Localization of residues that confer antibody binding specificity using human chorionic gonadotropin/luteinizing hormone β subunit chimeras and mutants

The glycoprotein hormones are a family of conserved heterodimeric proteins which share a common α subunit but differ in their hormone-specific β subunits. We used chimeras of human chorionic gonadotropin (hCG) and luteinizing hormone (hLH) β subunits to identify residues which enable monoclonal antibodies (mAb) to distinguish the two hormones. The LHβ-CGβ chimeras appeared to fold similar to hCGβ, since they combined with hCGα and, depending on their sequences, were recognized by hCG-selective mAbs. Amino acid residues Arg⁸-Arg¹⁰, Gly⁴⁷-Ala⁵¹, and Gln⁸⁹-Leu⁹² form a major epitope region and appear to be adjacent to each other on the surface of hCGβ. Gly⁴⁷-Ala⁵¹ and Gln⁸⁹-Leu⁹² are recognized by dimer-specific mAbs while Arg⁸-Arg¹⁰ is recognized by mAbs which have highest affinity for the free β subunit. These observations suggest that the conformation of this region of the β subunit changes when the α and β subunits combine. Residues which are C-terminal of Asp¹¹² form a second epitope domain. mAbs to the third domain distinguish hCGβ and hLHβ by the presence of Asn⁷⁷ in HcGβ and can be detected after hCG binds to receptors. These findings were used to develop a model of hCGβ which predicts the locations of these residues and their positions relative to the α subunit and receptor interfaces.