Terminal α2-6 Linked Sialic Acid Expression On VWF Specifically Enhances Proteolysis by ADAMTS13.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 30-30
Author(s):  
Rachel Therese McGrath ◽  
Thomas A J McKinnon ◽  
Barry Byrne ◽  
Richard O'Kennedy ◽  
Michael Laffan ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Blood Group ◽  
Conflicts Of Interest ◽  
Lectin Binding ◽  
Gel Filtration ◽  
Specific Effect ◽  
Cysteine Proteases ◽  
Binding Activity ◽  
Collagen Binding ◽  
Sodium Metaperiodate

Abstract Abstract 30 VWF is a large plasma sialoglycoprotein that mediates platelet tethering at sites of vascular injury. VWF function is dependent upon VWF multimeric composition, which is regulated in plasma by ADAMTS13. ABO(H) blood group determinants expressed on VWF N-linked glycans significantly influence susceptibility to ADAMTS13 proteolysis. In this study, we investigated whether terminal sialic acid residues expressed on the N- and O-linked glycans of VWF may also regulate proteolysis by ADAMTS13. VWF was initially purified from human plasma (pdVWF) by cryoprecipitation and gel filtration. Subsequently, VWF sialylation was modified using specific exoglycosidases and quantified by lectin-binding ELISA. The rate of glycosidase-treated VWF proteolysis by ADAMTS13 was determined by incubation with recombinant ADAMTS13 and subsequent measurement of residual VWF collagen binding activity. Complete VWF deglycosylation has been shown to enhance the rate of proteolysis by ADAMTS13. In contrast, enzymatic desialylation of VWF by α2-3,6,8,9 neuraminidase (Neu-VWF) markedly impaired the rate of ADAMTS13-mediated VWF proteolysis. Neu-VWF collagen binding activity was reduced to only 50±14% by ADAMTS13, compared to 11±7% for untreated VWF (p<0.01) at the same time point. Despite this, Neu-VWF exhibited increased susceptibility to proteases other than ADAMTS13 (trypsin, chymotrypsin and cathepsin B; all p<0.05). VWF sialylation is therefore a specific enhancer of ADAMTS13-mediated proteolysis. Consequently, quantification and molecular distribution of VWF sialylation was examined by sequential digestion and HPLC analysis. Total sialic acid expression on pdVWF was 167nmol/mg, of which the majority (133.4nmol/mg or 80.1%) was present on its N-linked glycan chains. Interestingly, despite the resistance to ADAMTS13 proteolysis observed upon complete desialylation, digestion of pdVWF with α2-3 neuraminidase to remove predominantly O-linked sialic acid did not influence the rate of ADAMTS13 proteolysis. Previous studies have demonstrated that VWF expressing different blood groups exhibit altered rates of proteolysis by ADAMTS13 (O ≥ B > A ≥ AB). Since α2-6 linked sialic acid and ABO(H) determinants are both expressed as terminal antigens on VWF N-linked glycans, the effect of desialylation upon blood group-specific VWF proteolysis by ADAMTS13 was determined. As expected, untreated group O VWF was cleaved significantly faster than group AB-VWF (p<0.05). However, the ability of ABO blood group to regulate ADAMTS13 proteolysis was completely ablated upon VWF desialylation, as both Neu-O-VWF and Neu-AB-VWF were cleaved by ADAMTS13 at identical rates. This indicates that VWF sialylation constitutes a more important determinant of susceptibility to ADAMTS13 proteolysis than ABO(H) expression. Sialic acid can mediate protein-protein interactions through either conformational and/or charge-mediated mechanisms. Despite this, sodium metaperiodate treatment of pdVWF to remove sialic acid anionic charge did not influence the rate of proteolysis by ADAMTS13. In contrast, the ability of sialic acid to specifically enhance ADAMTS13 proteolysis of VWF was significantly attenuated at high urea concentrations (≥2M), supporting the hypothesis that VWF sialylation enhances proteolysis by ADAMTS13 by promoting a ADAMTS13-specific permissive conformation. These novel data demonstrate that although sialic acid protects VWF against proteolysis by serine and cysteine proteases, it also specifically enhances susceptibility to proteolysis by ADAMTS13. Moreover, the magnitude of this sialic acid-specific effect on VWF proteolysis by ADAMTS13 is more marked than that attributable to N-linked ABO(H) blood group antigen expression. Therefore, quantitative variation in VWF sialylation represents a key regulator of VWF multimeric composition, and as such, is likely to be of clear patho-physiological significance. Disclosures: No relevant conflicts of interest to declare.

