title: N-linked glycan stabilization of the VWF A2 domain.
abstract: Shear forces in the blood trigger a conformational transition in the von Willebrand factor (VWF) A2 domain, from its native folded to an unfolded state, in which the cryptic scissile bond (Y1605-M1606) is exposed and can then be proteolysed by ADAMTS13. The conformational transition depends upon a Ca(2+)binding site and a vicinal cysteine disulfide bond. Glycosylation at N1574 has previously been suggested to modulate VWF A2 domain interaction with ADAMTS13 through steric hindrance by the bulky carbohydrate structure. We investigated how the N-linked glycans of the VWF A2 domain affect thermostability and regulate both the exposure of the ADAMTS13 binding sites and the scissile bond. We show by differential scanning fluorimetry that the N-linked glycans thermodynamically stabilize the VWF A2 domain. The essential component of the glycan structure is the first sugar residue (GlcNAc) at the N1574 attachment site. From its crystal structures, N1574-GlcNAc is predicted to form stabilizing intradomain interactions with Y1544 and nearby residues. Substitution of the surface-exposed Y1544 to aspartic acid is able to stabilize the domain in the absence of glycosylation and protect against ADAMTS13 proteolysis in both the VWF A2 domain and FLVWF. Glycan stabilization of the VWF A2 domain acts together with the Ca(2+)binding site and vicinal cysteine disulfide bond to control unfolding and ADAMTS13 proteolysis.
authors.affiliation: Centre for Haematology, Department of Medicine, Imperial College London, London, United Kingdom.
authors.forename: Christopher J
authors.identifier: http://orcid.org/0000-0002-4534-4865
authors.initials: CJ
authors.lastname: Lynch
authors.affiliation: Centre for Haematology, Department of Medicine, Imperial College London, London, United Kingdom.
authors.forename: David A
authors.identifier: http://orcid.org/0000-0003-4283-3695
authors.initials: DA
authors.lastname: Lane
mesh_terms: D051722:ADAM Proteins / Q000737:chemistry / Q000378:metabolism*; D000071120:ADAMTS13 Protein; D000117:Acetylglucosamine / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D055550:Protein Stability; D059748:Proteolysis; D014841:von Willebrand Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D011134:Polysaccharides; D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human; D003545:Cysteine; D002118:Calcium; D000117:Acetylglucosamine