The unfolded protein response in a dolichyl phosphate mannose-deficient Chinese hamster ovary cell line points out the key role of a demannosylation step in the quality-control mechanism of N-glycoproteins.
The CHO (Chinese hamster ovary) glycosylation mutant cell line, B3F7, transfers the truncated glycan Glc(3)Man(5)GlcNAc(2) on to nascent proteins. After deglucosylation, the resulting Man(5)GlcNAc(2) glycan is subjected to two reciprocal enzymic processes: the action of an endoplasmic-reticulum (ER) kifunensine-sensitive alpha1,2-mannosidase activity to yield a Man(4)GlcNAc(2) glycan, and the reglucosylation involved in the quality-control system which ensures that only correctly folded glycoproteins leave the ER. We show that the recombinant secreted alkaline phosphatase (SeAP) produced in stably transfected B3F7 cells, is co-immunoprecipitated with the GRP78 (glucose-regulated protein 78), a protein marker of the unfolded protein response (UPR). The level of GRP78 transcription has been evaluated by reverse transcription-PCR (RT-PCR) and we demonstrate that B3F7 cells present a constitutively higher level of UPR in the absence of inductors, compared with Pro(-5) cells. Interestingly, a decrease was observed in the UPR and an increase in SeAP secretion in the kifunensine-treated B3F7 cells. Altogether, these data highlight the relationships between the glycan structure, the quality control system and the UPR. Moreover, they support the idea that a specific demannosylation step is a key event of the glycoprotein quality control in B3F7 cells.
Foulquier
François
F
Laboratoire de Chimie Biologique, CNRS-UMR 8576, Université des Sciences et Technologies de Lille, F-59655 Villeneuve d'Ascq Cédex, France.
Harduin-Lepers
Anne
A
Duvet
Sandrine
S
Marchal
Ingrid
I
Mir
Anne Marie
AM
Delannoy
Philippe
P
Chirat
Frédéric
F
Cacan
René
R
D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004287:Dolichol Monophosphate Mannose / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D014158:Transcription, Genetic
D000199:Actins; D006023:Glycoproteins; D009844:Oligosaccharides; D004287:Dolichol Monophosphate Mannose