Imperial College London
Dr Tom McKinnon

Dr Tom McKinnon

Faculty of Medicine, Department of Immunology and Inflammation

Senior Lecturer in Immunology and Inflammation
 
 
 
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Contact

 

+44 (0)20 3313 2214t.mckinnon03

 
 
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Location

 

Commonwealth BuildingHammersmith Campus

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Summary

 

Publications

Citation

BibTex format

@article{Nowak:2017:10.1111/jth.13688,
author = {Nowak, AA and O'Brien, HER and Henne, P and Doerr, A and Vanhoorelbeke, K and Laffan, MA and McKinnon, TAJ},
doi = {10.1111/jth.13688},
journal = {Journal of Thrombosis and Haemostasis},
pages = {1155--1166},
title = {ADAMTS-13 glycans and conformation-dependent activity.},
url = {http://dx.doi.org/10.1111/jth.13688},
volume = {15},
year = {2017}
}
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RIS format (EndNote, RefMan)

TY  - JOUR
AB  - Background:ADAMTS-13 activity can be regulated by its conformation, whereby interactions between the C-terminal CUB domains and the spacer domain maintain ADAMTS-13 in a closed conformation. ADAMTS-13 contains 10 N-linked glycans, with four sites present in theTSP2 through to CUB domains that may contribute to its conformation.Objectives/Methods:We hypothesized that glycosylation contributes to ADAMTS-13 conformation and function. The proteolytic activity of glycan-modified ADAMTS-13 was assessed under static and shear stress conditions.Results:Enzymatic removal of terminal silaic acid or entire N-linked glycan chains decreased activity against FRETS-VWF73 at pH 7.4 and against full-length von Willebrand factor (VWF) under shear stress. Using truncated ADAMTS-13, we demonstrated that this was attributable to loss of sialic acid from the glycans in the metalloprotease domain and an effect of N-linked glycosylation in the TSP2 through to CUB domains. Mutation of the N-linked glycan sites in the MDTCS domains reduced or abolished protein expression. However, the N707Q, N828Q, N1235Q and N1354Q (TSP2, TSP4, CUB1, and CUB2 domains, respectively) variants were expressed normally. Interestingly, the N707Q and N828Q variants showed reduced activity against FRETS-VWF73, but normal activity under flow conditions. In contrast, the N1235Q and N1354Q variants had enhanced activity against FRETS-VWF73 and VWF under shear stress. Immunoprecipitation experiments confirmed that loss of N-linked glycans in the CUB domains significantly reduced the interaction with the spacer domain and enhanced binding to the 6A6 anti-ADAMTS-13 antibody, which recognizes a cryptic epitope in the metalloprotease domain.Conclusions:Together, these data demonstrate that the N-linked glycans of ADAMTS-13 play a crucial role in regulating ADAMTS-13 activity.
AU  - Nowak,AA
AU  - O'Brien,HER
AU  - Henne,P
AU  - Doerr,A
AU  - Vanhoorelbeke,K
AU  - Laffan,MA
AU  - McKinnon,TAJ
DO  - 10.1111/jth.13688
EP  - 1166
PY  - 2017///
SN  - 1538-7933
SP  - 1155
TI  - ADAMTS-13 glycans and conformation-dependent activity.
T2  - Journal of Thrombosis and Haemostasis
UR  - http://dx.doi.org/10.1111/jth.13688
UR  - http://www.ncbi.nlm.nih.gov/pubmed/28370891
UR  - http://hdl.handle.net/10044/1/46158
VL  - 15
ER  -
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