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1
Walker, Sierra A., Jesús S. Aguilar Díaz De león, Sara Busatto, Gregory A. Wurtz, Abba C. Zubair, Chad R. Borges, and Joy Wolfram. "Glycan Node Analysis of Plasma-Derived Extracellular Vesicles." Cells 9, no. 9 (August 22, 2020): 1946. http://dx.doi.org/10.3390/cells9091946.
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Blood plasma is a readily accessible source of extracellular vesicles (EVs), i.e., cell-secreted nanosized carriers that contain various biomolecules, including glycans. Previous studies have demonstrated that glycans play a major role in physiological and pathological processes, and certain plasma glycans have been associated with disease conditions. However, glycome studies have been limited by a lack of analytical techniques with the throughput capacity necessary to study hundreds of clinical samples. This study is the first to characterize the EV plasma glycome based on all major glycan classes. The results based on glycan node analysis revealed, as expected, that plasma-derived EVs have distinct glycan features from donor-matched whole plasma. Specifically, glycan nodes corresponding to those observed in chondroitin sulfate, dermatan sulfate, type I keratan sulfate, and type II keratan sulfate were enriched on EVs. The identification of specific differences in glycan features in plasma vs. plasma-derived EVs is relevant for understanding the physiological role of EVs and as a reference for future diagnostic studies. Additionally, the results indicate that EV glycan nodes do not substantially differ among a small set of healthy donors. These results lay the framework for the further evaluation of all EV glycan classes as diagnostic markers, therapeutic targets, and biologically active components in health and disease.
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Wopereis, Suzan, Stephanie Grünewald, Karin MLC Huijben, Éva Morava, Rosella Mollicone, Baziel GM van Engelen, Dirk J. Lefeber, and Ron A. Wevers. "Transferrin and Apolipoprotein C-III Isofocusing Are Complementary in the Diagnosis of N- and O-Glycan Biosynthesis Defects." Clinical Chemistry 53, no. 2 (February 1, 2007): 180–87. http://dx.doi.org/10.1373/clinchem.2006.073940.
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Abstract Background: Apolipoprotein C-III (apoC-III) isoelectric focusing (IEF) can be used to detect abnormalities in the biosynthesis of core 1 mucin-type O-glycans. Methods: We studied plasma samples from 55 patients with various primary defects in N- and/or O-glycosylation, 21 patients with secondary N-glycosylation defects, and 6 patients with possible glycosylation abnormalities. Furthermore, we analyzed 500 plasma samples that were sent to our laboratory for selective screening for inborn errors of metabolism. Results: Plasma samples from patients with congenital disorders of glycosylation (CDG) types –IIe and –IIf showed a hypoglycosylated apoC-III isoform profile, as did plasma samples from 75% of the patients with an unspecified CDG type II. Hyposialylated O-glycan profiles were also seen in plasma from 2 patients with hemolytic-uremic syndrome. In the 500 plasma samples from the selective screening, 3 patients were identified with a possible isolated defect in the biosynthesis of core 1 mucin-type O-glycans. Conclusions: To our knowledge this is the first study in which use of a plasma marker protein has identified patients in whom only O-glycan biosynthesis might be affected. The primary defect(s) remain as yet unknown. Plasma apoC-III IEF is complementary to transferrin isofocusing. In conjunction both tests identify biosynthesis defects in N-glycan and mucin-type core 1 O-glycan biosynthesis. The apoC-III IEF assay is likely to help metabolic laboratories to identify and unravel further subtypes of inborn errors of glycan biosynthesis.
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Eckardt, Veit, Christian Weber, and Philipp von Hundelshausen. "Glycans and Glycan-Binding Proteins in Atherosclerosis." Thrombosis and Haemostasis 119, no. 08 (July 2, 2019): 1265–73. http://dx.doi.org/10.1055/s-0039-1692720.
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AbstractComplex glycans are readily accessible on the endothelium and on cell and plasma components. They interact with glycan-binding proteins which translate their structure into function. Advanced analytical tools are available to investigate their structure and functional interactions. Modifications to glycan structures which alter their capacity to bind proteins are particularly relevant in atherosclerosis. We summarize the regulatory role of glycans and their binding partners in the development of the disease. Given their complexity, accessibility, and important functional role, glycans and glycan-binding proteins represent promising diagnostic tools and therapeutic targets.
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Chen, Jie, Xueli Li, Andrew Edmondson, Gail Ditewig Meyers, Kosuke Izumi, Amanda M. Ackermann, Eva Morava, Can Ficicioglu, Michael J. Bennett, and Miao He. "Increased Clinical Sensitivity and Specificity of Plasma Protein N-Glycan Profiling for Diagnosing Congenital Disorders of Glycosylation by Use of Flow Injection–Electrospray Ionization–Quadrupole Time-of-Flight Mass Spectrometry." Clinical Chemistry 65, no. 5 (May 1, 2019): 653–63. http://dx.doi.org/10.1373/clinchem.2018.296780.
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Abstract BACKGROUND Congenital disorders of glycosylation (CDG) represent 1 of the largest groups of metabolic disorders with >130 subtypes identified to date. The majority of CDG subtypes are disorders of N-linked glycosylation, in which carbohydrate residues, namely, N-glycans, are posttranslationally linked to asparagine molecules in peptides. To improve the diagnostic capability for CDG, we developed and validated a plasma N-glycan assay using flow injection–electrospray ionization–quadrupole time-of-flight mass spectrometry. METHODS After PNGase F digestion of plasma glycoproteins, N-glycans were linked to a quinolone using a transient amine group at the reducing end, isolated by a hydrophilic interaction chromatography column, and then identified by accurate mass and quantified using a stable isotope-labeled glycopeptide as the internal standard. RESULTS This assay differed from other N-glycan profiling methods because it was free of any contamination from circulating free glycans and was semiquantitative. The low end of the detection range tested was at 63 nmol/L for disialo-biantennary N-glycan. The majority of N-glycans in normal plasma had <1% abundance. Abnormal N-glycan profiles from 19 patients with known diagnoses of 11 different CDG subtypes were generated, some of which had previously been reported to have normal N-linked protein glycosylation by carbohydrate-deficient transferrin analysis. CONCLUSIONS The clinical specificity and sensitivity of N-glycan analysis was much improved with this method. Additional CDGs can be diagnosed that would be missed by carbohydrate-deficient transferrin analysis. The assay provides novel biomarkers with diagnostic and potentially therapeutic significance.
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Ashwood, Heather E., Christopher Ashwood, Anna P. Schmidt, Rebekah L. Gundry, Karin M. Hoffmeister, and Waseem Q. Anani. "Characterization and statistical modeling of glycosylation changes in sickle cell disease." Blood Advances 5, no. 5 (March 5, 2021): 1463–73. http://dx.doi.org/10.1182/bloodadvances.2020003376.
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AbstractSickle cell disease is an inherited genetic disorder that causes anemia, pain crises, organ infarction, and infections in 13 million people worldwide. Previous studies have revealed changes in sialic acid levels associated with red blood cell sickling and showed that stressed red blood cells bare surface-exposed clustered terminal mannose structures mediating hemolysis, but detailed glycan structures and anti-glycan antibodies in sickle cell disease remain understudied. Here, we compiled results obtained through lectin arrays, glycan arrays, and mass spectrometry to interrogate red blood cell glycoproteins and glycan-binding proteins found in the plasma of healthy individuals and patients with sickle cell disease and sickle cell trait. Lectin arrays and mass spectrometry revealed an increase in α2,6 sialylation and a decrease in α2,3 sialylation and blood group antigens displayed on red blood cells. Increased binding of proteins to immunogenic asialo and sialyl core 1, Lewis A, and Lewis Y structures was observed in plasma from patients with sickle cell disease, suggesting a heightened anti-glycan immune response. Data modeling affirmed glycan expression and plasma protein binding changes in sickle cell disease but additionally revealed further changes in ABO blood group expression. Our data provide detailed insights into glycan changes associated with sickle cell disease and refer glycans as potential therapeutic targets.
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Kratz, Ewa M., Anna Kałuża, Mariusz Zimmer, and Mirosława Ferens-Sieczkowska. "The Analysis of Sialylation,N-Glycan Branching, and Expression ofO-Glycans in Seminal Plasma of Infertile Men." Disease Markers 2015 (2015): 1–7. http://dx.doi.org/10.1155/2015/941871.
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Carbohydrates are known to mediate some events involved in successful fertilization. Although some studies on the glycosylation of seminal plasma proteins are available, the total glycan profile was rarely analyzed as a feature influencing fertilization potential. In this work we aimed to compare some glycosylation traits in seminal plasma glycoproteins of fertile and infertile men. The following findings emerge from our studies: (1) in human seminal plasma the presence and alterations ofO-linked glycans were observed; (2) the expression of SNA-reactive sialic acid significantly differs between asthenozoospermia and both normozoospermic (fertile and infertile) groups; (3) the expression of PHA-L-reactive highly branchedN-glycans was significantly lower in oligozoospermic patients than in both normozoospermic groups. Indication of the appropriate lectins that would enable the possibly precise determination of the glycan profile seems to be a good supplement to mass spectrum analysis. Extension of the lectin panel is useful for the further research.
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Canis, Kevin, Thomas A. J. McKinnon, Agata Nowak, Stuart M. Haslam, Maria Panico, Howard R. Morris, Mike A. Laffan, and Anne Dell. "Mapping the N-glycome of human von Willebrand factor." Biochemical Journal 447, no. 2 (September 26, 2012): 217–28. http://dx.doi.org/10.1042/bj20120810.
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vWF (von Willebrand factor) is a key component for maintenance of normal haemostasis, acting as the carrier protein of the coagulant Factor VIII and mediating platelet adhesion at sites of vascular injury. There is ample evidence that vWF glycan moieties are crucial determinants of its expression and function. Of particular clinical interest, ABH antigens influence vWF plasma levels according to the blood group of individuals, although the molecular mechanism underlying this phenomenon remains incompletely understood. The present paper reports analyses of the human plasma vWF N-glycan population using advanced MS. Glycomics analyses revealed approximately 100 distinct N-glycan compositions and identified a variety of structural features, including lactosaminic extensions, ABH antigens and sulfated antennae, as well as bisecting and terminal GlcNAc residues. We estimate that some 300 N-glycan structures are carried by human vWF. Glycoproteomics analyses mapped ten of the consensus sites known to carry N-glycans. Glycan populations were found to be distinct, although many structural features were shared across all sites. Notably, the H antigen is not restricted to particular N-glycosylation sites. Also, the Asn2635 site, previously designated as unoccupied, was found to be highly glycosylated. The delineation of such varied glycan populations in conjunction with current models explaining vWF activity will facilitate research aimed at providing a better understanding of the influence of glycosylation on vWF function.
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Chatterton, B. D., J. Mullington, H. Yang, M. Haack, R. Cummings, and S. D. Lehoux. "0058 Effects of Acute Total Sleep Deprivation on Human Plasma N-glycans." Sleep 43, Supplement_1 (April 2020): A23. http://dx.doi.org/10.1093/sleep/zsaa056.056.
