Detection of a glycosylated, incompletely folded form of chorionic gonadotropin beta subunit that is a precursor of hormone assembly in trophoblastic cells.

Abstract

The alpha and beta subunits of human chorionic gonadotropin are secreted both as a combined, noncovalently linked dimer form as well as uncombined, free forms by human trophoblastic cells. We have utilized the cultured choriocarcinoma cell line JAR to determine what regulates the combination of the two subunits. The human chorionic gonadotropin subunits produced by JAR cells were biosynthetically labeled with [35S] cysteine or [3H]mannose by a pulse-chase protocol, purified by immunoprecipitation with specific antisera that recognize free or combined subunits, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing or reducing conditions. Radioactively labeled bands were eluted from the gels and analyzed for total counts/minute incorporated, the ratio of free thiols to intramolecular cystine disulfides, and oligosaccharide composition. In some experiments, labeled gel bands were eluted with trypsin under nonreducing conditions, and the trypsin-released peptides were analyzed by high performance liquid chromatography. Using these procedures, the following results were obtained. The earliest, biosynthetically labeled form of the beta subunit detected in JAR cells contains high mannose N-linked oligosaccharides and has one-half of its incorporated cysteines present as free thiols. This form, termed pre-beta 1, has not yet combined with the alpha subunit even though the biosynthetically labeled alpha subunit is present in the cells at the same time. The pre-beta 1 form has a t1/2 of about 4 min and has a precursor-product relationship with a more completely disulfide-bonded form, termed pre-beta 2, which does combine with the alpha subunit to form a dimer. A subset of beta molecules produced in JAR cells does not attain the same disulfide bonding pattern as the pre-beta 2 form, does not combine with the alpha subunit, and is secreted as a free beta subunit into the culture medium. On the other hand, the earliest detectable form of the alpha subunit in JAR cells has all its thiols present as cystine disulfides, at a time when dimerization with the beta subunit has not yet taken place. These results strongly suggest that intramolecular disulfide bond formation in the beta subunit is the crucial and rate-limiting event in alpha beta dimer formation. The subset of beta molecules that remain free do not appear to form the appropriate intramolecular disulfides and thus do not achieve the correct conformation to combine with the alpha subunit.(ABSTRACT TRUNCATED AT 400 WORDS)