Abstract
The objective of this project was to study the function of O-glycosylations in von Willebrand factor (VWF) life cycle. In total, 14 different murine Vwf cDNAs mutated on one or several O-glycosylations sites were generated: 9 individual mutants, 2 doublets, 2 clusters and 1 mutant with all 9 murine glycosylation sites mutated (Del-O-Gly). We expressed each mutated cDNA in VWF deficient-mice by hydrodynamic injection. An immunosorbent assay with Peanut Agglutinin (PNA) was used to verify the O-glycosylation status. Wild-type (WT) VWF expressed by hepatocytes after hydrodynamic injection was able to bind PNA with slightly higher affinity than endothelial-derived VWF. In contrast, the Del-O-Gly VWF mutant did not bind PNA, demonstrating removal of O-linked glycans. All mutants displayed a normal multimeric pattern. Two mutants, Del-O-Gly and T1255A/T1256A, led to expression levels 50% lower than those induced by WT VWF and their half-life in vivo was significantly reduced. When testing the capacity of each mutant to correct the bleeding time of VWF-deficient mice, we found that S1486A, T1255A, T1256A and the doublet T1255A/T1256A were unable to do so. In conclusion we have shown that O-glycosylations are dispensable for normal VWF multimerization and biosynthesis. It also appears that some O-glycosylation sites, particularly the T1255 and T1256 residues, are involved in the maintenance of VWF plasma levels and are essential for normal haemostasis. As for the S1486 residue, it seems to be important for platelet binding as demonstrated in vitro using perfusion experiments.
Highlights
Von Willebrand Factor (VWF) is a large multimeric plasma glycoprotein essential for normal haemostasis
Considering that von Willebrand factor (VWF) produced after hydrodynamic injection is synthesized by hepatocytes and not by endothelial cells or megakaryocytes, we wanted to check whether similar glycosylation structures were present on the molecule
Considering the large number of O-glycosylation sites present on human VWF, we chose the hydrodynamic injection technique to express the different mutants in vivo [17,24,25]
Summary
Von Willebrand Factor (VWF) is a large multimeric plasma glycoprotein essential for normal haemostasis. Its main role is to mediate platelet adhesion to exposed subendothelial tissues at sites of vascular injury [1], and its accessory role is to act as a carrier molecule for procoagulant factor VIII, thereby protecting it from premature clearance [2]. During its synthesis which takes place exclusively in megakaryocytes and endothelial cells, VWF undergoes extensive posttranslational modifications such as dimerisation, removal of the propeptide, multimerization and addition of polysaccharidic chains [3]. Polysaccharidic chains have been shown to contribute to a number of cellular processes such as protein folding, stability and secretion but they can influence the biological activity and survival of the molecule [5,6,7]
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