Characterization of an alg2 mutant of the zygomycete fungus Rhizomucor pusillus

Author： Takeuchi Kyoko Yamazaki Haruka Shiraishi Norihiko Ohnishi Yasuo Nishikawa Yoshihisa Horinouchi Sueharu

ISSN： 1460-2423

Source： Glycobiology, Vol.9, Iss.12, 1999-12, pp. : 1287-1293

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Abstract

The zygomycete fungus Rhizomucor pusillus secretes an aspartic proteinase (MPP) that contains asparagine (N)-linked oligosaccharides at two sites. Mutant strain 1116 defective in N-glycosylation secretes MPP with truncated oligosaccharide chains. Lipid-linked oligosaccharides in mutant 1116 were labeled with [6-³H]glucosamine and [2-³H]mannose, prepared by cycles of solvent extraction, and analyzed by gel filtration chromatography on a Bio-Gel P-4 column after mild acid-hydrolysis. Mutant 1116 accumulated an intermediate, Man1GlcNAc2-dolichol pyrophosphate (PP-Dol), whereas wild-type strain F27 synthesized the fully assembled oligosaccharide precursor Glc3Man9GlcNAc2-PP-Dol. Consistent with this, alg2 encoding a mannosyltransferase in the lipid-linked oligosaccharide biosynthetic pathway in mutant 1116 had a 5 bp insertion that generated a stop codon in the middle of the coding sequence. Transformation of mutant 1116 with the intact alg2 gene on a pUC19-derived plasmid generated transformants that contained multicopies of alg2 at the alg2 locus. Glycosylation of the total proteins in the transformants was recovered to the same level as in strain F27, as determined with peroxidase-concanavalin A. These transformants produced MPP mainly with the same N-linked oligosaccharides as that produced by strain F27, but still with truncated oligosaccharides in small amounts. All of these data show that Alg2 is an α-1,3 or α-1,6 mannosyltransferase that elongates Man1GlcNAc2-PP-Dol to Man2GlcNAc2-PP-Dol. The slower growth of mutant 1116 was significantly recovered on introduction of alg2. The viability of the alg2 mutants of the zygomycete R.pusillus makes a contrast with the lethal effect of ALG2 mutations in the yeast Saccharomyces cerevisiae.