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Abstract Introduction There is a need for a novel biomarker that can be used to measure sleep sufficiency as it pertains to fitness for duty. As glycans (polysaccharides) are known to be involved in modifying protein effectiveness, we are exploring these as biomarkers that may be sensitive to differences between sleep deprivation and normal healthy adult sleep duration. We have measured one major class of glycans, called N-glycans, which are covalently linked to asparagine residues of polypeptide chains of membrane-bound and secreted proteins. We compared the plasma N-glycan profiles of participants before and after they participated in a total sleep deprivation protocol. Methods 10 healthy participants (6 male, 4 female) aged 30–44 went through 88 hours of total sleep deprivation. Hourly blood draws were taken via forearm catheter throughout the protocol. N-glycan analysis was performed using plasma samples collected at 17:35 prior to the first night of sleep deprivation and at 17:35 following 82.5 hours of continuous wakefulness. N-glycans were first cleaved from peptides and isolated from plasma, and profiles were then measured using Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) mass spectrometry. Results 66 N-glycans were observed in our profiles. Of these, the relative abundance of 17 N-glycans were significantly different following sleep deprivation (paired t-test, 13 with p<0.05, 4 with p<0.01). In each case, the relative abundance was lower in the sleep deprivation time point. We found two structures, Hex6HexNAc5NeuAc3 and Hex7HexNAc6NeuAc2, which were also significant in one of our previous chronic sleep restriction protocols. Conclusion While we observed that many N-glycans decreased in relative abundance, it is unclear whether these changes represent a shift in glycan synthesis or result from decreased expression of the proteins they are bound to. Our next steps involve exploring the functions of the proteins associated with Hex6HexNAc5NeuAc3 and Hex7HexNAc6NeuAc2, and measuring their expression levels. Support NIH/HL75501; NIH/National Center for Research Resources UL1-RR02758 and M01-RR01032 to the Harvard Clinical and Translational Science Center.
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Simunovic, Jelena, Marija Vilaj, Irena Trbojevic-Akmacic, Ana Momcilovic, Frano Vuckovic, Ivan Gudelj, Julija Juric, Natali Nakic, Gordan Lauc, and Marija Pezer. "Comprehensive N-glycosylation analysis of immunoglobulin G from dried blood spots." Glycobiology 29, no. 12 (May 30, 2019): 817–21. http://dx.doi.org/10.1093/glycob/cwz061.
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Abstract Immunoglobulin G (IgG) glycans are emerging as a new putative biomarker for biological age and different diseases, requiring a robust workflow for IgG glycome analysis, ideally beginning with a simple and undemanding sampling procedure. Here, we report the first comprehensive study on total N-glycans of IgG isolated from dried blood spots (DBSs), which was performed in a high-throughput mode. We compared the IgG N-glycan profiles originating from DBS with those originating from plasma, compared different media for DBS collection, evaluated analytical variation and assessed IgG N-glycan profile stability for different storage conditions. In conclusion, we show that DBSs are a good and stable source material for a robust IgG N-glycan analysis by ultra-performance liquid chromatography, suitable for blood sampling in conditions where no trained personnel and necessary laboratory equipment are available.
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Nakahara, Taku, Diane McCarthy, Yoshiaki Miura, and Hidehisa Asada. "High-throughput glycomics for discovery of cancer biomarkers." Journal of Clinical Oncology 30, no. 30_suppl (October 20, 2012): 9. http://dx.doi.org/10.1200/jco.2012.30.30_suppl.9.
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9 Background: While the importance of glycosylation in many cancers is well established, the use of glycomics in biomarker research has lagged behind genomics and proteomics. This is due, in part, to the lack of practical platforms capable of analyzing clinically relevant sample numbers. To address these challenges, we have developed a novel glycomics technology (the GlycanMap platform) that combines a high-throughput assay with custom bioinformatics and rapidly provides both biomarker candidates and information on the underlying biology. Methods: N-glycans were enzymatically released from their parent glycoproteins and captured on chemoselective beads. After washing to remove non-glycan components, purified glycans were derivatized to stabilize labile sialic acids and released from the beads. The steps described above were automated on a 96-well format robotics system to maximize throughput and reduce variability and can be performed in less than 24 hours. Released glycans were analyzed by MALDI-TOF MS using internal standards to facilitate quantitation. In addition to comparing individual glycans between groups, glycan changes were also analyzed with respect to known glycan biosynthetic pathways. Results: The automated assay was compatible with multiple biological sample types, including serum/plasma, tissue, and cell lysates. Human serum was used to assess assay performance and yielded 50-60 glycans with CVs of 10-15% and good linearity. The lower limit of detection was approximately 100 nM. The assay was applied to drug-treated colon cancer cells (HCT116) and revealed significant (> 2-fold) changes in 17 glycans. Projection of these glycan changes on the known N-glycan pathway showed that the most significant changes occurred in the medial-Golgi. Conclusions: We have developed and optimized a high-throughput glycomics platform to facilitate large-scale biomarker studies and assured its practical performance in terms of sensitivity, repeatability, and linearity. Application of this assay to drug-treated colon cancer cells demonstrated that projection of individual glycan changes against known glycan pathways provided additional information about biological mechanism and relevance.
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Verbij, Fabian, Eva Stokhuijzen, Floris van Alphen, Paul Kaijen, Alexander Meijer, and Jan Voorberg. "Analysis of the Glycan Composition on Plasma Derived ADAMTS13 Employing Tandem Mass Spectrometry." Blood 126, no. 23 (December 3, 2015): 1069. http://dx.doi.org/10.1182/blood.v126.23.1069.1069.
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Abstract Acquired thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder that results from the development of auto-antibodies against ADAMTS13 disrupting the binding of ADAMTS13 to von Willebrand factor and thereby preventing the proteinase activity and/or increasing the clearance from the circulation. Previous research from our department identified 9 O-linked glycosylation, 6 O-fucosylation and 2 C-mannosylation sites on plasma derived ADAMTS13. One of the N-linked glycosylation sites (N1354) is close to one of the previously identified peptides preferentially presented on HLA-DRB1*0301 and HLA-DRB1*1501 (ASYILIRD amino acid A1355-D1362) and also close to the HLA-DRB1*1101 peptide (FINVAPHAR amino acid F1328-R1336) suggesting a possible role for the glycosylation in the onset of acquired TTP. To study the glycosylation and glycan trees ADAMTS13 purified from cryosupernatant was reduced with dithiothreitol, alkylated with iodoacetamide and subsequently processed into peptides overnight with either trypsin or chymotrypsin. The peptides were then purified using ZIC-HILIC proteatips and finally analyzed by tandem mass spectrometry employing both higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD). The data files were analyzed using the BYONIC software package as well as manually. Using this approach we identified the glycan structure on 10 N-linked glycosylation. Nine out of 10 glycans contained complex carbohydrate structures terminating in sialic acid. The glycans at these N-linked sites were identified both with or without a fucose on the primary GlcNAc. We were unable to identify a GalNAc residue in the glycan linked to N614 in the spacer domain. This suggest that the glycan on N614 consist primarily of high mannose structures. Binding of ADAMTS13 to the mannose receptor on dendritic cells is most likely facilitated by the high mannose glycan on N614. Furthermore we identified 6 O-linked glycosylation sites either on a serine of a threonine. One O-linked glycan is located in the spacer domain, 2 were found in the thrombospondin type 1 repeat-6 (TSP6), another one was found in TSP8 and 1 O-linked glycosylation site was found in both of the CUB domains. Four out of 6 O-glycans contained terminal sialic acid of which 2 also contained a fucose attached to the GlcNAc. Several O-glycans contained a terminal galactose residue; one O-glycan in TSP6 terminated in both a GlcNAc and a GalNAc residue. O-fucosylation is a common post-translational modification of thrombospondin type 1 repeats. We identified 9 O-fucosylation sites in the TSP repeats. Seven out of 9 sites adhered to the consensus sequence previously defined for O-fucosylation. TSP1 and 2 contained an additional O-fucosylation site at residues T407 and S724; these sites did not match the consensus sequence for O-fucosylation. Interestingly, two additional O-fucosylation sites were identified in cysteine rich and spacer domain at residue S553 and S698. All these residues were predicted to contain a glucose-fucose modification. Next to these glucose-fucose modifications we also identified 2 fucose modification in both of the CUB domains at residues S1170 and T1344. These results show that ADAMTS13 is extensively modified by O-fucosylation. Evidence for C-mannosylation of 8 different tryptophans was obtained. In accordance with previous findings the W387 or W390 (TSP1) and W884 (TSP4) were found to be C-mannosylated. We also found C-mannosylated tryptophans at position and W730 (TSP2) and W1081 (TSP8). Four additional C-mannosylated tryptophans were detected at position W208 (metallo proteinase domain), W1307 (CUB1 domain) and W1379 and W1406 (CUB2 domain). These results show that C-mannosylation is a common post translational modification in ADAMTS13 that is also found outside the TSP domains. Taken together these findings highlight the extensive post translational modification of ADAMTS13 by diverse carbohydrate structures. We anticipate that our findings might be relevant for the clearance and/or immune recognition of ADAMTS13. Disclosures No relevant conflicts of interest to declare.
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Wopereis, Suzan, Stephanie Grünewald, Éva Morava, Johannes M. Penzien, Paz Briones, M. Teresa Garcı́a-Silva, Pierre N. M. Demacker, Karin M. L. C. Huijben, and Ron A. Wevers. "Apolipoprotein C-III Isofocusing in the Diagnosis of Genetic Defects in O-Glycan Biosynthesis." Clinical Chemistry 49, no. 11 (November 1, 2003): 1839–45. http://dx.doi.org/10.1373/clinchem.2003.022541.
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Abstract Background: Defects in the biosynthesis of N-glycans may be found by isoelectric focusing (IEF) of plasma transferrin. No test is available to demonstrate O-glycan biosynthesis defects. Methods: We used isoforms of apolipoprotein C-III (apoC-III) as a marker for the biosynthesis of core 1 mucin type O-glycans. Plasma samples from patients with primary defects and secondary alterations in N-glycan biosynthesis were studied by apoC-III isofocusing. Results: Age-related reference values for apoC-III were determined. Plasma samples from patients with the primary congenital disorders of glycosylation (CDG) types Ia–Ic, Ie, If, IIa, and IId all showed a normal apoC-III isofocusing profile. Plasma from two patients with CDG type IIx were tested: one showed a normal apoC-III distribution, whereas the other showed a hypoglycosylation profile. In plasma from patients with hemolytic uremic syndrome (HUS), a hypoglycosylation profile was obtained. Conclusions: IEF of apoC-III is a rapid and simple technique that may be used as a screening assay for abnormalities in core 1 mucin type O-glycans. Evidence that a patient in this study has a primary genetic defect affecting both N- and O-glycosylation provides the first example of an inborn error of metabolism affecting the biosynthesis of core 1 mucin type O-glycans. Our data narrow the options for the position of the primary defect in this patient down to a step in the biosynthesis, activation, or transfer of galactose or N-acetylneuraminic acid to both N- and O-glycans. Circulating neuraminidase excreted by Streptococcus pneumoniae caused the high percentage of asialo apoC-III in two HUS patients.
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Guillard, Maïlys, Eva Morava, Floris L. van Delft, Rosie Hague, Christian Körner, Maciej Adamowicz, Ron A. Wevers, and Dirk J. Lefeber. "Plasma N-Glycan Profiling by Mass Spectrometry for Congenital Disorders of Glycosylation Type II." Clinical Chemistry 57, no. 4 (April 1, 2011): 593–602. http://dx.doi.org/10.1373/clinchem.2010.153635.
