Inhibin A and B in vitro bioactivities are modified by their degree of glycosylation and their affinities to betaglycan.
Inhibin A and B, important regulators of normal function in tissues of the reproductive axis, are glycosylated at either Asn(268) or Asn(268) and Asn(302) in the alpha-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 (IC(50), 0.68 nM) than the 34-kDa isoform (IC(50), 8.2 nM) at displacing [(125)I]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 Asn(302) of the alpha-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.
Makanji
Yogeshwar
Y
Prince Henry's Institute of Medical Research, Monash University, PO Box 5152, Clayton, Victoria 3168, Australia.
Harrison
Craig A
CA
Stanton
Peter G
PG
Krishna
Radha
R
Robertson
David M
DM
D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D005640:Follicle Stimulating Hormone / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D007265:Inhibins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007536:Isomerism; D010902:Pituitary Gland / Q000166:cytology; D011485:Protein Binding; D011509:Proteoglycans / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D011509:Proteoglycans; D018125:Receptors, Transforming Growth Factor beta; D011994:Recombinant Proteins; C422362:inhibin A; C422364:inhibin B; C071736:betaglycan; D007265:Inhibins; D005640:Follicle Stimulating Hormone