the N-glycan profile of cancer cells in culture.

Characterization of Isomeric Glycans by Reversed Phase Liquid Chromatography-Electronic Excitation Dissociation Tandem Mass Spectrometry

Science.gov (United States)

Tang, Yang; Wei, Juan; Costello, Catherine E.; Lin, Cheng

2018-04-01

The occurrence of numerous structural isomers in glycans from biological sources presents a severe challenge for structural glycomics. The subtle differences among isomeric structures demand analytical methods that can provide structural details while working efficiently with on-line glycan separation methods. Although liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful tool for mixture analysis, the commonly utilized collision-induced dissociation (CID) method often does not generate a sufficient number of fragments at the MS2 level for comprehensive structural characterization. Here, we studied the electronic excitation dissociation (EED) behaviors of metal-adducted, permethylated glycans, and identified key spectral features that could facilitate both topology and linkage determinations. We developed an EED-based, nanoscale, reversed phase (RP)LC-MS/MS platform, and demonstrated its ability to achieve complete structural elucidation of up to five structural isomers in a single LC-MS/MS analysis. [Figure not available: see fulltext.

Development and Application of Multidimensional HPLC Mapping Method for O-linked Oligosaccharides

Directory of Open Access Journals (Sweden)

Koichi Kato

2011-12-01

Full Text Available Glycosylation improves the solubility and stability of proteins, contributes to the structural integrity of protein functional sites, and mediates biomolecular recognition events involved in cell-cell communications and viral infections. The first step toward understanding the molecular mechanisms underlying these carbohydrate functionalities is a detailed characterization of glycan structures. Recently developed glycomic approaches have enabled comprehensive analyses of N-glycosylation profiles in a quantitative manner. However, there are only a few reports describing detailed O-glycosylation profiles primarily because of the lack of a widespread standard method to identify O-glycan structures. Here, we developed an HPLC mapping method for detailed identification of O-glycans including neutral, sialylated, and sulfated oligosaccharides. Furthermore, using this method, we were able to quantitatively identify isomeric products from an in vitro reaction catalyzed by N-acetylglucosamine-6O-sulfotransferases and obtain O-glycosylation profiles of serum IgA as a model glycoprotein.

Mice lacking pituitary tumor transforming gene show elevated exposure of DGalNAc carbohydrate determinants

Directory of Open Access Journals (Sweden)

Lutsyk A. D.

2012-04-01

Full Text Available Aim. To investigate the influence of pituitary tumor transforming gene (pttg-1 knockout on glycome of parenchimal organs by means of lectin histochemistry. Methods. DGalNAc, DGlcNAc, NeuNAc carbohydrate determinants were labelled with soybean agglutinin (SBA and wheat germ agglutinin (WGA, conjugated to peroxidase, with subsequent visualization of the lectin-binding sites with diaminobenzidine. The testes and kidneys of murine strain BL6/C57 with the pttg-1 gene knockout (PTTG-KO were compared to the wild type (PTTG-WT animals, both groups 1 month of age. Results. Knockout of the pttg-1 gene was accompanied by enhanced exposure of the DGalNAc sugar residues within the Golgi complex of secondary spermatocytes, in a brush border of renal tubules and on the lumenal surface of collecting ducts. Conclusions. This study suggests that knockout of the pttg-1 gene may lead to the changes in carbohydrate processing in mammalian organism.

Integrative biological analysis for neuropsychopharmacology.

Science.gov (United States)

Emmett, Mark R; Kroes, Roger A; Moskal, Joseph R; Conrad, Charles A; Priebe, Waldemar; Laezza, Fernanda; Meyer-Baese, Anke; Nilsson, Carol L

2014-01-01

Although advances in psychotherapy have been made in recent years, drug discovery for brain diseases such as schizophrenia and mood disorders has stagnated. The need for new biomarkers and validated therapeutic targets in the field of neuropsychopharmacology is widely unmet. The brain is the most complex part of human anatomy from the standpoint of number and types of cells, their interconnections, and circuitry. To better meet patient needs, improved methods to approach brain studies by understanding functional networks that interact with the genome are being developed. The integrated biological approaches--proteomics, transcriptomics, metabolomics, and glycomics--have a strong record in several areas of biomedicine, including neurochemistry and neuro-oncology. Published applications of an integrated approach to projects of neurological, psychiatric, and pharmacological natures are still few but show promise to provide deep biological knowledge derived from cells, animal models, and clinical materials. Future studies that yield insights based on integrated analyses promise to deliver new therapeutic targets and biomarkers for personalized medicine.

Insights into cell wall structure of Sida hermaphrodita and its influence on recalcitrance.

Science.gov (United States)

Damm, Tatjana; Pattathil, Sivakumar; Günl, Markus; Jablonowski, Nicolai David; O'Neill, Malcolm; Grün, Katharina Susanne; Grande, Philipp Michael; Leitner, Walter; Schurr, Ulrich; Usadel, Björn; Klose, Holger

2017-07-15

The perennial plant Sida hermaphrodita (Sida) is attracting attention as potential energy crop. Here, the first detailed view on non-cellulosic Sida cell wall polysaccharide composition, structure and architecture is given. Cell walls were prepared from Sida stems and sequentially extracted with aqueous buffers and alkali. The structures of the quantitatively predominant polysaccharides present in each fraction were determined by biochemical characterization, glycome profiling and mass spectrometry. The amounts of glucose released by Accellerase-1500 ® treatment of the cell wall and the cell wall residue remaining after each extraction were used to assess the roles of pectin and hemicellulose in the recalcitrance of Sida biomass. 4-O-Methyl glucuronoxylan with a low proportion of side substitutions was identified as the major non-cellulosic glycan component of Sida stem cell walls. Pectic polysaccharides and xylans were found to be associated with lignin, suggesting that these polysaccharides have roles in Sida cell wall recalcitrance to enzymatic hydrolysis. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

The third dimension of reading the sugar code by lectins: design of glycoclusters with cyclic scaffolds as tools with the aim to define correlations between spatial presentation and activity.

Science.gov (United States)

Murphy, Paul V; André, Sabine; Gabius, Hans-Joachim

2013-04-04

Coding of biological information is not confined to nucleic acids and proteins. Endowed with the highest level of structural versatility among biomolecules, the glycan chains of cellular glycoconjugates are well-suited to generate molecular messages/signals in a minimum of space. The sequence and shape of oligosaccharides as well as spatial aspects of multivalent presentation are assumed to underlie the natural specificity/selectivity that cellular glycans have for endogenous lectins. In order to eventually unravel structure-activity profiles cyclic scaffolds have been used as platforms to produce glycoclusters and afford valuable tools. Using adhesion/growth-regulatory galectins and the pan-galectin ligand lactose as a model, emerging insights into the potential of cyclodextrins, cyclic peptides, calixarenes and glycophanes for this purpose are presented herein. The systematic testing of lectin panels with spatially defined ligand presentations can be considered as a biomimetic means to help clarify the mechanisms, which lead to the exquisite accuracy at which endogenous lectins select their physiological counterreceptors from the complexity of the cellular glycome.

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

Directory of Open Access Journals (Sweden)

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.

Improved hydrophilic interaction chromatography LC/MS of heparinoids using a chip with postcolumn makeup flow.

Science.gov (United States)

Staples, Gregory O; Naimy, Hicham; Yin, Hongfeng; Kileen, Kevin; Kraiczek, Karsten; Costello, Catherine E; Zaia, Joseph

2010-01-15

Heparan sulfate (HS) and heparin are linear, heterogeneous carbohydrates of the glycosaminoglycan (GAG) family that are modified by N-acetylation, N-sulfation, O-sulfation, and uronic acid epimerization. HS interacts with growth factors in the extracellular matrix, thereby modulating signaling pathways that govern cell growth, development, differentiation, proliferation, and adhesion. High-performance liquid chromatography (HPLC)-chip-based hydrophilic interaction liquid chromatography/mass spectrometry has emerged as a method for analyzing the domain structure of GAGs. However, analysis of highly sulfated GAG structures decasaccharide or larger in size has been limited by spray instability in the negative-ion mode. This report demonstrates that addition of postcolumn makeup flow to the amide-HPLC-chip configuration permits robust and reproducible analysis of extended GAG domains (up to degree of polymerization 18) from HS and heparin. This platform provides quantitative information regarding the oligosaccharide profile, degree of sulfation, and nonreducing chain termini. It is expected that this technology will enable quantitative, comparative glycomics profiling of extended GAG oligosaccharide domains of functional interest.

Loss of function of cinnamyl alcohol dehydrogenase 1 leads to unconventional lignin and a temperature-sensitive growth defect in Medicago truncatula.

Science.gov (United States)

Zhao, Qiao; Tobimatsu, Yuki; Zhou, Rui; Pattathil, Sivakumar; Gallego-Giraldo, Lina; Fu, Chunxiang; Jackson, Lisa A; Hahn, Michael G; Kim, Hoon; Chen, Fang; Ralph, John; Dixon, Richard A

2013-08-13

There is considerable debate over the capacity of the cell wall polymer lignin to incorporate unnatural monomer units. We have identified Tnt1 retrotransposon insertion mutants of barrel medic (Medicago truncatula) that show reduced lignin autofluorescence under UV microscopy and red coloration in interfascicular fibers. The phenotype is caused by insertion of retrotransposons into a gene annotated as encoding cinnamyl alcohol dehydrogenase, here designated M. truncatula CAD1. NMR analysis indicated that the lignin is derived almost exclusively from coniferaldehyde and sinapaldehyde and is therefore strikingly different from classical lignins, which are derived mainly from coniferyl and sinapyl alcohols. Despite such a major alteration in lignin structure, the plants appear normal under standard conditions in the greenhouse or growth chamber. However, the plants are dwarfed when grown at 30 °C. Glycome profiling revealed an increased extractability of some xylan and pectin epitopes from the cell walls of the cad1-1 mutant but decreased extractability of others, suggesting that aldehyde-dominant lignin significantly alters cell wall structure.

Glycomics meets artificial intelligence - Potential of glycan analysis for identification of seropositive and seronegative rheumatoid arthritis patients revealed.

Science.gov (United States)

Chocholova, Erika; Bertok, Tomas; Jane, Eduard; Lorencova, Lenka; Holazova, Alena; Belicka, Ludmila; Belicky, Stefan; Mislovicova, Danica; Vikartovska, Alica; Imrich, Richard; Kasak, Peter; Tkac, Jan

2018-06-01

In this study, one hundred serum samples from healthy people and patients with rheumatoid arthritis (RA) were analyzed. Standard immunoassays for detection of 10 different RA markers and analysis of glycan markers on antibodies in 10 different assay formats with several lectins were applied for each serum sample. A dataset containing 2000 data points was data mined using artificial neural networks (ANN). We identified key RA markers, which can discriminate between healthy people and seropositive RA patients (serum containing autoantibodies) with accuracy of 83.3%. Combination of RA markers with glycan analysis provided much better discrimination accuracy of 92.5%. Immunoassays completely failed to identify seronegative RA patients (serum not containing autoantibodies), while glycan analysis correctly identified 43.8% of these patients. Further, we revealed other critical parameters for successful glycan analysis such as type of a sample, format of analysis and orientation of captured antibodies for glycan analysis. Copyright © 2018 Elsevier B.V. All rights reserved.

Profiling analysis of low molecular weight heparins by multiple heart-cutting two dimensional chromatography with quadruple time-of-flight mass spectrometry.

