Purification and characterization of high molecular weight hCG from human first trimester placenta.

Abstract

Human chorionic gonadotropin (hCG) in first trimester placental cells is composed of immature alpha- and beta-subunits containing only N-linked high-mannose sugar chains. Intracellular immature intermediates are accumulated in rough endoplasmic reticulum in much greater quantity than mature hCG composed of mature subunits. We have previously shown that this immature hCG might be bound to other protein(s), including an ATP-binding protein, forming high molecular weight-hCG (HMW-hCG), which is not aggregate of immature hCG alone. To identify the ATP-binding protein forming the HMW-hCG in detail, proteins in HMW-hCG preparation were photoaffinity-labeled with 8-azido-[alpha-32P]ATP. Autoradiography followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the labeled protein with M(r) = 78000 was immunoprecipitated with any antibody against alpha-subunit, beta-subunit and hCG, indicating that this protein is bound to immature hCG. Furthermore, to determine whether some other proteins associate to form HMW-hCG, we purified HMW-hCG without breakdown to its components using columns of DE52, Heparin-Sepharose and Sephacryl S-300. As the final step of the purification, HMW-hCG was allowed to adsorb on a column of ATP-agarose and anti-hCG IgG-agarose, respectively. SDS-PAGE analysis of eluted proteins from the columns bound to the respective column via the constituent of HMW-hCG, such as ATP-binding protein or immature hCG, showed four common protein bands with molecular weights of 78000, 43000, 28000 and 20000. The protein with M(r) = 43000 was stained with any antibody against alpha-subunit, beta-subunit and hCG, indicating it to be immature hCG. The protein band with M(r) = 78000, which might correspond to the ATP-binding protein described above, was stained with anti-heat shock protein 70 (HSP70) monoclonal antibody. To confirm the association of immature hCG and HSP70-like protein, immature hCG preparation was incubated with HSP70-like protein purified from placental extracts. The molecular weight change of immature hCG appeared to increase by this incubation and was close to HMW-hCG, but not exactly the same. These results suggest that immature hCG intermediate exists as HMW-hCG containing HSP70-like protein, which has ATP-binding capacity, and two other proteins in first trimester placental cells.