Introduction of O-glycosidically linked mannose into proteins via mannosyl phosphoryl dolichol by microsomes from Fusarium soani f. pisi

Cutinase, a glycoprotein containing O-glycosidically linked carbohydrates, is induced in glucose-grown Fusarium solani f. pisi by cutin hydrolysate. Microsomal preparations from the induced cells catalyzed mannosyl transfer from GDP-mannose to glycolipid and glycoprotein fractions but not into oligosaccharide lipids. Maximal rates of mannosyl transfer into glycolipids and glycoproteins were obtained with 5 mm Mg²⁺ and 10 mm Mn²⁺, respectively. Mannosyl transfer into glycolipids and glycoproteins showed pH optima of 8.0 and 7.0, respectively, and both transfers showed an apparent K_m of about 2 μm for GDP-mannose. The mannosyl lipid was identified as β-d-mannosyl phosphoryl dolichol by thinlayer and ion-exchange chromatography, as well as by analyses of the products derived from it by acid and base treatments. The fungal microsomal preparation also catalyzed mannosyl transfer from GDP-mannose to exogenous dolichol phosphate. This transfer was stimulated maximally by 0.09% Triton X-100 and showed a pH optimum at pH 8.0. The apparent K_m values for dolichol phosphate and GDP-mannose were 120 and 2.3 μm, respectively. The product derived from exogenous dolichol phosphate was identified as β-d-mannosyl phosphoryl dolichol as indicated above. The endogenous mannosyl acceptor lipid from this fungus was isolated by DEAE-cellulose chromatography. Analysis of the p-nitrobenzoyl derivatives of the base hydrolysis products of this acceptor lipid by highperformance liquid chromatography showed that the major components of this dolichol were C₉₅ and C₁₀₀. The microsomal preparation also catalyzed the transfer of mannose from exogenous mannosyl phosphoryl dolichol to glycoproteins with a pH optimum of 7.5 and an apparent K_m of 1.7 μm. Analyses of the β-elimination products of the glycoproteins generated from both GDP-mannose and dolichol phosphoryl mannose showed that single mannosyl residues were transferred to hydroxyl groups of the endogenous proteins. Exogenous cutinase was not glycosylated even after denaturation, sulfitolysis, or removal of carbohydrates by HF hydrolysis. Sodium dodecyl sulfate electrophoresis indicated that cutinase and its possible precursors were among the in vitro glycosylation products. Bacitracin and amphomycin but not tunicamycin inhibited the mannosyl transfer reactions.