A kinetic comparison of the processing and secretion of the alpha beta dimer and the uncombined alpha and beta subunits of chorionic gonadotropin synthesized by human choriocarcinoma cells

Peters, B.P.; Krzesicki, R.F.; Hartle, R.J.; Perini, F.; Ruddon, R.W.

Journal of Biological Chemistry 259(24): 15123-15130

1984


ISSN/ISBN: 0021-9258
PMID: 6210286
Accession: 004579504

Download citation:  
Text
  |  
BibTeX
  |  
RIS

Article/Abstract emailed within 1 workday
Payments are secure & encrypted
Powered by Stripe
Powered by PayPal

Abstract
Human choriocarcinoma cells (JAR) synthesize the .alpha. and .beta. subunits of the glycoprotein hormone chorionic gonadotropin (hCG). In addition to the hCG dimer (.alpha.beta.), JAR cells secrete uncombined .alpha. and .beta. subunits into the culture medium. Pulse-chase studies with [35S]methionine or [3H]mannose were carried out to compare free .alpha., free .beta. and the .alpha.beta. dimer with regard to the kinetics of synthesis, N-linked oligosaccharide processing, and secretion and to determine the kinetics of .alpha.-.beta. subunit combination. A panel of 3 antisera was used to immunoprecipitate directly the free subunits and the .alpha.beta. dimer sequentially from the same cell lysates and culture media. The .alpha. subunit of hCG was synthesized in a slight molar excess (1.2-1.5-fold) over the .beta. subunit, and .alpha.beta. dimer was rapidly formed by combination of the intracellular .alpha. and .beta. precursors. Dimer formation was already apparent in JAR cells following a 10-min biosynthetic labeling incubation with [35S]methionine. The combination of subunits ceased by 30 min of chase even though 51% of .alpha. and 44% of .beta. remained free within the cells. Combination of the .alpha. and .beta. precursors had occurred before their N-linked oligosaccharides were processed beyond the Man8GlcNAc2 structure. The initial trimming of glucosyl and mannosyl units from the high-mannose oligosaccharides of the hCG precursors occurred more rapidly for free .alpha. and CG-.alpha. than for free .beta. and CG-.beta. JAR cells accumulated .alpha. precursors bearing mostly Man8GlcNAc2 units and .beta. precursors bearing Man9GlcNAc2 units that represent the substrates of the rate-limiting step in the secretory pathway. In spite of the fact that their N-linked oligosaccharides were trimmed at different rates, free .alpha., free .beta. and .alpha.beta. dimer were all secreted into the medium at the same rate with a half-time of 35 min. The secreted hCG forms were stable in the chase medium between 4-8 h, indicating that extracellular degradation, combination of free subunits to form dimer, or dissociation of dimer to form free subunits did not occur. The mature secreted forms of free .alpha., GC-.beta. and free .beta. contained predominantly endo-.beta.-N-acetylglucosaminidase H (endo H)-resistant N-linked oligosaccharides. In contrast, CG-.alpha. contained a more complicated array of oligosaccharide forms distributed equally between endo H-resistant glycopeptides and endo H-sensitive hybrid oligosaccharides. The different oligosaccharide content of secreted free .alpha. and CG-.alpha. may contribute to their apparent size difference on sodium dodecyl sulfate-polyacrylamide gels.