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Ouyang, Yilan; Zeng, Yangyang; Rong, Yinxiu; Song, Yue; Shi, Lv; Chen, Bo; Yang, Xinlei; Xu, Naiyu; Linhardt, Robert J; Zhang, Zhenqing

2015-09-01

Low molecular weight heparins (LMWHs) are polydisperse and microheterogenous mixtures of polysaccharides used as anticoagulant drugs. Profiling analysis is important for obtaining deeper insights into the structure of LMWHs. Previous oligosaccharide mapping methods are relatively low resolution and are unable to show an entire picture of the structural complexity of LMWHs. In the current study a profiling method was developed relying on multiple heart-cutting, two-dimensional, ultrahigh performance liquid chromatography with quadruple time-of-flight mass spectrometry. This represents an efficient, automated, and robust approach for profiling LMWHs. Using size-exclusion chromatography and ion-pairing reversed-phase chromatography in a two-dimensional separation, LMW components of different sizes and LMW components of the same size but with different charges and polarities can be resolved, providing a more complete picture of a LMWH. Structural information on each component was then obtained with quadrupole time-of-flight mass spectrometry. More than 80 and 120 oligosaccharides were observed and unambiguously assigned from the LMWHs, nadroparin and enoxaparin, respectively. This method might be useful for quality control of LMWHs and as a powerful tool for heparin-related glycomics.

Evolution of egg coats: linking molecular biology and ecology.

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Shu, Longfei; Suter, Marc J-F; Räsänen, Katja

2015-08-01

One central goal of evolutionary biology is to explain how biological diversity emerges and is maintained in nature. Given the complexity of the phenotype and the multifaceted nature of inheritance, modern evolutionary ecological studies rely heavily on the use of molecular tools. Here, we show how molecular tools help to gain insight into the role of egg coats (i.e. the extracellular structures surrounding eggs and embryos) in evolutionary diversification. Egg coats are maternally derived structures that have many biological functions from mediating fertilization to protecting the embryo from environmental hazards. They show great molecular, structural and functional diversity across species, but intraspecific variability and the role of ecology in egg coat evolution have largely been overlooked. Given that much of the variation that influences egg coat function is ultimately determined by their molecular phenotype, cutting-edge molecular tools (e.g. proteomics, glycomics and transcriptomics), combined with functional assays, are needed for rigorous inferences on their evolutionary ecology. Here, we identify key research areas and highlight emerging molecular techniques that can increase our understanding of the role of egg coats in the evolution of biological diversity, from adaptation to speciation. © 2015 John Wiley & Sons Ltd.

Aspergillus vaccines: Hardly worth studying or worthy of hard study?

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Levitz, Stuart M

2017-01-01

Vaccines rank among the greatest advances in the history of public health. Yet, despite the need, there are no licensed vaccines to protect humans against fungal diseases, including aspergillosis. In this focused review, some of the major scientific and logistical challenges to developing vaccines to protect at-risk individuals against aspergillosis are discussed. Approaches that have shown promise in animal models include vaccines that protect against multiple fungal genera and those that are specifically directed to Aspergillus Advances in proteomics and glycomics have facilitated identification of candidate antigens for use in subunit vaccines. Novel adjuvants and delivery systems are becoming available that can skew vaccine responses toward those associated with protection. Immunotherapy consisting of adoptive transfer of Aspergillus-specific T cells to allogeneic hematopoietic transplant recipients has advanced to human testing but is technically difficult and of unproven benefit. While progress has been impressive, much work still needs to be done if vaccines against aspergillosis are to become a reality. © The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Red blood cell (RBC) membrane proteomics--Part I: Proteomics and RBC physiology.

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Pasini, Erica M; Lutz, Hans U; Mann, Matthias; Thomas, Alan W

2010-01-03

Membrane proteomics is concerned with accurately and sensitively identifying molecules involved in cell compartmentalisation, including those controlling the interface between the cell and the outside world. The high lipid content of the environment in which these proteins are found often causes a particular set of problems that must be overcome when isolating the required material before effective HPLC-MS approaches can be performed. The membrane is an unusually dynamic cellular structure since it interacts with an ever changing environment. A full understanding of this critical cell component will ultimately require, in addition to proteomics, lipidomics, glycomics, interactomics and study of post-translational modifications. Devoid of nucleus and organelles in mammalian species other than camelids, and constantly in motion in the blood stream, red blood cells (RBCs) are the sole mammalian oxygen transporter. The fact that mature mammalian RBCs have no internal membrane-bound organelles, somewhat simplifies proteomics analysis of the plasma membrane and the fact that it has no nucleus disqualifies microarray based methods. Proteomics has the potential to provide a better understanding of this critical interface, and thereby assist in identifying new approaches to diseases. (c) 2009 Elsevier B.V. All rights reserved.

Fucosylation Is a Promising Target for Cancer Diagnosis and Therapy

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Shinichiro Shinzaki

2012-01-01

Full Text Available Oligosaccharides, sequences of carbohydrates conjugated to proteins and lipids, are arguably the most abundant and structurally diverse class of molecules. Fucosylation is one of the most important oligosaccharide modifications involved in cancer and inflammation. Recent advances in glycomics have identified several types of glyco-biomarkers containing fucosylation that are linked to certain types of cancer. Fucosylated alpha-fetoprotein (AFP is widely used in the diagnosis of hepatocellular carcinoma because it is more specific than alpha-fetoprotein. High levels of fucosylated haptoglobin have also been found in sera of patients with various carcinomas. We have recently established a simple lectin-antibody ELISA to measure fucosylated haptoglobin and to investigate its clinical use. Cellular fucosylation is dependent upon fucosyltransferase activity and the level of its donor substrate, guanosine diphosphate (GDP-fucose. GDP-mannose-4,6-dehydratase (GMDS is a key enzyme involved in the synthesis of GDP-fucose. Mutations of GMDS found in colon cancer cells induced a malignant phenotype, leading to rapid growth in athymic mice resistant to natural killer cells. This review describes the role of fucosylated haptoglobin as a cancer biomarker, and discusses the possible biological role of fucosylation in cancer development.

Isolation and characterization of heparan sulfate from various murine tissues.

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Warda, Mohamad; Toida, Toshihiko; Zhang, Fuming; Sun, Peilong; Munoz, Eva; Xie, Jin; Linhardt, Robert J

2006-11-01

Heparan sulfate (HS), is a proteoglycan (PG) found both in the extracellular matrix and on cell surface. It may represent one of the most biologically important glycoconjugates, playing an essential role in a variety of different events at molecular level. The publication of the mouse genome, and the intensive investigations aimed at understanding the proteome it encodes, has motivated us to initiate studies in mouse glycomics focused on HS. The current study is aimed at determining the quantitative and qualitative organ distribution of HS in mice. HS from brain, eyes, heart, lung, liver, kidney, spleen, intestine and skin was purified from 6-8 week old male and female mice. The recovered yield of HS from these organs is compared with the recovered whole body yield of HS. Structural characterization of the resulting HS relied on disaccharide analysis and (1)H-NMR spectroscopy. Different organs revealed a characteristic HS structure. These data begin to provide a structural understanding of the role of HS in cell-cell interactions, cell signaling and sub-cellular protein trafficking as well as a fundamental understanding of certain aspects of protein-carbohydrate interactions.

GlycoExtractor: a web-based interface for high throughput processing of HPLC-glycan data.

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Artemenko, Natalia V; Campbell, Matthew P; Rudd, Pauline M

2010-04-05

Recently, an automated high-throughput HPLC platform has been developed that can be used to fully sequence and quantify low concentrations of N-linked sugars released from glycoproteins, supported by an experimental database (GlycoBase) and analytical tools (autoGU). However, commercial packages that support the operation of HPLC instruments and data storage lack platforms for the extraction of large volumes of data. The lack of resources and agreed formats in glycomics is now a major limiting factor that restricts the development of bioinformatic tools and automated workflows for high-throughput HPLC data analysis. GlycoExtractor is a web-based tool that interfaces with a commercial HPLC database/software solution to facilitate the extraction of large volumes of processed glycan profile data (peak number, peak areas, and glucose unit values). The tool allows the user to export a series of sample sets to a set of file formats (XML, JSON, and CSV) rather than a collection of disconnected files. This approach not only reduces the amount of manual refinement required to export data into a suitable format for data analysis but also opens the field to new approaches for high-throughput data interpretation and storage, including biomarker discovery and validation and monitoring of online bioprocessing conditions for next generation biotherapeutics.

Inflammaging and human longevity in the omics era.

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Monti, Daniela; Ostan, Rita; Borelli, Vincenzo; Castellani, Gastone; Franceschi, Claudio

2017-07-01

Inflammaging is a recent theory of aging originally proposed in 2000 where data and conceptualizations regarding the aging of the immune system (immunosenescence) and the evolution of immune responses from invertebrates to mammals converged. This theory has received an increasing number of citations and experimental confirmations. Here we present an updated version of inflammaging focused on omics data - particularly on glycomics - collected on centenarians, semi-supercentenarians and their offspring. Accordingly, we arrived to the following conclusions: i) inflammaging has a structure where specific combinations of pro- and anti-inflammatory mediators are involved; ii) inflammaging is systemic and more complex than we previously thought, as many organs, tissues and cell types participate in producing pro- and anti-inflammatory stimuli defined "molecular garbage"; iii) inflammaging is dynamic, can be propagated locally to neighboring cells and systemically from organ to organ by circulating products and microvesicles, and amplified by chronic age-related diseases constituting a "local fire", which in turn produces additional inflammatory stimuli and molecular garbage; iv) an integrated Systems Medicine approach is urgently needed to let emerge a robust and highly informative set/combination of omics markers able to better grasp the complex molecular core of inflammaging in elderly and centenarians. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Nonreductive chemical release of intact N-glycans for subsequent labeling and analysis by mass spectrometry.

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Yuan, Jiangbei; Wang, Chengjian; Sun, Yujiao; Huang, Linjuan; Wang, Zhongfu

2014-10-01

A novel strategy is proposed, using cost-saving chemical reactions to generate intact free reducing N-glycans and their fluorescent derivatives from glycoproteins for subsequent analysis. N-Glycans without core α-1,3-linked fucose are released in reducing form by selective hydrolysis of the N-type carbohydrate-peptide bond of glycoproteins under a set of optimized mild alkaline conditions and are comparable to those released by commonly used peptide-N-glycosidase (PNGase) F in terms of yield without any detectable side reaction (peeling or deacetylation). The obtained reducing glycans can be routinely derivatized with 2-aminobenzoic acid (2-AA), 1-phenyl-3-methyl-5-pyrazolone (PMP), and potentially some other fluorescent reagents for comprehensive analysis. Alternatively, the core α-1,3-fucosylated N-glycans are released in mild alkaline medium and derivatized with PMP in situ, and their yields are comparable to those obtained using commonly used PNGase A without conspicuous peeling reaction or any detectable deacetylation. Using this new technique, the N-glycans of a series of purified glycoproteins and complex biological samples were successfully released and analyzed by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS), demonstrating its general applicability to glycomic studies. Copyright © 2014 Elsevier Inc. All rights reserved.

Top-down approach for the direct characterization of low molecular weight heparins using LC-FT-MS.

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Li, Lingyun; Zhang, Fuming; Zaia, Joseph; Linhardt, Robert J

2012-10-16

Low molecular heparins (LMWHs) are structurally complex, heterogeneous, polydisperse, and highly negatively charged mixtures of polysaccharides. The direct characterization of LMWH is a major challenge for currently available analytical technologies. Electrospray ionization (ESI) liquid chromatography-mass spectrometry (LC-MS) is a powerful tool for the characterization complex biological samples in the fields of proteomics, metabolomics, and glycomics. LC-MS has been applied to the analysis of heparin oligosaccharides, separated by size exclusion, reversed phase ion-pairing chromatography, and chip-based amide hydrophilic interaction chromatography (HILIC). However, there have been limited applications of ESI-LC-MS for the direct characterization of intact LMWHs (top-down analysis) due to their structural complexity, low ionization efficiency, and sulfate loss. Here we present a simple and reliable HILIC-Fourier transform (FT)-ESI-MS platform to characterize and compare two currently marketed LMWH products using the top-down approach requiring no special sample preparation steps. This HILIC system relies on cross-linked diol rather than amide chemistry, affording highly resolved chromatographic separations using a relatively high percentage of acetonitrile in the mobile phase, resulting in stable and high efficiency ionization. Bioinformatics software (GlycReSoft 1.0) was used to automatically assign structures within 5-ppm mass accuracy.