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BACKGROUND Determination of the genetic defect in patients with a congenital disorder of glycosylation (CDG) is challenging because of the wide clinical presentation, the large number of gene products involved, and the occurrence of secondary causes of underglycosylation. Transferrin isoelectric focusing has been the method of choice for CDG screening; however, improved methods are required for the molecular diagnosis of patients with CDG type II. METHODS Plasma samples with a typical transferrin isofocusing profile were analyzed. N-glycans were released from these samples by PNGase F [peptide-N4-(acetyl-β-glucosaminyl)-asparagine amidase] digestion, permethylated and purified, and measured on a MALDI linear ion trap mass spectrometer. A set of 38 glycans was used for quantitative comparison and to establish reference intervals for such glycan features as the number of antennae, the level of truncation, and fucosylation. Plasma N-glycans from control individuals, patients with known CDG type II defects, and patients with a secondary cause of underglycosylation were analyzed. RESULTS CDGs due to mannosyl (α-1,6-)-glycoprotein β-1,2-N-acetylglucosaminyltransferase (MGAT2), β-1,4-galactosyltransferase 1 (B4GALT1), and SLC35C1 (a GDP-fucose transporter) defects could be diagnosed directly from the N-glycan profile. CDGs due to defects in proteins involved in Golgi trafficking, such as subunit 7 of the conserved oligomeric Golgi complex (COG7) and subunit V0 a2 of the lysosomal H+-transporting ATPase (ATP6V0A2) caused a loss of triantennary N-glycans and an increase of truncated structures. Secondary causes with liver involvement were characterized by increased fucosylation, whereas the presence of plasma sialidase produced isolated undersialylation. CONCLUSIONS MALDI ion trap analysis of plasma N-glycans documents features that discriminate between primary and secondary causes of underglycosylation and should be applied as the first step in the diagnostic track of all patients with an unsolved CDG type II.
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Adua, Eric, Elham Memarian, Alyce Russell, Irena Trbojević-Akmačić, Ivan Gudelj, Julija Jurić, Peter Roberts, Gordan Lauc, and Wei Wang. "Utilization of N-glycosylation profiles as risk stratification biomarkers for suboptimal health status and metabolic syndrome in a Ghanaian population." Biomarkers in Medicine 13, no. 15 (October 2019): 1273–87. http://dx.doi.org/10.2217/bmm-2019-0005.
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Aim: The study sought to apply N-glycosylation profiles to understand the interplay between suboptimal health status (SHS) and metabolic syndrome (MetS). Materials & methods: In this study, 262 Ghanaians were recruited from May to July 2016. After completing a health survey, plasma samples were collected for clinical assessments while ultra performance liquid chromatography was used to measure plasma N-glycans. Results: Four glycan peaks were found to predict case status (MetS and SHS) using a step-wise Akaike’s information criterion logistic regression model selection. This model yielded an area under the curve of MetS: 83.1% (95% CI: 78.0–88.1%) and SHS: 67.1% (60.6–73.7%). Conclusion: Our results show that SHS is a significant, albeit modest, risk factor for MetS and N-glycan complexity was associated with MetS.
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McKinnon, Thomas A. J., Alain C. K. Chion, and Mike A. Laffan. "The Effect of Von Willebrand Factor Glycans on the Interaction with ADAMTS13." Blood 108, no. 11 (November 16, 2006): 1701. http://dx.doi.org/10.1182/blood.v108.11.1701.1701.
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Abstract Von Willebrand factor (VWF) is a large plasma glycoprotein that mediates platelet tethering at sites of vascular injury. This function is dependent upon its multimeric size, which is modulated in plasma through proteolysis by ADAMTS13. Terminal ABO blood group sugars presented on the N-linked glycans of VWF alter the susceptibility of VWF to cleavage by ADAMTS13. Two predicted N-linked glycan sites (N1515 & N1574) are in close proximity to the ADAMTS13 cleavage site (Y1605-M1606). However, is it not known whether these predicted sites are occupied, or indeed, present the ABO sugars. In this study, purified plasma VWF was digested with trypsin, and the resulting fragments separated by ion-exchange chromatography. A 55kDa fragment observed under reducing SDS-PAGE was identified as VWF residues 1493–1849 by mass spectrocosopy and N-terminal sequencing. This fragment encompassed the predicted N-linked glycan sites at N1515 & N1574. Incomplete PNGase F digestion produced three protein bands, indicating that both glycosylation sites were occupied. Furthermore, analysis with blood group specific lectins demonstrated presentation of the ABO sugars on these glycan chains. This data suggests that the N1515 and N1574 glycosylation sites and subsequent ABO(H) sugar modification may influence VWF proteolysis. To further elucidate the role of other VWF glycan structures on susceptibility to ADAMTS13 cleavage, purified VWF was treated with neuraminidase to remove terminal sialic acid residues, producing asialo VWF (As-VWF), and PNGase F to remove whole N-linked glycan chains. (Nless-VWF). Lectin analysis demonstrated removal of >95% of terminal sialic residues and >75% whole N-linked glycan chains. Both AsVWF and Nless-VWF bound to type III collagen with similar affinity to wild type VWF (KD 1.9nM, 1.6nM, 1.4nM respectively) and demonstrated similar multimeric composition. Interestingly, As-VWF had decreased susceptibility to ADAMTS13 cleavage, but bound ADAMTS13 with comparable affinity to wild type VWF (KD 11nM & 12nM respectively). Nless-VWF was cleaved faster by ADAMTS13 and bound ADAMTS13 with ~ 4-fold increased affinity (KD 3.4nM). This data demonstrates that the glycan moiety of VWF plays an important role in mediating the interaction with ADAMTS13.
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Wibmer, Constantinos Kurt, Jason Gorman, Colin S. Anthony, Nonhlanhla N. Mkhize, Aliaksandr Druz, Talita York, Stephen D. Schmidt, et al. "Structure of an N276-Dependent HIV-1 Neutralizing Antibody Targeting a Rare V5 Glycan Hole Adjacent to the CD4 Binding Site." Journal of Virology 90, no. 22 (August 31, 2016): 10220–35. http://dx.doi.org/10.1128/jvi.01357-16.
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ABSTRACT All HIV-1-infected individuals develop strain-specific neutralizing antibodies to their infecting virus, which in some cases mature into broadly neutralizing antibodies. Defining the epitopes of strain-specific antibodies that overlap conserved sites of vulnerability might provide mechanistic insights into how broadly neutralizing antibodies arise. We previously described an HIV-1 clade C-infected donor, CAP257, who developed broadly neutralizing plasma antibodies targeting an N276 glycan-dependent epitope in the CD4 binding site. The initial CD4 binding site response potently neutralized the heterologous tier 2 clade B viral strain RHPA, which was used to design resurfaced gp120 antigens for single-B-cell sorting. Here we report the isolation and structural characterization of CAP257-RH1, an N276 glycan-dependent CD4 binding site antibody representative of the early CD4 binding site plasma response in donor CAP257. The cocrystal structure of CAP257-RH1 bound to RHPA gp120 revealed critical interactions with the N276 glycan, loop D, and V5, but not with aspartic acid 368, similarly to HJ16 and 179NC75. The CAP257-RH1 monoclonal antibody was derived from the immunoglobulin-variable IGHV3-33 and IGLV3-10 genes and neutralized RHPA but not the transmitted/founder virus from donor CAP257. Its narrow neutralization breadth was attributed to a binding angle that was incompatible with glycosylated V5 loops present in almost all HIV-1 strains, including the CAP257 transmitted/founder virus. Deep sequencing of autologous CAP257 viruses, however, revealed minority variants early in infection that lacked V5 glycans. These glycan-free V5 loops are unusual holes in the glycan shield that may have been necessary for initiating this N276 glycan-dependent CD4 binding site B-cell lineage. IMPORTANCE The conserved CD4 binding site on gp120 is a major target for HIV-1 vaccine design, but key events in the elicitation and maturation of different antibody lineages to this site remain elusive. Studies have shown that strain-specific antibodies can evolve into broadly neutralizing antibodies or in some cases act as helper lineages. Therefore, characterizing the epitopes of strain-specific antibodies may help to inform the design of HIV-1 immunogens to elicit broadly neutralizing antibodies. In this study, we isolate a narrowly neutralizing N276 glycan-dependent antibody and use X-ray crystallography and viral deep sequencing to describe how gp120 lacking glycans in V5 might have elicited these early glycan-dependent CD4 binding site antibodies. These data highlight how glycan holes can play a role in the elicitation of B-cell lineages targeting the CD4 binding site.
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Hino, Shingo, Takayasu Mizushima, Katsunori Kaneko, Erika Kawai, Takashi Kondo, Tomomi Genda, Takahiro Yamada, Koji Hase, Naomichi Nishimura, and Tatsuya Morita. "Mucin-Derived O-Glycans Act as Endogenous Fiber and Sustain Mucosal Immune Homeostasis via Short-Chain Fatty Acid Production in Rat Cecum." Journal of Nutrition 150, no. 10 (April 14, 2020): 2656–65. http://dx.doi.org/10.1093/jn/nxaa097.
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ABSTRACT Background Intestinal mucins escape digestion and enter the large bowel where they are degraded by the microbiota. To what extent and how mucins impact large-bowel physiology remain unclear. Objective This study examined the large-bowel fermentation characteristics of mucins and mucin-derived O-glycan sugars and whether they affect gut immunity. Methods Mucin secretion from the terminal ileum was determined from feces of ileorectostomized male Wistar rats (age 6 wk) fed an AIN76-based control diet (CD) for 15 d (experiment 1). Normal male Wistar rats (age 6 wk; 4 wk for experiment 4) were fed CD ± porcine stomach mucin (PM) at 6 or 12 g/kg diet, equivalent to 1.5 and 3 times the daily mucin secretion, for 14 d (experiment 2); CD ± N-acetylglucosamine (GlcNAc), fucose, or N-acetylneuraminic acid at 10 g/kg diet for 14 d (experiment 3); or CD ± PM (15 g/kg diet) or GlcNAc (10 g/kg diet) for 29 d (experiment 4). SCFAs, microbial composition, and cecal O-glycan content were assessed. IgA+ plasma cells and regulatory T cells and inflammatory cytokine expression in the cecum were evaluated (experiment 4). Results Daily mucin secretion corresponded to 43.2 μmol of O-glycans. Cecal O-glycan contents were comparable between CD- and PM-fed rats. PM-fed rats harbored more mucin-degrading bacteria. Cecal concentrations of acetate (+37%) and n-butyrate (+73%) were higher in 12-g/kg PM diet–fed rats versus CD (P < 0.05). Among O-glycan sugars, only GlcNAc produced higher n-butyrate concentrations (+68%) versus CD (P < 0.05), with increased numbers of butyrate-producing bacteria. GlcNAc increased the abundance of IgA+ plasma cells (+29%) and regulatory T cells (+33%) versus CD, whereas PM increased IgA+ plasma cells (+25%) (all P < 0.05). GlcNAc and PM decreased expression of Tnfa (−30%, −40%) and Ifng (−30%, −70%) versus CD (all P < 0.05). Conclusions Mucin-derived O-glycans act as endogenous fiber and maintain mucosal immune homeostasis via large-bowel SCFA production in rats.
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Elola, María T., Ada G. Blidner, Fátima Ferragut, Candelaria Bracalente, and Gabriel A. Rabinovich. "Assembly, organization and regulation of cell-surface receptors by lectin–glycan complexes." Biochemical Journal 469, no. 1 (June 19, 2015): 1–16. http://dx.doi.org/10.1042/bj20150461.
