Protein folding and assembly in vitro parallel intracellular folding and assembly. Catalysis of folding and assembly of the human chorionic gonadotropin alpha beta dimer by protein disulfide isomerase

• Published in: The Journal of biological chemistry 1993-08, Vol.268 (22), p.16472-16482
• Main Authors: Huth, J R, Perini, F, Lockridge, O, Bedows, E, Ruddon, R W
• Format: Article
• Language: English
• Subjects: Animals; Catalysis; Chorionic Gonadotropin - chemistry; Chorionic Gonadotropin - metabolism; Chromatography, High Pressure Liquid; Disulfides - chemistry; Dogs; Electrophoresis, Polyacrylamide Gel; Humans; Isomerases - metabolism; Kinetics; Peptide Mapping; Protein Disulfide-Isomerases; Protein Folding; Tumor Cells, Cultured
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(19)85444-3

Although purified proteins will refold and assemble in vitro, it is not known if cellular factors change the mechanisms of these processes. Based on the gel migration of folding intermediates, the kinetic relationships between these intermediates, and on the order of formation of six disulfide bonds, we have found that the in vitro folding pathway of the human chorionic gonadotropin beta subunit (hCG-beta) is indistinguishable from the intracellular folding pathway. The same rate-limiting event was found in both folding environments; however, the t1/2 for this step in a cell is 4 min, whereas in vitro the t1/2 was > or = 80 min. Protein disulfide isomerase (PDI) increased the in vitro rate of this event (t1/2 = 25 min) without changing the order of disulfide bond formation. PDI also catalyzed the in vitro rate of assembly of hCG subunits. In intact cells, assembly of the alpha beta heterodimer occurs before all of the intramolecular disulfide bonds of beta are formed. In vitro, assembly was increased after reduction of two of the carboxyl-terminal disulfide bonds of hCG-beta by PDI. These results strongly suggest that both in intact cells and in vitro, partially unfolded hCG-beta is more assembly-competent than is fully folded hCG-beta. The comparison of in vitro and intracellular hCG-beta folding and hCG subunit assembly which is shown in this report indicates that the assisted folding and assembly pathway that occurs in cells, where proteins such as PDI play a role, differs only in rate but not in the order of disulfide bond formation or in the precursor-product relationships among the folding intermediates.