Galectin-3 Binds to Lubricin and Reinforces the Lubricating Boundary Layer of Articular Cartilage.

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Reesink, Heidi L; Bonnevie, Edward D; Liu, Sherry; Shurer, Carolyn R; Hollander, Michael J; Bonassar, Lawrence J; Nixon, Alan J

2016-05-09

Lubricin is a mucinous, synovial fluid glycoprotein that enables near frictionless joint motion via adsorption to the surface of articular cartilage and its lubricating properties in solution. Extensive O-linked glycosylation within lubricin's mucin-rich domain is critical for its boundary lubricating function; however, it is unknown exactly how glycosylation facilitates cartilage lubrication. Here, we find that the lubricin glycome is enriched with terminal β-galactosides, known binding partners for a family of multivalent lectins called galectins. Of the galectin family members present in synovial fluid, we find that galectin-3 is a specific, high-affinity binding partner for lubricin. Considering the known ability of galectin-3 to crosslink glycoproteins, we hypothesized that galectins could augment lubrication via biomechanical stabilization of the lubricin boundary layer. We find that competitive inhibition of galectin binding results in lubricin loss from the cartilage surface, and addition of multimeric galectin-3 enhances cartilage lubrication. We also find that galectin-3 has low affinity for the surface layer of osteoarthritic cartilage and has reduced affinity for sialylated O-glycans, a glycophenotype associated with inflammatory conditions. Together, our results suggest that galectin-3 reinforces the lubricin boundary layer; which, in turn, enhances cartilage lubrication and may delay the onset and progression of arthritis.

Study of cnidarian-algal symbiosis in the "omics" age.

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Meyer, Eli; Weis, Virginia M

2012-08-01

The symbiotic associations between cnidarians and dinoflagellate algae (Symbiodinium) support productive and diverse ecosystems in coral reefs. Many aspects of this association, including the mechanistic basis of host-symbiont recognition and metabolic interaction, remain poorly understood. The first completed genome sequence for a symbiotic anthozoan is now available (the coral Acropora digitifera), and extensive expressed sequence tag resources are available for a variety of other symbiotic corals and anemones. These resources make it possible to profile gene expression, protein abundance, and protein localization associated with the symbiotic state. Here we review the history of "omics" studies of cnidarian-algal symbiosis and the current availability of sequence resources for corals and anemones, identifying genes putatively involved in symbiosis across 10 anthozoan species. The public availability of candidate symbiosis-associated genes leaves the field of cnidarian-algal symbiosis poised for in-depth comparative studies of sequence diversity and gene expression and for targeted functional studies of genes associated with symbiosis. Reviewing the progress to date suggests directions for future investigations of cnidarian-algal symbiosis that include (i) sequencing of Symbiodinium, (ii) proteomic analysis of the symbiosome membrane complex, (iii) glycomic analysis of Symbiodinium cell surfaces, and (iv) expression profiling of the gastrodermal cells hosting Symbiodinium.

Infection Dynamics Vary between Symbiodinium Types and Cell Surface Treatments during Establishment of Endosymbiosis with Coral Larvae

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Bette Lynn Willis

2011-07-01

Full Text Available Symbioses between microbes and higher organisms underpin high diversity in many ecosystems, including coral reefs, however mechanisms underlying the early establishment of symbioses remain unclear. Here we examine the roles of Symbiodinium type and cell surface recognition in the establishment of algal endosymbiosis in the reef-building coral, Acropora tenuis. We found 20–70% higher infection success (proportion of larvae infected and five-fold higher Symbiodinium abundance in larvae exposed to ITS-1 type C1 compared to ITS-1 type D in the first 96 h following exposure. The highest abundance of Symbiodinium within larvae occurred when C1-type cells were treated with enzymes that modified the 40–100 kD glycome, including glycoproteins and long chain starch residues. Our finding of declining densities of Symbiodinium C1 through time in the presence of intact cell surface molecules supports a role for cell surface recognition molecules in controlling post-phagocytosis processes, leading to rejection of some Symbiodinium types in early ontogeny. Reductions in the densities of unmodified C1 symbionts after 96 h, in contrast to increases in D symbionts may suggest the early initiation of a winnowing process contributing to the establishment of Symbiodinium D as the dominant type in one-month old juveniles of A. tenuis.

The O-Linked Glycome and Blood Group Antigens ABO on Mucin-Type Glycoproteins in Mucinous and Serous Epithelial Ovarian Tumors.

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Varvara Vitiazeva

Full Text Available Mucins are heavily O-glycosylated proteins where the glycosylation has been shown to play an important role in cancer. Normal epithelial ovarian cells do not express secreted mucins, but their abnormal expression has previously been described in epithelial ovarian cancer and may relate to tumor formation and progression. The cyst fluids were shown to be a rich source for acidic glycoproteins. The study of these proteins can potentially lead to the identification of more effective biomarkers for ovarian cancer.In this study, we analyzed the expression of the MUC5AC and the O-glycosylation of acidic glycoproteins secreted into ovarian cyst fluids. The samples were obtained from patients with serous and mucinous ovarian tumors of different stages (benign, borderline, malignant and grades. The O-linked oligosaccharides were released and analyzed by negative-ion graphitized carbon Liquid Chromatography (LC coupled to Electrospray Ionization tandem Mass Spectrometry (ESI-MSn. The LC-ESI-MSn of the oligosaccharides from ovarian cyst fluids displayed differences in expression of fucose containing structures such as blood group ABO antigens and Lewis-type epitopes.The obtained data showed that serous and mucinous benign adenomas, mucinous low malignant potential carcinomas (LMPs, borderline and mucinous low-grade carcinomas have a high level of blood groups and Lewis type epitopes. In contrast, this type of fucosylated structures were low abundant in the high-grade mucinous carcinomas or in serous carcinomas. In addition, the ovarian tumors that showed a high level of expression of blood group antigens also revealed a strong reactivity towards the MUC5AC antibody. To visualize the differences between serous and mucinous ovarian tumors based on the O-glycosylation, a hierarchical cluster analysis was performed using mass spectrometry average compositions (MSAC.Mucinous benign and LMPs along with mucinous low-grade carcinomas appear to be different from serous and high-grade mucinous carcinomas based on their O-glycan profiles.

Glycomics expression analysis of sulfated glycosaminoglycans of human colorectal cancer tissues and non-neoplastic mucosa by electrospray ionization mass spectrometry.

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Marolla, Ana Paula Cleto; Waisberg, Jaques; Saba, Gabriela Tognini; Waisberg, Daniel Reis; Margeotto, Fernando Beani; Pinhal, Maria Aparecida da Silva

2015-01-01

To determine the presence of glycosaminoglycans in the extracellular matrix of connective tissue from neoplastic and non-neoplastic colorectal tissues, since it has a central role in tumor development and progression. Tissue samples from neoplastic and non-neoplastic colorectal tissues were obtained from 64 operated patients who had colorectal carcinoma with no distant metastases. Expressions of heparan sulphate, chondroitin sulphate, dermatan sulphate and their fragments were analyzed by electrospray ionization mass spectrometry, with the technique for extraction and quantification of glycosaminoglycans after proteolysis and electrophoresis. The statistical analysis included mean, standard deviation, and Student'st test. The glycosaminoglycans extracted from colorectal tissue showed three electrophoretic bands in agarose gel. Electrospray ionization mass spectrometry showed characteristic disaccharide fragments from glycosaminoglycans, indicating their structural characterization in the tissues analyzed. Some peaks in the electrospray ionization mass spectrometry were not characterized as fragments of sugars, indicating the presence of fragments of the protein structure of proteoglycans generated during the glycosaminoglycan purification. The average amount of chondroitin and dermatan increased in the neoplastic tissue compared to normal tissue (p=0.01). On the other hand, the average amount of heparan decreased in the neoplastic tissue compared to normal tissue (p= 0.03). The method allowed the determination of the glycosaminoglycans structural profile in colorectal tissue from neoplastic and non-neoplastic colorectal tissue. Neoplastic tissues showed greater amounts of chondroitin sulphate and dermatan sulphate compared to non-neoplastic tissues, while heparan sulphate was decreased in neoplastic tissues.

Glycomics expression analysis of sulfated glycosaminoglycans of human colorectal cancer tissues and non-neoplastic mucosa by electrospray ionization mass spectrometry

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Marolla, Ana Paula Cleto [Universidade Federal de São Paulo, São Paulo, SP (Brazil); Waisberg, Jaques [Hospital do Servidor Público Estadual, São Paulo, SP (Brazil); Faculdade de Medicina do ABC, Santo André, SP (Brazil); Saba, Gabriela Tognini [Faculdade de Medicina do ABC, Santo André, SP (Brazil); Waisberg, Daniel Reis [Faculdade de Medicina da Universidade de São Paulo, São Paulo, SP (Brazil); Margeotto, Fernando Beani; Pinhal, Maria Aparecida da Silva [Faculdade de Medicina do ABC, Santo André, SP (Brazil)

2015-07-01

To determine the presence of glycosaminoglycans in the extracellular matrix of connective tissue from neoplastic and non-neoplastic colorectal tissues, since it has a central role in tumor development and progression. Tissue samples from neoplastic and non-neoplastic colorectal tissues were obtained from 64 operated patients who had colorectal carcinoma with no distant metastases. Expressions of heparan sulphate, chondroitin sulphate, dermatan sulphate and their fragments were analyzed by electrospray ionization mass spectrometry, with the technique for extraction and quantification of glycosaminoglycans after proteolysis and electrophoresis. The statistical analysis included mean, standard deviation, and Student’s t test. The glycosaminoglycans extracted from colorectal tissue showed three electrophoretic bands in agarose gel. Electrospray ionization mass spectrometry showed characteristic disaccharide fragments from glycosaminoglycans, indicating their structural characterization in the tissues analyzed. Some peaks in the electrospray ionization mass spectrometry were not characterized as fragments of sugars, indicating the presence of fragments of the protein structure of proteoglycans generated during the glycosaminoglycan purification. The average amount of chondroitin and dermatan increased in the neoplastic tissue compared to normal tissue (p=0.01). On the other hand, the average amount of heparan decreased in the neoplastic tissue compared to normal tissue (p= 0.03). The method allowed the determination of the glycosaminoglycans structural profile in colorectal tissue from neoplastic and non-neoplastic colorectal tissue. Neoplastic tissues showed greater amounts of chondroitin sulphate and dermatan sulphate compared to non-neoplastic tissues, while heparan sulphate was decreased in neoplastic tissues.