A Critical Role for N- and O-Linked Carbohydrates In Modulating Von Willebrand Factor Clearance In Vivo

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 382-382 ◽  
Author(s):  
Emily McRae ◽  
Orla Rawley ◽  
Hendrik Nel ◽  
Rachel Therese McGrath ◽  
Gudmundur Bergsson ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Half Life ◽  
Conflicts Of Interest ◽  
Lectin Binding ◽  
Critical Role ◽  
Gel Filtration ◽  
Macrophage Depletion ◽  
Plasma Half Life

Abstract Abstract 382FN2 VWF is a multimeric plasma sialoglycoprotein essential for normal haemostasis. Although the biosynthesis, structure and functional properties of VWF have been well characterized, the molecular mechanism(s) underlying its clearance remain poorly understood. Nevertheless, enhanced VWF clearance is important in the pathophysiology of VWD. Moreover, emerging data suggest that variation in VWF glycosylation (notably ABO blood group) may constitute an important regulator of in vivo clearance rates. To define the role of VWF glycans in modulating clearance, VWF was purified from human plasma (pdVWF) by cryoprecipitation and gel filtration. Subsequently, VWF glycosylation was modified using exoglycosidases and quantified by specific lectin-binding ELISAs. Finally, the effect of altered glycosylation on VWF plasma half-life was characterized by administration of VWF glycan variants to VWF−/− mice. Wild type pdVWF was cleared in biphasic manner, characterized by a rapid initial phase followed by a slower secondary phase (t1/2 = 46.9 min). Enzymatic desialylation of VWF with α2–3,6,8,9 neuraminidase (Neu-VWF) markedly enhanced VWF clearance (t1/2 = 3.7 min; p<0.01). Digestion of pdVWF with α2–3 neuraminidase to remove predominantly O-linked sialic acid (which constitutes less than 20% total VWF sialylation) was also sufficient to markedly enhance VWF clearance (t1/2 = 13.1 min; p<0.05). In the presence of the asialoglycoprotein receptor (ASGPR)-antagonist ASOR, the mean residence time of Neu-VWF was identical to that of pd-VWF. Recent studies have shown that macrophages may be important in VWF clearance. Since the ASGPR is expressed on both hepatocytes and macrophages, the effect of macrophage depletion on VWF clearance was assessed. Pre-treatment with liposome-encapsulated clodronate depleted F4/80+CD11b+ murine macrophages by 75%, and significantly prolonged Neu-VWF survival. However Neu-VWF survival was not corrected to that observed in the presence of ASOR. For example, plasma Neu-VWF survival after 5 mins was corrected from 30±6% to 92±7% in the presence of ASOR, compared to 78±10% following clodronate macrophage-depletion. Cumulatively, these findings demonstrate that both N- and O-linked sialylation are critical in protecting VWF against ASGPR-mediated clearance. Moreover, ASGPR-modulated clearance is at least in part macrophage-dependent. ß-galactose residues exposed following removal of capping sialic acid are recognised by the ASGPR. To further define the role of specific sugars in regulating VWF clearance, the effect of terminal sialic acid and sub-terminal galactose removal by sequential neuraminidase and galactosidase digestions was studied. Surprisingly, VWF exposed to sequential neuraminidase and galactosidase digestions (NeuGal-VWF) was cleared rapidly from the plasma in a monophasic fashion (t1/2 = 4.8 min). Moreover, treatment with PNGase F to completely remove N-linked carbohydrate structures also markedly decreased the plasma half-life (PNG-VWF; t1/2 = 2.1 min). In keeping with their lack of exposed galactose residues, the enhanced clearance of NeuGal-VWF and PNG-VWF were not mediated via the ASGPR (ASOR had no significant effect). In contrast, macrophage depletion by liposomal clodronate significantly inhibited the enhanced clearance of both NeuGal-VWF and PNG-VWF respectively. These data suggest that the ASGPR is not the only macrophage receptor involved in modulating VWF clearance, which is consistent with the relatively minor prolongation in VWF survival previously reported in Asgpr1−/− mice. These novel data demonstrate that variation in the N- or O-linked carbohydrate structures significantly modulate VWF half-life in vivo. Moreover, VWF clearance is not mediated solely through the ASGPR, but may also require additional as yet unidentified macrophage receptors for full clearance. Therefore, qualitative and quantitative variation in VWF glycosylation represents a key regulator of VWF clearance, and as such is likely to be of direct pathophysiological significance. Disclosures: No relevant conflicts of interest to declare.

