title: Site-specific analysis of von Willebrand factor O-glycosylation.
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.
  
  
  OBJECTIVE
  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.
authors.affiliation: Molecular Biochemistry, Octapharma, Berlin, Germany.
authors.forename: B A
authors.initials: BA
authors.lastname: Solecka
authors.affiliation: Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Weise
authors.affiliation: Department of Haematology, Faculty of Medicine, Hammersmith Hospital Campus, Imperial College, London, UK.
authors.forename: M A
authors.initials: MA
authors.lastname: Laffan
authors.affiliation: Molecular Biochemistry, Octapharma, Berlin, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Kannicht
mesh_terms: D015295:Antigens, Tumor-Associated, Carbohydrate / Q000737:chemistry*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D006020:Glycopeptides / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D037102:Lectins / Q000737:chemistry; D013058:Mass Spectrometry; D037121:Plant Lectins / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry*; D000072417:Protein Domains; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014841:von Willebrand Factor / Q000737:chemistry*
chemical_list: D015295:Antigens, Tumor-Associated, Carbohydrate; D006020:Glycopeptides; D037102:Lectins; D037121:Plant Lectins; D011134:Polysaccharides; C040286:Tn antigen; C043696:jacalin; D014841:von Willebrand Factor; D006026:Glycoside Hydrolases
keywords: Jacalin; glycopeptides; glycosylation; matrix-assisted laser desorption-ionization mass spectrometry; von Willebrand factor