ISOLATION AND CHARACTERIZATION OF A CHINESE-HAMSTER OVARY (CHO) MUTANT DEFECTIVE IN THE 2ND STEP OF GLYCOSYLPHOSPHATIDYLINOSITOL BIOSYNTHESIS

Mutant cell lines defective in the biosynthesis of glycosylphosphatidy linositol (GPI) described to date were isolated by selecting cells whi ch no longer expressed one or more endogenous GPI-anchored proteins on their surface, In this study, a new mutant in this pathway was isolat ed from ethylmethane-sulphonate-mutagenized Chinese hamster ovary cell s stably transfected with human placental alkaline phosphatase (FLAP) as a marker of GPI-anchored proteins. A three-step protocol was employ ed, In the first step, cells with decreased surface expression of FLAP were selected by four rounds of complement-mediated lysis with an ant i-(alkaline phosphatase) antibody, The surviving cells were cloned by limiting dilution and those with low levels of total alkaline phosphat ase activity were selected in the second step, Finally, the ability of each clone to synthesize the first three intermediates in GPI biosynt hesis in vitro was assessed to determine which cells with low alkaline phosphatase activity harboured a defect in one of these reactions. Of 230 potential mutants, one was defective in the second step of GPI bi osynthesis. Microsomes from this mutant, designated G9PLAP.85, were co mpletely unable to deacetylate either endogenous GlcNAc-phosphatidylin ositol (PI) synthesized from UDP[6-H-3]GlcNAc or exogenous GlcNAc-PI a dded directly to the membranes. Complementation analysis with the Thy- 1-deficient murine lymphoma cells demonstrated that G9PLAP.85 has a mo lecular defect distinct from these previously described mutants, There fore, these results suggest that mutants in GPI biosynthesis could be selected from almost any cell line expressing a GPI-anchored marker pr otein.