DETERMINATION OF SUBUNIT CONTACT-ASSOCIATED EPITOPES OF THE BETA-SUBUNIT OF HUMAN FOLLICLE-STIMULATING-HORMONE

Three different experimental approaches were used to assess the regions on the beta-subunit of human FSH (hFSH-beta) that may be altered or masked by its association with the alpha-subunit of hFSH (hFSH-alpha) in the heterodimeric hFSH molecule. In a direct approach, we tested whether synthetic peptides corresponding to hFSH-beta sequences 1-20, 16-36, 33-53, 49-67, 66-85, 81-100, and 98-111 inhibited association of hFSH-alpha and hFSH-beta in an enzyme-linked immunosorbent assay. Synthetic peptides-(81-100), -(98-111), and -(66-85) caused greater than 50% inhibition of subunit association, whereas other peptides showed 26% or less inhibition. These data suggested that the C-terminal sequences of hFSH-beta, particualrly 81-100, are at a subunit interface with hFSH-alpha in heterodimeric hFSH. In another approach we reasoned that antibodies with a higher affinity for free hFSH-beta than for heterodimeric hFSH bind to epitopes on hFSH-beta that are masked or altered by hFSH-alpha subunit. To test this hypothesis, epitopes of hFSH-beta were mapped using synthetic peptides of hFSH-beta sequences, three monoclonal antibodies (3G3, 4D5, and 4G8), and a polyclonal antiserum (NIDDK anti-hFSH-beta). Compared to 3G3 all the other antibodies exhibited minimal reactivity with hFSH, but bound strongly to hFSH-beta. The epitope-mapping data with both 4D5 and NIDDK anti-hFSH-beta identified peptide 81-100, which was not recognized by 3G3. The epitope map with 4G8 identified the same three peptides as with 3G3. However, in the case of 4G8 its reactivity with peptide 33-53 was the least, whereas it was ranked first for 3G3. Since both 3G3 and 4G8 had an identical affinity for hFSH-beta, it was hypothesized that sequences in peptide 33-53 may be altered or masked by hFSH-alpha. To test this, we determined the specificity of anti-hFSH-beta-(33-53) peptide antiserum for hFSH-beta and hFSH in an enzyme-linked immunosorbent assay. The antipeptide antiserum bound strongly to free hFSH-beta and weakly to hFSH, suggesting that part of the sequence in peptide-(33-53) was masked or altered by association with hFSH-alpha in heterodimeric hFSH. Taken together, the subunit association studies, the epitope-mapping data, and the specificity of anti-hFSH-beta-(33-53) peptide antiserum have suggested that sequences in petpide-(81-100) and -(33-53) are masked or conformationally altered by hFSH-alpha in heterodimeric hFSH.