Compositional analysis of Glucosaminyl(acyl)phosphatidylinositol Accumulated in HeLa S3 Cells

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Abstract
GlcN(acyl)PtdIns, a derivative of phosphatidylinositol (PtdIns) in which glucosamine and a fatty acid are linked to inositol hydroxyl groups, has been proposed to be an intermediate in the mammalian biosynthetic pathway for glycosylphosphatidylinositol (glycosyl-PtdIns) anchors of membrane proteins. In this report, GlcN(acyl)PtdIns metabolically labeled with [3H]inositol is shown to accumulate in a HeLa S3 cell subline. The amount of GlcN(acyl)PtdIns in these HeLa S3 cells is about 107 molecules/cell, a level comparable to those of the most abundant glycosyl-PtdIns-containing molecules reported to date. GlcN(acyl)PtdIns was purified by a two-step procedure involving octyl-Sepharose and thin-layer chromatography. Octyl-Sepharose separated phospholipids according to their number of hydrocarbon chains: one in 2-lysoPtdIns, two in PtdIns, and three in GlcN(acyl)PtdIns. Purification also was aided by prior treatment of lipid extracts with bee venom phospholipase A2, an enzyme that did not cleave GlcN(acyl)PtdIns. The GlcN-inositol head group in purified GlcN(acyl)PtdIns was confirmed by a number of procedures, including cation-exchange chromatography and mass spectrometry; after radiomethylation, an equal molar ratio of GlcN(Me)2/inositol was measured. Fatty acid analysis indicated an overall stoichiometry of 2.3 mol fatty acid/mol inositol with palmitic (16:0), stearic (18:0) and oleic (18:1) acids being predominant. Analysis of GlcN(acyl)inositol produced by HF fragmentation showed that palmitate was the acyl group attached to inositol and indicated that stearic and oleic acids were in the glycerolipid. Base methanolysis revealed that about 15% of the purified GlcN(acyl)PtdIns contained alkylglycerol. A substantial conversion of GlcN(acyl)PtdIns to a slightly more polar lipid occurred after overnight incubation in even mildly alkaline buffers. Although the current data do not allow proposal of a structure for this lipid, its formation from GlcN(acyl)PtdIns may be important because the conversion appeared to occur in vivo.