Expression of terminal α2-6–linked sialic acid on von Willebrand factor specifically enhances proteolysis by ADAMTS13

Blood ◽  
2010 ◽  
Vol 115 (13) ◽  
pp. 2666-2673 ◽  
Author(s):  
Rachel T. McGrath ◽  
Thomas A. J. McKinnon ◽  
Barry Byrne ◽  
Richard O'Kennedy ◽  
Virginie Terraube ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Von Willebrand Factor ◽  
High Performance ◽  
Cysteine Proteases ◽  
Binding Activity ◽  
Chromatography Analysis ◽  
Total Sialic Acid ◽  
Sequential Digestion ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract von Willebrand factor (VWF) multimeric composition is regulated in plasma by ADAMTS13. VWF deglycosylation enhances proteolysis by ADAMTS13. In this study, the role of terminal sialic acid residues on VWF glycans in mediating proteolysis by ADAMTS13 was investigated. Quantification and distribution of VWF sialylation was examined by sequential digestion and high-performance liquid chromatography analysis. Total sialic acid expression on VWF was 167nmol/mg, of which the majority (80.1%) was present on N-linked glycan chains. Enzymatic desialylation of VWF by α2-3,6,8,9 neuraminidase (Neu-VWF) markedly impaired ADAMTS13-mediated VWF proteolysis. Neu-VWF collagen binding activity was reduced to 50% (± 14%) by ADAMTS13, compared with 11% (± 7%) for untreated VWF. Despite this, Neu-VWF exhibited increased susceptibility to other proteases, including trypsin, chymotrypsin, and cathepsin B. VWF expressing different blood groups exhibit altered ADAMTS13 proteolysis rates (O ≥ B > A ≥ AB). However, ABO blood group regulation of ADAMTS13 proteolysis was ablated on VWF desialylation, as both Neu-O-VWF and Neu-AB-VWF were cleaved by ADAMTS13 at identical rates. These novel data show that sialic acid protects VWF against proteolysis by serine and cysteine proteases but specifically enhances susceptibility to ADAMTS13 proteolysis. Quantitative variation in VWF sialylation therefore represents a key determinant of VWF multimeric composition and, as such, may be of pathophysiologic significance.

scholarly journals Phagocytosis stimulates alternative glycosylation of macrosialin (mouse CD68), a macrophage-specific endosomal protein

1999 ◽  
Vol 338 (3) ◽  
pp. 687-694 ◽  
Author(s):  
Rosângela P. da SILVA ◽  
Siamon GORDON
Keyword(s):  
Protein Synthesis ◽  
Sialic Acid ◽  
Membrane Protein ◽  
Lectin Binding ◽  
Peritoneal Macrophages ◽  
Binding Activity ◽  
Amaranthus Caudatus ◽  
Galanthus Nivalis ◽  
Particle Internalization

