Mannosidase action, independent of glucose trimming, is essential for proteasome-mediated degradation of unassembled glycosylated Ig light chains.
In order to study the role of N-glycans in the ER-associated degradation of unassembled immunoglobulin light (Ig L) chains, we introduced N-glycan acceptor sites into the variable domain of the murine Ig L chain kappaNS1, which is unfolded in unassembled molecules. We investigated the fate of kappaNS1 glycosylated at position 70 (K70) and of a double mutant (kappa18/70) in stably transfected HeLa cells. Degradation of both chains was impaired by lactacystin, a specific inhibitor of the proteasome. The mannosidase inhibitor dMNJ also blocked degradation in a step preceding proteasome action, as did two protein synthesis inhibitors, cycloheximide and puromycin. In contrast, ER glucosidase inhibitors dramatically accelerated the degradation of the chains when added either pre- or posttranslationally. The accelerated degradation was sensitive to lactacystin, dMNJ and cycloheximide, too. None of these drugs, except lactacystin, affected the degradation of unglycosylated kappaNS1 chains. We conclude that ER mannosidases and proteasome activities, but not glucose trimming (and therefore, most likely not the calnexin/calreticulin UDP:glucose glycoprotein glucosyl transferase cycle), are essential for ER-associated degradation (ERAD) of soluble glycoproteins. A role for a short-lived protein, acting before or simultaneously to ER mannosidases, is suggested.
Chillarón
J
J
Biochemie-Zentrum Heidelberg (BZH), Germany.
Adán
C
C
Haas
I G
IG
D000818:Animals; D003546:Cysteine Endopeptidases / Q000378:metabolism / Q000494:pharmacology*; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism; D007887:Fabaceae / Q000201:enzymology; D005803:Genes, Immunoglobulin; D005947:Glucose / Q000378:metabolism; D005959:Glucosidases / Q000037:antagonists & inhibitors / Q000494:pharmacology; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D007147:Immunoglobulin Light Chains / Q000187:drug effects / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000187:drug effects / Q000378:metabolism; D008361:Mannosidases / Q000378:metabolism / Q000494:pharmacology*; D051379:Mice; D009097:Multienzyme Complexes / Q000378:metabolism / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins; D010946:Plants, Medicinal; D046988:Proteasome Endopeptidase Complex; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D014162:Transfection; D014178:Translocation, Genetic
D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D009097:Multienzyme Complexes; D010940:Plant Proteins; D011500:Protein Synthesis Inhibitors; D005959:Glucosidases; D008361:Mannosidases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D005947:Glucose