Disulfide bond formation is not required for human chorionic gonadotropin subunit association. Studies with dithiothreitol in JEG-3 cells.
To study the influence of disulfide bridge formation on the assembly of the subunits of human chorionic gonadotropin in JEG-3 choriocarcinoma cells, dithiothreitol (DTT) was used to create a reducing milieu in the endoplasmic reticulum (ER) in vivo. In the presence of 5 mM DTT during pulse-chase experiments all of the beta-subunit precursors observed in unperturbed cells (pbeta(0), pbeta(1), pbeta(2), and beta(*)) collapsed into the pbeta(0) form. The reducing milieu of the ER was reoxidized in less than 5 min after removal of DTT from the medium. DTT markedly increased the half-life of the pbeta(0) precursor from 8.8 to 65.2 min. Under reoxidation conditions, the beta-subunit precursors folded back from pbeta(0) in less than 5 min. In unperturbed JEG-3 cells, the alpha-subunit was present in both fully glycosylated and monoglycosylated precursor (pre-alpha) forms. The attachment of the second N-linked glycan residue of the alpha-subunit was accelerated in the presence of DTT, and consequently pre-alpha-subunit was missing from the DTT-treated cultures. The formation of alphabeta-dimers appeared to be at least partially independent of the oxidation state in the ER. The alphabeta-dimer was present under conditions in which disulfide bridge formation was prevented by exposure to 5 mM DTT before and during the pulse period. This clearly suggests that the human chorionic gonadotropin subunits may acquire association-competent conformations even when no disulfide bridge formation has taken place.
Singh
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V
Biochemie-Zentrum Heidelberg, University of Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany.
Merz
W E
WE
D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000737:chemistry / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism*; D004229:Dithiothreitol / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015292:Glycoprotein Hormones, alpha Subunit / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D011487:Protein Conformation; D014407:Tumor Cells, Cultured
D018997:Chorionic Gonadotropin, beta Subunit, Human; D004220:Disulfides; D015292:Glycoprotein Hormones, alpha Subunit; D004229:Dithiothreitol