PubMed1980
The clonal human choriocarcinoma cell line JEG-3 secretes hCG and heterogeneous forms of its free alpha-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 in Sephadex G-100 or, after reduction, electrophoresed on 12-20% gradient sodium dodecyl sulfate-polyacrylamide slab gels (SDS-PAG:), were analyzed by immunoprecipitation with antisera to hCT-alpha and hCG-beta. The intracellular labeled free alpha-subunit observed in lysates at all time points of either continuous or pulse-chase experiments was the same size or was slightly smaller on G-100 (apparent mol wt, 21,600 +/- 3,900) than urinary standard alpha-subunit (CR119 alpha; apparent mol wt, 22,700 +/- 1,500) and also somewhat smaller on SDS-PAGE (apparent mol wt, 19,400 +/- 600) than urinary standard alpha-subunit (apparent mol wt, 20,200 +/- 1,100). However, the predominant form of secreted free alpha-subunit, 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 alpha-subunit in the cell lysate, and our data suggest that the small intracellular alpha-subunit is a precursor of the larger secreted alpha-subunit and not vice versa. The beta-subunit of secreted hCG was somewhat larger (apparent mol wt, 31,500 +/- 1,000) on SDS-PAGE than standard beta-subunit (CR119-2 beta and CR115 beta; 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 alpha-subunit as well as a large beta-subunit in hCG may be due to posttranslational oligosaccharide addition during the secretory process.