Topographic Analysis of the α-Subunit of Human Follicle-Stimulating Hormone using Site-Specific Antipeptide Antisera*

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Abstract
Structure-function investigations were undertaken to increase understanding of the surface topology of the .alpha.-subunit of human FSH (hFSH). The objectives were to determine which sequences of the .alpha.-subunit of hFSH are surface-oriented (exposed to antibody) and to identify which of these surface-oriented sequences are in contact with the .beta.-subunit of hFSH in the .alpha./.beta. heterodimer. Seven overlapping synthetic peptides spanning the primary structure of hFSH.alpha. were used for immunizing rabbits to generate site-specific antipeptide antisera. The antisera were characterized with respect to their reactivity to the seven synthetic peptides, as well as hFSH, hFSH.alpha., hFSH.beta., and hFSH.alpha.r/a (reduced and alkylated), using an enzyme-linked immunosorbent assay. All of the peptides successfully generated antipeptide antisera with titers that ranged from 1:1,600 to 1:80,000. Anti 1-15 bound exclusively to hFSH.alpha.. Anti 11-27 and anti-33-58 bound to hFSH.alpha. to a much greater extent that to hFSH. In contrast, anti-73-92 had only slightly higher binding to hFSH.alpha. than to hFSH. Anti-22-39, anti-51-65, and anti-61-78 all failed to bind to either hFSH or hFSH.alpha.. With the exception of anti-22-39, all of the remaining antisera bound to hFSH.alpha.r/a. None of the antisera bound to hFSH.beta.. These data strongly suggest the following. Sequences 1-15, 11-27, and 33-58 contain residues that are masked by hFSH.beta. and are thus in or near the .alpha./.beta.-subunit interface. In addition, sequences 11-27 and 33-58 contain other distinct residues that are surface-oriented in the hFSH heterodimer. In contrast, sequence 73-92 appears to be surface-oriented in the hFSH heterodimer. Lastly, sequences 51-65 and 51-78 appear to be buried with the native .alpha.-subunit and, thus, are unable to interact with antibodies. These results agree with and extent previous findings and will prove useful to those currently investigating the surface topology and structure-function relationships of the glycoprotein hormones.