Site‐specific analysis of von Willebrand factor O‐glycosylation. (17th February 2016)

• Record Type: Journal Article
• Authors: Solecka, B. A.; Weise, C.; Laffan, M. A.; Kannicht, C.
• Is Part Of: Journal of thrombosis and haemostasis. Volume 14:Number 4(2016:Apr.)
• Page Start: 733
• Page End: 746
• Publication Date: 2016-02-17
• Subjects: glycopeptides -- glycosylation -- Jacalin -- matrix‐assisted laser desorption‐ionization mass spectrometry -- von Willebrand factor; Thrombosis -- Periodicals; Hemostasis -- Periodicals; Blood coagulation disorders -- Periodicals; 616.1
• DOI: 10.1111/jth.13260
• Languages: English
• ISSNs: 1538-7933

Abstract

Abstract : Essentials O ‐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.