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

摘要

pThe CHO (Chinese hamster ovary) glycosylation mutant cell line, B3F7, transfers the truncated glycan Glcsub3/subMansub5/subGlcNAcsub2/sub on to nascent proteins. After deglucosylation, the resulting Mansub5/subGlcNAcsub2/sub glycan is subjected to two reciprocal enzymic processes: the action of an endoplasmic-reticulum (ER) kifunensine-sensitive α1,2-mannosidase activity to yield a Mansub4/subGlcNAcsub2/sub 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 Prosup?5/sup 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./p