Intracellular folding pathway of human chorionic gonadotropin beta subunit.
Few experimental models have been used to investigate how proteins fold inside a cell. Using the formation of disulfide bonds as an index of conformational changes during protein folding, we have developed a unique system to determine the intracellular folding pathway of the beta subunit of human chorionic gonadotropin (hCG). Three folding intermediates of the beta subunit were purified from [35S]cysteine-labeled JAR choriocarcinoma cells by immunoprecipitation and by reverse-phase high performance liquid chromatography (HPLC). To identify unformed disulfide bonds, nonreduced folding intermediates were treated with trypsin to liberate non-disulfide-bound, [35S]cysteine-containing peptides from the disulfide-linked peptides. Released peptides were purified by HPLC and identified by amino acid sequencing. The amount of a peptide that was released indicated the extent of disulfide bond formation involving the cysteine in that peptide. Of the six disulfide bonds in hCG-beta, bonds 34-88 and 38-57 form first. The rate-limiting event of folding involves the formation of the S-S bonds between cysteines 23 and 72 and cysteines 9 and 90. Disulfide bond 93-100, the formation of which appears to be necessary for assembly with the alpha subunit of the hCG heterodimer, forms next. Finally, disulfide bond 26-110 forms after assembly with the alpha subunit, suggesting that completion of folding of the COOH terminus in the beta subunit occurs after assembly with the alpha subunit.
Huth
J R
JR
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha 68198-6805.
Mountjoy
K
K
Perini
F
F
Ruddon
R W
RW
D000595:Amino Acid Sequence; D002822:Choriocarcinoma / Q000378:metabolism; D006063:Chorionic Gonadotropin / Q000737:chemistry* / Q000235:genetics; D002852:Chromatography, Ion Exchange; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011487:Protein Conformation; D014357:Trypsin / Q000378:metabolism; D014407:Tumor Cells, Cultured
D006063:Chorionic Gonadotropin; D004220:Disulfides; D014357:Trypsin