Laffan, M

McKinnon, TAJ

Nowak, AA

Blood

Spiral - Imperial College Digital Repository

The N-Linked Glycans of ADAMTS13 Are Critical Determinants of Conformation and Proteolytic ActivityAgata Anna Nowak, MSc, PhD  Michael Laffan, MD Thomas Anthony Jude McKinnon, PhD Introduction: The multimeric size and haemostatic potential of Von Willebrand Factor (VWF) is regulated by the metalloproteinase ADAMTS13 that cleaves VWF within its A2 domain. Under conditions of shear stress, ADAMTS13 cleavage mediates VWF collagen and platelet binding function and therefore regulates the size of the growing thrombus. It has also recently been shown that an intra-domain interaction between the Spacer and CUB domains is present in ADAMST13 resulting in both "closed" and "open" ADAMTS13 molecules. A significant portion of the ADAMTS13 molecule is comprised of glycan structures with 10 N-linked glycosylation sites spaced throughout the molecule, while the exact functional role of these is not determined we hypothesised that they may affect ADAMTS13 conformation and proteolytic activity.Methods: Wild type and ADAMTS13 mutants lacking specific N-linked glycosylation sites were expressed in HEK293T cells as were ADAMTS13 variants truncated after the spacer domain (MDTCS), cysteine rich domain (MDTC) and the disintegrin-like domain (MD) and the isolated CUB domains. Terminal silaic acid residues and whole N-linked glycan chains were removed enzymatically with neuraminidase (As-AD13) and PNGase F (PNG-AD13) respectively. The ability of ADAMTS13 and its variants to cleave VWF was assessed under both static and flow conditions and binding between the spacer and CUB domains determined by immunoprecipitation based assays.Results: As-AD13 and PNG-AD13 both demonstrated significantly reduced cleavage of the small FRETS-76 substrate and full length VWF under static conditions and importantly under conditions of physiological shear stress. Removal of sialic acid or N-linked glycan chains from the MDTCS, MDTC and MD variants also reduced proteolysis, indicating that silylation of one or both of the N-linked glycans in the metalloproteinase domain is required for proper ADAMTS13 function. Neuraminidase digestion did not affect the binding of ADAMTS13 to VWF; however PNG-AD13 had significantly reduced binding to VWF but interestingly PNG-MDTCS bound VWF with normal affinity. Using co-immunoprecipitation we demonstrated altered interactions between the Spacer and CUB domains depending on their respective glycosylation status. We hypothesise that the N-linked glycans modulate the recently determined intra-molecular interaction within ADAMTS13, and their absence alters the conformation of ADAMTS13 in solution. To further investigate this we expressed ADAMTS13 mutants lacking specific N-linked glycosylation sites. Mutation of the conserved glycan sites in the CUB 1 (N1235) and CUB 2 (N1354) domains to glutamine did not affect the expression of ADAMTS13 but in contrast to PNG-AD13, both these mutants were gain-of-function, having significantly enhanced ability to interact with and proteolysis VWF under static and flow conditions. To assess the potential therapeutic use of these gain-of-function mutants and wild type ADAMTS13 in a thrombotic setting, we pre-formed thrombi on collagen surfaces under both anti and pro-coagulant conditions then perfused wild type or mutant ADAMTS13 over the thrombi surface and monitored the reduction in thrombi size. Surprisingly, neither wild type ADAMTS13 nor the N1235Q or N1354Q mutants were able to reduce the size of pre-formed thrombi even at high concentrations (200nM) and furthermore the presence of ADAMTS13 did not enhance the tissue plasminogen mediated reduction in fibrin rich thrombi.Conclusion: The N-linked glycans of ADAMTS13 have critical effects on ADAMTS13 function; with terminal sialic acid residues in the metalloproteinase domain being essential for proper proteolytic function and glycosylation of the Spacer and CUB domains required to modulate the open/closed conformation of ADAMTS13. In addition, our data also demonstrates that while ADAMTS13 is effective at controlling the size of the growing thrombus it is ineffective at cleaving VWF once a thrombus is formed and is thus unable to reduce the size of pre-formed thrombi. Together these data highlight the importance of N-linked glycosylation for proper ADAMTS13 function.

The N-linked glycans of ADAMTS13 are critical determinants of conformation and proteolytic activity

Abstract

Similar works

Full text

Available Versions

Spiral - Imperial College Digital Repository