Abstract
Glycosylation of Asn-52 of the alpha-subunit (alphaAsn-52) is required for bioactivity of the alphabeta-dimeric human chorionic gonadotropin (hCG), although at a molecular level the effect of the glycan at alphaAsn-52 is not yet understood. To study the role of this glycan for heterodimer stability, the beta-subunit was recombined in solution with either the alpha-subunit or the alpha-subunit enzymically deglycosylated at alphaAsn-52. Enzymic deglycosylation avoids modification of the glycans at alphaAsn-78 and disturbing the protein folding. The efficiency of recombination after 16 h is 80%, independent of whether alphaAsn-52 is glycosylated or not. The dissociation constant of the hCG complex, with or without the glycan at alphaAsn-52, is less than 1 x 10(-5) s(-1), indicating that the glycan at alphaAsn-52 does not contribute significantly to the stability of the dimer. CD and NMR spectra indicate a local conformational difference between both alphabeta-dimeric hCG variants, most probably involving amino acids of the hCG beta-subunit close to the glycan at alphaAsn-52. These data explain the native-like receptor-binding abilities of hCG lacking the glycan at alphaAsn-52. It is proposed that for bioactivity the glycan at alphaAsn-52 is necessary for inducing and stabilizing a conformational change in hCG upon binding to the receptor, resulting in activation of the signal-transduction pathway.