Characterization of the intracellular and secreted forms of the glycoprotein hormone chorionic gonadotropin produced by human malignant cells.

Abstract

Cultured human choriocarcinoma (JAR) cells synthesize high mannose oligosaccharide-containing Mr = 15,000 and 18,000 forms of the a subunit and Mr = 18,000 and 24,000 forms of the P3 subunit of human chorionic gonadotropin (hCG).These intracellular forms have a long half-life (-1 h) and remain sensitive to endoglycosidase H (endo H) and resistant to neuraminidase digestion for as long as they are detectable intracellularly during pulse-chase experiments.The "mature," secreted forms of the subunits, however, do not accumulate intracellularly prior to secretion and are neuraminidase sensitive.Thus, secretion of hCG subunits from JAR choriocarcinoma cells occurs con- tinuously, indicating that the concentration and stor- age steps of the secretory pathway are abbreviated or lacking for hCG in these cells.Pretreatment of JAR cells with tunicamycin (5 ,tg/ml) or digestion of the immunoprecipitated, intracellular forms of hCG sub- units with endo H indicates that the apoprotein forms are Mr = 12,000 for a subunit and Mr = 15,000 for /3 subunit.Partial NH2-terminal sequences of the 15,000, 18,000, and 12,000 (apoprotein) forms of JAR a are identical to that of placental hCG-a.Similarly, the partial NH2-terminal sequences of the 24,000 and 15,000 (apoprotein) forms of JAR /8 are identical to that of placental hCG-,B.The predominant endo H-sensitive, high mannose oligosaccharide derived from the intra- cellular a subunit has the general structure (Man)8-GleNAc, suggesting that the rate-limiting step in the processing of a subunit is at an a-mannosidase step.Free a produced by JAR cells differs from placental hCG-a in that the relative molecular weight of the intracellular apoprotein of free a is higher than that expected for hCG-a, and secreted free a migrates slower than hCG-a on sodium dodecyl sulfate-polyacrylamide gels and contains fucose.However, both the intracel- lular and secreted forms of free a appear to have the same NH2-terminal sequence as hCG-a.These data suggest that free a subunit produced by JAR cells is a larger polypeptide and has a different carbohydrate composition than placental hCG-a.We have previously shown that cultured human malignant cells derived from a variety of tumors synthesize and secrete