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Galectins are a family of β-galactoside-binding lectins carrying at least one consensus sequence in the carbohydrate-recognition domain. Properties of glycosylated ligands, such as N- and O-glycan branching, LacNAc (N-acetyl-lactosamine) content and the balance of α2,3- and α2,6-linked sialic acid dramatically influence galectin binding to a preferential set of counter-receptors. The presentation of specific glycans in galectin-binding partners is also critical, as proper orientation and clustering of oligosaccharide ligands on multiple carbohydrate side chains increase the binding avidity of galectins for particular glycosylated receptors. When galectins are released from the cells, they typically concentrate on the cell surface and the local matrix, raising their local concentration. Thus galectins can form their own multimers in the extracellular milieu, which in turn cross-link glycoconjugates on the cell surface generating galectin–glycan complexes that modulate intracellular signalling pathways, thus regulating cellular processes such as apoptosis, proliferation, migration and angiogenesis. Subtle changes in receptor expression, rates of protein synthesis, activities of Golgi enzymes, metabolite concentrations supporting glycan biosynthesis, density of glycans, strength of protein–protein interactions at the plasma membrane and stoichiometry may modify galectin–glycan complexes. Although galectins are key contributors to the formation of these extended glycan complexes leading to promotion of receptor segregation/clustering, and inhibition of receptor internalization by surface retention, when these complexes are disrupted, some galectins, particularly galectin-3 and -4, showed the ability to drive clathrin-independent mechanisms of endocytosis. In the present review, we summarize the data available on the assembly, hierarchical organization and regulation of conspicuous galectin–glycan complexes, and their implications in health and disease.
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Mittermayr, Stefan, Jonathan Bones, Giao N. Le, and Peter O'Gorman. "N-Glycan Analysis of Polyclonal IgG from Patients with Multiple Myeloma Enables Classification of Stage Specific Pathologies." Blood 126, no. 23 (December 3, 2015): 1761. http://dx.doi.org/10.1182/blood.v126.23.1761.1761.
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Abstract Background Multiple myeloma (MM) is an incurable plasma cell malignancy, with eventual disease refractory and relapse. Its benign precursor, monoclonal gammopathy of undetermined significance (MGUS), has an annual transformation rate of 1%, while that of the asymptomatic smouldering myeloma (SMM) is 10%. The pathognomonic feature is the presence abnormal monoclonal immunoglobulin, of which immunoglobulin G (IgG) paraprotein is the most common. All subclasses of Igs are post-translationally modified by the addition of N-glycans, reportedly influencing structure, stability, and biological function. Previous studies in MM IgG had suggested an increase in the glycosylation of the antigen-binding fragment (Fab), and an overall elevation of sialylation. Using glycomic platforms, we aimed to investigate and characterise the IgG N-glycosylation profiles across the myeloma disease spectrum. Methods Polyclonal IgG was extracted from sera of patients with MM, SMM, MGUS, and age-matched control, using Protein G affinity chromatography. The quantity of extracted IgG was determined using the Bradford assay. N-glycans were enzymatically liberated from a normalised quantity of purified IgG, fluorescently labelled and profiled using hydrophilic interaction ultra-performance liquid chromatography. A combination of exoglycosidase digestions and mass spectrometry were used to elucidate the glycan structures with full linkage and positional specificity. Localisation analyses were performed to determine the distribution of N-glycans at asparagine 297 in the Fc region of the antibody and those present at any additional glycosylation sites present in the Fab region using a combination of enzymatic digestion using the commercially available IdeS enzyme and chemical reduction followed by SDS-PAGE separation of the resulting protein fragments and subsequent in-gel digestion of the N-glycans. Non-supervised principal component analysis (PCA) was employed to detect distinguishable chromatographic features among the studied groups. Longitudinal analyses of samples from individual patients collected during multiple clinical assessments were also performed. Results N-glycan analysis of polyclonal IgG showed unique N-glycan peaks with statistically significant chromatogram variation across the 4 studied groups. PCA identified specific patterns of glycosylation present in the glycan profiles, thus demonstrating the ability to distinguish between MGUS, SMM, MM and control. Sialylated biantennary N-glycans and N-glycans containing bisecting GlcNAc residues contributed most to the PCA separation. Further characterisation of the glycans using sialic acid linkage specific derivatisation and LC-MS analysis confirmed that sialic acids were present in an α2-6 linked configuration. Localisation analysis revealed N-glycans present in the Fc region of the extracted polyclonal antibodies consisted of the standard biantennary type glycans with core fucosylation, variable degrees of galactosylation and low levels of sialylation. Such oligosaccharide structures suggest that the Fc regions of polyclonal IgG present in patients with varying stages of plasma cell disorder maintain a correct orientation to facilitate interaction with Fcγ receptors. Sialylated biantennary N-glycans, identified during global polyclonal IgG glycosylation profiling, were found to be located predominantly in the Fab region of the antibody. The formation of these larger highly sialylated N-glycan structures is likely due to the removal of steric hindrance resulting in more facilitated access by the associated glycosyltransferases required for oligosaccharide biosynthesis. The presence of these charged oligosaccharide structures, with their inherent structural dynamics, are likely to affect the ability of the antibody to recognise antigen and form an immune complex. Conclusions Glycan analysis of polyclonal IgG extracted from the sera of patients with varying stages of myeloma progression is reported. Differential glycosylation on polyclonal IgG was observed between the patients with MGUS, SMM, and MM, with alterations in the levels of sialylated biantennary structures and glycans bearing bisecting GlcNAc residues capable of distinguishing between the patient groups in the spectrum of plasma cell disorders. Disclosures No relevant conflicts of interest to declare.
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Wu, Di, Weston B. Struwe, David J. Harvey, Michael A. J. Ferguson, and Carol V. Robinson. "N-glycan microheterogeneity regulates interactions of plasma proteins." Proceedings of the National Academy of Sciences 115, no. 35 (August 15, 2018): 8763–68. http://dx.doi.org/10.1073/pnas.1807439115.
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Altered glycosylation patterns of plasma proteins are associated with autoimmune disorders and pathogenesis of various cancers. Elucidating glycoprotein microheterogeneity and relating subtle changes in the glycan structural repertoire to changes in protein–protein, or protein–small molecule interactions, remains a significant challenge in glycobiology. Here, we apply mass spectrometry-based approaches to elucidate the global and site-specific microheterogeneity of two plasma proteins: α1-acid glycoprotein (AGP) and haptoglobin (Hp). We then determine the dissociation constants of the anticoagulant warfarin to different AGP glycoforms and reveal how subtle N-glycan differences, namely, increased antennae branching and terminal fucosylation, reduce drug-binding affinity. Conversely, similar analysis of the haptoglobin–hemoglobin (Hp–Hb) complex reveals the contrary effects of fucosylation and N-glycan branching on Hp–Hb interactions. Taken together, our results not only elucidate how glycoprotein microheterogeneity regulates protein–drug/protein interactions but also inform the pharmacokinetics of plasma proteins, many of which are drug targets, and whose glycosylation status changes in various disease states.
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Pismenetskaya, I. U., and T. D. Butters. "Chromatographic profiles of blood plasma free oligosaccharides in patients with cardiovascular disease." Visnyk of Dnipropetrovsk University. Biology, medicine 6, no. 1 (March 26, 2015): 51–56. http://dx.doi.org/10.15421/021510.
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Free oligosaccharides (FOS) are unbound structural analogs of glycans of glycoconjugates. There are several sources of them inside the cell: 1) multistep pathways of N-glycosylation, 2) the cell quality control and endoplastic reticulum-associated degradation of mis-glycosylated and/or misfolded glycoproteins, 3) lysosomal degradation of mature glycoconjugates. Some of these FOS are the earliest indicators of potential glycosylation alterations that would be revealed in the course of the cell quality control and the endoplastic reticulum-associated degradation. Ischemia and hypertension cause stress of intracellular organelles leading to disruption of their functions. The main objective of the work was the characterization of free oligosaccharides (FOS) in plasma obtained from patients with cardiovascular diseases compared to those from healthy subjects to evaluate the potential of these compounds for diagnostics. Chromatographic profiles of FOS composed of 4–12 monosaccharides were obtained and analyzed for quantitative and qualitative differences between the samples. After plasma deproteinization and FOS purification the oligosaccharides were labelled with anthranilic acid (2-AA), separated into the neutral and charged with QAE Sephadex (Q25-120) chromatography and analysed using high-performance liquid chromatography (HPLC). Glucose unit values were determined following comparison with a 2-AA-labelled glucose oligomer ladder derived from a partial hydrolysate of dextran as an external standard. The data were collected and processed using Empower software. The charged FOS were digested with the sialidase from Arthrobacter ureafaciens. 2-AA – labelled free oligosaccharides from transferrin were used as an external standard for the structure decoding. The profiles obtained were compared with intracellular free oligosaccharides of known structures and with the glycan structures and their descriptions in the databases GlycoBase and EUROCarbDB. These approaches allowed predicting a range of glycan structures for each of the main peaks of HPLC profiles of plasma free oligosaccharides and managing ways for their future experimental analysis. In the case of cardiovascular disorders, HPLC profiles of FOS revealed a changing pattern of heterogeneity, depending on the severity of the disease. Three main enlarged glycan species in the netral fraction and one peak in the charged fraction distinguished the FOS of the patients from those of the healthy volunteers. It has been revealed that the neutral marker peaks were represented by polimannose glycans with 5–7 mannose residues and 1–2 residues of N-acetylglucosamine, and one of the major peaks of the charged fraction – by two-antennary complex N-glycan with two sialic acid residues. The study of free oligosaccharides of blood plasma is a new field of glycobiology allowing an evaluation of an organism state at the level of the cell organelle functional status and openning up broad prospects for finding early diagnostic and prognostic markers of cardiac insufficiency.
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Maric, Tihana, Ana Katusic Bojanac, Ana Matijevic, Marcello Ceppi, Marco Bruzzone, Evangelini Evgeni, Tea Petrovic, et al. "Seminal Plasma Protein N-Glycan Peaks Are Potential Predictors of Semen Pathology and Sperm Chromatin Maturity in Men." Life 11, no. 9 (September 20, 2021): 989. http://dx.doi.org/10.3390/life11090989.
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Background: Male infertility is increasingly becoming a health and demographic problem. While it may originate from congenital or acquired diseases, it can also result from environmental exposure. Hence, the complexity of involved molecular mechanisms often requires a multiparametric approach. This study aimed to associate semen parameters with sperm DNA fragmentation, chromatin maturity and seminal plasma protein N-glycosylation. Methods: The study was conducted with 166 participants, 20–55 y old, 82 normozoospermic and 84 with pathological diagnosis. Sperm was analyzed by Halosperm assay and aniline blue staining, while seminal plasma total protein N-glycans were analyzed by ultra-high-performance liquid chromatography. Results: Sperm DNA fragmentation was significantly increased in the pathological group and was inversely correlated with sperm motility and viability. Seminal plasma total protein N-glycans were chromatographically separated in 37 individual peaks. The pattern of seminal plasma N-glycan peaks (SPGP) showed that SPGP14 significantly differs between men with normal and pathological semen parameters (p < 0.001). The multivariate analysis showed that when sperm chromatin maturity increases by 10%, SPGP17 decreases by 14% while SPGP25 increases by 25%. Conclusion: DNA integrity and seminal plasma N-glycans are associated with pathological sperm parameters. Specific N-glycans are also associated with sperm chromatin maturity and have a potential in future fertility research and clinical diagnostics.