Glycomics expression analysis of sulfated glycosaminoglycans of human colorectal cancer tissues and non-neoplastic mucosa by electrospray ionization mass spectrometry

International Nuclear Information System (INIS)

Marolla, Ana Paula Cleto; Waisberg, Jaques; Saba, Gabriela Tognini; Waisberg, Daniel Reis; Margeotto, Fernando Beani; Pinhal, Maria Aparecida da Silva

2015-01-01

To determine the presence of glycosaminoglycans in the extracellular matrix of connective tissue from neoplastic and non-neoplastic colorectal tissues, since it has a central role in tumor development and progression. Tissue samples from neoplastic and non-neoplastic colorectal tissues were obtained from 64 operated patients who had colorectal carcinoma with no distant metastases. Expressions of heparan sulphate, chondroitin sulphate, dermatan sulphate and their fragments were analyzed by electrospray ionization mass spectrometry, with the technique for extraction and quantification of glycosaminoglycans after proteolysis and electrophoresis. The statistical analysis included mean, standard deviation, and Student’s t test. The glycosaminoglycans extracted from colorectal tissue showed three electrophoretic bands in agarose gel. Electrospray ionization mass spectrometry showed characteristic disaccharide fragments from glycosaminoglycans, indicating their structural characterization in the tissues analyzed. Some peaks in the electrospray ionization mass spectrometry were not characterized as fragments of sugars, indicating the presence of fragments of the protein structure of proteoglycans generated during the glycosaminoglycan purification. The average amount of chondroitin and dermatan increased in the neoplastic tissue compared to normal tissue (p=0.01). On the other hand, the average amount of heparan decreased in the neoplastic tissue compared to normal tissue (p= 0.03). The method allowed the determination of the glycosaminoglycans structural profile in colorectal tissue from neoplastic and non-neoplastic colorectal tissue. Neoplastic tissues showed greater amounts of chondroitin sulphate and dermatan sulphate compared to non-neoplastic tissues, while heparan sulphate was decreased in neoplastic tissues

Noninvasive biomarkers in non-alcoholic fatty liver disease: Current status and a glimpse of the future

Science.gov (United States)

Fitzpatrick, Emer; Dhawan, Anil

2014-01-01

The development of non invasive biomarkers of disease has become a major focus of interest in nonalcoholic fatty liver disease (NAFLD). The large prevalence of the disease and the invasive nature of the investigation means that screening with liver biopsy is impractical. In addition to screening, the differentiation of those with simple steatosis vs steatohepatitis and fibrosis is clinically important as the prognosis of each differs. Serum biomarkers may be a combination of simple markers derived from large data sets or direct markers of disease activity. Serum markers of inflammation, apoptosis and oxidative stress in addition to fibrosis have been extensively studied in patients with NAFLD. Other techniques such as transient elastography, magnetic resonance elastography and acoustic radiation force imaging are becoming more established as noninvasive methods of detecting fibrosis in a variety of chronic liver conditions in addition to NAFLD. Newer high throughput methods such as proteomics and glycomics allow the nonhypothesis-driven identification of novel markers and may also potentially contribute to our understanding of the pathogenesis of the condition. This review addresses some of the methodological issues which need to be considered in the search for the ideal biomarker. It is likely that a combination of serum biomarkers and techniques such as transient elastography may provide the optimal diagnostic discrimination however this remains to be proven in large studies. PMID:25152587

Cell cultures for schistosomes - Chances of success or wishful thinking?

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Quack, T; Wippersteg, V; Grevelding, C G

2010-08-01

Due to their worldwide importance for human and animal health, schistosomes are in the focus of national and international research activities. Their aims are to elucidate the genome, the transcriptome, the proteome and the glycome of schistosomes with the expectation to understand the biology of these blood flukes and to identify new candidate antigens for the development of a vaccine, or target molecules for the design of novel pharmaceutical compounds. All of these efforts have delivered a vast amount of information about the genetic equipment of schistosomes. In the emerging era of post-genomic research, however, methods and tools are necessary to interpret all available data and to characterise molecules of interest in more detail. In addition to transgenesis, it is generally accepted that cell lines for schistosomes are among the requirements to overcome present research limitations. In our commentary the prospect of establishing cell cultures for schistosomes is discussed. To this end we summarise the comprehensive endeavours made in the past regarding the establishment of invertebrate cell lines pointing to critical parameters that should be considered when making new attempts towards schistosome cell culturing. Furthermore, based on preliminary data with pilot-character, we discuss recent advances indicating the possibility of overcoming existing restrictions with respect to the 'immortalisation' of cells by oncogenes. Copyright 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

S-nitrosoglutathione promotes cell wall remodelling, alters the transcriptional profile and induces root hair formation in the hairless root hair defective 6 (rhd6) mutant of Arabidopsis thaliana.

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Moro, Camila Fernandes; Gaspar, Marilia; da Silva, Felipe Rodrigues; Pattathil, Sivakumar; Hahn, Michael G; Salgado, Ione; Braga, Marcia Regina

2017-03-01

Nitric oxide (NO) exerts pleiotropic effects on plant development; however, its involvement in cell wall modification during root hair formation (RHF) has not yet been addressed. Here, mutants of Arabidopsis thaliana with altered root hair phenotypes were used to assess the involvement of S-nitrosoglutathione (GSNO), the primary NO source, in cell wall dynamics and gene expression in roots induced to form hairs. GSNO and auxin restored the root hair phenotype of the hairless root hair defective 6 (rhd6) mutant. A positive correlation was observed between increased NO production and RHF induced by auxin in rhd6 and transparent testa glabra (ttg) mutants. Deposition of an epitope within rhamnogalacturonan-I recognized by the CCRC-M2 antibody was delayed in root hair cells (trichoblasts) compared with nonhair cells (atrichoblasts). GSNO, but not auxin, restored the wild-type root glycome and transcriptome profiles in rhd6, modulating the expression of a large number of genes related to cell wall composition and metabolism, as well as those encoding ribosomal proteins, DNA and histone-modifying enzymes and proteins involved in post-translational modification. Our results demonstrate that NO plays a key role in cell wall remodelling in trichoblasts and suggest that it also participates in chromatin modification in root cells of A. thaliana. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

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

Science.gov (United States)

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.

Structural Feature Ions for Distinguishing N- and O-Linked Glycan Isomers by LC-ESI-IT MS/MS

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Everest-Dass, Arun V.; Abrahams, Jodie L.; Kolarich, Daniel; Packer, Nicolle H.; Campbell, Matthew P.

2013-06-01

Glycomics is the comprehensive study of glycan expression in an organism, cell, or tissue that relies on effective analytical technologies to understand glycan structure-function relationships. Owing to the macro- and micro-heterogeneity of oligosaccharides, detailed structure characterization has required an orthogonal approach, such as a combination of specific exoglycosidase digestions, LC-MS/MS, and the development of bioinformatic resources to comprehensively profile a complex biological sample. Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) has emerged as a key tool in the structural analysis of oligosaccharides because of its high sensitivity, resolution, and robustness. Here, we present a strategy that uses LC-ESI-MS/MS to characterize over 200 N- and O-glycans from human saliva glycoproteins, complemented by sequential exoglycosidase treatment, to further verify the annotated glycan structures. Fragment-specific substructure diagnostic ions were collated from an extensive screen of the literature available on the detailed structural characterization of oligosaccharides and, together with other specific glycan structure feature ions derived from cross-ring and glycosidic-linkage fragmentation, were used to characterize the glycans and differentiate isomers. The availability of such annotated mass spectrometric fragmentation spectral libraries of glycan structures, together with such substructure diagnostic ions, will be key inputs for the future development of the automated elucidation of oligosaccharide structures from MS/MS data.

Accounting for undetected compounds in statistical analyses of mass spectrometry 'omic studies.

Science.gov (United States)

Taylor, Sandra L; Leiserowitz, Gary S; Kim, Kyoungmi

2013-12-01

Mass spectrometry is an important high-throughput technique for profiling small molecular compounds in biological samples and is widely used to identify potential diagnostic and prognostic compounds associated with disease. Commonly, this data generated by mass spectrometry has many missing values resulting when a compound is absent from a sample or is present but at a concentration below the detection limit. Several strategies are available for statistically analyzing data with missing values. The accelerated failure time (AFT) model assumes all missing values result from censoring below a detection limit. Under a mixture model, missing values can result from a combination of censoring and the absence of a compound. We compare power and estimation of a mixture model to an AFT model. Based on simulated data, we found the AFT model to have greater power to detect differences in means and point mass proportions between groups. However, the AFT model yielded biased estimates with the bias increasing as the proportion of observations in the point mass increased while estimates were unbiased with the mixture model except if all missing observations came from censoring. These findings suggest using the AFT model for hypothesis testing and mixture model for estimation. We demonstrated this approach through application to glycomics data of serum samples from women with ovarian cancer and matched controls.

Binary boronic acid-functionalized attapulgite with high adsorption capacity for selective capture of nucleosides at acidic pH values

International Nuclear Information System (INIS)

Li, Huihui; Zhu, Shuqiang; Cheng, Ting; Wang, Shuxia; Zhu, Bin; Liu, Xiaoyan; Zhang, Haixia

2016-01-01

Boronate affinity materials have been widely used for selective capture of cis-diols such as nucleosides. Adsorbents with features of low binding pH and high adsorption capacity are highly desired. However, most reported materials only possess one of the two features. We have synthesized a 1,3,5-triazine-containing binary boronic acid by reacting cyanuric chloride with 3-amino phenylboronic acid, and the product was then grafted onto attapulgite (a fibrous aluminum-magnesium silicate). The resulting functionalized attapulgite exhibit low binding pH (5.0) and display high adsorption capacity (19.5 ± 1.1 mg⋅g"−"1 for adenosine). The material exhibits high selectivity for cis-diols even in the presence of a 1000-fold excess of interferences. It was applied to the selective extraction of nucleosides from human urine. Typical features of the method include (a) limits of detection in the range from 4 to 17 ng⋅mL"−"1, (b) limits of quantification between 13 and 57 ng⋅mL"−"1, (c) relative standard deviations of ≤9.1 %, and (d) recoveries of nucleosides from spiked human urine between 85.0 and 112.9 %. In our perception, the material and method offer a promising strategy for selective capture of cis-diols in the areas of proteomics, metabolomics and glycomics. (author)

Glycomic Analysis of Life Stages of the Human Parasite Schistosoma mansoni Reveals Developmental Expression Profiles of Functional and Antigenic Glycan Motifs

NARCIS (Netherlands)

Smit, C.H.; van Diepen, A.; Nguyen, D.L.; Wuhrer, M.; Hoffmann, K.F.; Deelder, A.M.; Hokke, C.H.

2015-01-01

Glycans present on glycoproteins and glycolipids of the major human parasite Schistosoma mansoni induce innate as well as adaptive immune responses in the host. To be able to study the molecular characteristics of schistosome infections it is therefore required to determine the expression profiles

Comprehensive peptidomic and glycomic evaluation reveals that sweet whey permeate from colostrum is a source of milk protein-derived peptides and oligosaccharides

NARCIS (Netherlands)

Dallas, D.C.; Weinborn, V.; Moura Bell, de J.M.L.N.; Wang, M.; Parker, E.A.; Guerrero, A.; Hettinga, K.A.; Lebrilla, C.B.; German, J.B.; Barile, D.

2014-01-01

Whey permeate is a co-product obtained when cheese whey is passed through an ultrafiltration membrane to concentrate whey proteins. Whey proteins are retained by the membrane, whereas the low-molecular weight compounds such as lactose, salts, oligosaccharides and peptides pass through the membrane

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.

Discrimination of Isomers of Released N- and O-Glycans Using Diagnostic Product Ions in Negative Ion PGC-LC-ESI-MS/MS

Science.gov (United States)

Ashwood, Christopher; Lin, Chi-Hung; Thaysen-Andersen, Morten; Packer, Nicolle H.

2018-03-01

Profiling cellular protein glycosylation is challenging due to the presence of highly similar glycan structures that play diverse roles in cellular physiology. As the anomericity and the exact linkage type of a single glycosidic bond can influence glycan function, there is a demand for improved and automated methods to confirm detailed structural features and to discriminate between structurally similar isomers, overcoming a significant bottleneck in the analysis of data generated by glycomics experiments. We used porous graphitized carbon-LC-ESI-MS/MS to separate and detect released N- and O-glycan isomers from mammalian model glycoproteins using negative mode resonance activation CID-MS/MS. By interrogating similar fragment spectra from closely related glycan isomers that differ only in arm position and sialyl linkage, product fragment ions for discrimination between these features were discovered. Using the Skyline software, at least two diagnostic fragment ions of high specificity were validated for automated discrimination of sialylation and arm position in N-glycan structures, and sialylation in O-glycan structures, complementing existing structural diagnostic ions. These diagnostic ions were shown to be useful for isomer discrimination using both linear and 3D ion trap mass spectrometers when analyzing complex glycan mixtures from cell lysates. Skyline was found to serve as a useful tool for automated assessment of glycan isomer discrimination. This platform-independent workflow can potentially be extended to automate the characterization and quantitation of other challenging glycan isomers. [Figure not available: see fulltext.

Multiomics Data Triangulation for Asthma Candidate Biomarkers and Precision Medicine.

