Site‐specific analysis of von Willebrand factor O‐glycosylation

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 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.