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Structure of asparagine-linked oligosaccharides of an aspartic proteinase from the zygomycete fungus Rhizomucor pusillus

Kohji Murakami^1,

Kyoko Takeuchi^1,

Teruhiko Beppu^1,

Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113, Japan

ABSTRACT

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 Man₅ GlcNAc₂, and that bound to residue Asn¹⁸⁸ was Man₅ GlcNAc₂ and Man₅GlcNAc₂. These observations suggest that the processing of mannose residues in asparagine-linked oligosaccharides in the Golgi apparatus of Rhizomucor resembles that in mammalian cells.

^*Author for correspondence: Sueharu Horinouchi Tel: +81 3 3812 2111, ext. 5123. Fax: +81 3 5802 2931. e-mail: asuhori@hongo.ecc.u-tokyo.ac.jp

Present address: Department of Applied Biological Science, College of Bioresource Sciences, Nihon University, Fujisawa-shi, Kanagawa 252, Japan.

Present address: Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Machida-shi, Tokyo 194, Japan.

Present address: Research Division, The Green Cross Corporation, Hirakata-shi, Osaka 573, Japan.

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