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Biochem. J. (2006) 400 (33–41) (Printed in Great Britain)

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Man2C1, an a-mannosidase, is involved in the trimming of free oligosaccharides in the cytosol

Tadashi SUZUKI*‡§¹, Izumi HARA*§, Miyako NAKANO*‡, Masaki SHIGETA*, Takatoshi NAKAGAWA†, Akihiro KONDO†‡§, Yoko FUNAKOSHI*§ and Naoyuki TANIGUCHI‡

*Department of Biochemistry, Osaka University Graduate School of Medicine, Osaka, 565-0871, Japan, †Department of Glycotherapeutics, Osaka University Graduate School of Medicine, Osaka, 565-0871, Japan, ‡21st COE (Center of Excellence) Program, Osaka University Graduate School of Medicine, Osaka, 565-0871, Japan, §CREST (Core Research for Evolutionary Science and Technology), JST (Japan Science and Technology Agency), Kawaguchi 332-0012, Japan, and Department of Disease Glycomics, Research Institute for Microbial Diseases, Osaka University, Japan

The endoplasmic-reticulum-associated degradation of misfolded (glyco)proteins ensures that only functional, correctly folded proteins exit from the endoplasmic reticulum and that misfolded ones are degraded by the ubiquitin–proteasome system. During the degradation of misfolded glycoproteins, they are deglycosylated by the PNGase (peptide:N-glycanase). The free oligosaccharides released by PNGase are known to be further catabolized by a cytosolic a-mannosidase, although the gene encoding this enzyme has not been identified unequivocally. The findings in the present study demonstrate that an a-mannosidase, Man2C1, is involved in the processing of free oligosaccharides that are formed in the cytosol. When the human Man2C1 orthologue was expressed in HEK-293 cells, most of the enzyme was localized in the cytosol. Its activity was enhanced by Co²⁺, typical of other known cytosolic a-mannosidases so far characterized from animal cells. The down-regulation of Man2C1 activity by a small interfering RNA drastically changed the amount and structure of oligosaccharides accumulating in the cytosol, demonstrating that Man2C1 indeed is involved in free oligosaccharide processing in the cytosol. The oligosaccharide processing in the cytosol by PNGase, endo-b-N-acetylglucosaminidase and a-mannosidase may represent the common ‘non-lysosomal’ catabolic pathway for N-glycans in animal cells, although the molecular mechanism as well as the functional importance of such processes remains to be determined.

Key words: a-mannosidase, cytosol, free oligosaccharide, non-lysosomal degradation, N-glycan, peptide:N-glycanase (PNGase).

Abbreviations used: 2-AA, 2-aminobenzoic acid; DMM, deoxymannojirimycin; EGFP, enhanced green fluorescent protein; ENGase, endo-b-N-acetylglucosaminidase; ER, endoplasmic reticulum; ERAD, ER-associated degradation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KIF, kifunensine; MALDI–TOF MS, matrix-assisted laser desorption ionization–time-of-flight MS; PA, pyridylamino; PDI, protein disulfide-isomerase; PNGase, peptide:N-glycanase; pNP-a-Man, p-nitrophenyl-a-D-mannoside; RNAi, RNA interference; RNase B, ribonuclease B; siRNA, small interfering RNA; SW, swainsonine; TFA, trifluoroacetic acid.

¹To whom correspondence should be addressed (email tsuzuki@biochem.med.osaka-u.ac.jp).

Received 22 June 2006/13 July 2006; accepted 18 July 2006

Published as BJ Immediate Publication 18 July 2006, doi:10.1042/BJ20060945

The Biochemical Society, London ©2006