Tyrosine sulfation of the glycoprotein Ib-IX complex: identification of sulfated residues and effect on ligand binding.
Here, we present evidence that glycoprotein (GP) Ib alpha, one of three polypeptides that make up the GP Ib--IX complex--the receptor for von Willebrand factor (vWf) on the surface of unactivated platelets--is modified by sulfation of tyrosine residues. Only GP Ib alpha was found to incorporate 35S when the GP Ib--IX complex was immunoprecipitated from [35S]sulfate metabolically labeled L and CHO cells that express the recombinant complex. The occurrence of sulfation on tyrosine residues of the polypeptide backbone was determined by removing O- and N-linked oligosaccharides. Limited proteolytic digestion of metabolically labeled GP Ib alpha revealed that sulfated tyrosine residues are located in the 45-kDa globular region containing the vWf binding site. By mutating potentially sulfated tyrosine residues to phenylalanine and comparing the stoichiometry of sulfate incorporation of these mutants to the incorporation in wild-type GP Ib alpha, three clustered tyrosine residues--Tyr-276, Tyr-278, and Tyr-279-were identified that undergo the modification. Culturing cells in sulfate-depleted medium containing sodium chlorate and guaiacol completely inhibited GP Ib alpha sulfation but did not decrease GP Ib-IX expression on the cell surface. Similarly, transiently transfected CHO cells expressed the mutant GP Ib alpha polypeptide on their surfaces at the same levels as they expressed wild-type GP Ib alpha. These results suggest that tyrosine sulfation of GP Ib alpha has little or no effect on the synthesis, assembly, and surface expression of the GP Ib-IX complex. Nevertheless, inhibiting sulfation of GP Ib alpha reduced the binding of 125I-labeled vWf in the presence of ristocetin by up to 37%.(ABSTRACT TRUNCATED AT 250 WORDS)
Dong
J F
JF
Gladstone Institute of Cardiovascular Disease, Cardiovascular Research Institute, San Francisco, California 94141-9100.
Li
C Q
CQ
López
J A
JA
D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D007739:L Cells; D008024:Ligands; D051379:Mice; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011485:Protein Binding; D013463:Sulfuric Acid Esters / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*; D014841:von Willebrand Factor / Q000378:metabolism*
D008024:Ligands; D010980:Platelet Membrane Glycoproteins; D013463:Sulfuric Acid Esters; D014841:von Willebrand Factor; D014443:Tyrosine