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Gornik, O., J. Wagner, M. Pucic, A. Knezevic, I. Redzic, and G. Lauc. "Stability of N-glycan profiles in human plasma." Glycobiology 19, no. 12 (September 2, 2009): 1547–53. http://dx.doi.org/10.1093/glycob/cwp134.
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Vogt, Marian Samuel, Gesa Felicitas Schmitz, Daniel Varón Silva, Hans-Ulrich Mösch, and Lars-Oliver Essen. "Structural base for the transfer of GPI-anchored glycoproteins into fungal cell walls." Proceedings of the National Academy of Sciences 117, no. 36 (August 24, 2020): 22061–67. http://dx.doi.org/10.1073/pnas.2010661117.
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The correct distribution and trafficking of proteins are essential for all organisms. Eukaryotes evolved a sophisticated trafficking system which allows proteins to reach their destination within highly compartmentalized cells. One eukaryotic hallmark is the attachment of a glycosylphosphatidylinositol (GPI) anchor to C-terminal ω-peptides, which are used as a zip code to guide a subset of membrane-anchored proteins through the secretory pathway to the plasma membrane. In fungi, the final destination of many GPI-anchored proteins is their outermost compartment, the cell wall. Enzymes of the Dfg5 subfamily catalyze the essential transfer of GPI-anchored substrates from the plasma membrane to the cell wall and discriminate between plasma membrane-resident GPI-anchored proteins and those transferred to the cell wall (GPI-CWP). We solved the structure of Dfg5 from a filamentous fungus and used in crystallo glycan fragment screening to reassemble the GPI-core glycan in a U-shaped conformation within its binding pocket. The resulting model of the membrane-bound Dfg5•GPI-CWP complex is validated by molecular dynamics (MD) simulations and in vivo mutants in yeast. The latter show that impaired transfer of GPI-CWPs causes distorted cell-wall integrity as indicated by increased chitin levels. The structure of a Dfg5•β1,3-glycoside complex predicts transfer of GPI-CWP toward the nonreducing ends of acceptor glycans in the cell wall. In addition to our molecular model for Dfg5-mediated transglycosylation, we provide a rationale for how GPI-CWPs are specifically sorted toward the cell wall by using GPI-core glycan modifications.
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Suhre, Karsten, Irena Trbojević-Akmačić, Ivo Ugrina, Dennis Mook-Kanamori, Tim Spector, Johannes Graumann, Gordan Lauc, and Mario Falchi. "Fine-Mapping of the Human Blood Plasma N-Glycome onto Its Proteome." Metabolites 9, no. 7 (June 26, 2019): 122. http://dx.doi.org/10.3390/metabo9070122.
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Most human proteins are glycosylated. Attachment of complex oligosaccharides to the polypeptide part of these proteins is an integral part of their structure and function and plays a central role in many complex disorders. One approach towards deciphering this human glycan code is to study natural variation in experimentally well characterized samples and cohorts. High-throughput capable large-scale methods that allow for the comprehensive determination of blood circulating proteins and their glycans have been recently developed, but so far, no study has investigated the link between both traits. Here we map for the first time the blood plasma proteome to its matching N-glycome by correlating the levels of 1116 blood circulating proteins with 113 N-glycan traits, determined in 344 samples from individuals of Arab, South-Asian, and Filipino descent, and then replicate our findings in 46 subjects of European ancestry. We report protein-specific N-glycosylation patterns, including a correlation of core fucosylated structures with immunoglobulin G (IgG) levels, and of trisialylated, trigalactosylated, and triantennary structures with heparin cofactor 2 (SERPIND2). Our study reveals a detailed picture of protein N-glycosylation and suggests new avenues for the investigation of its role and function in the associated complex disorders.
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Shajahan, Asif, Nitin T. Supekar, Digantkumar Chapla, Christian Heiss, Kelley W. Moremen, and Parastoo Azadi. "Simplifying Glycan Profiling through a High-Throughput Micropermethylation Strategy." SLAS TECHNOLOGY: Translating Life Sciences Innovation 25, no. 4 (May 4, 2020): 367–79. http://dx.doi.org/10.1177/2472630320912929.
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Glycoproteins play key roles in various molecular and cellular functions and are among the most difficult to analyze biomolecules on account of their microheterogeneity, non-template-driven synthesis, and low abundances. The stability, serum half-life, immunogenicity, and biological activity of therapeutic glycoproteins, including antibodies, vaccines, and biomarkers, are regulated by their glycosylation profile. Thus, there is increasing demand for the qualitative and quantitative characterization and validation of glycosylation on glycoproteins. One of the most important derivatization processes for the structural characterization of released glycans by mass spectrometry (MS) is permethylation. We have recently developed a permethylation strategy in microscale that allows facile permethylation of glycans and permits the processing of large sample sets in nanogram amounts through high-throughput sample handling. Here, we are reporting the wide potential of micropermethylation-based high-throughput structural analysis of glycans from various sources, including human plasma, mammalian cells, and purified glycoproteins, through an automated tandem electrospray ionization–mass spectrometry (ESI-MSn) platform. The glycans released from the plasma, cells, and glycoproteins are permethylated in microscale in a 96-well plate or microcentrifuge tube and isolated by a C18 tip-based cleanup through a shorter and simple process. We have developed a workflow to accomplish an in-depth automated structural characterization MS program for permethylated N/O-glycans through an automated high-throughput multistage tandem MS acquisition. We have demonstrated the utility of this workflow using the examples of sialic acid linkages and bisecting GlcNAc ( N-acetylglucosamine) on the glycans. This approach can automate the high-throughput screening of glycosylation on large sample sets of glycoproteins, including clinical glycan biomarkers and glycoprotein therapeutics.
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Hoffmeister, Karin M. "Novel Glycan Dependent Platelet Clearance Mechanisms." Blood 130, Suppl_1 (December 7, 2017): SCI—31—SCI—31. http://dx.doi.org/10.1182/blood.v130.suppl_1.sci-31.sci-31.
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Platelet numbers are intricately regulated to avoid spontaneous bleeding or arterial occlusion and organ damage. The growth factor thrombopoietin (TPO) promotes platelet biogenesis by controlling megakaryocyte maturation and differentiation. Recently, we identified a feedback mechanism by which clearance of aged, desialylated platelets stimulates TPO synthesis by hepatocytes in mice. The Ashwell-Morell receptor (AMR) of hepatocytes, originally termed the hepatic asialoglycoprotein receptor, was the first cellular receptor to be identified and isolated and the first lectin (carbohydrate-binding) to be detected in mammals. Our work showed that the AMR recognizes and removes senescent, sialic acid-depleted platelets (desialylated) under steady state conditions. Desialylated platelets and the AMR are the physiological ligand-receptor pair regulating hepatic TPO mRNA production. The AMR-mediated removal of desialylated platelets regulates hepatic TPO synthesis by recruiting JAK2 and STAT3 to increase thrombopoiesis. Recent genetics studies have revealed that AMR haplodeficiency provides protection from atherosclerosis by regulating plasma glycolipids and platelets. The potential interactions of AMR with LDL receptor may regulate the rate of LDL uptake and as a result may lower plasma non-HDL cholesterol. Taken together, the AMR appears to be a multifaceted receptor, specializing in the clearance of desialylated platelets and plasma glycolipids. Thus, platelet clearance, its biogenesis via TPO, and vascular integrity appear to be regulated by intricately interwoven mechanisms dependent on the AMR. Disclosures Hoffmeister: Amgen: Consultancy.
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Hipgrave Ederveen, Agnes L., Noortje de Haan, Melissa Baerenfaenger, Dirk J. Lefeber, and Manfred Wuhrer. "Dissecting Total Plasma and Protein-Specific Glycosylation Profiles in Congenital Disorders of Glycosylation." International Journal of Molecular Sciences 21, no. 20 (October 15, 2020): 7635. http://dx.doi.org/10.3390/ijms21207635.
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Protein N-glycosylation is a multifactorial process involved in many biological processes. A broad range of congenital disorders of glycosylation (CDGs) have been described that feature defects in protein N-glycan biosynthesis. Here, we present insights into the disrupted N-glycosylation of various CDG patients exhibiting defects in the transport of nucleotide sugars, Golgi glycosylation or Golgi trafficking. We studied enzymatically released N-glycans of total plasma proteins and affinity purified immunoglobulin G (IgG) from patients and healthy controls using mass spectrometry (MS). The applied method allowed the differentiation of sialic acid linkage isomers via their derivatization. Furthermore, protein-specific glycan profiles were quantified for transferrin and IgG Fc using electrospray ionization MS of intact proteins and glycopeptides, respectively. Next to the previously described glycomic effects, we report unprecedented sialic linkage-specific effects. Defects in proteins involved in Golgi trafficking (COG5-CDG) and CMP-sialic acid transport (SLC35A1-CDG) resulted in lower levels of sialylated structures on plasma proteins as compared to healthy controls. Findings for these specific CDGs include a more pronounced effect for α2,3-sialylation than for α2,6-sialylation. The diverse abnormalities in glycomic features described in this study reflect the broad range of biological mechanisms that influence protein glycosylation.
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Tudor, Lucija, Gordana Nedic Erjavec, Matea Nikolac Perkovic, Marcela Konjevod, Dubravka Svob Strac, Suzana Uzun, Oliver Kozumplik, Tanja Jovanovic, Gordan Lauc, and Nela Pivac. "N-glycomic Profile in Combat Related Post-Traumatic Stress Disorder." Biomolecules 9, no. 12 (December 6, 2019): 834. http://dx.doi.org/10.3390/biom9120834.
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Post-traumatic stress disorder (PTSD) develops in a portion of individuals exposed to extreme trauma. Glycosylation is a post-translational modification that affects protein functions and is altered in various pathophysiological states and aging. There are still no validated biomarkers of PTSD. The aim of this study was to evaluate the N-glycomic profile in 543 male Caucasian individuals (299 veterans with PTSD and 244 control subjects). The study included discovery (N = 233) and replication (N = 310) cohort. Hydrophilic interaction HPLC and ultra-performance liquid chromatography were used to separate and detect 39 plasma and 24 IgG N-glycan species, respectively. All results were corrected for the effects of age and multiple testing. Significant results included only significantly altered N-glycans in cases/controls in both cohorts, in the same direction. Results showed that six plasma N-glycans (four increased and two decreased) were altered in PTSD vs. controls in both cohorts, but IgG N-glycans were similar between groups. The severity of PTSD was not associated with different plasma N-glycans. This is the first study detecting alterations in plasma N-glycans in PTSD. These N-glycans are also associated with other neuropsychiatric disorders and inflammation, suggesting possible shared glycosylation mechanisms.
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Hino, Shingo, Takayasu Mizushima, Katsunori Kaneko, Erika Kawai, Takashi Kondo, Tomomi Genda, Takahiro Yamada, Koji Hase, Naomichi Nishimura, and Tatsuya Morita. "Mucin-Derived O-Glycans Act as Endogenous Fiber and Sustain Mucosal Immune Homeostasis via Short-Chain Fatty Acid Production in Rat Cecum." Current Developments in Nutrition 4, Supplement_2 (May 29, 2020): 1563. http://dx.doi.org/10.1093/cdn/nzaa062_020.
