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Inhibin A and B in Vitro Bioactivities Are Modified by Their Degree of Glycosylation and Their Affinities to Betaglycan

Prince Henry’s Institute of Medical Research (Y.M., C.A.H., P.G.S., D.M.R.) and Department of Obstetrics and Gynaecology (Y.M.), Monash University, Clayton, Victoria 3168, Australia; and DSL-Beckman Coulter (R.K.), Webster, Texas 77598

Address all correspondence and requests for reprints to: David M. Robertson, Ph.D., Prince Henry’s Institute of Medical Research, PO Box 5152, Clayton, Victoria 3168, Australia. E-mail: david.robertson{at}princehenrys.org.

Inhibin A and B, important regulators of normal function in tissues of the reproductive axis, are glycosylated at either Asn²⁶⁸ or Asn²⁶⁸ and Asn³⁰² in the -subunit to produce 31- and 34-kDa isoforms, respectively. In this study, glycosylated isoforms of recombinant human inhibin A and B were purified from conditioned medium using immunoaffinity chromatography and reversed-phase HPLC. The masses of the purified inhibin preparations were determined by several inhibin immunoassays, and their in vitro bioactivities were based on suppression of FSH release by rat pituitary cells in culture. Based on a ratio of in vitro bioactivity to immunoactivity (B:I ratio), the monoglycosylated 31-kDa inhibin A was 5-fold more potent than the diglycosylated 34-kDa inhibin A (B:I ratio, 1.22 ± 0.15 vs. 0.24 ± 0.05; P < 0.001, respectively). The 31-kDa inhibin B was significantly (P < 0.001) more potent (1.75 ± 0.29) than the 34-kDa form (1.08 ± 0.20). Because inhibin biological activity is dependent upon interactions with the coreceptor betaglycan, the effect of inhibin glycosylation on betaglycan binding was assessed. Analogous to the pattern of in vitro bioactivity, 31-kDa inhibin A was 12-fold more active (IC₅₀, 0.68 nM) than the 34-kDa isoform (IC₅₀, 8.2 nM) at displacing [¹²⁵I]inhibin A from COS7 cells expressing betaglycan. However, the 1.6-fold difference in bioactivity of the inhibin B isoforms was not matched by differences in their affinities for betaglycan. It is concluded that glycosylation of Asn³⁰² of the -subunit of inhibin A and B results in a decrease in bioactivity, and the effect on inhibin A, at least, is explained by its reduced affinity to betaglycan.

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