Phosphatidylinositol-anchored glycoproteins of PC12 pheochromocytoma cells and brain.
PC12 pheochromocytoma cells and cultures of early postnatal rat cerebellum were labeled with [3H]glucosamine, [3H]fucose, [3H]leucine, [3H]ethanolamine, or sodium [35S]sulfate and treated with a phosphatidylinositol-specific phospholipase C. Enzyme treatment of [3H]glucosamine- or [3H]fucose-labeled PC12 cells led to a 15-fold increase in released glycoproteins. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, most of the released material migrated as a broad band with an apparent molecular size of 32,000 daltons (Da), which was specifically immunoprecipitated by a monoclonal antibody to the Thy-1 glycoprotein. A second glycoprotein, with an apparent molecular size of 158,000 Da, was also released. After treatment with endo-beta-galactosidase, 40-45% of the [3H]glucosamine or [3H]fucose radioactivity in the phospholipase-released glycoproteins was converted to products of disaccharide size, and the molecular size of the 158-kDa glycoprotein decreased to 145 kDa, demonstrating that it contains fucosylated poly-(N-acetyllactosaminyl) oligosaccharides. The phospholipase also released labeled Thy-1 and the 158-kDa glycoprotein from PC12 cells cultured in the presence of [3H]ethanolamine, which specifically labels this component of the phosphatidylinositol membrane-anchoring sequence, while in the lipid-free protein residue of cells not treated with phospholipase, Thy-1 and a doublet at 46/48 kDa were the only labeled proteins. At least eight early postnatal rat brain glycoproteins also appear to be anchored to the membrane by phosphatidylinositol. Sulfated glycoproteins of 155, 132/134, 61, and 21 kDa are the predominant species released by phospholipase, which does not affect a major 44-kDa protein seen in [3H]ethanolamine-labeled brain cultures. The 44-48- and 155/158-kDa proteins may be common to both PC12 cells and brain.
Margolis
R K
RK
Department of Pharmacology, State University of New York Health Science Center, Brooklyn 11203.
Goossen
B
B
Margolis
R U
RU
D000310:Adrenal Gland Neoplasms; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D002531:Cerebellum / Q000378:metabolism*; D019856:Ethanolamine; D004983:Ethanolamines / Q000378:metabolism; D005944:Glucosamine / Q000378:metabolism; D007930:Leucine / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008970:Molecular Weight; D010673:Pheochromocytoma; D010716:Phosphatidylinositols / Q000378:metabolism*; D051381:Rats; D013431:Sulfates / Q000378:metabolism
D004983:Ethanolamines; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D010716:Phosphatidylinositols; D013431:Sulfates; D019856:Ethanolamine; D007930:Leucine; D005944:Glucosamine