Macrosialin (mouse CD68), a macrophage-specific member of the lysosomal-associated membrane protein family, displays N-linked glycosylation and a heavily sialylated, mucin-like domain. We show that phagocytosis of zymosan by inflammatory peritoneal macrophages potently alters glycan processing of macrosialin in vitro. The phagocytic glycoform is not induced by other forms of endocytosis and depends on particle internalization. Zymosan uptake does not influence macrosialin protein synthesis, but increases the specific incorporation of d-[2-3H]mannose, d-[6-3H]galactose, N-acetyl-d-[1-3H]glucosamine and l-[5,6-3H]fucose by 2–15-fold. The phagocytic glycoform displays increased binding of agglutinins from peanut, Amaranthus caudatus and Galanthus nivalis, whereas binding of the sialic-acid-specific Maakia amurensis agglutinin is slightly reduced. Digestion by N-Glycanase abolishes the incorporation of [3H]mannose label and Galanthus nivalis agglutinin binding activity, but preserves the incorporation of galactose and N-acetylglucosamine and specific lectin binding. We also show that phagocytosis increases the complexity and length of O-linked chains. The data presented highlight the importance of differential glycosylation in the biology of macrosialin, phagosomes and macrophages in general.

scholarly journals Autoantibody Mediated Desialylation Impairs Human Thrombopoiesis and Platelet Life Span

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2346-2346
Author(s):  
Irene Marini ◽  
Jan Zlamal ◽  
Lisann Pelzel ◽  
Wolfgang Bethge ◽  
Christoph Faul ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Life Span ◽  
Conflicts Of Interest ◽  
Lectin Binding ◽  
Cell Interaction ◽  
Bleeding Tendency ◽  
Sialidase Inhibitor ◽  
Von Willebrand ◽  
The Impact

Background: The low platelet count in autoimmune thrombocytopenia (ITP) is caused by enhanced destruction of opsonised platelets in the spleen upon binding of the anti-platelet autoantibodies (AAbs) to the glycoproteins (GPs) express on PLT's surface. Data from animal model suggested that desialylation may contribute to PLT destruction in ITP. However, accumulating evidence suggests that reduction of PLT generation from megakaryocytes (MKs) in bone morrow is also responsible thrombocytopenia in ITP. Based on these considerations, we hypothesized that AAb-mediated desialylation of the GPs expressed on PLT and MKs may interfere with PLT formation and life span. Methods: Sera from 100 ITP patients were investigated in this study. AAb-induced desialylation was detected using a lectin binding assay (LBA) by flow cytometry (FC). To investigate the impact of desialylation on the life-span of human PLTs, the NSG mouse model was used. PLTs and MKs functions were assessed after AAb treatment using proplatelet formation test and adhesion assays on different surfaces. Results: Sera from 35/100 (35%) ITP patients induced cleavage of sialic acid from PLT surface. Injection of desialylating AAbs in vivo resulted in accelerated clearance of human PLTs which was significantly reduced by a specific sialidase inhibitor that prevents desialylation on the PLT surface (survival after 5h: 29%, range 22-40% vs. 48%, range 41-53%, p=0.014, respectively). Desialylating AAbs caused a significant reduction in PLT adhesion to fibrinogen and von Willebrand factor (mean of % adherent PLTs compared to control IgG: 34±6%, p=0.004 and 26±2%, p=0.001, respectively). Interestingly, PLT adhesion was recovered in the presence of a sialidase inhibitor (mean of % adherent PLTs: 86±6%, p=0.001 and 67±10, p=0.020, respectively). IgG fractions from 7/10 (70%) ITP-sera were able to cleave sialic acid and induce exposure of ß-galactose residues on CD34+-derived MKs. Desialylating AAbs induced lower ability to form proplatelet extensions compared to control IgG, which was significantly increased in the presence of the sialidase inhibitor (mean of % proplatelet forming MKs: 42±11% vs. 90±9%, p=0.032, respectively). Conclusion: Our findings show that AAbs from a subgroup of ITP patients are not only able to cleave sialic acid on surface of human PLTs, but also on MKs leading to accelerate PLT destruction and impaired thrombopoiesis, respectively. In addition, we observed that AAb-mediated receptor desialyation interferes with cell interaction with extracellular matrix proteins leading to impaired PLT adhesion, MK differentiation and thrombopoiesis. These novel findings highlight the multiple effects of AAbs in ITP and add to the existing evidence that ITP is rather a group of disorders sharing common characteristics, namely loss of immune tolerance toward PLT and MK antigens and increased bleeding tendency. Disclosures No relevant conflicts of interest to declare.

