Unique recognition of activin and inhibin by polyclonal antibodies to inhibin subunits.

Inhibin-A is a glycoprotein composed of an alpha subunit containing a glycosylation site and a beta A subunit, whereas activin-A is a homodimer of two inhibin beta A subunits. We examined the recognition of activin-A and inhibin-A by several antisera to the alpha or beta A subunit, and factors affecting the recognition. A total of six polyclonal antibodies to inhibin subunits, i.e., two antisera to a peptide fragment of the alpha subunit [alpha (1-19) and alpha (1-26)], and four antisera to the beta A subunit [beta A (1-10), beta A (70-79), beta A (87-99), and beta A (94-105)], was generated. On Western blot analysis, the anti-beta A (87-99) and beta A (94-105) sera recognized recombinant human activin-A but not inhibin-A under non-reducing conditions. When inhibin-A was deglycosylated with N-glycosidase-F, inhibin-A could be recognized by the anti-beta A (87-99) and beta A (94-105) sera. In addition, when activin-A bound to a nitrocellulose membrane was pre-incubated with recombinant human follistatin, the recognition of activin-A by the anti- beta A (87-99) and beta A (94-105) sera was decreased. These results suggested that the lower affinity of follistatin to inhibin-A than to activin-A might be likely explained as reflecting a site associated with the glycosylation of inhibin-A. However, the exposure of amino acids 87-105 of the inhibin beta A subunit on the molecular surface through deglycosylation did not increase the affinity of inhibin-A for follistatin but rather resulted in poor binding with follistatin. The present data suggest that (1) amino acids 87-105 of the inhibin/activin beta A subunit are located on the molecular surface, although this region of inhibin-A is concealed by the carbohydrate chain of the alpha subunit, (2) the region responsible for follistatin binding within the activin beta A subunit is spanned by amino acids 87-105, and (3) the mode of binding of inhibin-A to follistatin is quite different from that of activin-A to follistatin, and the former may be influenced by glycosylation.