Science.gov (United States)

Pecak, Matija; Korošec, Peter; Kunej, Tanja

2018-06-01

Asthma is a common complex disorder and has been subject to intensive omics research for disease susceptibility and therapeutic innovation. Candidate biomarkers of asthma and its precision treatment demand that they stand the test of multiomics data triangulation before they can be prioritized for clinical applications. We classified the biomarkers of asthma after a search of the literature and based on whether or not a given biomarker candidate is reported in multiple omics platforms and methodologies, using PubMed and Web of Science, we identified omics studies of asthma conducted on diverse platforms using keywords, such as asthma, genomics, metabolomics, and epigenomics. We extracted data about asthma candidate biomarkers from 73 articles and developed a catalog of 190 potential asthma biomarkers (167 human, 23 animal data), comprising DNA loci, transcripts, proteins, metabolites, epimutations, and noncoding RNAs. The data were sorted according to 13 omics types: genomics, epigenomics, transcriptomics, proteomics, interactomics, metabolomics, ncRNAomics, glycomics, lipidomics, environmental omics, pharmacogenomics, phenomics, and integrative omics. Importantly, we found that 10 candidate biomarkers were apparent in at least two or more omics levels, thus promising potential for further biomarker research and development and precision medicine applications. This multiomics catalog reported herein for the first time contributes to future decision-making on prioritization of biomarkers and validation efforts for precision medicine in asthma. The findings may also facilitate meta-analyses and integrative omics studies in the future.

The A0 blood group genotype modifies the jejunal glycomic binding pattern profile of piglets early associated with a simple or complex microbiota

NARCIS (Netherlands)

Priori, D.; Colombo, M.; Koopmans, S.J.; Jansman, A.J.M.; Meulen, van der J.; Trevisi, P.; Bosi, P.

2016-01-01

The intestinal epithelium glycocalyx sugar motif is an important determinant of the bacterial-host interaction and may be affected in pigs by gut microbiota and by blood group genotype. The aim was to study the effect of intestinal association with different microbiota and A0 blood group

Combined N-glycome and N-glycoproteome analysis of the Lotus japonicus seed globulin fraction shows conservation of protein structure and glycosylation in legumes

DEFF Research Database (Denmark)

Dam, Svend Secher; Thaysen-Andersen, Morten; Stenkjær, Eva

2013-01-01

Legume food allergy, such as allergy toward peanuts and soybeans, is a health issue predicted to worsen as dietary advice recommends higher intake of legume-based foods. Lotus japonicus (Lotus) is an established legume plant model system for studies of symbiotic and pathogenic microbial...... interactions and, due to its well characterized genotype/phenotype and easily manipulated genome, may also be suitable for studies of legume food allergy. Here we present a comprehensive study of the Lotus N-glycoproteome. The global and site-specific N-glycan structures of Lotus seed globulins were analyzed...

Horizon Scanning: How Will Metabolomics Applications Transform Food Science, Bioengineering, and Medical Innovation in the Current Era of Foodomics?

Science.gov (United States)

Bayram, Mustafa; Gökırmaklı, Çağlar

2018-03-01

Food and engineering sciences have tended to neglect the importance of human nutrition sciences and clinical study of new molecules discovered by food engineering community, and vice versa. Yet, the value of systems thinking and use of omics technologies in food engineering are rapidly emerging. Foodomics is a new concept and practice to bring about "precision nutrition" and integrative bioengineering studies of food composition, quality, and safety, and applications to improve health of humans, animals, and other living organisms on the planet. Foodomics signals a three-way convergence among (1) food engineering; (2) omics systems science technologies such as proteomics, metabolomics, glycomics; and (3) medical/life sciences. This horizon scanning expert review aims to challenge the current practices in food sciences and bioengineering so as to adopt foodomics and systems thinking in foodstuff analysis, with a focus on possible applications of metabolomics. Among the omics biotechnologies, metabolomics is one of the prominent analytical platforms of interest to both food engineers and medical researchers engaged in nutritional sciences, precision medicine, and systems medicine diagnostics. Medical and omics system scientists, and bioengineering scholars can mutually learn from their respective professional expertise. Moving forward, establishment of "Foodomics Think Tanks" is one conceivable strategy to integrate medical and food sciences innovation at a systems scale. With its rich history in food sciences and tradition of interdisciplinary scholarship, the Silk Road countries offer notable potential for synthesis of diverse knowledge strands necessary to realize the prospects of foodomics from Asia and Middle East to Europe.

Fucosyltransferase Induction during Influenza Virus Infection Is Required for the Generation of Functional Memory CD4+ T Cells

Science.gov (United States)

Carrette, Florent; Henriquez, Monique L.; Fujita, Yu

2018-01-01

T cells mediating influenza viral control are instructed in lymphoid and nonlymphoid tissues to differentiate into memory T cells that confer protective immunity. The mechanisms by which influenza virus–specific memory CD4+ T cells arise have been attributed to changes in transcription factors, cytokines and cytokine receptors, and metabolic programming. The molecules involved in these biosynthetic pathways, including proteins and lipids, are modified to varying degrees of glycosylation, fucosylation, sialation, and sulfation, which can alter their function. It is currently unknown how the glycome enzymatic machinery regulates CD4+ T cell effector and memory differentiation. In a murine model of influenza virus infection, we found that fucosyltransferase enzymatic activity was induced in effector and memory CD4+ T cells. Using CD4+ T cells deficient in the Fut4/7 enzymes that are expressed only in hematopoietic cells, we found decreased frequencies of effector cells with reduced expression of T-bet and NKG2A/C/E in the lungs during primary infection. Furthermore, Fut4/7−/− effector CD4+ T cells had reduced survival with no difference in proliferation or capacity for effector function. Although Fut4/7−/− CD4+ T cells seeded the memory pool after primary infection, they failed to form tissue-resident cells, were dysfunctional, and were unable to re-expand after secondary infection. Our findings highlight an important regulatory axis mediated by cell-intrinsic fucosyltransferase activity in CD4+ T cell effectors that ensure the development of functional memory CD4+ T cells. PMID:29491007

Quantitative twoplex glycan analysis using 12C6 and 13C6 stable isotope 2-aminobenzoic acid labelling and capillary electrophoresis mass spectrometry.

Science.gov (United States)

Váradi, Csaba; Mittermayr, Stefan; Millán-Martín, Silvia; Bones, Jonathan

2016-12-01

Capillary electrophoresis (CE) offers excellent efficiency and orthogonality to liquid chromatographic (LC) separations for oligosaccharide structural analysis. Combination of CE with high resolution mass spectrometry (MS) for glycan analysis remains a challenging task due to the MS incompatibility of background electrolyte buffers and additives commonly used in offline CE separations. Here, a novel method is presented for the analysis of 2-aminobenzoic acid (2-AA) labelled glycans by capillary electrophoresis coupled to mass spectrometry (CE-MS). To ensure maximum resolution and excellent precision without the requirement for excessive analysis times, CE separation conditions including the concentration and pH of the background electrolyte, the effect of applied pressure on the capillary inlet and the capillary length were evaluated. Using readily available 12/13 C 6 stable isotopologues of 2-AA, the developed method can be applied for quantitative glycan profiling in a twoplex manner based on the generation of extracted ion electropherograms (EIE) for 12 C 6 'light' and 13 C 6 'heavy' 2-AA labelled glycan isotope clusters. The twoplex quantitative CE-MS glycan analysis platform is ideally suited for comparability assessment of biopharmaceuticals, such as monoclonal antibodies, for differential glycomic analysis of clinical material for potential biomarker discovery or for quantitative microheterogeneity analysis of different glycosylation sites within a glycoprotein. Additionally, due to the low injection volume requirements of CE, subsequent LC-MS analysis of the same sample can be performed facilitating the use of orthogonal separation techniques for structural elucidation or verification of quantitative performance.

Exploiting fluorescence for multiplex immunoassays on protein microarrays

International Nuclear Information System (INIS)

Herbáth, Melinda; Balogh, Andrea; Matkó, János; Papp, Krisztián; Prechl, József

2014-01-01

Protein microarray technology is becoming the method of choice for identifying protein interaction partners, detecting specific proteins, carbohydrates and lipids, or for characterizing protein interactions and serum antibodies in a massively parallel manner. Availability of the well-established instrumentation of DNA arrays and development of new fluorescent detection instruments promoted the spread of this technique. Fluorescent detection has the advantage of high sensitivity, specificity, simplicity and wide dynamic range required by most measurements. Fluorescence through specifically designed probes and an increasing variety of detection modes offers an excellent tool for such microarray platforms. Measuring for example the level of antibodies, their isotypes and/or antigen specificity simultaneously can offer more complex and comprehensive information about the investigated biological phenomenon, especially if we take into consideration that hundreds of samples can be measured in a single assay. Not only body fluids, but also cell lysates, extracted cellular components, and intact living cells can be analyzed on protein arrays for monitoring functional responses to printed samples on the surface. As a rapidly evolving area, protein microarray technology offers a great bulk of information and new depth of knowledge. These are the features that endow protein arrays with wide applicability and robust sample analyzing capability. On the whole, protein arrays are emerging new tools not just in proteomics, but glycomics, lipidomics, and are also important for immunological research. In this review we attempt to summarize the technical aspects of planar fluorescent microarray technology along with the description of its main immunological applications. (topical review)

Evidence for Differential Glycosylation of Trophoblast Cell Types*

Science.gov (United States)

Chen, Qiushi; Pang, Poh-Choo; Cohen, Marie E.; Longtine, Mark S.; Schust, Danny J.; Haslam, Stuart M.; Blois, Sandra M.; Dell, Anne; Clark, Gary F.

2016-01-01

Human placental villi are surfaced by the syncytiotrophoblast (STB), with a layer of cytotrophoblasts (CTB) positioned just beneath the STB. STB in normal term pregnancies is exposed to maternal immune cells in the placental intervillous space. Extravillous cytotrophoblasts (EVT) invade the decidua and spiral arteries, where they act in conjunction with natural killer (NK) cells to convert the spiral arteries into flaccid conduits for maternal blood that support a 3–4 fold increase in the rate of maternal blood flow into the placental intervillous space. The functional roles of these distinct trophoblast subtypes during pregnancy suggested that they could be differentially glycosylated. Glycomic analysis of these trophoblasts has revealed the expression of elevated levels of biantennary N-glycans in STB and CTB, with the majority of them bearing a bisecting GlcNAc. N-glycans terminated with polylactosamine extensions were also detected at low levels. A subset of the N-glycans linked to these trophoblasts were sialylated, primarily with terminal NeuAcα2–3Gal sequences. EVT were decorated with the same N-glycans as STB and CTB, except in different proportions. The level of bisecting type N-glycans was reduced, but the level of N-glycans decorated with polylactosamine sequences were substantially elevated compared with the other types of trophoblasts. The level of triantennary and tetraantennary N-glycans was also elevated in EVT. The sialylated N-glycans derived from EVT were completely susceptible to an α2–3 specific neuraminidase (sialidase S). The possibility exists that the N-glycans associated with these different trophoblast subpopulations could act as functional groups. These potential relationships will be considered. PMID:26929217

Evidence for Differential Glycosylation of Trophoblast Cell Types.

