FREE ALPHA-SUBUNIT OF HUMAN CHORIONIC-GONADOTROPIN - MOLECULAR-BASIS OF IMMUNOLOGICALLY AND BIOLOGICALLY-ACTIVE DOMAINS

Immunochemical studies were undertaken to identify surface-orientated epitopes of the free alpha subunit of human chorionic gonadotrophin (h CG-alpha) at the amino acid sequence level. We investigated the molecu lar organization of these epitopes, resolved the immunological topogra phy in terms of spatial arrangement of antigenic domains and related s tructures to functions such as subunit association or receptor binding . Overlapping synthetic peptides covering the entire amino acid sequen ce of hCG-alpha, an enzymatically digested hCG-alpha subunit, and a re duced and alkylated hCG-alpha preparation were assayed in a solid-phas e one-site enzyme-linked immunoassay, and in a solution-phase competit ive radioimmunoassay (RIA). The antigenic topography was mapped by mon oclonal antibodies (MCAs) in two-site binding assays (sandwich RIA). O n the surface of hCG-alpha, seven different epitopes (alpha(1)-alpha(7 )), arranged in four spatially distinct domains, could be distinguishe d: A, alpha(1,2,4); B, alpha(3,5); C, alpha(6); D, alpha(7). The pepti des spanning hCG-alpha(13-18), hCG-alpha(17-22) and hCG-alpha(33-42) a ppeared to contribute to the formation of epitopes alpha(2), alpha(4) and alpha(6) respectively. Since epitope alpha(6) is present only on t he free non-assembled subunit of different species, we concluded that the region hCG-alpha(33-42), which is evolutionarily highly conserved, represents a subunit assembly site. All but one epitope (alpha(7)) ar e destroyed by reducing and alkylating hCG-alpha. In contrast, chymotr yptic digestion of hCG-alpha, leading to release of the heptapeptide h CG-alpha(41-47), did not affect epitope expression, indicating that th is sequence is not involved in the formation of antigenic determinants . Addressing the biological properties of hCG-alpha epitopes by radior eceptor assay revealed that the three hCG-alpha peptides corresponding to epitopes alpha(2), alpha(4) and alpha(6) did not displace radiolab elled hCG from its receptor, whereas any of the MCAs directed against determinants (alpha(1)-alpha(5)), shared by hCG and hCG-alpha, totally inhibited binding. Consistent with this, the antibodies neutralized t he biological activity of hCG in terms of testosterone production in a mouse Leydig cell in vitro bioassay. We therefore concluded that horm one antibody-binding sites differ from those of hormone receptor bindi ng, revealing no essential congruence of immunologically and biologica lly active domains.