Metabolism of exogenous sn-1-alkyl-sn-2-lyso-glucosaminyl-phosphatidylinositol in HeLa D cells

Accumulation of glucosaminyl(acyl)phosphatidylinositol in a metabolically inert compartment

A. Wongkajornsilp, D. Sevlever, T. L. Rosenberry

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

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 24 h 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.

Original languageEnglish (US)
Pages (from-to)305-313
Number of pages9
JournalBiochemical Journal
Volume359
Issue number2
DOIs
StatePublished - Oct 15 2001

Fingerprint

Somatostatin-Secreting Cells
Phosphatidylinositols
HeLa Cells
Metabolism
Glycosylphosphatidylinositols
Inositol
Lipids
Paroxysmal Hemoglobinuria
Biosynthetic Pathways
Uridine Diphosphate
Isotopes
Labeling
Fatty Acids
Cell Culture Techniques

Keywords

  • Glycolipid uptake
  • Glycosylphosphatidylinositols
  • Paroxysmal nocturnal haemoglobinuria

ASJC Scopus subject areas

  • Biochemistry

Cite this

Metabolism of exogenous sn-1-alkyl-sn-2-lyso-glucosaminyl-phosphatidylinositol in HeLa D cells : Accumulation of glucosaminyl(acyl)phosphatidylinositol in a metabolically inert compartment. / Wongkajornsilp, A.; Sevlever, D.; Rosenberry, T. L.

In: Biochemical Journal, Vol. 359, No. 2, 15.10.2001, p. 305-313.

Research output: Contribution to journalArticle

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abstract = "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 24 h 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.",
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AB - 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 24 h 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.

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