N-linked glycan stabilisation of the VWF A2 domain.

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Title: N-linked glycan stabilisation of the VWF A2 domain.
Authors: Lynch, CJ
Lane, DA
Item Type: Journal Article
Abstract: Shear forces in the blood trigger a conformational transition in the 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 disulphide 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 stabilise 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 stabilising intradomain interactions with Y1544 and nearby residues. Substitution of the surface exposed Y1544 to aspartic acid is able to stabilise the domain in the absence of glycosylation and to protect against ADAMTS13 proteolysis in both the VWF A2 domain and FLVWF. Glycan stabilisation of the VWF A2 domain acts together with the Ca(2+) binding site and vicinal cysteine disulphide bond to control unfolding and ADAMTS13 proteolysis.
Issue Date: 31-Mar-2016
Date of Acceptance: 13-Jan-2016
URI: http://hdl.handle.net/10044/1/53223
DOI: https://dx.doi.org/10.1182/blood-2015-09-672014
ISSN: 0006-4971
Publisher: American Society of Hematology
Start Page: 1711
End Page: 1718
Journal / Book Title: Blood
Volume: 127
Copyright Statement: © 2016 by The American Society of Hematology
Sponsor/Funder: British Heart Foundation
Funder's Grant Number: FS/14/21/30733
Keywords: Science & Technology
Life Sciences & Biomedicine
Hematology
VON-WILLEBRAND-FACTOR
FACTOR A1 DOMAIN
GLYCOPROTEIN IB
SCISSILE BOND
3D STRUCTURE
ADAMTS13
BINDING
GLYCOSYLATION
STABILITY
CLASSIFICATION
ADAM Proteins
ADAMTS13 Protein
Acetylglucosamine
Binding Sites
Calcium
Crystallography, X-Ray
Cysteine
HEK293 Cells
Humans
Models, Molecular
Polysaccharides
Protein Binding
Protein Folding
Protein Interaction Domains and Motifs
Protein Stability
Proteolysis
von Willebrand Factor
1102 Cardiovascular Medicine And Haematology
1103 Clinical Sciences
1114 Paediatrics And Reproductive Medicine
Immunology
Publication Status: Published
Appears in Collections:Department of Medicine
Faculty of Medicine



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