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Vol. 16, Issue 10, 4714-4724, October 2005

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Characterization of Schizosaccharomyces pombe ER -Mannosidase: A Reevaluation of the Role of the Enzyme on ER-associated Degradation

Laboratory of Glycobiology, Fundación Instituto Leloir, C1405BWE Buenos Aires, Argentina

Submitted March 23, 2005; Revised July 12, 2005; Accepted July 26, 2005
Monitoring Editor: Reid Gilmore

It has been postulated that creation of Man₈GlcNAc₂ isomer B (M8B) by endoplasmic reticulum (ER) -mannosidase I constitutes a signal for driving irreparably misfolded glycoproteins to proteasomal degradation. Contrary to a previous report, we were able to detect in vivo (but not in vitro) an extremely feeble ER -mannosidase activity in Schizosaccharomyces pombe. The enzyme yielded M8B on degradation of Man₉GlcNAc₂ and was inhibited by kifunensin. Live S. pombe cells showed an extremely limited capacity to demannosylate Man₉GlcNAc₂ present in misfolded glycoproteins even after a long residence in the ER. In addition, no preferential degradation of M8B-bearing species was detected. Nevertheless, disruption of the -mannosidase encoding gene almost totally prevented degradation of a misfolded glycoprotein. This and other conflicting reports may be best explained by assuming that the role of ER mannosidase on glycoprotein degradation is independent of its enzymatic activity. The enzyme, behaving as a lectin binding polymannose glycans of varied structures, would belong together with its enzymatically inactive homologue Htm1p/Mnl1p/EDEM, to a transport chain responsible for delivering irreparably misfolded glycoproteins to proteasomes. Kifunensin and 1-deoxymannojirimycin, being mannose homologues, would behave as inhibitors of the ER mannosidase or/and Htm1p/Mnl1p/EDEM putative lectin properties.

View larger version (13K): [in this window] [in a new window]   Figure 1. Glycan structures. The figure represents the glycan transferred to proteins in wild-type S. pombe cells (Glc3Man9GlcNAc2). Lettering corresponds to the order of residue addition in the synthesis of the dolichol-P-P derivative. Numbers between monosaccharides correspond to the carbon atoms involved in the respective linkages. M8A lacks residues g and l-n, M8B residues j and l-n, and M8C residues k-n. The Man7GlcNAc2 isomer formed in S. pombe lacks residues i and k-n. The glycan transferred in MadIA214cells (Man5GlcNAc2) lacks residues h-n.

Abbreviations used: CNX, calnexin; CRT, calreticulin; CPY, carboxypeptidase Y; CPY^*, mutant CPY unable to properly fold; DMJ, 1-deoxymannojirimycin; DTT, dithiothreitol; EDEM, endoplasmic reticulum degradation enhancing -mannosidase-like protein; Endo H, endo--N-acetylglucosaminidase H; ER, endoplasmic reticulum; ERAD, endoplasmic reticulum-associated degradation; GT, UDP-Glc:glycoprotein glucosyltransferase; KFN, kifunensin; M8A, M8B, and M8C, Man₈GlcNAc₂ isomers as defined in Figure 1; MG132, N-carboxybenzoxyl-leucinyl-leucinyl-leucinal; NMDNJ, N-methyldeoxynojirimycin.

Address correspondence to: Armando J. Parodi (aparodi{at}leloir.org.ar).

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