Submitted June 21, 2007
Accepted October 17, 2007
N-linked glycosylation of VWF modulates its interaction with ADAMTS13
Thomas A J McKinnon*, Alain CK Chion, Alexander J Millington, David A. Lane, and Mike A Laffan
Haematology Department, Imperial College, Hammersmith Hospital Campus, London, United Kingdom
* Corresponding author; email: t.mckinnon03{at}imperial.ac.uk.
We examined the role of N-linked glycan structures of VWF on its interaction with ADAMTS13. PNGase F digestion of plasma derived VWF followed by lectin analysis demonstrated that >90% of VWF N-linked glycan chains could be removed from the molecule (PNG-VWF) under non-denaturing conditions without disruption of its multimeric structure or its ability to bind to collagen. PNG-VWF was shown to have a ~4 fold increased affinity for ADAMTS13 compared to control VWF. PNG-VWF was cleaved by ADAMTS13 appreciably faster than control VWF and was also proteolysed in the absence of urea. Occupancy of the two predicted N-linked glycan sites on VWF at N1515 and N1574 and their presentation of ABO(H) blood group sugars was confirmed with an isolated tryptic fragment encompassing these two sites. 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 susceptibility to ADAMTS13 proteolysis. Together these data demonstrate that the N-linked glycans of VWF have a marked modulatory effect on the interaction with ADAMTS13. At least part of this effect is conformational, but a marked contribution from the glycan at N1574 suggests that steric hindrance may also be important.
