RESUMO
The zygomycete fungus Rhizomucor pusillus (previously called Mucor pusillus) secretes an aspartic proteinase containing two asparagine-linked, high-mannose type oligosaccharide chains at Asn79 and Asn188. For structural elucidation of the carbohydrate moieties, the protein was divided into two portions, an N-terminal portion containing Asn79 and a C-terminal portion containing Asn188, by a specific autocatalytic cleavage under alkaline conditions. Each of the asparagine-linked oligosaccharides was then released by peptide-N-glycosidase F digestion and pyridylaminated with a fluorescent reagent, 2-aminopyridine, at the reducing end. High-performance liquid chromatography analyses showed that the structure of the asparagine-linked oligosaccharide chain attached to residue Asn79 was Man5GlcNAc2, and that bound to residue Asn188 was Man5GlcNAc2 and Man6GlcNAc2. These observations suggest that the processing of mannose residues in asparagine-linked oligosaccharides in the Golgi apparatus of Rhizomucor resembles that in mammalian cells.
