TOPOGRAPHIC ANALYSIS OF THE ALPHA-SUBUNIT OF HUMAN FOLLICLE-STIMULATING-HORMONE USING SITE-SPECIFIC ANTIPEPTIDE ANTISERA

Structure-function investigations were undertaken to increase understanding of the surface topology of the cv-subunit of human FSH (hFSH). The objectives were to determine which sequences of the a-subunit of hFSH are surfaceoriented (exposed to antibody) and to identify which of these surface-oriented sequences are in contact with the β-subunit of hFSH in the α/β heterodimer. Seven overlapping synthetic peptides spanning the primary structure of hFSHα 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, hFSHα, hFSHβ, and hFSHα β(reduced and alkylated), using an enzyme-linked immunosorbent assay. All of the peptides successfully generated antipeptide antisera with titers that range from 1:1, 600 to 1:80, 000. Anti-1-15 bound exclusively to hFSHα. Anti-11-27 and anti-33-58 bound to hFSHα to a much greater extent than to hFSH. In contrast, anti-73-92 had only slightly higher binding to hFSHα than to hFSH. Anti-22-39, anti-51-65, and anti-61-78 all failed to bind to either hFSH or hFSHα. With the exception of anti-22-39, all of the remaining antisera bound to hFSHα β. None of the antisera bound to hFSHβ. These data strongly suggest the following. Sequences 1-15, 11-27, and 33-58 contain residues that are masked by hFSHβ and are thus in or near the α/β-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 61-78 appear to be buried within the native a-subunit and, thus, are unable to interact with antibodies. These results agree with and extend previous findings and will prove useful to those currently investigating the surface topology and structure-function relationships of the glycoprotein hormones. © 1990 by The Endocrine Society.