Effect of the disulfide isomerase PDIa4 on the antibody production of Chinese hamster ovary cells.

Therapeutic monoclonal antibodies recognize and bind specific molecules on the surface of target cells, stimulating the immune system, which can attack these targeted cells. These antibodies are produced by mammalian cells, including Chinese hamster ovary (CHO) cells, because the formation of antibodies requires complicated posttranslational modifications, including peptidyl-prolyl cis/trans isomerization, disulfide bond formation, and glycosylation. Currently, it is thought that the efficient production of secretory proteins is limited by posttranslational processes. The ER is the biosynthesis site of all secreted and membrane proteins. The accumulation of unfolded proteins in the ER causes the ER stress response. During the ER stress state, various molecular chaperones are expressed to prevent proteins from the aggregate formation. The molecular chaperone involved in ER stress likely plays an essential role in the production of secretory proteins. The purpose of this study was to improve the production of monoclonal antibodies by cells. We elucidated the function of ER chaperones in the production of a monoclonal antibody. First, we quantitatively measured the mRNA expression levels of protein disulfide-isomerase family members. In CHO HcD6 cells treated with tunicamycin, the expression level of pdia4 was significantly increased. Second, we investigated the relationship between PDIa4 and antibody productivity in pdia4-knockdown cells. Both a decrease in the amount of secreted antibody and the accumulation of immature antibodies inside the cells were observed. Recombinant PDIa4 was able to refold the antibodies and Fabs. These results indicate that PDIa4 affects the production of monoclonal antibodies by catalyzing disulfide bond formation in these antibodies in CHO cells.

Inter-ring communication is dispensable in the reaction cycle of group II chaperonins.

Chaperonins are ubiquitous molecular chaperones with the subunit molecular mass of 60kDa. They exist as double-ring oligomers with central cavities. An ATP-dependent conformational change of the cavity induces the folding of an unfolded protein that is captured in the cavity. In the group I chaperonins, which are present in eubacteria and eukaryotic organelles, inter-ring communication takes important role for the reaction cycle. However, there has been limited study on the inter-ring communication in the group II chaperonins that exist in archaea and the eukaryotic cytosol. In this study, we have constructed the asymmetric ring complex of a group II chaperonin using circular permutated covalent mutants. Although one ring of the asymmetric ring complex lacks ATPase or ATP binding activity, the other wild-type ring undergoes an ATP-dependent conformational change and maintains protein-folding activity. The results clearly demonstrate that inter-ring communication is dispensable in the reaction cycle of group II chaperonins.

Dissection of the ATP-dependent conformational change cycle of a group II chaperonin.

Group II chaperonin captures an unfolded protein while in its open conformation and then mediates the folding of the protein during ATP-driven conformational change cycle. In this study, we performed kinetic analyses of the group II chaperonin from a hyperthermophilic archaeon, Thermococcus sp. KS-1 (TKS1-Cpn), by stopped-flow fluorometry and stopped-flow small-angle X-ray scattering to reveal the reaction cycle. Two TKS1-Cpn variants containing a Trp residue at position 265 or position 56 exhibit nearly the same fluorescence kinetics induced by rapid mixing with ATP. Fluorescence started to increase immediately after the start of mixing and reached a maximum at 1-2s after mixing. Only in the presence of K(+) that a gradual decrease in fluorescence was observed after the initial peak. Similar results were obtained by stopped-flow small-angle X-ray scattering. A rapid fluorescence increase, which reflects nucleotide binding, was observed for the mutant containing a Trp residue near the ATP binding site (K485W), irrespective of the presence or absence of K(+). Without K(+), a small, rapid fluorescence decrease followed the initial increase, and then a gradual decrease was observed. In contrast, with K(+), a large, rapid fluorescence decrease occurred just after the initial increase, and then the fluorescence gradually increased. Finally, we observed ATP binding signal and also subtle conformational change in an ATPase-deficient mutant with K485W mutation. Based on these results, we propose a reaction cycle model for group II chaperonins.