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Abstract Objectives Intestinal mucins escape digestion and enter the large bowel where they are degraded by microbiome. To what extent and how mucins impact large-bowel physiology remain unclear. This study examined the large-bowel fermentation characteristics of mucins and mucin-derived O-glycan sugars and whether mucins and N-acetylglucosamine (GlcNAc) affect gut immunity. Methods Mucin secretion from the terminal ileum was determined from feces of ileorectostomized male Wistar rats (age 6 weeks) fed AIN76-based control diet (CD) for 15 d (Expt. 1). Normal male Wistar rats (age 6 weeks; 4 weeks for Expt. 4) were fed CD ± porcine stomach mucin (PM) at 6 or 12g/kg diet, equivalent to 1.5 and 3 times daily mucin secretion, for 14 d (Expt. 2); CD ± GlcNAc, fucose, or N-acetylneuraminic acid at 10g/kg diet for 14 d (Expt. 3); or CD ± PM (15 g/kg diet) or GlcNAc (10 g/kg diet) for 29 d (Expt. 4). Short-chain fatty acids (SCFAs), microbial composition, and cecal O-glycan content were assessed. IgA+ plasma and regulatory T cells and inflammatory cytokine expression in the cecum were evaluated (Expt. 4). Results Daily mucin secretion corresponded to 43.2 mmol of O-glycans. PM was efficiently fermented in the cecum, as evidenced by comparable amounts of cecal O-glycans between groups. PM-fed rats harbored more mucin-degrading bacteria. Cecal SCFA concentrations, particularly n-butyrate, were higher in 12g/kg PM diet-fed rats versus CD (P < 0.05). Among O-glycan sugars, only GlcNAc produced higher n-butyrate concentrations versus CD (P < 0.05), with increased numbers of several butyrate-producing bacteria. GlcNAc increased the abundance of IgA + plasma and regulatory T cells versus CD (P < 0.05). PM produced a similar but less-significant trend. GlcNAc and PM feeding decreased expression of Tnfa and Ifng versus CD (P < 0.05). Conclusions Mucin-derived O-glycans act as endogenous fiber and maintain mucosal immune homeostasis via large-bowel SCFA production in rats. Funding Sources JSPS KAKENHI.
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Pochechueva, Tatiana, Alexander Chinarev, Andreas Schoetzau, André Fedier, Nicolai V. Bovin, Neville F. Hacker, Francis Jacob, and Viola Heinzelmann-Schwarz. "Blood Plasma-Derived Anti-Glycan Antibodies to Sialylated and Sulfated Glycans Identify Ovarian Cancer Patients." PLOS ONE 11, no. 10 (October 20, 2016): e0164230. http://dx.doi.org/10.1371/journal.pone.0164230.
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Fang, Fei, Qun Zhao, Zhigang Sui, Yu Liang, Hao Jiang, Kaiguang Yang, Zhen Liang, Lihua Zhang, and Yukui Zhang. "Glycan Moieties as Bait to Fish Plasma Membrane Proteins." Analytical Chemistry 88, no. 10 (April 27, 2016): 5065–71. http://dx.doi.org/10.1021/acs.analchem.6b01082.
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Ferdosi, Shadi, Thai H. Ho, Erik P. Castle, Melissa L. Stanton, and Chad R. Borges. "Behavior of blood plasma glycan features in bladder cancer." PLOS ONE 13, no. 7 (July 24, 2018): e0201208. http://dx.doi.org/10.1371/journal.pone.0201208.
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Dotz, Viktoria, Roosmarijn F. H. Lemmers, Karli R. Reiding, Agnes L. Hipgrave Ederveen, Aloysius G. Lieverse, Monique T. Mulder, Eric J. G. Sijbrands, Manfred Wuhrer, and Mandy van Hoek. "Plasma protein N-glycan signatures of type 2 diabetes." Biochimica et Biophysica Acta (BBA) - General Subjects 1862, no. 12 (December 2018): 2613–22. http://dx.doi.org/10.1016/j.bbagen.2018.08.005.
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Greto, Valentina L., Ana Cvetko, Tamara Štambuk, Niall J. Dempster, Domagoj Kifer, Helena Deriš, Ana Cindrić, et al. "Extensive weight loss reduces glycan age by altering IgG N-glycosylation." International Journal of Obesity 45, no. 7 (May 3, 2021): 1521–31. http://dx.doi.org/10.1038/s41366-021-00816-3.
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Abstract Background Obesity, a major global health problem, is associated with increased cardiometabolic morbidity and mortality. Protein glycosylation is a frequent posttranslational modification, highly responsive to inflammation and ageing. The prospect of biological age reduction, by changing glycosylation patterns through metabolic intervention, opens many possibilities. We have investigated whether weight loss interventions affect inflammation- and ageing-associated IgG glycosylation changes, in a longitudinal cohort of bariatric surgery patients. To support potential findings, BMI-related glycosylation changes were monitored in a longitudinal twins cohort. Methods IgG N-glycans were chromatographically profiled in 37 obese patients, subjected to low-calorie diet, followed by bariatric surgery, across multiple timepoints. Similarly, plasma-derived IgG N-glycan traits were longitudinally monitored in 1680 participants from the TwinsUK cohort. Results Low-calorie diet induced a marked decrease in the levels of IgG N-glycans with bisecting GlcNAc, whose higher levels are usually associated with ageing and inflammatory conditions. Bariatric surgery resulted in extensive alterations of the IgG N-glycome that accompanied progressive weight loss during 1-year follow-up. We observed a significant increase in digalactosylated and sialylated glycans, and a substantial decrease in agalactosylated and core fucosylated IgG N-glycans (adjusted p value range 7.38 × 10−04–3.94 × 10−02). This IgG N-glycan profile is known to be associated with a younger biological age and reflects an enhanced anti-inflammatory IgG potential. Loss of BMI over a 20 year period in the TwinsUK cohort validated a weight loss-associated agalactosylation decrease (adjusted p value 1.79 × 10−02) and an increase in digalactosylation (adjusted p value 5.85 × 10−06). Conclusions Altogether, these findings highlight that weight loss substantially affects IgG N-glycosylation, resulting in reduced glycan and biological age.
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Singh, Richa, Mohammed Mashari Almutairi, Romario Pacheco-Andrade, Mohamed Y. Mahmoud Almiahuob, and Mauricio Di Fulvio. "Impact of Hybrid and Complex N-Glycans on Cell Surface Targeting of the Endogenous Chloride CotransporterSlc12a2." International Journal of Cell Biology 2015 (2015): 1–20. http://dx.doi.org/10.1155/2015/505294.
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The Na+K+2Cl−cotransporter-1 (Slc12a2, NKCC1) is widely distributed and involved in cell volume/ion regulation. Functional NKCC1 locates in the plasma membrane of all cells studied, particularly in the basolateral membrane of most polarized cells. Although the mechanisms involved in plasma membrane sorting of NKCC1 are poorly understood, it is assumed that N-glycosylation is necessary. Here, we characterize expression, N-glycosylation, and distribution of NKCC1 in COS7 cells. We show that ~25% of NKCC1 is complex N-glycosylated whereas the rest of it corresponds to core/high-mannose and hybrid-type N-glycosylated forms. Further, ~10% of NKCC1 reaches the plasma membrane, mostly as core/high-mannose type, whereas ~90% of NKCC1 is distributed in defined intracellular compartments. In addition, inhibition of the first step of N-glycan biosynthesis with tunicamycin decreases total and plasma membrane located NKCC1 resulting in almost undetectable cotransport function. Moreover, inhibition of N-glycan maturation with swainsonine or kifunensine increased core/hybrid-type NKCC1 expression but eliminated plasma membrane complex N-glycosylated NKCC1 and transport function. Together, these results suggest that (i) NKCC1 is delivered to the plasma membrane of COS7 cells independently of its N-glycan nature, (ii) most of NKCC1 in the plasma membrane is core/hybrid-type N-glycosylated, and (iii) the minimal proportion of complex N-glycosylated NKCC1 is functionally active.
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Chion, Alain, Jamie O'Sullivan, Gudmundur Bergsson, Sean Keyes, Orla Rawley, Padraic Fallon, Nico van Rooijen, et al. "N-Linked Glycans within the A1A2A3 Domains of VWF Play a Critical Role in Modulating Macrophage-Mediated Clearance." Blood 124, no. 21 (December 6, 2014): 469. http://dx.doi.org/10.1182/blood.v124.21.469.469.
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Abstract Enhanced plasma clearance of von Willebrand factor (VWF) plays an important role in the etiology of both type 1 and type 2 VWD. Nevertheless, although significant progress has been achieved in understanding the structure and functional properties of VWF, the mechanism(s) responsible for modulating VWF clearance from the plasma remain poorly understood. Accumulating recent data suggests that hepatic and splenic macrophages play key roles in modulating VWF clearance. A number of putative macrophage receptors for VWF have been also been described, including LRP1, β2-integrins and Siglec-5. In addition, it is well recognised that variation in VWF glycan expression significantly influences its clearance rate. In particular, terminal ABO(H) blood group determinants which are predominantly expressed on the N-linked glycans of human VWF significantly modulate its rate of clearance. Critically however, the molecular mechanisms through which specific macrophage receptors interact with particular regions of the complex VWF glycoprotein have not been defined. To investigate the role of VWF glycans and specific VWF domains in regulating VWF clearance, we expressed and purified a series of recombinant VWF variants and truncations with/without specific glycan sites. In addition, VWF glycosylation was modified using specific exoglycosidase digestions. Subsequently, recombinant VWF variants and glycoforms thereof were injected into VWF-/-mice, and plasma VWF clearance rates determined by ELISA. VWF-macrophage interactions were also quantified in vitro using phorbol ester-differentiated monocytic THP-1 cells, and primary human monocytes, in a High Content Analysis Imaging system. In keeping with previous reports, we observed that clearance of a truncated VWFA1A2A3 fragment in VWF-/-mice was very similar to that of full-length wild type (WT-) VWF (VWFA1A2A3; t1/2 = 6.3 min versus rWT-VWF; t1/2 = 7.9 min). Furthermore, chemical depletion of macrophages using clodronate liposomes administration significantly inhibited A1A2A3 clearance in vivo (1.7-fold at 10 min time point) to a similar extent to that observed with full length VWF. In vitro binding experiments confirmed that A1A2A3 bound to differentiated THP-1 cells in a dose- and time- dependent manner. Interestingly, this binding was significantly enhanced in the presence of ristocetin. Cumulatively, these data demonstrate that the A1A2A3 domains of VWF contain a critical receptor-binding site for macrophage-mediated clearance. Interestingly, we observed that the half-life of infused human plasma-derived VWF and recombinant VWF expressed in HEK293T cells in VWF-/- mice were significantly different. Furthermore, treatment with PNGase F to completely remove N-linked glycan structures markedly enhanced the clearance of full length VWF (t1/2 2.1 min; p<0.05). Collectively, these findings highlight the essential roles played by N-glycans in regulating VWF survival. Two N-linked glycan sites are located within A1A2A3 at N1515 and N1574 respectively. Importantly, we found that PNGase digestion of A1A2A3 resulted in markedly enhanced macrophage binding in vitro. Consequently we hypothesized that the two N-glycans located within the A2 domain might be important in regulating VWF clearance by macrophages. Targeted disruption of these individual N-glycan sites by site-directed mutagenesis (A1A2A3-N1515Q and A1A2A3-N1574Q respectively) resulted in significantly enhanced macrophage binding in vitro compared to wild type A1A2A3. Furthermore, following tail vein infusion in VWF-/-mice, full length VWFN1515Q and VWFN1574Q both demonstrated markedly reduced half-lives compared to wild type VWF (VWFN1515Q; t1/2 = 3.7 min, VWFN1574Q; t1/2 = 5.5 min). Finally, introduction of the N1515Q point mutation into truncated A1A2A3 also served to significantly enhance plasma clearance, (A1A2A3N1515Q-VWF; t1/2 = 3.1 min versus A1A2A3-VWF; t1/2 = 6.3 min). In conclusion, our novel data identify a crucial role of the VWF A domains in regulating macrophage-mediated VWF clearance. In addition, we further demonstrate that the N-linked glycans structures located at N1515 and N1574 within the A2 domain play specific roles in protecting VWF against in vivo clearance by macrophages. Given the important role played by enhanced VWF clearance in the etiology of type I VWD, these findings are of direct clinical importance. Disclosures No relevant conflicts of interest to declare.
