Site-specific analysis of von Willebrand factor O-glycosylation
File(s)
Accepted version
Author(s)
Solecka, BA
Weise, C
Laffan, MA
Kannicht, C
Type
Journal Article
Abstract
Background
O-glycosylation of von Willebrand factor (VWF) affects many of its functions; however, there is currently no information on the occupancy of the 10 putative O-glycosylation sites.
Objectives
The aim of this study was the site-specific analysis of VWF O-glycosylation.
Methods
Tryptic VWF-O-glycopeptides were isolated by lectin affinity chromatography and/or by reverse-phase high-performance liquid chromatography. Subsequently, the purified glycopeptides were analyzed by glycosidase digestion and mass spectrometry.
Results
We found that all 10 predicted O-glycosylation sites in VWF are occupied. The majority of the glycan structures on all glycosylation sites is represented by disialyl core 1 O-glycan. The presence of core 2 O-glycan was also confirmed; interestingly, this structure was not evenly distributed among all 10 glycosylation sites. Analysis of the glycopeptides flanking the A1 domain revealed that generally more core-2-type O-glycan was present on the C-terminal Cluster 2 glycopeptide (encompassing T1468, T1477, S1486 and T1487) compared with the N-terminal Cluster 1 glycopeptide (encompassing T1248, T1255, T1256 and S1263). Disialosyl motifs were present on both glycopeptides flanking the A1 domain and on the glycosylation site T2298 in the C1 domain. In addition, we identify sulfation of core 2 O-glycans and the presence of the rare Tn antigen.
Conclusions
This is the first study to describe the qualitative and semi-quantitative distribution of O-glycan structures on all 10 O-glycosylation sites, which will provide a valuable starting point for further studies exploring the functional and structural implications of O-glycosylation in VWF.
O-glycosylation of von Willebrand factor (VWF) affects many of its functions; however, there is currently no information on the occupancy of the 10 putative O-glycosylation sites.
Objectives
The aim of this study was the site-specific analysis of VWF O-glycosylation.
Methods
Tryptic VWF-O-glycopeptides were isolated by lectin affinity chromatography and/or by reverse-phase high-performance liquid chromatography. Subsequently, the purified glycopeptides were analyzed by glycosidase digestion and mass spectrometry.
Results
We found that all 10 predicted O-glycosylation sites in VWF are occupied. The majority of the glycan structures on all glycosylation sites is represented by disialyl core 1 O-glycan. The presence of core 2 O-glycan was also confirmed; interestingly, this structure was not evenly distributed among all 10 glycosylation sites. Analysis of the glycopeptides flanking the A1 domain revealed that generally more core-2-type O-glycan was present on the C-terminal Cluster 2 glycopeptide (encompassing T1468, T1477, S1486 and T1487) compared with the N-terminal Cluster 1 glycopeptide (encompassing T1248, T1255, T1256 and S1263). Disialosyl motifs were present on both glycopeptides flanking the A1 domain and on the glycosylation site T2298 in the C1 domain. In addition, we identify sulfation of core 2 O-glycans and the presence of the rare Tn antigen.
Conclusions
This is the first study to describe the qualitative and semi-quantitative distribution of O-glycan structures on all 10 O-glycosylation sites, which will provide a valuable starting point for further studies exploring the functional and structural implications of O-glycosylation in VWF.
Date Issued
2016-02-17
Date Acceptance
2015-12-30
Citation
Journal of Thrombosis and Haemostasis, 2016, 14 (4), pp.733-746
ISSN
1538-7933
Publisher
Wiley
Start Page
733
End Page
746
Journal / Book Title
Journal of Thrombosis and Haemostasis
Volume
14
Issue
4
Copyright Statement
© 2016 International Society on Thrombosis and Haemostasis. This is the accepted version of the following article: Solecka BA, Weise C, Laffan MA, Kannicht C. Site-specific analysis of von Willebrand factor O-glycosylation. J Thromb Haemost 2016; 14: 733–46, which has been published in final form at http://dx.doi.org/10.1111/jth.13260
Sponsor
British Heart Foundation
Grant Number
PG/11/50/28984
Subjects
Jacalin
glycopeptides
glycosylation
matrix-assisted laser desorption-ionization mass spectrometry
von Willebrand factor
Cardiovascular System & Hematology
1102 Cardiovascular Medicine And Haematology
1103 Clinical Sciences
Publication Status
Published