Plasma Derived von Willebrand Factor Preparations: Collagen Binding and Ristocetin Cofactor Activities

1997 ◽  
Vol 78 (02) ◽  
pp. 930-933 ◽  
Author(s):  
Ping Chang ◽  
D L Aronson
Keyword(s):  
Von Willebrand Factor ◽  
Functional Activity ◽  
Binding Activity ◽  
Collagen Binding ◽  
Binding Function ◽  
Cofactor Activity ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Ristocetin Cofactor ◽  
Ristocetin Cofactor Activity

SummaryFive plasma preparations (11 lots) used in the treatment of von Willebrand’s disease (vWD) were evaluated. The collagen binding function of von Willebrand factor (vWF) containing preparations was compared with the ristocetin cofactor activity and the vWF antigen. Some preparations have higher ratio of functional activity (ristocetin cofactor and collagen binding) relative to the antigen than is found in normal plasma. The ristocetin cofactor activity and the collagen binding activity are tightly correlated (r = .95). Ultracentrifugal (UCF) analysis was used to compare the size distribution of vWf antigen, ristocetin cofactor and collagen binding activity. The sedimentation of all of the vWF parameters in the plasma products was slower than in plasma. In plasma products the ristocetin cofactor activity sediments the most rapidly, the collagen binding activity is slower and the antigen the slowest. The collagen/antigen ratio decreases with decreasing vWF size. Assignment of potency to vWF containing preparations utilizing the collagen binding activity may be more precise and as accurate as with the traditional ristocetin cofactor assay.

scholarly journals The role of sialic acid in the expression of human MN blood group antigens.

1979 ◽  
Vol 254 (6) ◽  
pp. 2112-2119 ◽  
Author(s):  
J.E. Sadler ◽  
J.C. Paulson ◽  
R.L. Hill
Keyword(s):  
Sialic Acid ◽  
Blood Group ◽  
Blood Group Antigens ◽  
Mn Blood Group

scholarly journals Characterization of glycoconjugates of human gastrointestinal mucosa by lectins. II. Lectin binding to the isolated glycoproteins of normal and malignant gastric mucosa.

1984 ◽  
Vol 32 (7) ◽  
pp. 690-696 ◽  
Author(s):  
J Fischer ◽  
G Uhlenbruck ◽  
P J Klein ◽  
M Vierbuchen ◽  
R Fischer
Keyword(s):  
Molecular Weight ◽  
Gastric Mucosa ◽  
High Molecular Weight ◽  
Lectin Binding ◽  
Gel Filtration ◽  
Molar Ratio ◽  
Gastric Cancer Cells ◽  
Tissue Sections ◽  
Gradient Gel Electrophoresis ◽  
Triton X

Using affinity chromatography on HPA-, PNA-, Con A, and WGA-agarose columns only a part (10-30%) of the high molecular weight mucous glycoproteins could be isolated from the Triton X-100 solubilized components of normal as well as carcinomatous gastric mucosa. The main part of the mucus was not bound by the lectins, which corresponds to our earlier lectin histochemical observations on paraffin-embedded tissue sections. The lectin-bound mucous glycoproteins had a relatively lower molecular weight, ranging from about 250-1,000 kilodaltons, as indicated by polyacrylamide gradient gel electrophoresis and by gel filtration on Biogel A 1.5 m column. In gas chromatographic analysis the molar ratio of aminohexoses to galactose was found to be much higher (3:1) in the lectin-bound mucous substances than in the whole high molecular weight mucus (1:1). This finding indicates that lectins have a higher affinity to the hexosamine rich components of mucus, which may be special forms of mucous glycoprotein molecules or the incompletely glycosylated core and backbone regions of the oligosaccharide chains of mucus. Extremely high hexosamine values (10:1) were found in the PNA isolated mucus of gastric adenocarcinoma. Since it is known that PNA binds to the terminal disaccharide, beta-galactose-(1-3)-N-acetylgalactosamine, which is localized at the reducing end of the oligosaccharide chains of mucus, it is highly probable that the elongation of the oligosaccharide side chains is disturbed in gastric cancer cells.

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