Mannolipid donor specificity of glycosylphosphatidylinositol mannosyltransferase-I (GPIMT-I) determined with an assay system utilizing mutant CHO-K1 cells

Alex W. Deluca, Jeffrey S. Rush, Mark A. Lehrman, C. J. Waechter

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19 Scopus citations


The microsomal enzyme glycosylphosphatidylinositol mannosyltransferase I (GPIMT-I) catalyses the transfer of a mannosyl residue from β-mannosylphosphoryldolichol (β-Man-P-Dol) to glucosamine-α(1,6)(acyl)phosphatidylinositol (GlcN-aPI) to form Manα(1,4)GlcN-aPI (ManGlcN-aPI), an intermediate in glycosylphosphatidylinositol (GPI) synthesis. While the transfer of [3H]mannosyl units to endogenous GlcN-aPI was not seen when membrane fractions from normal Chinese hamster ovary (CHO) K1 cells were incubated with exogenous [3H]Man-P-Dol, GPIMT-I activity could be characterized with an in vitro enzyme assay system employing membrane fractions from Lec15 or Lec35 cells. These CHO cell mutants apparently contain elevated levels of endogenous GlcN-aPI due to the inability to synthesize (Lec15) or utilize (Lec35) β-Man-P-Dol in vivo. The presence of a saturated α-isoprene unit in the dolichyl moiety is required for optimal GPIMT-I activity since β-mannosylphosphorylpolyprenol (β-Man-P-Poly), which contains a fully unsaturated polyisoprenyl chain, was only 50% as effective as β-[3H]Man-P-Dol as a mannosyl donor. When β-[3H]- Man-P-Dol and α-[3H]Man-P-Dol were compared as substrates, GPIMT-I exhibited a strict stereospecificity for the mannolipid containing the β-mannosyl-phosphoryl linkage. β-[3H]Man-P-dolichols containing 11 or 19 isoprenyl units were equally effective substrates for GPIMT-I. Membrane fractions from Lec 9, a CHO mutant that apparently lacks polyprenol reductase activity and synthesizes very little β-Man-P-Dol, but accumulates β-Man-P-Poly, synthesized no detectable Man-GlcN-aPI when incubated with β-[3H]Man-P-Dol in vitro. This indirect assay suggests that GlcN-aPI does not accumulate in Lec 9 cells, possibly because it is mannosylated via β-Man-P-Poly, or perhaps the small amount of Man-P-Dol formed by the mutant in vivo. These experiments demonstrate that: (i) membrane fractions from the CHO mutants, Lec15 and Lec 35, provide a useful system for the characterization of GPIMT-I activity; (ii) GPIMT-I utilizes Man-P-Dol or Man-P-Poly as direct mannosyl donors for Man-GlcN-aPI synthesis, although Man-P-Poly is used less efficiently; and (iii) the transfer of mannosyl residues from Man-P-Dol to GlcN-aPI is stereospecific for mannolipid substrates containing mannosyl-phosphoryl linkages of the β-configuration.

Original languageEnglish
Pages (from-to)909-915
Number of pages7
Issue number6
StatePublished - Dec 1994

Bibliographical note

Funding Information:
We thank Dr Rene Frenkel (UT Southwestern) for the generous use of his Bioscan apparatus. Mr Biswanath Pramanik provided valuable assistance with cell culture experiments, and Ms Felicia Ware assisted with several of the GPFMT-I assays. This work was supported by NIH grant GM38545 (awarded to M.A.L.), NIH grant GM36065 (awarded to C.J.W.) and grant 1-1168 (awarded to M.A.L.) from the Robert Welch Foundation.


  • CHO mutants
  • Glycosylphosphatidylinositol
  • Mannosylphosphoryldolichol
  • Mannosyltransferase

ASJC Scopus subject areas

  • Biochemistry


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