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

A soluble form of ribophorin I (RI332) is rapidly degraded in Hela and Chin ese 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 pr ocess. Specifically, it has been suggested that endoplasmic reticulum (ER) mannosidase I could trigger the targeting of improperly folded glycoprotein s to degradation. We used a CHO-derived glycosylation-defective cell line, MadIA214, for investigating the role of mannosidase(s) as a signal for glyc oprotein degradation. Glycoproteins in MadIA214 cells carry truncated Glc(1 )Man(5)GlcNAc(2) N-glycans, This oligomannoside structure interferes with p rotein maturation and folding, leading to an alteration of the ER morpholog y and the detection of high levels of soluble oligomannoside species caused by glycoprotein degradation. An HA-epitope-tagged soluble variant of ribop horin I (RI332-3HA) expressed in MadIA214 cells was rapidly degraded, compa rable to control cells with the complete Glc(3)Man(9)GlcNAc(c) N-glycan, ER -associated degradation (ERAD) of RI332-SHA was also proteasome-mediated in MadIA214 cells, as demonstrated by inhibition of RI332-3HA degradation wit h agents specifically blocking proteasomal activities. Two inhibitors of al pha1,2-mannosidase activity also stabilized RI332-3HA in the glycosylation- defective cell line. This is striking, because the major mannosidase activi ty in the ER is the one of mannosidase I, specific for a mannose alpha1,2-l inkage that is absent from the truncated Man(5) structure. Interestingly, t hough the Man, derivative was present in large amounts in the total protein pool, the two major species linked to RI352-3HA shortly after synthesis co nsisted of Glc(1)Man(5) and Man(4), being replaced by Man(4) and Man(3) whe n proteasomal degradation was inhibited. In contrast, the untrimmed interme diate of RI332-3HA was detected in mutant cells treated with mannosidase in hibitors. Our results unambiguously demonstrate that an al,2-mannosidase th at is not ER mannosidase I is involved in ERAD of RI332-3HA in the glycosyl ation-defective cell line, MadIA214.