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

Abstract

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 ␤-subunit precursors observed in unperturbed cells (p␤ 0 , p␤ 1 , p␤ 2 , and ␤ * ) collapsed into the p␤ 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 p␤ 0 precursor from 8.8 to 65.2 min. Under reoxidation conditions, the ␤-subunit precursors folded back from p␤ 0 in less than 5 min. In unperturbed JEG-3 cells, the ␣-subunit was present in both fully glycosylated and monoglycosylated precursor (pre-␣) forms. The attachment of the second N-linked glycan residue of the ␣-subunit was accelerated in the presence of DTT, and consequently pre-␣-subunit was missing from the DTT-treated cultures. The formation of ␣␤-dimers appeared to be at least partially independent of the oxidation state in the ER. The ␣␤-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.Secretory and membrane glycoproteins of eukaryotic cells are co-translationally translocated into the lumen of the endoplasmic reticulum (ER) 1 from where they travel to Golgi complex on the secretory pathway and to other destinations. Recently, it became evident that the transport of proteins out of the ER is limited by a unique "quality control" system that involves recognition and retention of misfolded or misassembled proteins. If further attempts of acquiring the correct folding fail, these proteins may be directed into a degradation pathway (1-4). In the case of oligomeric proteins, the correct formation of disulfide bonds plays an important role in the assembly of secretory and membrane proteins (5-7), which in turn determines stability, intracellular transport, maturation, and function.The disulfide bonds are generated through oxidation in the ER. The ER lumen is unique among the various compartments in the eukaryotic cells because it provides an oxidizing environment for the disulfide bond formation with the help of the protein disulfide isomerase that promotes the disulfide bond formation (8,9). Recently, it was demonstrated that the cotranslational disulfide bond formation, folding, and oligomerization of proteins within the ER can be reversibly inhibited by the addition of the disulfide bridge disrupting agent dithiothreitol (DTT) to living cells (10 -12). Interestingly, upon the removal of DTT, the disulfide bond formation, folding via normal ER folding intermediates, and oligomerization seems to take place (10 -17). Moreover, DTT does not inhibit the transport within t...

Epithelial Human Chorionic Gonadotropin Is Expressed and Produced in Human Secretory Endometrium During the Normal Menstrual Cycle1

“…Restriction enzyme examination excluded LHB expression. Various molecular forms of the CGB and CGA subunits (such as dimeric hCG) were also observed for endometrial tissue, which demonstrates a molecular pattern similar to that exhibited by placental hCG [34][35][36][37]. Human chorionic gonadotropin is expressed and released maximally in secretory-transformed endometrium with the highest evaluated scores.…”

“…The main bands were approximately 31 and 29 kDa for endometrial CGB and 24 and 21 kDa for CGA. In addition, numerous hCG dimeric bands of 44, 38, and 35 kDa and CGB subunits of 17 and 15 kDa were detected depending on the desialyzation grade [34][35][36][37]. The molecular expression profiles correlate with known molecular isoforms of placental hCG such as those found in serum during pregnancy.…”

The Gonadotropin Hormones and Their Receptors∗

“…C, C-terminus; N, N-terminus. [38][39][40][41] In addition to the heterodimeric nature of the hormones, homodimers have also been found for LHβ, 42,43 and for the α subunit. [28][29][30][31][32] Moreover, it was posited that subunit association occurred before the seatbelt was latched by closure of Cys26 and Cys110.…”

Folding and Maturation of Tyrosinase-related Protein-1 Are Regulated by the Post-translational Formation of Disulfide Bonds and by N-Glycan Processing

“…The folding pathway and the final conformation of the protein were altered, despite the collapse of the chain to a compact conformation stabilized by disulfide bridges. Similarly, the ␤-subunit precursors of the human chorionic gonadotropin, despite incorrect folding (39), could collapse to a conformation competent for dimerization with the ␣-subunit in presence of DTT (40). On the contrary, disulfide bond formation in the acetylcholine receptor occurs post-translationally, preceding the chain conformational maturation and subunit assembly (41).…”