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 (GPLMT-I) catalyses the transfer of a mannosyl residue from beta-mannosylphosphoryldolichol (beta-Man-P-Dol) to glucosamine-alpha(1,6)(acyl)phosphatidylinositol (GlcN-aPI) to form Man alpha(1,4)GlcN-aPI (ManGlcN-aPI), an intermediate in glycosylphosphatidylinositol (GPI) synthesis, While the transfer of [H-3]mannosyl units to endogenous GlcN-aPI was not seen when membrane fractions from normal Chinese hamster ovary (CHO) K1 cells were incubated with exogenous [H-3]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) beta-Man-P-Dol in vivo. The presence of a saturated a-isoprene unit in the dolichyl moiety is required for optimal GPIMT-I activity since beta-mannosylphosphorylpolyprenol (beta-Man-P-Poly), which contains a fully unsaturated polyisoprenyl chain, was only 50% as effective as beta-[H-3]Man-P-Dol as a mannosyl donor, When beta-[H-3]Man-P-Dol and alpha-[H-3]Man-P-Dol were compared as substrates, GPIMT-I exhibited a strict stereospecificity for the mannolipid containing the beta-mannosyl-phosphoryl linkage. beta-[H-3]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 beta-Man-P-Dol, but accumulates beta-Man-P-Poly, synthesized no detectable Man-GlcN-aPI when incubated with beta-[H-3]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 beta-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 Lec35, 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 beta-configuration.