Site-directed mutagenesis of platelet glycoprotein Ib alpha demonstrating residues involved in the sulfation of tyrosines 276, 278, and 279.
The interaction between platelet glycoprotein (GP) Ib alpha and von Willebrand factor (VWF) is essential for initiation of hemostasis. The sulfation of the 3 tyrosine residues 276, 278, and 279 in GPIb alpha is an important posttranslational modification that seems to promote the interaction with VWF. The environment where sulfation of tyrosines occurs has been proposed to contain highly acidic residues. This investigation has examined the highly acidic region from Asp249 to Asp287 in the mature GPIb alpha protein. Changes to most of the carboxylic acids in this region resulted in decreased reactivity to VWF. Only 3 mutants (Glu270Gln, Asp283Asn, Asp283Asn/Glu285Gln/Asp287Asn) resulted in the abolition of sulfation. Two novel mutations were also created. First, a deletion of the 7 amino acids from Tyr276 to Glu282 led to a loss of sulfation and totally abolished VWF binding in the presence of botrocetin. This confirms that it is these 3 tyrosines that undergo sulfation and that this region is crucial for botrocetin-mediated VWF binding. The second mutation involves changing the lysine residues at 253, 258, and 262 to alanine. This also led to distinct changes in VWF binding and abolition of sulfation.
Tait
A Sasha
AS
Department of Haematology, Prince of Wales Hospital, New South Wales, Australia. s.tait@unsw.edu.au
Dong
Jing-Fei
JF
López
José A
JA
Dawes
Ian W
IW
Chong
Beng H
BH
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003435:Crotalid Venoms / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D011499:Protein Processing, Post-Translational; D012310:Ristocetin / Q000494:pharmacology; D013431:Sulfates / Q000378:metabolism*; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism*; D014841:von Willebrand Factor / Q000378:metabolism
D003435:Crotalid Venoms; D019038:Platelet Glycoprotein GPIb-IX Complex; D013431:Sulfates; D014841:von Willebrand Factor; D012310:Ristocetin; D014443:Tyrosine; C030961:botrocetin