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Site‐specific analysis of von Willebrand factor O‐ glycosylation
Author(s) -
Solecka B. A.,
Weise C.,
Laffan M. A.,
Kannicht C.
Publication year - 2016
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/jth.13260
Subject(s) - glycosylation , glycan , glycopeptide , chemistry , von willebrand factor , biochemistry , glycoprotein , microbiology and biotechnology , biology , platelet , immunology , antibiotics
EssentialsO ‐glycosylation in von Willebrand factor (VWF) is important for its proper function. We report the first detailed site‐specific analysis of the O ‐glycosylation in plasma‐derived VWF. All 10 O ‐glycosylation sites are occupied, with the disialyl core 1 O ‐glycan as the major structure. Core 2 O ‐glycan is unevenly distributed and, sulfated O ‐glycans and Tn antigen have been found.Summary 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 T 1468 , T 1477 , S 1486 and T 1487 ) compared with the N‐terminal Cluster 1 glycopeptide (encompassing T 1248 , T 1255 , T 1256 and S 1263 ). Disialosyl motifs were present on both glycopeptides flanking the A1 domain and on the glycosylation site T 2298 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 .