The somatic genetic defect in paroxysmal nocturnal haemoglobinuria (PNH) involves a block in the transfer of GlcNAc from UDP-GlcNAc to phosphatidylinositol (PI), the first step in the biosynthetic pathway for glycosylphosphatidylinositols (GPIs). We asked whether an exogenous lipid corresponding to an early intermediate in this pathway can be taken up by cells in culture and proceed through the GPI pathway. This approach could offer a strategy to bypass the block in PNH. To address this question we incubated HeLa D cells with sn-1-alkyl-sn-2-lyso-GlcN-[3H]PI (lyso-alkyl-GlcN-[3H]PI) for 24h and analysed the cellular lipids. We found three lipid products: unaltered lyso-alkyl-GlcN-[3H]PI, GlcN-[3H]PI and GlcN(acyl)[3H]PI (GlcN-PI with a fatty acid acyl group on inositol). Since the latter two lipids are intermediates in the GPI biosynthetic pathway, this observation demonstrates that an exogenous lipid can enter and proceed partially through this pathway. However, the conversion of GlcN(acyl)PI to downstream mannosylated GPI intermediates in the GPI pathway was inefficient, both for GlcN(acyl)PI produced from the exogenous lipid as well as from that obtained by metabolic labelling with [3H]inositol. We investigated this poor conversion by examining whether GlcN(acyl)PI, radioactively labelled sequentially by [14C]inositol and [3H]inositol, resided in one compartment and could be readily metabolized to downstream intermediates. Isotope ratios indicated that the turnover of GlcN(acyl)PI was slower than those of either downstream mannosylated GPIs or even GPI anchors on proteins, the final products of GPI pathway. This result is incompatible with the one-compartment model and indicates that GlcN(acyl)PI in HeLa D cells accumulates largely in a compartment that is inert to subsequent mannosylation.
Abbreviations used: PI, phosphatidylinositol; lyso-PI, sn-1-acyl-2-lyso-PI; GlcN-PI, glucosaminyl-PI; lyso-alkyl-GlcN-PI, sn-1-alkyl-sn-2-lyso-GlcN-PI; GlcN(acyl)PI, glucosaminyl(acyl)PI; PI-PLC, PI-specific phospholipase C; GPI, glycosylphosphatidylinositol; PNH, paroxysmal nocturnal haemoglobinuria; DMEM, Dulbecco's modified Eagle's medium.
Present address: Research Department, Mayo Clinic Jacksonville, 4500 San Pablo Road, Jacksonville, FL 32224, U.S.A.