Engineering the Ovarian Hormones Inhibin A and Inhibin B to Enhance Synthesis and Activity

Abstract

Ovarian-derived inhibin A and inhibin B (heterodimers of common α- and differing β-subunits) are secreted throughout the menstrual cycle in a discordant pattern, with smaller follicles producing inhibin B, whereas the dominant follicle and corpus luteum produce inhibin A. The classical function for endocrine inhibins is to block signalling by activins (homodimers of β-subunits) in gonadotrope cells of the anterior pituitary and, thereby, inhibit the synthesis of FSH. Whether inhibin A and inhibin B have additional physiological functions is unknown, primarily because producing sufficient quantities of purified inhibins, in the absence of contaminating activins, for preclinical studies has proven extremely difficult. Here, we describe novel methodology to enhance inhibin A and inhibin B activity and to produce these ligands free of contaminating activins. Using computational modeling and targeted mutagenesis, we identified a point mutation in the activin β A-subunit, A347H, which completely disrupted activin dimerization and activity. Importantly, this β A-subunit mutation had minimal effect on inhibin A bioactivity. Mutation of the corresponding residue in the inhibin β B-subunit, G329E, similarly disrupted activin B synthesis/activity without affecting inhibin B production. Subsequently, we enhanced inhibin A potency by modifying the binding site for its co-receptor, betaglycan. Introducing a point mutation into the α-subunit (S344I) increased inhibin A potency ~12-fold. This study has identified a means to eliminate activin A/B interference during inhibin A/B production, and has facilitated the generation of potent inhibin A and inhibin B agonists for physiological exploration.