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, d ithiothreitol (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 exp eriments all of the beta-subunit precursors observed in unperturbed cells ( p beta(0), p beta(1), p beta(2), and beta*) collapsed into the p beta(0) fo rm. The reducing milieu of the ER was reoxidized in less than 5 min after r emoval of DTT from the medium. DTT markedly increased the half-life of the p beta(0) precursor from 8.8 to 65.2 min. Under reoxidation conditions, the beta-subunit precursors folded back from p beta(0) in less than 5 min. In unperturbed JEG-3 cells, the alpha-subunit was present in both fully glycos ylated 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 th e DTT-treated cultures. The formation of alpha beta-dimers appeared to be a t least partially independent of the oxidation state in the ER. The alpha b eta-dimer was present under conditions in which disulfide bridge formation was prevented by exposure to 5 mM DTT before and during the pulse period. T his clearly suggests that the human chorionic gonadotropin subunits may acq uire association-competent conformations even when no disulfide bridge form ation has taken place.