title: N-linked glycosylation of VWF modulates its interaction with ADAMTS13.
abstract: We examined the role of N-linked glycan structures of VWF on its interaction with ADAMTS13. PNGase F digestion followed by lectin analysis demonstrated that more than 90% of VWF N-linked glycan chains could be removed from the molecule (PNG-VWF) without disruption of its multimeric structure or its ability to bind to collagen. PNG-VWF had an approximately 4-fold increased affinity for ADAMTS13 compared with control VWF. PNG-VWF was cleaved by ADAMTS13 faster than control VWF and was also proteolysed in the absence of urea. Occupancy of the N-linked glycan sites at N1515 and N1574 and their presentation of ABO(H) blood group sugars were confirmed with an isolated tryptic fragment. Recombinant VWF was mutated to prevent glycosylation at these sites. Mutation of N1515 did not alter ADAMTS13 binding or increase rate of ADAMTS13 proteolysis. Mutation of N1574 increased the susceptibility of VWF to ADAMTS13 proteolysis and allowed cleavage in the absence of urea. Mutation of N1574 in the isolated recombinant VWF-A2 domain also increased binding and ADAMTS13 proteolysis. These data demonstrate that the N-linked glycans of VWF have a modulatory effect on the interaction with ADAMTS13. At least part of this effect is conformational, but steric hindrance may also be important.
authors.affiliation: Haematology Department, Imperial College of London, Hammersmith Hospital Campus, London, United Kingdom. t.mckinnon03@imperial.ac.uk
authors.forename: Thomas A J
authors.initials: TA
authors.lastname: McKinnon
authors.forename: Alain C K
authors.initials: AC
authors.lastname: Chion
authors.forename: Alexander J
authors.initials: AJ
authors.lastname: Millington
authors.forename: David A
authors.initials: DA
authors.lastname: Lane
authors.forename: Mike A
authors.initials: MA
authors.lastname: Laffan
mesh_terms: D051722:ADAM Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003094:Collagen / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014841:von Willebrand Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D014841:von Willebrand Factor; D003094:Collagen; D051722:ADAM Proteins; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase