Metabolic labeling of glycosylphosphatidylinositol-anchor of heparan sulfate proteoglycans in rat ovarian granulosa cells.
The glycosylphosphatidylinositol (GPI)-anchor of the plasma membrane-associated heparan sulfate (HS) proteoglycan was metabolically radiolabeled with [3H]myristic acid, [3H]palmitic acid, [3H]inositol, [3H]ethanolamine, or [32P]phosphate in rat ovarian granulosa cell culture. Cell cultures labeled with [3H]myristic acid or [3H]palmitic acid were extracted with 4 M guanidine HCl buffer containing 2% Triton X-100 and the proteoglycans were purified by ion exchange chromatography after extensive delipidation. Specific incorporation of 3H into GPI-anchor was demonstrated by removing the label with a phosphatidylinositol-specific phospholipase C (PI-PLC). Incorporation of 3H activity into glycosaminoglycans and core glycoproteins was also demonstrated. However, the specific activity of 3H in these structures was approximately 2 orders of magnitude lower than that in the GPI-anchor, suggesting that 3H label was the result of the metabolic utilization of catabolic products of the 3H-labeled fatty acids. PI-PLC treatment of cell cultures metabolically labeled with [3H]inositol, [3H]ethanolamine, or [32P]phosphate specifically released radiolabeled cell surface-associated HS proteoglycans indicating the presence of GPI-anchor in these proteoglycans. GPI-anchored HS proteoglycans accounted for 20-30% of the total cell surface-associated HS proteoglycans and virtually all of them were removed by PI-PLC. These results further substantiate the presence of GPI-anchored heparan sulfate proteoglycan in ovarian granulosa cells and its cell surface localization.
Yanagishita
M
M
Bone Research Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892.
D000818:Animals; D002478:Cells, Cultured; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D019856:Ethanolamine; D004983:Ethanolamines / Q000378:metabolism; D005260:Female; D006017:Glycolipids / Q000096:biosynthesis*; D017261:Glycosylphosphatidylinositols; D006107:Granulosa Cells / Q000378:metabolism*; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000096:biosynthesis* / Q000302:isolation & purification; D007294:Inositol / Q000378:metabolism; D007700:Kinetics; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010716:Phosphatidylinositols / Q000096:biosynthesis*; D010761:Phosphorus Radioisotopes; D011509:Proteoglycans / Q000096:biosynthesis* / Q000302:isolation & purification; D051381:Rats; D011919:Rats, Inbred Strains; D013462:Sulfur Radioisotopes; D014316:Tritium
D019856:Ethanolamine; D004983:Ethanolamines; D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D007294:Inositol; D019814:Myristic Acid; D009227:Myristic Acids; D019308:Palmitic Acid; D010169:Palmitic Acids; D010710:Phosphates; D010716:Phosphatidylinositols; D010761:Phosphorus Radioisotopes; D011509:Proteoglycans; D013462:Sulfur Radioisotopes; D014316:Tritium