INTRACELLULAR IMMATURE SUBUNITS OF HUMAN CHORIONIC-GONADOTROPIN IN 1ST TRIMESTER PLACENTAL CELLS - PURIFICATION AND CHARACTERIZATION

As we previously reported [Sakakibara et al. (1986) Biochem. Biophys. Res. Commun. 137, 443-452; and Tominaga et al. (1989) J. Biochem. 105, 992-997], subunits of human chorionic gonadotropin (hCG) containing immature N-linked sugar chains (immature subunits), i.e., the 21 kDa form of alpha-subunit and the 23 and 19 kDa forms of beta-subunit, are present predominantly in first trimester placental cells. The molecular mass of intracellular hCG consisting of these subunits, based on gel filtration, was approximately 200 kDa, suggesting homo- or hetero-oligomerization of intracellular hCG. In the present study, we purified the 21 kDa form of alpha-subunit as well as the 23 and 19 kDa forms of beta-subunit from fresh normal first trimester placental tissues by gel filtration and reverse-phase high-performance liquid chromatography. Purified subunits were hydrolyzed (with a decrease in their molecular weighs) by endoglycosidase H and alpha-mannosidase but not by sialidase or sialidase followed by O-glycanase, indicating that those forms have presumably only high-mannose-type N-linked sugar chains but not O-linked sugar chains of the type present in mature beta-subunit. Fifteen cycles of Edman degradation of the purified forms of the subunits were performed. Only one phenylthiohydantoin amino acid, which was the same amino acid as in the urinary-beta-subunit, was detected at each step for the mixture of 23 and 19 kDa forms of beta-subunit, indicating that the protein backbones of both forms are identical to each other as well as to the urinary-beta-subunit. Thus, it is estimated that the 23 and 19 kDa forms bind two and one high-mannose sugar chains, respectively. The N-terminal sequence of the 21 kDa form of alpha-subunit was also the same as that of the urinary-alpha-subunit. The purified immature subunits were able to combine with each other. hCG, reconstituted with immature subunits, stimulated testosterone production in rat Leydig cells with an activity of about 10% that of urinary hCG. When hCG, reconstituted with immature subunits, was filtered by a column of YMC pack 200 diol, three peaks of hCG were obtained. The major peak was obtained at a similar elution position to urinary hCG but the other fractions were obtained at positions of much higher molecular weight, estimated as 100 and 200 kDa. These results indicate that immature hCG tends to aggregate and may exist as a homo-oligomer in the cells.