Kinetics of folding and assembly of the human chorionic gonadotropin beta subunit in transfected Chinese hamster ovary cells.
We have employed Chinese hamster ovary (CHO) cell lines transfected with either the wild type human chorionic gonadotropin beta (hCG-beta) gene alone (CHO beta cells) or in conjunction with the gene expressing the alpha subunit (CHO alpha,beta cells) to study the folding pathway of the hCG-beta subunit. In both CHO beta and CHO alpha,beta cells, the earliest detectable hCG-beta precursor, p beta 1, which had two of six potential disulfide bonds (34-88 and 38-57) formed, was converted to p beta 2, a form that, following the formation of disulfide bonds between cysteines 9-90 and 23-72, migrated more slowly than p beta 1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. The t1/2 for the conversion of p beta 1 to p beta 2 in CHO alpha,beta and CHO beta cells was 5 min, demonstrating that the alpha subunit had no effect on the rate of this conversion. Furthermore, the tryptic-releasable peptides generated from nonreduced p beta 1 or p beta 2 were the same in both CHO alpha,beta and CHO beta cells. Thus, both the rate and order of disulfide bond formation during the conversion of the folding intermediate p beta 1 into p beta 2 were the same whether or not the alpha subunit was present. A comparison between cell types expressing different alpha/beta subunit ratios revealed that the higher the glycoprotein hormone alpha subunit to beta subunit ratio, the greater the rate and extent of hCG heterodimer assembly.
Bedows
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Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha 68198-6805.
Huth
J R
JR
Ruddon
R W
RW
D000818:Animals; D016466:CHO Cells; D006063:Chorionic Gonadotropin / Q000737:chemistry* / Q000235:genetics; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D010449:Peptide Mapping; D011233:Precipitin Tests; D011487:Protein Conformation; D014162:Transfection; D014357:Trypsin / Q000378:metabolism
D006063:Chorionic Gonadotropin; D004220:Disulfides; D014357:Trypsin