Science.gov (United States)

Chen, Qiushi; Pang, Poh-Choo; Cohen, Marie E; Longtine, Mark S; Schust, Danny J; Haslam, Stuart M; Blois, Sandra M; Dell, Anne; Clark, Gary F

2016-06-01

Human placental villi are surfaced by the syncytiotrophoblast (STB), with a layer of cytotrophoblasts (CTB) positioned just beneath the STB. STB in normal term pregnancies is exposed to maternal immune cells in the placental intervillous space. Extravillous cytotrophoblasts (EVT) invade the decidua and spiral arteries, where they act in conjunction with natural killer (NK) cells to convert the spiral arteries into flaccid conduits for maternal blood that support a 3-4 fold increase in the rate of maternal blood flow into the placental intervillous space. The functional roles of these distinct trophoblast subtypes during pregnancy suggested that they could be differentially glycosylated. Glycomic analysis of these trophoblasts has revealed the expression of elevated levels of biantennary N-glycans in STB and CTB, with the majority of them bearing a bisecting GlcNAc. N-glycans terminated with polylactosamine extensions were also detected at low levels. A subset of the N-glycans linked to these trophoblasts were sialylated, primarily with terminal NeuAcα2-3Gal sequences. EVT were decorated with the same N-glycans as STB and CTB, except in different proportions. The level of bisecting type N-glycans was reduced, but the level of N-glycans decorated with polylactosamine sequences were substantially elevated compared with the other types of trophoblasts. The level of triantennary and tetraantennary N-glycans was also elevated in EVT. The sialylated N-glycans derived from EVT were completely susceptible to an α2-3 specific neuraminidase (sialidase S). The possibility exists that the N-glycans associated with these different trophoblast subpopulations could act as functional groups. These potential relationships will be considered. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Novel Magnetic Microprobe with Benzoboroxole-Modified Flexible Multisite Arm for High-Efficiency cis-Diol Biomolecule Detection.

Science.gov (United States)

Chen, Guosheng; Huang, Siming; Kou, Xiaoxue; Zhang, Jin'ge; Wang, Fuxin; Zhu, Fang; Ouyang, Gangfeng

2018-03-06

With regard to regulating a variety of biological events, including molecular recognition, signal transduction, cell adhesion, and immune response, cis-diol biomolecules, such as saccharides and glycoproteins, play vital roles. However, saccharides and glycoproteins in living systems usually exist in very low abundance, along with abundant interfering components. High-efficiency detection of saccharides and glycoproteins is a challenging yet highly impactful area of research. Herein, we reported a novel magnetic microprobe with a benzoboroxole-modified flexible multisite arm (PEG 2000-grafted PAMAM dendrimers; the microprobe was denoted as BFMA-MNP) for high-efficiency saccharides detection. The extraction capacity was significantly improved by ∼2 orders of magnitude, because of the integration of the enhanced hydrophilicity and multivalency effects in benzoboroxoles and the enhanced accessibility of the binding sites within the PEG 2000-grafted PAMAM dendrimers. As a result, the proposed approach possessed several advantages, compared with previous boronic acid-based methods, including ultrahigh sensitivity (limit of detection was <1 ng/mL), wide linear range (ranged from 0.5 μM to 2000 μM), and applicable in physiological pH condition. Furthermore, we established a general BFMA-MNP/glycoproteins/AuNPs sandwich assay to realize the visual glycoprotein qualitative screening for the first time. The unique sandwich assay possessed the dual nature of the magnetic separation by BFMA-MNPs and specific coloration by citrate-coated AuNPs. This visual sandwich assay enabled fast differentiation of the existence of glycoproteins in complicated samples without any advanced instruments. We believe the proposed BFMA-MNP microprobe herein will advance the ideas to detect and identify trace saccharides and glycoproteins in important fields such as glycomics and glycoproteomics.

The role of the clinician in the multi-omics era: are you ready?

Science.gov (United States)

van Karnebeek, Clara D M; Wortmann, Saskia B; Tarailo-Graovac, Maja; Langeveld, Mirjam; Ferreira, Carlos R; van de Kamp, Jiddeke M; Hollak, Carla E; Wasserman, Wyeth W; Waterham, Hans R; Wevers, Ron A; Haack, Tobias B; Wanders, Ronald J A; Boycott, Kym M

2018-01-23

Since Garrod's first description of alkaptonuria in 1902, and newborn screening for phenylketonuria introduced in the 1960s, P4 medicine (preventive, predictive, personalized, and participatory) has been a reality for the clinician serving patients with inherited metabolic diseases. The era of high-throughput technologies promises to accelerate its scale dramatically. Genomics, transcriptomics, epigenomics, proteomics, glycomics, metabolomics, and lipidomics offer an amazing opportunity for holistic investigation and contextual pathophysiologic understanding of inherited metabolic diseases for precise diagnosis and tailored treatment. While each of the -omics technologies is important to systems biology, some are more mature than others. Exome sequencing is emerging as a reimbursed test in clinics around the world, and untargeted metabolomics has the potential to serve as a single biochemical testing platform. The challenge lies in the integration and cautious interpretation of these big data, with translation into clinically meaningful information and/or action for our patients. A daunting but exciting task for the clinician; we provide clinical cases to illustrate the importance of his/her role as the connector between physicians, laboratory experts and researchers in the basic, computer, and clinical sciences. Open collaborations, data sharing, functional assays, and model organisms play a key role in the validation of -omics discoveries. Having all the right expertise at the table when discussing the diagnostic approach and individualized management plan according to the information yielded by -omics investigations (e.g., actionable mutations, novel therapeutic interventions), is the stepping stone of P4 medicine. Patient participation and the adjustment of the medical team's plan to his/her and the family's wishes most certainly is the capstone. Are you ready?

Protein glycosylation in cancers and its potential therapeutic applications in neuroblastoma

Directory of Open Access Journals (Sweden)

Wan-Ling Ho

2016-09-01

Full Text Available Abstract Glycosylation is the most complex post-translational modification of proteins. Altered glycans on the tumor- and host-cell surface and in the tumor microenvironment have been identified to mediate critical events in cancer pathogenesis and progression. Tumor-associated glycan changes comprise increased branching of N-glycans, higher density of O-glycans, generation of truncated versions of normal counterparts, and generation of unusual forms of terminal structures arising from sialylation and fucosylation. The functional role of tumor-associated glycans (Tn, sTn, T, and sLea/x is dependent on the interaction with lectins. Lectins are expressed on the surface of immune cells and endothelial cells or exist as extracellular matrix proteins and soluble adhesion molecules. Expression of tumor-associated glycans is involved in the dysregulation of glycogenes, which mainly comprise glycosyltransferases and glycosidases. Furthermore, genetic and epigenetic mechanisms on many glycogenes are associated with malignant transformation. With better understanding of all aspects of cancer-cell glycomics, many tumor-associated glycans have been utilized for diagnostic, prognostic, and therapeutic purposes. Glycan-based therapeutics has been applied to cancers from breast, lung, gastrointestinal system, melanomas, and lymphomas but rarely to neuroblastomas (NBs. The success of anti-disialoganglioside (GD2, a glycolipid antigen antibodies sheds light on glycan-based therapies for NB and also suggests the possibility of protein glycosylation-based therapies for NB. This review summarizes our understanding of cancer glycobiology with a focus of how protein glycosylation and associated glycosyltransferases affect cellular behaviors and treatment outcome of various cancers, especially NB. Finally, we highlight potential applications of glycosylation in drug and cancer vaccine development for NB.

Nanoelectrospray high capacity ion trap multiple stage mass spectrometry for the structural analysis of human brain gangliosides

International Nuclear Information System (INIS)

Vukelic, Zeljka; Ratiu, Cornelia; Grozescu, Ioan; Zamfir, Alina Diana

2006-01-01

Full text: A novel protocol based on electrospray ionization (ESI) multiple stage high capacity ion trap (HCT) mass spectrometry (MS) was developed for glycosphingolipidomic surveys. The method was optimized for detailed structural elucidation of human brain gangliosides and particularly applied to human hippocampus-associated structures. The multiple stage MS experiments allowed for a complete structural characterization of GM1 ganglioside species, which was achieved by elucidation of the oligosaccharide sequence, identification of the GM1 a structural isomer from the data upon sialic acid localization along the sugar backbone and determination of the d18:1/18:0 of fatty acid/sphingoid base composition of the ceramide moiety. The methodology developed here is of general practical applicability for glycolipids and represents a step forward in the implementation of the advanced and most modern MS methods in glycomics. Gangliosides are glycosphingolipids, which consist of a mono- to polysialylated oligosaccharide chain of variable length attached to a ceramide portion of different composition with respect to the type of sphingoid base and fatty acid residues. Among all body systems, the central nervous system (CNS) possesses the highest content of gangliosides and they are playing a particularly important biological role at this level. Specific changes in the ganglioside expression and type of the expressed structures were observed to occur during brain development, maturation, and aging, and due to diseases or neurodegeneration processes. Gangliosides represent, therefore, an important class of biomarkers, carriers of information upon various CNS processes and events. Though in the human brain, their expression was observed to have a regional and tissue development induced specificity, the differences in ganglioside structure, composition and quantity were not systematically investigated or rigorously determined so far. (authors)

N-glycosylation profile of undifferentiated and adipogenically differentiated human bone marrow mesenchymal stem cells: towards a next generation of stem cell markers.

Science.gov (United States)

Hamouda, Houda; Ullah, Mujib; Berger, Markus; Sittinger, Michael; Tauber, Rudolf; Ringe, Jochen; Blanchard, Véronique

2013-12-01

Mesenchymal stem cells (MSCs) are multipotent cells that are easy to isolate and expand, develop into several tissues, including fat, migrate to diseased organs, have immunosuppressive properties and secrete regenerative factors. This makes MSCs ideal for regenerative medicine. For application and regulatory purposes, knowledge of (bio)markers characterizing MSCs and their development stages is of paramount importance. The cell surface is coated with glycans that possess lineage-specific nature, which makes glycans to be promising candidate markers. In the context of soft tissue generation, we aimed to identify glycans that could be markers for MSCs and their adipogenically differentiated progeny. MSCs were isolated from human bone marrow, adipogenically stimulated for 15 days and adipogenesis was verified by staining the lipid droplets and quantitative real time polymerase chain reaction of the marker genes peroxisome proliferator-activated receptor gamma (PPARG) and fatty acid binding protein-4 (FABP4). Using matrix-assisted laser desorption-ionization-time of flight mass spectrometry combined with exoglycosidase digestions, we report for the first time the N-glycome of MSCs during adipogenic differentiation. We were able to detect more than 100 different N-glycans, including high-mannose, hybrid, and complex N-glycans, as well as poly-N-acetyllactosamine chains. Adipogenesis was accompanied by an increased amount of biantennary fucosylated structures, decreased amount of fucosylated, afucosylated tri- and tetraantennary structures and increased sialylation. N-glycans H6N5F1 and H7N6F1 were significantly overexpressed in undifferentiated MSCs while H3N4F1 and H5N4F3 were upregulated in adipogenically differentiated MSCs. These glycan structures are promising candidate markers to detect and distinguish MSCs and their adipogenic progeny.

N-linked (N-) glycoproteomics of urinary exosomes. [Corrected].

Science.gov (United States)

Saraswat, Mayank; Joenväära, Sakari; Musante, Luca; Peltoniemi, Hannu; Holthofer, Harry; Renkonen, Risto

2015-02-01

Epithelial cells lining the urinary tract secrete urinary exosomes (40-100 nm) that can be targeted to specific cells modulating their functionality. One potential targeting mechanism is adhesion between vesicle surface glycoproteins and target cells. This makes the glycopeptide analysis of exosomes important. Exosomes reflect the physiological state of the parent cells; therefore, they are a good source of biomarkers for urological and other diseases. Moreover, the urine collection is easy and noninvasive and urinary exosomes give information about renal and systemic organ systems. Accordingly, multiple studies on proteomic characterization of urinary exosomes in health and disease have been published. However, no systematic analysis of their glycoproteomic profile has been carried out to date, whereas a conserved glycan signature has been found for exosomes from urine and other sources including T cell lines and human milk. Here, we have enriched and identified the N-glycopeptides from these vesicles. These enriched N-glycopeptides were solved for their peptide sequence, glycan composition, structure, and glycosylation site using collision-induced dissociation MS/MS (CID-tandem MS) data interpreted by a publicly available software GlycopeptideId. Released glycans from the same sample was also analyzed with MALDI-MS. We have identified the N-glycoproteome of urinary exosomes. In total 126 N-glycopeptides from 51 N-glycosylation sites belonging to 37 glycoproteins were found in our results. The peptide sequences of these N-glycopeptides were identified unambiguously and their glycan composition (for 125 N-glycopeptides) and structures (for 87 N-glycopeptides) were proposed. A corresponding glycomic analysis with released N-glycans was also performed. We identified 66 unique nonmodified N-glycan compositions and in addition 13 sulfated/phosphorylated glycans were also found. This is the first systematic analysis of N-glycoproteome of urinary exosomes. © 2015 by The

Comparative analysis of native and permethylated human milk oligosaccharides by liquid chromatography coupled to high resolution mass spectrometry.

