Glycobiology, 2001, Vol. 11, No. 7 565-576
© 2001 Oxford University Press
N-glycan structure of a short-lived variant of ribophorin I expressed in the MadIA214 glycosylation-defective cell line reveals the role of a mannosidase that is not ER mannosidase I in the process of glycoprotein degradation
3URA CNRS 1960, Département d’Immunologie Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France, 4Institute of Medical Biochemistry, Department of Molecular Genetics, University and Biocenter Vienna, Dr. Bohr-Gasse 9/2, A-1030 Vienna, Austria, and 5Laboratoire de Chimie Biologique, UMR CNRS 111, Université des Sciences et Technologies de Lille, 59655 Villeneuve d’Ascq Cedex, France
A soluble form of ribophorin I (RI332) is rapidly degraded in Hela and Chinese hamster ovary (CHO) cells by a cytosolic proteasomal pathway, and the N-linked glycan present on the protein may play an important role in this process. Specifically, it has been suggested that endoplasmic reticulum (ER) mannosidase I could trigger the targeting of improperly folded glycoproteins to degradation. We used a CHO-derived glycosylation-defective cell line, MadIA214, for investigating the role of mannosidase(s) as a signal for glycoprotein degradation. Glycoproteins in MadIA214 cells carry truncated Glc1Man5GlcNAc2 N-glycans. This oligomannoside structure interferes with protein maturation and folding, leading to an alteration of the ER morphology and the detection of high levels of soluble oligomannoside species caused by glycoprotein degradation. An HA-epitope-tagged soluble variant of ribophorin I (RI332-3HA) expressed in MadIA214 cells was rapidly degraded, comparable to control cells with the complete Glc3Man9GlcNAc2 N-glycan. ER-associated degradation (ERAD) of RI332-3HA was also proteasome-mediated in MadIA214 cells, as demonstrated by inhibition of RI332-3HA degradation with agents specifically blocking proteasomal activities. Two inhibitors of 
1,2-mannosidase activity also stabilized RI332-3HA in the glycosylation-defective cell line. This is striking, because the major mannosidase activity in the ER is the one of mannosidase I, specific for a mannose 1,2-linkage that is absent from the truncated Man5 structure. Interestingly, though the Man5 derivative was present in large amounts in the total protein pool, the two major species linked to RI332-3HA shortly after synthesis consisted of Glc1Man5 and Man4, being replaced by Man4 and Man3 when proteasomal degradation was inhibited. In contrast, the untrimmed intermediate of RI332-3HA was detected in mutant cells treated with mannosidase inhibitors. Our results unambiguously demonstrate that an 1,2-mannosidase that is not ER mannosidase I is involved in ERAD of RI332–3HA in the glycosylation-defective cell line, MadIA214.
1 To whom correspondence should be addressed
2 Present address: Différenciation Cellulaire et Prions, UPR 1983 CNRS, Institut André Lwoff, 7 rue Guy Moguet, BP 8, 94800 Villejuif, France
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