Structural and biosynthetic studies of the granule membrane protein, GMP-140, from human platelets and endothelial cells.
GMP-140 is an integral membrane glycoprotein of apparent Mr = 140,000 located in secretory storage granules of platelets and vascular endothelial cells. When these cells are activated, GMP-140 redistributes from the membrane of the granules to the plasma membrane. To gain insight into the potential function of GMP-140, we examined aspects of its structure and biosynthesis. The amino acid composition of platelet GMP-140 revealed elevated numbers of cystinyl (6.1%), prolinyl (7.2%), and tryptophanyl (2.1%) residues. GMP-140 contained 28.8% carbohydrate by weight, distributed among N-acetylneuraminic acid, neutral sugar, and N-acetylglucosamine residues. Enzymatic removal of N-linked oligosaccarides reduced the protein's apparent Mr by more than 50,000. The biosynthesis of GMP-140 in HEL cells, which share biochemical features with megakaryocytes, was studied by pulse-chase labeling with [35S]cysteine followed by immunoprecipitation. HEL cells synthesized a heterogeneous GMP-140 precursor of 98-125 kDa which converted to a mature 140-kDa form within 40-60 min. Removal of high mannose oligosaccarides by endo-beta-N-acetylglucosaminidase H treatment reduced the apparent Mr of the precursor but not the mature protein. Tunicamycin-treated HEL cells synthesized three to four precursors of 80-92 kDa, suggesting the possibility of heterogeneity of GMP-140 at the protein level. Exposure of activated platelets to proteases followed by Western blotting indicated that most of the mass of GMP-140 was located on the extracytoplasmic side of the membrane. Our studies indicate that GMP-140 is a cysteine-rich, heavily glycosylated protein with a large extracytoplasmic domain. These features are compatible with a receptor function for the molecule when it is exposed on the surface of activated platelets and endothelial cells.
Johnston
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GI
St. Francis Medical Research Institute, University of Oklahoma Health Sciences Center, Oklahoma City 73104.
Kurosky
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A
McEver
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RP
D000596:Amino Acids / Q000032:analysis; D001792:Blood Platelets / Q000032:analysis*; D004220:Disulfides / Q000032:analysis; D004730:Endothelium, Vascular / Q000032:analysis*; D006596:Hexosaminidases / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D009844:Oligosaccharides / Q000032:analysis; D019007:P-Selectin; D010980:Platelet Membrane Glycoproteins / Q000032:analysis* / Q000096:biosynthesis; D014415:Tunicamycin / Q000494:pharmacology
D000596:Amino Acids; D004220:Disulfides; D009844:Oligosaccharides; D019007:P-Selectin; D010980:Platelet Membrane Glycoproteins; D014415:Tunicamycin; D006596:Hexosaminidases