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Oursel, Stéphanie; Cholet, Sophie; Junot, Christophe; Fenaille, François

2017-12-15

Human milk oligosaccharides (HMOs) represent the third most abundant components of milk after lactose and lipids. HMOs are indigestible by the suckling infant but can act as prebiotics and have significant biological functions regarding the organism defense against pathogens (such as bacteria or viruses) by preventing interactions with their receptors. Although constituted of only five distinct monosaccharide building blocks, HMOs are highly structurally diverse compounds with many co-existing structural isomers. Here we report the development and comparison of two distinct glycomic platforms based on liquid chromatography coupled to high resolution mass spectrometry (LC-MS) for analyzing HMOs. We have implemented and thoroughly compared the LC-MS of permethylated and native HMOs on reversed phase (RP) and porous graphitic carbon (PGC) columns for their ability to resolve the natural heterogeneity of milk oligosaccharides at the highest sensitivity. Our data essentially underlines the usefulness of analyzing HMOs as permethylated derivatives especially for getting more precise structural information at high sensitivity. For instance, permethylation annihilates gas-phase fucose migration during MS/MS experiments, thus facilitating spectra interpretation and giving access to relevant information regarding oligosaccharide branching and isomer distinction. At the opposite, LC-MS profiling of native HMOs (using PGC) in milk performed best in terms of detected species, while also being much faster in terms of sample preparation. Although less efficient than PGC chromatography, RPLC proved successful for separating pairs of permethylated isomeric HMOs. A key advantage of RP over PGC liquid chromatography is that retention times can be correlated to molecular weights, which can greatly facilitate further HMO identification using retention time prediction. Altogether these data lead us to think that LC-MS analysis of native HMOs (using PGC) can be used as first

Innate Immunity and Breast Milk.

Science.gov (United States)

Cacho, Nicole Theresa; Lawrence, Robert M

2017-01-01

Human milk is a dynamic source of nutrients and bioactive factors; unique in providing for the human infant's optimal growth and development. The growing infant's immune system has a number of developmental immune deficiencies placing the infant at increased risk of infection. This review focuses on how human milk directly contributes to the infant's innate immunity. Remarkable new findings clarify the multifunctional nature of human milk bioactive components. New research techniques have expanded our understanding of the potential for human milk's effect on the infant that will never be possible with milk formulas. Human milk microbiome directly shapes the infant's intestinal microbiome, while the human milk oligosaccharides drive the growth of these microbes within the gut. New techniques such as genomics, metabolomics, proteomics, and glycomics are being used to describe this symbiotic relationship. An expanded role for antimicrobial proteins/peptides within human milk in innate immune protection is described. The unique milieu of enhanced immune protection with diminished inflammation results from a complex interaction of anti-inflammatory and antioxidative factors provided by human milk to the intestine. New data support the concept of mucosal-associated lymphoid tissue and its contribution to the cellular content of human milk. Human milk stem cells (hMSCs) have recently been discovered. Their direct role in the infant for repair and regeneration is being investigated. The existence of these hMSCs could prove to be an easily harvested source of multilineage stem cells for the study of cancer and tissue regeneration. As the infant's gastrointestinal tract and immune system develop, there is a comparable transition in human milk over time to provide fewer immune factors and more calories and nutrients for growth. Each of these new findings opens the door to future studies of human milk and its effect on the innate immune system and the developing infant.

Online combination of reversed-phase/reversed-phase and porous graphitic carbon liquid chromatography for multicomponent separation of proteomics and glycoproteomics samples.

Science.gov (United States)

Lam, Maggie P Y; Lau, Edward; Siu, S O; Ng, Dominic C M; Kong, Ricky P W; Chiu, Philip C N; Yeung, William S B; Lo, Clive; Chu, Ivan K

2011-11-01

In this paper, we describe an online combination of reversed-phase/reversed-phase (RP-RP) and porous graphitic carbon (PGC) liquid chromatography (LC) for multicomponent analysis of proteomics and glycoproteomics samples. The online RP-RP portion of this system provides comprehensive 2-D peptide separation based on sequence hydrophobicity at pH 2 and 10. Hydrophilic components (e.g. glycans, glycopeptides) that are not retained by RP are automatically diverted downstream to a PGC column for further trapping and separation. Furthermore, the RP-RP/PGC system can provide simultaneous extension of the hydropathy range and peak capacity for analysis. Using an 11-protein mixture, we found that the system could efficiently separate native peptides and released N-glycans from a single sample. We evaluated the applicability of the system to the analysis of complex biological samples using 25 μg of the lysate of a human choriocarcinoma cell line (BeWo), confidently identifying a total of 1449 proteins from a single experiment and up to 1909 distinct proteins from technical triplicates. The PGC fraction increased the sequence coverage through the inclusion of additional hydrophilic sequences that accounted for up to 6.9% of the total identified peptides from the BeWo lysate, with apparent preference for the detection of hydrophilic motifs and proteins. In addition, RP-RP/PGC is applicable to the analysis of complex glycomics samples, as demonstrated by our analysis of a concanavalin A-extracted glycoproteome from human serum; in total, 134 potentially N-glycosylated serum proteins, 151 possible N-glycosylation sites, and more than 40 possible N-glycan structures recognized by concanavalin A were simultaneously detected. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

HEK293T cell lines defective for O-linked glycosylation.

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James M Termini

Full Text Available Here we describe derivatives of the HEK293T cell line that are defective in their ability to generate mucin-type O-linked glycosylation. Using CRISPR/Cas9 and a single-cell GFP-sorting procedure, the UDP-galactose-4-epimerase (GALE, galactokinase 1 (GALK1, and galactokinase 2 (GALK2 genes were knocked out individually and in combinations with greater than 90% of recovered clones having the desired mutations. Although HEK293T cells are tetraploid, we found this approach to be an efficient method to target and disrupt all 4 copies of the target gene. Deficient glycosylation in the GALE knockout cell line could be rescued by the addition of galactose and N-acetylgalactosamine (GalNAc to the cell culture media. However, when key enzymes of the galactose/GalNAc salvage pathways were disrupted in tandem (GALE+GALK1 or GALE+GALK2, O-glycosylation was eliminated and could not be rescued by the addition of either galactose plus GalNAc or UDP-galactose plus UDP-GalNAc. GALK1 and GALK2 are key enzymes of the galactose/GalNAc salvage pathways. Mass spectrometry was performed on whole cell lysate of the knockout cell lines to verify the glycosylation phenotype. As expected, the GALE knockout was almost completely devoid of all O-glycosylation, with minimal glycosylation as a result of functional salvage pathways. However, the GALE+GALK1 and GALE+GALK2 knockout lines were devoid of all O-glycans. Mass spectrometry analysis revealed that the disruption of GALE, GALK1, and GALE+GALK2 had little effect on the N-glycome. But when GALE was knocked out in tandem with GALK1, N-glycans were exclusively of the high mannose type. Due to the well-characterized nature of these five knockout cell lines, they will likely prove useful for a wide variety of applications.

Loss of Arabidopsis GAUT12/IRX8 causes anther indehiscence and leads to reduced G lignin associated with altered matrix polysaccharide deposition

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Zhangying eHao

2014-07-01

Full Text Available GAUT12 (GAlactUronosylTransferase12/IRX8 (IRregular Xylem8 is a putative glycosyltransferase involved in Arabidopsis secondary cell wall biosynthesis. Previous work showed that Arabidopsis irregular xylem8 (irx8 mutants have collapsed xylem due to a reduction in xylan and a lesser reduction in a subfraction of homogalacturonan (HG. We now show that male sterility in the irx8 mutant is due to indehiscent anthers caused by reduced deposition of xylan and lignin in the endothecium cell layer. The reduced lignin content was demonstrated by histochemical lignin staining and pyrolysis Molecular Beam Mass Spectrometry (pyMBMS and is associated with reduced lignin biosynthesis in irx8 stems. Examination of sequential chemical extracts of stem walls using 2D 13C-1H Heteronuclear Single-Quantum Correlation (HSQC NMR spectroscopy and antibody-based glycome profiling revealed a reduction in G lignin in the 1 M KOH extract and a concomitant loss of xylan, arabinogalactan and pectin epitopes in the ammonium oxalate, sodium carbonate, and 1 M KOH extracts from the irx8 walls compared with wild-type walls. Immunolabeling of stem sections using the monoclonal antibody CCRC-M138 reactive against an unsubstituted xylopentaose epitope revealed a bi-lamellate pattern in wild-type fiber cells and a collapsed bi-layer in irx8 cells, suggesting that at least in fiber cells, GAUT12 participates in the synthesis of a specific layer or type of xylan or helps to provide an architecture framework required for the native xylan deposition pattern. The results support the hypothesis that GAUT12 functions in the synthesis of a structure required for xylan and lignin deposition during secondary cell wall formation.

Identification of multiple isomeric core chitobiose-modified high-mannose and paucimannose N-glycans in the planarian Schmidtea mediterranea.

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Subramanian, Sabarinath Peruvemba; Babu, Ponnusamy; Palakodeti, Dasaradhi; Subramanian, Ramaswamy

2018-05-04

Cell surface-associated glycans mediate many cellular processes, including adhesion, migration, signaling, and extracellular matrix organization. The galactosylation of core fucose (GalFuc epitope) in paucimannose and complex-type N -glycans is characteristic of protostome organisms, including flatworms (planarians). Although uninvestigated, the structures of these glycans may play a role in planarian regeneration. Whole-organism MALDI-MS analysis of N -linked oligosaccharides from the planarian Schmidtea mediterranea revealed the presence of multiple isomeric high-mannose and paucimannose structures with unusual mono-, di-, and polygalactosylated ( n = 3-5) core fucose structures; the latter structures have not been reported in other systems. Di- and trigalactosylated core fucoses were the most dominant glycomers. N -Glycans showed extensive, yet selective, methylation patterns, ranging from non-methylated to polymethylated glycoforms. Although the majority of glycoforms were polymethylated, a small fraction also consisted of non-methylated glycans. Remarkably, monogalactosylated core fucose remained unmethylated, whereas its polygalactosylated forms were methylated, indicating structurally selective methylation. Using database searches, we identified two potential homologs of the Galβ1-4Fuc-synthesizing enzyme from nematodes (GALT-1) that were expressed in the prepharyngeal, pharyngeal, and mesenchymal regions in S. mediterranea. The presence of two GALT-1 homologs suggests different requirements for mono- and polygalactosylation of core fucose for the formation of multiple isomers. Furthermore, we observed variations in core fucose glycosylation patterns in different planarian strains, suggesting evolutionary adaptation in fucose glycosylation. The various core chitobiose modifications and methylations create >60 different glycoforms in S. mediterranea. These results contribute greatly to our understanding of N -glycan biosynthesis and suggest the presence of a

Hyper-O-GlcNAcylation of YB-1 affects Ser102 phosphorylation and promotes cell proliferation in hepatocellular carcinoma

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Liu, Qingqing [Department of Gastroenterology, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu Province (China); Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, 19 Qi-xiu Road, Nantong 226001, Jiangsu Province (China); Tao, Tao [Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, 19 Qi-xiu Road, Nantong 226001, Jiangsu Province (China); Liu, Fang [Key Laboratory of Neuroregeneration, Nantong University, Nantong 226001, Jiangsu Province (China); Ni, Runzhou [Department of Gastroenterology, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu Province (China); Lu, Cuihua, E-mail: lch1516@yeah.net [Department of Gastroenterology, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu Province (China); Shen, Aiguo, E-mail: shag@ntu.edu.cn [Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, 19 Qi-xiu Road, Nantong 226001, Jiangsu Province (China); Key Laboratory of Neuroregeneration, Nantong University, Nantong 226001, Jiangsu Province (China)

