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Impact of O ‐linked glycosylation of the VWF‐A1‐domain flanking regions on platelet interaction
Author(s) -
Esch Jan Schulte am,
Robson Simon C.,
Knoefel Wolfram T.,
Eisenberger Claus F.,
Peiper Matthias,
Rogiers Xavier
Publication year - 2005
Publication title -
british journal of haematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.907
H-Index - 186
eISSN - 1365-2141
pISSN - 0007-1048
DOI - 10.1111/j.1365-2141.2004.05253.x
Subject(s) - glycosylation , von willebrand factor , mutant , glycoprotein , chemistry , threonine , platelet membrane glycoprotein , serine , biochemistry , platelet glycoprotein gpib ix complex , platelet , amino acid , n linked glycosylation , alanine , mutation , membrane glycoproteins , microbiology and biotechnology , biology , gene , phosphorylation , glycan , immunology
Summary This study investigated the functional impact of O ‐linked glycosylation of von Willebrand Factor (VWF) A1 domains on the interaction with platelet receptors. Native or mutant VWF‐A1‐domains were transiently overexpressed on COS‐7 cells as membrane glycosylphosphatidylinositol (GPI)‐anchored FLAG‐tagged fusion proteins. Cytofluometric analysis assured comparable levels of A1‐domain expression among native and mutant homologues as well as for different culture conditions. Expressing native VWF‐A1‐domains under O ‐linked glycosylation blocking conditions increased the platelet aggregatory responses observed for fully glycosylated forms. Utilizing a neuronal network for prediction of O ‐linked glycosylation of mammalian proteins, threonine (T) and serine (S) residues located in the VWF‐A1‐loop flanking regions – not in the loop itself – were determined to be glycosylated n‐terminal at amino acids T485, S490, T492 and T493 and c‐terminal at T705. Simultaneous selective charge‐to‐alanine mutation of S490, T492 and T493 led to gain in aggregatory responses. When compared with native forms, equivalent alterations of T485 did not dictate functional differences. Any alanine‐substitution for T705 revealed a substantial loss in aggregatory effects – possibly as a result of structural desintegration of the VWF‐A1‐binding site for glycoprotein (GP) Ib. These data suggest specific O ‐linked glycosylation of the amino‐terminal VWF‐A1‐loop‐flanking region to have a negative regulatory impact on the A1‐domain affinity of non‐activated human VWF for human platelet‐GPIb.