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Garner, Omai B., and Linda G. Baum. "Galectin–glycan lattices regulate cell-surface glycoprotein organization and signalling." Biochemical Society Transactions 36, no. 6 (November 19, 2008): 1472–77. http://dx.doi.org/10.1042/bst0361472.
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The formation of multivalent complexes of soluble galectins with glycoprotein receptors on the plasma membrane helps to organize glycoprotein assemblies on the surface of the cell. In some cell types, this formation of galectin–glycan lattices or scaffolds is critical for organizing plasma membrane domains, such as lipid rafts, or for targeted delivery of glycoproteins to the apical or basolateral surface. Galectin–glycan lattice formation is also involved in regulating the signalling threshold of some cell-surface glycoproteins, including T-cell receptors and growth factor receptors. Finally, galectin–glycan lattices can determine receptor residency time by inhibiting endocytosis of glycoprotein receptors from the cell surface, thus modulating the magnitude or duration of signalling from the cell surface. This paper reviews recent evidence in vitro and in vivo for critical physiological and cellular functions that are regulated by galectin–glycoprotein interactions.
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Pučić, Maja, Sofia Pinto, Mislav Novokmet, Ana Knežević, Olga Gornik, Ozren Polašek, Kristian Vlahoviček, et al. "Common aberrations from the normal human plasma N-glycan profile." Glycobiology 20, no. 8 (April 8, 2010): 970–75. http://dx.doi.org/10.1093/glycob/cwq052.
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Srinivasan, Karunya, Sucharita Roy, Nathaniel Washburn, Sandra F. Sipsey, Robin Meccariello, James W. Meador, Leona E. Ling, Anthony M. Manning, and Ganesh V. Kaundinya. "A Quantitative Microtiter Assay for Sialylated Glycoform Analyses Using Lectin Complexes." Journal of Biomolecular Screening 20, no. 6 (April 7, 2015): 768–78. http://dx.doi.org/10.1177/1087057115577597.
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Fidelity of glycan structures is a key requirement for biotherapeutics, with carbohydrates playing an important role for therapeutic efficacy. Comprehensive glycan profiling techniques such as liquid chromatography (LC) and mass spectrometry (MS), while providing detailed description of glycan structures, require glycan cleavage, labeling, and paradigms to deconvolute the considerable data sets they generate. On the other hand, lectins as probes on microarrays have recently been used in orthogonal approaches for in situ glycoprofiling but require analyte labeling to take advantage of the capabilities of automated microarray readers and data analysis they afford. Herein, we describe a lectin-based microtiter assay (lectin–enzyme-linked immunosorbent assay [ELISA]) to quantify terminal glycan moieties, applicable to in vitro and in-cell glycan-engineered Fc proteins as well as intact IgGs from intravenous immunoglobulin (IVIG), a blood product containing pooled polyvalent IgG antibodies extracted from plasma from healthy human donors. We corroborate our findings with industry-standard LC-MS profiling. This “customizable” ELISA juxtaposes readouts from multiple lectins, focusing on a subset of glycoforms, and provides the ability to discern single- versus dual-arm glycosylation while defining levels of epitopes at sensitivities comparable to MS. Extendable to other biologics, this ELISA can be used stand-alone or complementary to MS for quantitative glycan analysis.
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Misevic, Gradimir, and Emanuela Garbarino. "Glycan-to-Glycan Binding: Molecular Recognition through Polyvalent Interactions Mediates Specific Cell Adhesion." Molecules 26, no. 2 (January 13, 2021): 397. http://dx.doi.org/10.3390/molecules26020397.
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Glycan-to-glycan binding was shown by biochemical and biophysical measurements to mediate xenogeneic self-recognition and adhesion in sponges, stage-specific cell compaction in mice embryos, and in vitro tumor cell adhesion in mammals. This intermolecular recognition process is accepted as the new paradigm accompanying high-affinity and low valent protein-to-protein and protein-to-glycan binding in cellular interactions. Glycan structures in sponges have novel species-specific sequences. Their common features are the large size >100 kD, polyvalency >100 repeats of the specific self-binding oligosaccharide, the presence of fucose, and sulfated and/or pyruvylated hexoses. These structural and functional properties, different from glycosaminoglycans, inspired their classification under the glyconectin name. The molecular mechanism underlying homophilic glyconectin-to-glyconectin binding relies on highly polyvalent, strong, and structure-specific interactions of small oligosaccharide motifs, possessing ultra-weak self-binding strength and affinity. Glyconectin localization at the glycocalyx outermost cell surface layer suggests their role in the initial recognition and adhesion event during the complex and multistep process. In mammals, Lex-to-Lex homophilic binding is structure-specific and has ultra-weak affinity. Cell adhesion is achieved through highly polyvalent interactions, enabled by clustering of small low valent structure in plasma membranes.
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Cvetko, Ana, Domagoj Kifer, Olga Gornik, Lucija Klarić, Elizabeth Visser, Gordan Lauc, James F. Wilson, and Tamara Štambuk. "Glycosylation Alterations in Multiple Sclerosis Show Increased Proinflammatory Potential." Biomedicines 8, no. 10 (October 13, 2020): 410. http://dx.doi.org/10.3390/biomedicines8100410.
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Multiple sclerosis (MS) is an inflammatory autoimmune disorder affecting the central nervous system (CNS), with unresolved aetiology. Previous studies have implicated N-glycosylation, a highly regulated enzymatic attachment of complex sugars to targeted proteins, in MS pathogenesis. We investigated individual variation in N-glycosylation of the total plasma proteome and of IgG in MS. Both plasma protein and IgG N-glycans were chromatographically profiled and quantified in 83 MS cases and 88 age- and sex-matched controls. Comparing levels of glycosylation features between MS cases and controls revealed that core fucosylation (p = 6.96 × 10−3) and abundance of high-mannose structures (p = 1.48 × 10−2) were the most prominently altered IgG glycosylation traits. Significant changes in plasma protein N-glycome composition were observed for antennary fucosylated, tri- and tetrasialylated, tri- and tetragalactosylated, high-branched N-glycans (p-value range 1.66 × 10−2–4.28 × 10−2). Classification performance of N-glycans was examined by ROC curve analysis, resulting in an AUC of 0.852 for the total plasma N-glycome and 0.798 for IgG N-glycome prediction models. Our results indicate that multiple aspects of protein glycosylation are altered in MS, showing increased proinflammatory potential. N-glycan alterations showed substantial value in classification of the disease status, nonetheless, additional studies are warranted to explore their exact role in MS development and utility as biomarkers.
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Itsara, Andy, Jill M. Johnsen, Kerry W. Lannert, Katherine Odem-Davis, Ayse Bilge Ozel, Karl C. Desch, and Thomas Walsh. "Complex Relationship of Blood Group H, ABO Glycans, and Von Willebrand Factor Levels." Blood 126, no. 23 (December 3, 2015): 2242. http://dx.doi.org/10.1182/blood.v126.23.2242.2242.
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Abstract Background: ABO(H) is a carbohydrate blood group system expressed in multiple tissues including red blood cells, blood vessels, and mucosal surfaces. ABO is the largest known genetic modifier of plasma VWF level (VWF:Ag). It has been hypothesized that the effect of ABO on VWF is mediated by H glycan density. FUT2, the gene underlying Secretor phenotype, encodes a glycosyltransferase synthesizing H antigen in mucosal tissues, and variation in the gene has also previously been associated with VWF:Ag, but past studies have been conflicting. To clarify these relationships, we studied the relationship between VWF:Ag, ABH glycans, and FUT2 genotype. Methods: The primary study group was a representative cohort of US blood donors from the Retrovirus Epidemiology Donor Study (REDS, N=499). A validation cohort of unrelated individuals was created from the Genes and Blood Clotting Study (GABC), healthy siblings between ages 14 and 35 years from University of Michigan, Ann Arbor, by choosing a random individual from each family (N=488). VWF:Ag was determined by ELISA in platelet poor plasma. Forensic techniques were adapted to detect ABH glycans in whole blood (REDS) or RBC-rich, frozen buffy coat (GABC). A and B glycans were detected using anti-A or anti-B (Immucor). A biotinylated Ulex europaeus agglutinin (UEA lectin, Vector Labs) was used to detect H. Relative A, B, and H antigen density was quantified on dot blots with ImageQuant (GE). In REDS, functional FUT2 alleles (Secretor) were determined by Sanger sequencing of FUT2 exon 2. FUT2 copy-number was assayed with real-time quantitative PCR. In GABC, FUT2 genotypes were determined through SNP genotyping (Illumina). In REDS, genotype data was phased (BEAGLE) to identify functional haplotypes. In GABC, functional alleles were inferred from the genotypes of two SNPs (rs601338, rs1047781) that determined secretor status in nearly all samples in REDS (see below). Multivariate regression was applied to VWF:Ag as a function H antigen density and O vs. non-O blood group, both separately and within the same model. All models were adjusted for age, gender, and self-reported ethnicity. Results: ABO blood group frequencies in both cohorts were similar to that of the US population. VWF:Ag differed significantly between ABO blood groups with lower values in blood group O versus non-O (REDS: ratio = 0.75, 95% CI [0.71, 0.80], p<2.2e-16, log-linear regression). H glycan density also differed between ABO blood group (p<2.2e-16, Kruskal-Wallis; O vs. non-O, ratio = 2.03, 95% CI [1.88, 2.19], p<2.2e-16). A significant fraction of H glycan density variation was attributable to ABO (REDS r2 =0.45; GABC r2 =0.34), but a wide range of H glycan density was observed in all blood groups, including within blood group O. In multivariate regression, both H glycan density and O versus non-O blood group, considered separately or in the same model, were significantly associated with VWF:Ag (REDS combined model: p = 2.9e-5 and 2.7e-4 for H glycan and blood group, respectively). In GABC but not in REDS, regression was also significant for an interaction between H glycan density and blood group (p = 0.031). Both cohorts estimated a stronger association of VWF:Ag with H in non-O blood groups. FUT2 sequencing in the REDS cohort identified 22 distinct FUT2 haplotypes. 99.5% (497/499) of individuals could be accurately assigned FUT2 genotypes based on the common W154* (rs601338) polymorphism alone. In GABC, this decreased to 94% (476/488) due to higher frequency of a hypomorphic allele (rs1047781) more commonly found in Asian individuals. In REDS, quantitative PCR did not identify copy-number variation at FUT2. There was no significant association between FUT2 genotype-predicted function and VWF:Ag or H glycan density in either cohort. Conclusion: H glycan density correlated with VWF:Ag in both cohorts. H glycan density could mediate the association between ABO blood group and VWF:Ag. If so, this suggests a non-linear, complex relationship between H and VWF and a separate ABO mechanism cannot be excluded. In both cohorts, there was no association of FUT2 (Secretor) genotype with VWF. Our data suggest prior focused approaches to FUT2 genotyping are at risk to undercall non-functional alleles, particularly in cohorts with non-European ancestries. Taken together, these indicate blood group H may be a significant modifier of VWF:Ag, and that this effect is not due to the influence of Secretor phenotype. Disclosures Johnsen: Octapharma: Other: Speaker; Biogen: Research Funding; CSL Behring: Other: Speaker.
