Disulfide Bond Formation Is Not Required for Human Chorionic Gonadotropin Subunit Association

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.