Truncated N-glycans affect protein folding in the ER of CHO-derived mutant cell lines without preventing calnexin binding.
The involvement of N-glycans in the folding of influenza virus hemagglutinin (HA) was analyzed in two CHO-derived glycosylation mutants exhibiting a thermosensitive defect for secretion of human placental alkaline phosphatase. Truncated Man(5)GlcNAc(2)oligosaccharides with one or three glucose residues are attached to proteins of the MadIA214 and B3F7AP2-1 mutant cells, respectively. Newly synthesized proteins retained in these cells carry a Man(4)trimmed glycan generated by a mannosidase different from the ER mannosidases I and II and suggesting a recycling through the Golgi complex. The glucosidase inhibitor castanospermine affects the binding of HA folding intermediates to the lectin-like chaperone calnexin in B3F7AP2-1 but not in MadIA214 cells. We demonstrated that calnexin interacts in vivo with truncated Man(5)derivatives. In MadIA214 cells, this is only possible when Man(5)GlcNAc(2)on protein becomes reglucosylated. The pattern of intermediates seen during the folding of HA in the MadIA214 and B3F7AP2-1 mutant cell lines is different than in control cells. We also observed a variable occupancy of the seven glycosylation-sites. However, even under conditions that restore glycosylation of all sites, the folding intermediates of HA in the mutant cells still remain heterogeneous. Our results demonstrate that addition of truncated N-glycans interferes extensively with the folding of newly synthesized proteins in vivo.
Ermonval
M
M
Unité de Génétique Somatique, URA CNRS 1960, Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France,
Duvet
S
S
Zonneveld
D
D
Cacan
R
R
Buttin
G
G
Braakman
I
I
D000818:Animals; D016466:CHO Cells; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D037281:Calnexin; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006031:Glycosylation; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000378:metabolism*; D006801:Humans; D008358:Mannose / Q000378:metabolism; D009154:Mutation; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding
D002135:Calcium-Binding Proteins; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D011134:Polysaccharides; D037281:Calnexin; D008358:Mannose