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Critcher, Meg, Timothy O'Leary, and Mia L. Huang. "Glycoengineering: scratching the surface." Biochemical Journal 478, no. 4 (February 18, 2021): 703–19. http://dx.doi.org/10.1042/bcj20200612.
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At the surface of many cells is a compendium of glycoconjugates that form an interface between the cell and its surroundings; the glycocalyx. The glycocalyx serves several functions that have captivated the interest of many groups. Given its privileged residence, this meshwork of sugar-rich biomolecules is poised to transmit signals across the cellular membrane, facilitating communication with the extracellular matrix and mediating important signalling cascades. As a product of the glycan biosynthetic machinery, the glycocalyx can serve as a partial mirror that reports on the cell's glycosylation status. The glycocalyx can also serve as an information-rich barrier, withholding the entry of pathogens into the underlying plasma membrane through glycan-rich molecular messages. In this review, we provide an overview of the different approaches devised to engineer glycans at the cell surface, highlighting considerations of each, as well as illuminating the grand challenges that face the next era of ‘glyco-engineers’. While we have learned much from these techniques, it is evident that much is left to be unearthed.
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Lowe, M. E. "Site-specific mutations in the COOH-terminus of placental alkaline phosphatase: a single amino acid change converts a phosphatidylinositol-glycan-anchored protein to a secreted protein." Journal of Cell Biology 116, no. 3 (February 1, 1992): 799–807. http://dx.doi.org/10.1083/jcb.116.3.799.
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Placental alkaline phosphatase (PLAP) is anchored in the plasma membrane by a phosphatidylinositol-glycan moiety (PI-glycan). PI-glycan is added posttranslationally to the nascent peptide chain after the removal of 29 amino acids from the COOH-terminus. The contribution of selected COOH-terminal amino acids to the signal for PI-glycan addition was tested by creating a fusion protein with the COOH-terminus of PLAP and a secreted protein and by mutagenesis of specific PLAP COOH-terminal amino acids. The cDNA encoding the COOH-terminus of PLAP was fused in frame to the cDNA for human clotting Factor X and expressed in transfected COS-1 cells. Fusion proteins containing 32 amino acids of the PLAP COOH-terminus were modified by PI-glycan addition. Thus, the signal for PI-glycan modification must reside in these amino acids. Next, the region between the hydrophobic domain and the cleavage site was examined for additional determinants. Mutations of the hydrophilic residues in the spacer region demonstrated that these amino acids do not contribute to the signal for PI-glycan addition. Deletion of amino acids in the spacer region prevented the addition of PI-glycan suggesting that the length of the spacer domain or the amino acids around the cleavage site are important determinants. Finally, we demonstrated that interruption of the hydrophobic domain by a charged residue prevents PI-glycan addition and results in a protein that is secreted into the medium. The finding that a single Leu to Arg substitution in the hydrophobic domain converts a PI-glycan anchored, membrane protein to a secreted protein suggests that an essential signal for the correct sorting of PI-glycan anchored proteins versus secreted proteins resides in the hydrophobic domain. Substitution of a charged amino acid for a hydrophobic amino acid may be a mechanism for producing membrane bound and secreted forms of the same protein.
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PAPANASTASIOU, Philemon, Malcolm J. McCONVILLE, Julie RALTON, and Peter KÖHLER. "The variant-specific surface protein of Giardia, VSP4A1, is a glycosylated and palmitoylated protein." Biochemical Journal 322, no. 1 (February 15, 1997): 49–56. http://dx.doi.org/10.1042/bj3220049.
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The variant-specific surface proteins (VSPs) of the ancient protist Giardia duodenalis(syn.: Giardia intestinalis, Giardia lamblia) are cysteine- and threonine-rich polypeptides that can vary considerably in sequence and size. In the present study, we have purified a VSP (VSP4A1, formerly called CRISP-90) from a cloned Giardiaisolate, derived from a sheep, by Triton X-114 phase partitioning and anion-exchange chromatography. Analysis of the purified VSP4A1 showed that this protein is post-translationally modified with both glycans and lipid. The glycans of VSP4A1 were detected and partially characterized by (1) compositional analysis, which indicated the presence of GlcNAc and Glc (0.5 and 1.0 mol/mol of protein respectively), and (2) the specific labelling of VSP4A1 with galactosyltransferase/UDP-[3H]Gal. The glycans were released by β-elimination, suggesting that they are O-linked to the protein. Bio-Gel P4 chromatography of the released galactosylated glycans and further compositional analysis suggested that the major glycan on the VSP is a trisaccharide with Glc at the reducing terminus. These and other results indicate the absence of any N-linked glycans on the VSP and suggest instead that it is elaborated with a novel type of short O-linked glycan. Compositional analysis and radiolabelling experiments also indicated that VSP4A1 is modified with covalently linked palmitate (1 mol/mol of protein). Hydroxylamine treatment at neutral pH of [3H]palmitate-labelled VSP4A1 indicated that the acyl chain may be attached by a thioester linkage. A likely location for the lipid modification appears to be in the region of the C-terminal domain where it may facilitate association of the protein with the plasma membrane.
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Teare, John M., David S. Kates, Anita Shah, and Stephen Garger. "Improved Pharmacokinetic Profile for BAY 81-8973 Due to Increased Branching and Sialylation of N-Linked Glycans of Recombinant Factor VIII." Blood 132, Supplement 1 (November 29, 2018): 1209. http://dx.doi.org/10.1182/blood-2018-99-113061.
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Abstract The circulatory half-life of recombinant factor VIII (rFVIII) products is affected by glycosylation of the FVIII protein, including N-linked glycan branching and terminal sialic acid occupancy, primarily through receptor-mediated hepatic clearance (eg, asialoglycoprotein receptor [ASGPR] and lipoprotein receptor-related protein [LRP]). BAY 81-8973 (Kovaltry®, Bayer, Berkeley, CA) is an unmodified full-length rFVIII for treatment of hemophilia A. The BAY 81-8973 manufacturing process results in a product of enhanced purity with a consistently high degree of branching and sialylation of N-linked glycans. This study evaluated whether a relationship exists between N-linked glycosylation patterns and pharmacokinetic (PK) characteristics of BAY 81-8973 and 2 other rFVIII products (sucrose-formulated rFVIII [rFVIII-FS; Kogenate® FS, Bayer] and antihemophilic factor (recombinant) plasma/albumin-free method [rAHF-PFM; Advate®, Shire, Westlake Village, CA]). N-linked glycans or terminal carbohydrates were enzymatically removed from immobilized BAY 81-8973, rFVIII-FS, and rAHF-PFM proteins and analyzed using high-performance liquid chromatography to determine the percentage of individual N-linked glycan structures and degree of sialylation of each structure. PK data were available from 2 separate phase 1 crossover studies in which the PK profile of BAY 81-8973 was compared with that of rFVIII-FS (n=26) and rAHF-PFM (n=18) in patients with severe hemophilia A who received a single 50-IU/kg dose of each product. BAY 81-8973 and rFVIII-FS had increased N-linked glycan branching with higher levels of sialylation compared with rAHF-PFM. Levels of trisialylated glycans were 29.0% for BAY 81-8973 versus 11.5% for rFVIII-FS and 4.8% to 5.5% for rAHF-PFM; tetrasialylated glycans were 12.0% versus 2.8% and 0.6%, respectively. Degree of sialylation was 96% for BAY 81-8973, 94% for rFVIII-FS, and 78% to 81% for rAHF-PFM. Based on chromogenic assay results from the single-dose phase 1 PK studies, BAY 81-8973 half-life was 15% longer than that for rFVIII-FS and 16% longer than rAHF-PFM. Increases in the percentage of sialylated tri-antennary and tetra-antennary N-glycans correlated well with longer half-life of rFVIII in humans (adjusted R2=0.978 and 0.892 for tri-antennary and tetra-antennary N-glycans, respectively). Higher percentages of sialylation (ie, sialic acid capping) correlated with a longer half-life (adjusted R2=0.697), but the relationship was not as strong as that between glycan branching and half-life. Improved PK for BAY 81-8973 relative to rFVIII-FS and rAHF-PFM as seen in single-dose crossover PK studies might be related to this greater level of branching and sialylation, which may prolong the time BAY 81-8973 remains in the circulation. Disclosures Teare: Bayer: Employment. Kates:Bayer: Employment. Shah:Bayer: Employment. Garger:Bayer: Employment.
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Thanabalasingham, G., J. E. Huffman, J. J. Kattla, M. Novokmet, I. Rudan, A. L. Gloyn, C. Hayward, et al. "Mutations in HNF1A Result in Marked Alterations of Plasma Glycan Profile." Diabetes 62, no. 4 (December 28, 2012): 1329–37. http://dx.doi.org/10.2337/db12-0880.
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Mun, Ji-Young, Kyung Jin Lee, Hoon Seo, Min-Sun Sung, Yee Sook Cho, Seung-Goo Lee, Ohsuk Kwon, and Doo-Byoung Oh. "Efficient Adhesion-Based Plasma Membrane Isolation for Cell SurfaceN-Glycan Analysis." Analytical Chemistry 85, no. 15 (July 23, 2013): 7462–70. http://dx.doi.org/10.1021/ac401431u.
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Regoeczi, Erwin, J. Michael Kay, Paul A. Chindemi, Ouahida Zaimi, and Kaye L. Suyama. "Transferrin glycosylation in hypoxia." Biochemistry and Cell Biology 69, no. 4 (April 1, 1991): 239–44. http://dx.doi.org/10.1139/o91-036.
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The aim of this study was to examine the effect of reduced O2 tension on the glycosylation of transferrin. Rats were placed in a hypobaric chamber (380 mmHg) that corresponded to an altitude of 5486 m above sea level for 21 days. The animals responded with marked increases in hematocrit (from 44 to 76%) and cardiac weight, and with reductions in the concentration of plasma transferrin averaging 15%. Analyses of their plasma transferrin by serial anion-exchange and lectin affinity chromatography revealed no changes in the extent of glycan branching. However, there was a moderate rise in the proportion of fucosylated transferrin molecules (fucosylation index) and a slight decrease in the transferrin fraction bearing a tetrasialylated biantennary glycan. The fucosylation index correlated positively with plasma transferrin concentrations in the test animals, but not in the controls. In contradistinction to the situation with transferrin, hypoxic rats exhibited a reduced fucosylation index of immunoglobulin G.Key words: fucosylation index, hypoxia, immunoglobulin G, lectin affinity chromatography, transferrin.