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

Abstract

GlcN(acyl)Ptdlns, 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-Ptdlns) anchors of membrane proteins. In this report, GlcN(acy1)Ptdlns metabolically labeled with [ 'Hlinositol is shown to accumulate in a HeLa S3 cell subline. The amount of GlcN(acy1)PtdIns in these HeLa S3 cells is about 10' molecules/cell, a level comparable to those of the most abundant glycosyl-Ptdlns-containing molecules reported to date. GlcN(acy1)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-lysoPtdlns, two in PtdIns, and three in GlcN(acy1)Ptdlns. Purification also was aided by prior treatment of lipid extracts with bee venom phospholipase A2, an enzyme that did not cleave GlcN(acy1)PtdTns. The GlcN-inositol head group in purified GlcN(acy1)Ptdlns was confirmed by a number of procedures, including cation-exchange chromatography and mass spectrometry ; after radiomethylation, an equal molar ratio of GlcN(Me)Jinositol was measured. Fatty acid analysis indicated an overall stoichiometry of 2.3 mol fatty acidimol inositol with palmitic (16:0), stearic (18:O) and oleic ( 1 8 : l ) acids being predominant. Analysis of GlcN(acy1)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(acy1)Ptdlns contained alkylglycerol. A substantial conversion of GlcN(acy1)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(acy1)Ptdlns may be important because the conversion appeared to occur in vivo.

Changes in molecular species profiles of glycosylphosphatidylinositol anchor precursors in early stages of biosynthesis

“…A similar increase in alk/acyl types was seen in murine lymphocytes, suggesting that it is a general phenomenon in mammalian cells. There is a report that ?15% of GlcN-acyl-PI from HeLa S3 cells was Changes in molecular species of GPI anchor in biosynthesis alk/acyl type (30). Together, these results indicate that selection of alkyl/acyl species occurs either in the flip-flop step of GlcN-PI or in the catalytic process of inositol acylation (Fig.…”

“…In another report, it was found that GlcN-acyl-PI contained myristic acids as minor components (apart from palmitic acids, the major components) at the C-2 position of inositol in malaria parasites (34). It was reported previously that GlcN-acyl-PI contained only palmitic acid at this position in mammalian cells (30). However, based on our detection of [GlcN-Ins(-myristoyl)-P] 2 fragments (m/z 612) (Fig.…”

Substrate specificity of the N-acetylglucosaminyl-phosphatidylinositol de-N-acetylase of glycosylphosphatidylinositol membrane anchor biosynthesis in African trypanosomes and human cells

“…This suggests that there may be some heterogeneity in the acyl chain attached to the inositol ring, as has been observed in human erythrocyte acetylcholinesterase [24], T. brucei glycolipid C [25] and T. brucei procyclic acid repetitive protein [26]. This conclusion would be broadly consistent with the ability of the mammalian inositolacyltransferase to use different acyl-CoA donors [27] but it is at variance with the study of Sevlever et al [28] on the GlcN-(acyl)PI that accumulates in HeLa S3 cells, which suggests that, in that case, the acyl chain is exclusively palmitate.…”

Effect of glycosylphosphatidylinositol (GPI)-phospholipase D overexpression on GPI metabolism

“…As shown in Figure 1, when 3 H-inositol-labelled HeLa D cells were lysed in acidic pH, GlcN-(acyl)PI was decreased and a species migrating at the position of GlcN-(acyl)Inos was detected. This fragment has been extensively characterized in a previous study by a variety of methods, including hydrophobic chromatography and MS [29]. Moreover, GlcN-(acyl)Inos was not present when these lysates were incubated in the presence of PNT (an inhibitor of GPI-PLD) or at pH 8.0 (Figure 1).…”

“…HeLa D is a subline of HeLa S3 cells that accumulates GlcN-(acyl)PI [27], the third intermediate of the GPI biosynthetic pathway. The advantage of using this approach is that GlcN-(acyl)Inos, the GPI-PLD cleavage product of GlcN-(acyl)PI, can be easily identified, because it moves further away from the origin of the TLC plate [29], in contrast with the GPI-PLD products of mannosylated GPI anchor intermediates that stay at or very close to the origin. As shown in Figure 1, when 3 H-inositol-labelled HeLa D cells were lysed in acidic pH, GlcN-(acyl)PI was decreased and a species migrating at the position of GlcN-(acyl)Inos was detected.…”