Mannosylation of a synthetic gpi precursor by hamster membranes requires prior palmttoyl-CoA dependent acylation

GlcN-PI(CS) (di-octanoyl ghicosamine-α-phosphatidylinositol), a short chain analog of an intermediate in the synthesis of glycosylphosphatidylinositol (GPI) anchois was used in an in vitro assay with Chinese hamster ovary cell membranes and pHJ-Mannose-PDolichol (generated with exogenous GDP-[3H]-Mannose) to investigate conditions required for its mannosylation. Mannosylation of GicN-Pl(C8) required palnritoyl-CoA either added exogenousry or synthesized in vitro by adding ATP and free CoA. Nitrous acid, HF, a-mannosidase, GPIPLD and PI-PLC treatments and Bio-Gel P-2 chromatography showed that the structure of major mannosylated product was consistent with Man-G)cN-(acyl)-PI(C8). Pataritoyl-CoA was an acyl donor as different chain length acyl-CoA's gave products with different mobilities on TLC. At higher concentrations, GlcN-PI(C8) was an inhibitor of its own mannosylation as well as mannosylation of endogenous GlcN(acyl)-PI. This suggests that palmitoyl-CoA dependent acylation converted GlcNPI(C8) from an inhibitor into an acceptor of the mannosyltransferase.