Identification, Characterization, and Biosynthesis of a Novel N-Glycan Modification in the Fruiting Body of the Basidiomycete Coprinopsis cinerea

Abstract

Coprinopsis cinerea is a model organism for fruiting body development in homobasidiomycetes. Here, we focused on Nlinked oligosaccharides (NLO) of cell wall proteins in the hyphae of two developmental stages, vegetative mycelium and fruiting body. High mannose-type glycans were the most commonly found structures. In addition, we observed a novel glycan, predominantly present in fruiting body. This oligosaccharide structure was of the high mannose type with at least five mannoses and a bisecting ␣1-4 N-acetylglucosamine (GlcNAc) at the ␤-mannose of the N-glycan core. The transferase responsible for this modification, CcGnt1 (C. cinerea GlcNAc transferase 1), was identified and expressed in insect cells. In vitro activity of CcGnt1 was demonstrated. This novel glycosyltransferase belongs to the glycosyltransferase family 8 (GT8) and is predicted to be a type II membrane protein. Expression of the CcGnt1 locus was up-regulated in fruiting body, but down-regulation of expression by means of RNAi decreased the level of bisected NLO; however had no apparent effect on fruiting body formation.

Unraveling Unique Structure and Biosynthesis Pathway of N-Linked Glycans in Human Fungal Pathogen Cryptococcus neoformans by Glycomics Analysis

“…Moreover, although the exact order is not yet clear, a single ␤1,2-xylose residue is added to the first mannose at the trimannosyl core of N-glycan during the early processing stages in the Golgi. As in the case of the xylose addition to C. neoformans N-glycans, the modification of N-glycans by bisecting GlcNAc at the ␤-mannose of the N-glycan core was previously reported, and the corresponding transferase has been characterized in the basidiomycete Coprinopsis cinerea (10). Then, similar to the addition of mannosyl phosphate in S. cerevisiae, the xylose phosphate might be added to some N-glycans at the core and outer chain regions, generating acidic N-glycans with negative charges during the late processing stages.…”

A Combined System for Engineering Glycosylation Efficiency and Glycan Structure in Saccharomyces cerevisiae

“…2-AB labeling was performed as described previously (27). For permethylation, purified and dried N-glycans were dissolved in a slurry of finely ground NaOH pellets in dimethyl sulfoxide (ϳ0.2 ml), followed by addition of 0.1 ml of methyl iodide.…”

“…Reaction mixtures were incubated overnight at 37°C. Glycans were desalted and purified from enzymes with a small-scale carbon column (27).…”

Mannoside recognition and degradation by bacteria

“…Moreover, an additional N-acetylglucosaminyl residue α-1,4 bound to the β-1,4-linked mannosyl of the core pentasaccharide has been reported in the fruiting body of the basidiomycete Coprinopsis cinerea strain AmutBmut (Buser et al, 2010). This observation indicates that the substitution on this mannosyl is taxon dependent: plants add β-xylose, animals add β-GlcpNAc, and fungi add α-GlcpNAc.…”

“…The long α-1,6 side chain of fungal N-glycans seems to be shorter than that in yeast or even absent, since that of the pathogenic yeast C. albicans is 100-150 mannosyl residues long, compared to the 150 residue-long one found in S. cerevisiae (Masuoka, 2004). Finally, fungal N-glycans are long, and more similar to those in yeast when in a budding, unicellular form, while vegetative mycelium forms harbour mainly HMNGs, closer to those in other eukaryotes (Buser et al, 2010).…”