2016-12-10

As an essential post-translational modification, O-GlcNAcylation has been thought to be able to modulate various nuclear and cytoplasmic proteins and is emerging as a key regulator of multiple biological processes, such as transcription, cell growth, signal transduction, and cell motility. Recently, authoritative glycomics analyses have reported extensive crosstalk between O-GlcNAcylation and phosphorylation, which always dynamically interplay with each other and regulate signaling, transcription, and other cellular processes. Also, plentiful studies have shown close correlation between YB-1 phosphorylation and tumorigenesis. Therefore, our study aimed to determine whether YB-1 was O-GlcNAc modified and whether such modification could interact with its phosphorylation during the process of HCC development. Western blot and immunohistochemistry were firstly conducted to reveal obvious up-regulation of YB-1, OGT and O-GlcNAc modification in HCC tissues. What is more, not only YB-1 was identified to be O-GlcNAcylated but hyper-O-GlcNAcylation was demonstrated to facilitate HCC cell proliferation in a YB-1 dependent manner. Moreover, we detected four specific O-GlcNAc sites and confirmed T126A to be the most effective mutant in HCC cell proliferation via close O-GlcNAcylation-phosphorylation interaction. Even more interestingly, we discovered that T126A-induced HCC cell retardation and subdued transcriptional activity of YB-1 could be partially reversed by T126A/S102E mutant. From all above, it is not difficult to find that glycosylated-YB-1 mainly enhanced cell proliferation through congenerous actions with YB-1 phosphorylation and thus played indispensable roles in fine-tuning cell proliferation and procession of HCC. - Highlights: • YB-1 and OGT are associated with HCC prognosis. • YB-1 is O-GlcNAc modified in HCC. • Hyper-O-GlcNAcylation promotes HCC cell proliferation in dependent of YB-1. • The proliferating role of O-GlcNAcylation is based on Ser102

A Comprehensive, Open-source Platform for Mass Spectrometry-based Glycoproteomics Data Analysis.

Science.gov (United States)

Liu, Gang; Cheng, Kai; Lo, Chi Y; Li, Jun; Qu, Jun; Neelamegham, Sriram

2017-11-01

Glycosylation is among the most abundant and diverse protein post-translational modifications (PTMs) identified to date. The structural analysis of this PTM is challenging because of the diverse monosaccharides which are not conserved among organisms, the branched nature of glycans, their isomeric structures, and heterogeneity in the glycan distribution at a given site. Glycoproteomics experiments have adopted the traditional high-throughput LC-MS n proteomics workflow to analyze site-specific glycosylation. However, comprehensive computational platforms for data analyses are scarce. To address this limitation, we present a comprehensive, open-source, modular software for glycoproteomics data analysis called GlycoPAT (GlycoProteomics Analysis Toolbox; freely available from www.VirtualGlycome.org/glycopat). The program includes three major advances: (1) "SmallGlyPep," a minimal linear representation of glycopeptides for MS n data analysis. This format allows facile serial fragmentation of both the peptide backbone and PTM at one or more locations. (2) A novel scoring scheme based on calculation of the "Ensemble Score (ES)," a measure that scores and rank-orders MS/MS spectrum for N- and O-linked glycopeptides using cross-correlation and probability based analyses. (3) A false discovery rate (FDR) calculation scheme where decoy glycopeptides are created by simultaneously scrambling the amino acid sequence and by introducing artificial monosaccharides by perturbing the original sugar mass. Parallel computing facilities and user-friendly GUIs (Graphical User Interfaces) are also provided. GlycoPAT is used to catalogue site-specific glycosylation on simple glycoproteins, standard protein mixtures and human plasma cryoprecipitate samples in three common MS/MS fragmentation modes: CID, HCD and ETD. It is also used to identify 960 unique glycopeptides in cell lysates from prostate cancer cells. The results show that the simultaneous consideration of peptide and glycan

Innate Immunity and Breast Milk

Directory of Open Access Journals (Sweden)

Nicole Theresa Cacho

2017-05-01

Full Text Available Human milk is a dynamic source of nutrients and bioactive factors; unique in providing for the human infant’s optimal growth and development. The growing infant’s immune system has a number of developmental immune deficiencies placing the infant at increased risk of infection. This review focuses on how human milk directly contributes to the infant’s innate immunity. Remarkable new findings clarify the multifunctional nature of human milk bioactive components. New research techniques have expanded our understanding of the potential for human milk’s effect on the infant that will never be possible with milk formulas. Human milk microbiome directly shapes the infant’s intestinal microbiome, while the human milk oligosaccharides drive the growth of these microbes within the gut. New techniques such as genomics, metabolomics, proteomics, and glycomics are being used to describe this symbiotic relationship. An expanded role for antimicrobial proteins/peptides within human milk in innate immune protection is described. The unique milieu of enhanced immune protection with diminished inflammation results from a complex interaction of anti-inflammatory and antioxidative factors provided by human milk to the intestine. New data support the concept of mucosal-associated lymphoid tissue and its contribution to the cellular content of human milk. Human milk stem cells (hMSCs have recently been discovered. Their direct role in the infant for repair and regeneration is being investigated. The existence of these hMSCs could prove to be an easily harvested source of multilineage stem cells for the study of cancer and tissue regeneration. As the infant’s gastrointestinal tract and immune system develop, there is a comparable transition in human milk over time to provide fewer immune factors and more calories and nutrients for growth. Each of these new findings opens the door to future studies of human milk and its effect on the innate immune system

Molecular galactose-galectin association in neuroblastoma cells: An unconventional tool for qualitative/quantitative screening.

Science.gov (United States)

Pastorino, Fabio; Ponzoni, Mirco; Simone, Giuseppina

2017-05-01

Galectin decorates the cell membrane and forms an extracellular molecular association with galactoside units. Here, galactoside probes have been used to study galectin expression in neuroblastoma cells. The hypothesis behind this investigation has been that the molecular mechanisms by which glycans modulate neural metastatic cells involve a protein-carbohydrate association, galectin-galactose. Preliminary screening to validate the hypothesis has been performed with galactose moieties anchored to beads. The molecular association has been studied by FACS. In vitro experiments reveal the molecular binding preferences of the metastatic neuroblastoma cells. Ex vivo, the galactose probes discriminate healthy tissues. The unconventional assay in microfluidics used in this study displayed results analogous to the above (GI-LI-N cell capture efficiency overcomes IMR-32). At the point of equilibrium of shear and binding forces, the capture yield inside the chamber was measured to 60 ± 4.4% in GI-LI-N versus 40 ± 2.1% in IMR-32. Staining of the fished cells and subsequent conjugation with red beads bearing the galactose also have evidenced that microfluidics can be used to study and quantify the molecular association of galectin-galactose. Most importantly, a crucial insight for obtaining single-cell qualitative/quantitative glycome analysis has been achieved. Finally, the specificity of the assay performed in microfluidics is demonstrated by comparing GI-LI-N fishing efficiency in galactose and fucose environments. The residual adhesion to fucose confirmed the existence of receptors for this glycan and that its eventual unspecific binding (i.e. due to electrostatic interactions) is insignificant compared with the molecular binding. Identification and understanding of this mechanism of discrimination can be relevant for diagnostic monitoring and for producing probes tailored to interfere with galectin activities associated with the malignant phenotype. Besides, the given

The emerging functionality of endogenous lectins: A primer to the concept and a case study on galectins including medical implications.

Science.gov (United States)

Gabius, Hans-Joachim; Wu, Albert M

2006-01-01

Biochemistry textbooks commonly make it appear that it is a foregone conclusion that the hardware of biological information storage and transfer is confined to nucleotides and amino acids, the letters of the genetic code. However, the remarkable talents of a third class of biomolecules are often overlooked. For example, one of them far surpasses the building blocks of nucleic acids and proteins in terms of theoretical coding capacity by oligomer formation. Although often exclusively assigned to duties in energy metabolism, carbohydrates as part of cellular glycoconjugates (glycoproteins, proteoglycans, glycolipids) have, in fact, other important tasks. Currently, they are increasingly gaining recognition as an operative high-density information coding system. An elaborate enzymatic machinery enables cells to be versatile enough to produce a glycan profile (glycome) that is as characteristic as a fingerprint. Moreover, swift modifications during dynamic processes, such as differentiation or malignant transformation, are readily possible. The translation of the information presented in oligosaccharide determinants to biological responses is carried out by lectins. Recognition of foreign glycosignatures in innate immunity, regulation of cell-cell/matrix interactions, cell migration or growth, and intra- and intercellular glycan routing etc represent physiologically far-reaching lectin-carbohydrate functionality. The classification of endogenous lectins is guided by sequence alignments and conservation of distinct structural traits. For example, a jelly-roll-like folding pattern and maintenance of key residue positioning involved in stacking and C-H/pi-interactions as well as directional hydrogen bonds to the 1-galactoside ligands are common denominators among galectins. Biochemical and biophysical studies are beginning to unravel the intricacies of the selection of a limited set of endogenous ligands, such as certain integrins or ganglioside GM1, and combined with

Quantitative analysis of N-glycans from human alfa-acid-glycoprotein using stable isotope labeling and zwitterionic hydrophilic interaction capillary liquid chromatography electrospray mass spectrometry as tool for pancreatic disease diagnosis

International Nuclear Information System (INIS)

Giménez, Estela; Balmaña, Meritxell; Figueras, Joan; Fort, Esther; Bolós, Carme de; Sanz-Nebot, Victòria; Peracaula, Rosa; Rizzi, Andreas

2015-01-01

candidate structure worth to be corroborated by an extended study including more clinical cases; especially those with chronic pancreatitis and initial stages of pancreatic cancer. Importantly, the results demonstrate that the presented methodology combining an enrichment of a target protein by IAC with isotope coded relative quantitation of N-glycans can be successfully used for targeted glycomics studies. The methodology is assumed being suitable as well for other such studies aimed at finding novel cancer associated glycoprotein biomarkers

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

Quantitative analysis of N-glycans from human alfa-acid-glycoprotein using stable isotope labeling and zwitterionic hydrophilic interaction capillary liquid chromatography electrospray mass spectrometry as tool for pancreatic disease diagnosis

Energy Technology Data Exchange (ETDEWEB)

Giménez, Estela, E-mail: estelagimenez@ub.edu [Department of Analytical Chemistry, University of Barcelona, Diagonal 647, E-08028 Barcelona (Spain); Balmaña, Meritxell [Biochemistry and Molecular Biology Unit, Department of Biology, University of Girona, Campus Montilivi s/n, 17071 Girona (Spain); Figueras, Joan [Department of Surgery, Dr. Josep Trueta University Hospital, IdlBGi, 17007 Girona (Spain); Fort, Esther [Digestive Unit, Dr. Josep Trueta University Hospital, 17007 Girona (Spain); Bolós, Carme de [Gastroesophagic Cancer Research Group, Research Programme in Cancer, Hospital del Mar Medical Research Institute (IMIM), Dr. Aiguader, 88, 08003 Barcelona (Spain); Sanz-Nebot, Victòria [Department of Analytical Chemistry, University of Barcelona, Diagonal 647, E-08028 Barcelona (Spain); Peracaula, Rosa [Biochemistry and Molecular Biology Unit, Department of Biology, University of Girona, Campus Montilivi s/n, 17071 Girona (Spain); Rizzi, Andreas [Institute of Analytical Chemistry, University of Vienna, Währinger Straße 38, A-1090 Vienna (Austria)

2015-03-25

AGP as a candidate structure worth to be corroborated by an extended study including more clinical cases; especially those with chronic pancreatitis and initial stages of pancreatic cancer. Importantly, the results demonstrate that the presented methodology combining an enrichment of a target protein by IAC with isotope coded relative quantitation of N-glycans can be successfully used for targeted glycomics studies. The methodology is assumed being suitable as well for other such studies aimed at finding novel cancer associated glycoprotein biomarkers.