Impact of O-linked glycosylation of the VWF-A1-domain flanking regions on platelet interaction.
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.
Schulte am Esch
Jan
J
Clinic for General and Visceral Surgery, Heinrich Heine University Duesseldorf, Germany. jan.schulteamesch@uni-duesseldorf.de
Robson
Simon C
SC
Knoefel
Wolfram T
WT
Eisenberger
Claus F
CF
Peiper
Matthias
M
Rogiers
Xavier
X
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism*; D019556:COS Cells; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010974:Platelet Aggregation; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D014841:von Willebrand Factor / Q000235:genetics* / Q000378:metabolism
D000596:Amino Acids; D019038:Platelet Glycoprotein GPIb-IX Complex; D014841:von Willebrand Factor