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 [H-3] myristic acid, [H-3]palmitic acid, [H-3]inositol, [H-3]ethanolamine, or [P-32]phosphate in rat ovarian granulosa cell culture. Cell cultures labeled with [H-3]myristic acid or [H-3]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 H-3 into GPI-anchor was demonstrated by removing the label with a phosphatidylinositol-specific phospholipase C (PI-PLC). Incorporation of H-3 activity into glycosaminoglycans and core glycoproteins was also demonstrated. However, the specific activity of H-3 in these structures was approximately 2 orders of magnitude lower than that in the GPI-anchor, suggesting that H-3 label was the result of the metabolic utilization of catabolic products of the H-3-labeled fatty acids. PI-PLC treatment of cell cultures metabolically labeled with [H-3]inositol, [H-3]ethanolamine, or [P-32]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.