Characterization ofSchizosaccharomyces pombeER α-Mannosidase: A Reevaluation of the Role of the Enzyme on ER-associated Degradation

Abstract

It has been postulated that creation of Man 8 GlcNAc 2 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 Man 9 GlcNAc 2 and was inhibited by kifunensin. Live S. pombe cells showed an extremely limited capacity to demannosylate Man 9 GlcNAc 2 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.

The evolution of N-glycan-dependent endoplasmic reticulum quality control factors for glycoprotein folding and degradation

“…Mns1, Golgi mannosidases, and EDEM were distinguished by phylogenetic methods (Fig. 3B) (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)30). Alignments of protein sequences were made by using ClustalW, and manual adjustments and trimming of the alignments were performed with Jalview (39).…”

“…To our surprise, Trichomonas, which makes a Man 5 GlcNAc 2 N-glycan precursor and so was not expected to have N-glycandependent ERAD, contains putative Mns1-like mannosidases, Yos9, and cytosolic PNGases (Figs. 1 E and F and 3B and Table 1) (1)(2)(3)(4)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26).…”

“…One model is that Mns1 removes a single Man residue from the middle arm of Man 9 GlcNAc 2 to make Man 8B GlcNAc 2 , which in turn interacts with EDEM that has chaperone and/or lectin activity. This model is complicated by evidence that Mns1 has weak mannosidase activity in S. pombe (17); more than one Man residue may be removed from N-glycans of misfolded proteins in mammalian cells (13); N-glycan-dependent ERAD occurs in mutant cell lines that are missing the middle and upper Man arms on their N-glycans (18); and there are multiple EDEM paralogs, at least two of which (EDEM1 and EDEM3) have mannosidase activity in vivo (19)(20)(21)(22)(23). Yos9, which resembles the Man-6-P receptor, has also been implicated in N-glycandependent ERAD, likely as a lectin (24,25).…”

Purification and biochemica characterisation of endoplasmic reticulum α 1,2-mannosidase from Sporothrix schenckiil

“…Enzymes from family 47 are α1,2-mannosidases that participate in the trimming of N-glycans and in ER-associated degradation (Herscovics 1999a, b, 2001, Helenius & Aebi 2004. Two subgroups of enzymes comprise family 47: the ER α1,2-mannosidases that trim only one mannose residue from Man 9 GlcNAc 2 (M 9 ) generating Man 8 GlcNAc isomer B (M 8 B) (Ziegler & Trimble 1991, Tremblay & Herscovics 1999, Mora-Montes et al 2004, Movsichoff et al 2005; and Golgi α1,2-mannosidases from mammalian cells and filamentous fungi, which act after the ER enzyme to remove three α1,2-linked mannose units, generating Man 5 GlcNAc 2 , an intermediate required for the biosynthesis of complex and hybrid N-glycans (Yoshida et al 1993, Lal et al 1998, Ichishima et al 1999, Eades & Hintz 2000, Tremblay & Herscovics 2000, Lobsanov et al 2002, Akao et al 2006. Lower eukaryotes, such as Saccharomyces cerevisiae, lack Golgi α1,2-mannosidases and synthesise only high-mannose N-glycans (Herscovics 1999b).…”

Galactose-Specific Recognition System in the Fission Yeast <i>Schizosaccharomyces pombe</i>

“…N-Glycosylation begins with the assembly of a precursor, dolichol pyrophosphatelinked Man 5 GlcNAc 2 glycan (Dol-PP-Man 5 GlcNAc 2 ) at the cytoplasmic face of the ER (4). The precursor is then flipflopped into the lumen of the ER by a process that is not well understood in S. pombe.…”