De NovoSynthesis and Secretion of Heterogeneous Forms of Human Chorionic Gonadotropin and Its Freeα-Subunit in the Human Choriocarcinoma Clonal Cell Line JEG-3

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Abstract
The clonal human choriocarcinoma cell line JEG-3 secretes hCG and heterogeneous forms of its free α-subunit. We have studied the relationship of these forms in de novo biosynthesis experiments. Cells at near confluence were labeled with [35S]methionine by continuous (10-min to 24-h exposure) and by pulse-chase (5-min exposure, 10-min to 4-h chase) techniques. Media and cell lysates, chromatographed on Sephadex G-100 or, after reduction, electrophoresed on 12–20% gradient sodium dodecyl sulfate-polyacrylamide slab gels (SDSPAGE), were analyzed by immunoprecipitation with antisera to hCG-α and hCG-β. The intracellular labeled free α-subunit observed in lysates at all time points of either continuous or pulsechase experiments was the same size or was slightly smaller on G-100 (apparent mol wt, 21,600 ± 3,900) than urinary standard α-subunit (CR119α; apparent mol wt, 22,700 ± 1,500) and also somewhat smaller on SDS-PAGE (apparent mol wt, 19,400 ± 600) than urinary standard α-subunit (apparent mol wt, 20,200 ± 1,100). However, the predominant form of secreted free asubunit, at chase times as early as 30 min, migrated with a higher apparent molecular weight in both systems (SDS-PAGE, 22,100 ± 400; G-100,29,300 ± 2,700). We have not observed this secreted large free α-subunit in the cell lysate, and our data suggest that the small intracellular α-subunit is a precursor of the larger secreted α-subunit and not vice versa. The β-subunit of secreted hCG was somewhat larger (apparent mol wt, 31,500 ± 1,000) on SDS-PAGE than standard β-subunit (CR119-2β- and CR115β-; apparent mol wt, 29,900 ± 1,900). Secreted intact hCG migrated with urinary standard hCG (CR119) on G-100, but analysis of 35S-labeled intracellular hCG was complicated by co-precipitating large mol wt proteins. De novo synthesis and secretion of a large form of free α-subunit as well as a large β-subunit in hCG may be due to posttranslational oligosaccharide addition during the secretory process. (Endocrinology106: 849, 1980)