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

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.