Formation of A hCG variant with truncated peptide moiety as a biosynthetic product in vitro

Abstract

Summary In serum of tumor patients variants of human chorionic gonadotropin (hCG) or of the free β-subunit with lower molecular weights may be present as well as proteolytically nicked forms. The origin of these variants, secreted by cells or formed in the periphery, is unknown. In the present study, brefeldin A (BFA) was used to block transiently the anterograde transport from the ER to the Golgi apparatus in JAr cells to increase the residency of hCG in the ER and thus to amplify possible proteolytic processes taking place in this cellular compartment. In JAr cells, BFA (2 hours, 0.05-0.4 μg/ml) diminished the amount of secreted free β-subunit (up to 90%) in a dose-dependent and reversible manner; hCG secretion was less affected. In the BFA cultures, the molar ratio hCG/free was increased up to five-fold. Pulse-chase experiments, using [ 3 H]-Leu for labeling of newly synthesized protein, showed the presence of lower molecular weight variants of the hCG-α and β subunits when hCG was arrested in the ER in presence of BFA. This could be observed most clearly in 8-bromo-cAMP (1 mM) stimulated cultures. In BFA treated cultures, the free β-subunit as well as the β-subunit contained in hCG showed and about 3 kD lower apparent molecular weight in comparison to the β-subunits of controls (34 kD). The lower molecular weight variants of the β-subunits still contained the carboxy-terminal peptide (CTP) since they were bound to anti-CTP-Sepharose. The carbohydrate part could also not account for the difference in the apparent molecular weight since it was not abolished by removal of the N-linked carbohydrate residues. The results demonstrate the capability of the ER to produce truncated variants of the free β-subunit and a truncated β-subunit contained in hCG which are secreted in vitro . During a transient arrest of the de novo synthesized proteins in the ER, the free β-subunit as well as hCG may be most probably proteolytic cleaved in the amino-terminal domain of the β-subunit or in the core part of the molecule.