The molecular basis for epitopes on the free beta-subunit of human chorionic gonadotrophin (hCG), its carboxyl-terminal peptide and the hCG beta-core fragment.

Abstract

The molecular basis for antigenic determinants on the free beta-subunit of human chorionic gonadotrophin (hCG beta), its carboxyl-terminal peptide (hCG beta CTP) and the hCG beta-core fragment (hCG beta cf) was elucidated by means of monoclonal antibodies (MCAs). The objective of the present study was to resolve the antigenic topography of these three molecules in terms of epitope identification at different levels of structural organization as well as analysis of their spatial arrangement. An hCG beta cf preparation, a synthetic peptide corresponding to the hCG beta CTP (beta 109-145), overlapping synthetic peptides spanning the entire amino acid sequence of hCG beta, and a reduced and alkylated hCG beta preparation were assayed in a solid-phase one-site enzyme-linked immunoassay and in a soluble-phase direct-binding radioimmunoassay (RIA) or competitive RIA. The antigenic topography was mapped by incorporating the MCAs into two-site binding assays. On the surface of free hCG beta, nine different epitopes (beta 1-beta 9), arranged in three spatially distinct domains, could be distinguished. Epitopes beta 1-beta 7 were located in a single large domain on both hCG beta and the hCG beta cf whereas hCG beta CTP contained two topographically distant determinants, designated beta 8 and beta 9 respectively. All but the two epitopes located on hCG beta CTP (beta 8 and beta 9) were destroyed by reducing and alkylating hCG beta, suggesting that most antigenic determinants are predominantly non-contiguous and require an intact tertiary structure whereas the molecular structure of hCG beta CTP is linear. At a molecular level, amino acid residues spanning hCG beta 45-52, hCG beta 137-144 and hCG beta 113-116 contributed to the formation of epitopes beta 5, beta 8 and beta 9 respectively. We have also shown that the hCG beta cf represents the immunodominant part of the free beta-subunit of hCG, containing seven mainly conformationally determined epitopes, one of which has a share of the sequence beta 45-52. The hCG beta CTP does not play a critical role in the immunologically important tertiary structure of hCG beta and was itself found to be a predominantly continuous sequence also within the native hormone, expressing two spatially distant antigenic determinants located within residues beta 113-116 and beta 137-144 respectively.