Hormone-Specific Inhibitory Influence of α-Subunit Asn56 Oligosaccharide on In Vitro Subunit Association and Follicle-Stimulating Hormone Receptor Binding of Equine Gonadotropins1

Abstract

Hybrid hormones were created using combinations of equine (e) LH, eFSH, and eCG alpha- and beta-subunit preparations. The efficiency of eFSH beta association was highest with eLH alpha (64-72%) and was lowest with eCG alpha (37-50%). Selective removal of alphaAsn56 oligosaccharide increased heterodimerization efficiency by 9-20% for eLH alpha, by 21-28% for eFSH alpha, and by 28-41% for eCG alpha. Both alpha and beta subunits contributed significantly to FSH receptor-binding activities of the hybrids. Purified hybrid hormone preparations consisting of either eFSH beta or eLH beta combined with eLH alpha, eFSH alpha, or eCG alpha were prepared. Equine FSH beta hybrids were more active in the FSH radioreceptor assay than eLH beta hybrids; within each beta-subunit group the eLH alpha hybrids were the most active, followed by eFSH alpha hybrids, while the least active were eCG alpha hybrids. A truncated, des(121-149) eLH beta derivative (eLH beta t) combined with native alpha-subunit preparations exhibited the same effect of alpha-subunit type on FSH receptor binding. Hybrids combining the eLH beta t derivative with Asn56-deglycosylated (N56dg-)eLH alpha, N56dg-eFSH alpha, and N56dg-eCG alpha preparations possessed 2.2- to 4.3-fold increased FSH receptor-binding activities as compared with the same hybrid preparations possessing the Asn56 carbohydrate. Granulosa cell bioassay of purified native eFSH beta and eLH beta hybrid hormones indicated no significant effect of the alpha-subunit carbohydrate differences on progesterone production. The alpha-subunit Asn56 oligosaccharide exerts a hormone-specific inhibitory influence on in vitro subunit reassociation and FSH receptor binding related to the size of its Man(alpha1-6)Man antenna.

Glycoprotein profiling by electrospray mass spectrometry

“…Oligosaccharides attached to the human ␣-subunit at Asn 78 are important for proper folding of the protein [14]. In addition, the type of oligosaccharides attached to a second glycosylation site (Asn 52 ) have been shown to influence receptor-binding affinity and are essential for full expression of biological activity at the cellular level [15]. (This site is numbered Asn 56 in ovine and other mammalian ␣-subunits.)…”

Truncated equine LH beta and asparagine(56)-deglycosylated equine LH alpha combine to produce a potent FSH antagonist

“…Following 24-h incubation at 37 C, the preparations were placed in a P-10 Centricon, the Tris-acetate buffer replaced with 0·126 M ammonium bicarbonate buffer by ultrafiltration, and protein recovered by lyophilization. The dried protein was dissolved in 200 µl 0·126 M NH 4 HCO 3 and purified by Superdex 75 column chromatography (Butnev et al 1998).…”

“…Testosterone released into the culture medium following 3-h incubation at 37 C was determined by radioimmunoassay. FSH biological activity was determined by incubating hormone samples in 16-mm wells of 24-well tissue culture plates containing 300 000 granulosa cells obtained from diethylstilbestrol (DES)-primed immature rat ovaries (Butnev et al 1998). The progesterone content of the medium was measured after 72 h and the amount of estradiol converted from testosterone present in the medium was determined after 96 h. All steroid RIAs employed antisera provided by Dr Gordon Niswender (Colorado State University, Fort Collins, CO, USA) (Korenman et al 1974).…”

“…The -subunit carbohydrate attached to Asn 56 is essential for in vitro signal transduction activity, modulates receptor-binding potency, and may contribute to heterodimer stability (Matzuk et al 1989, Bishop et al 1994, Flack et al 1994, Heikoop et al 1998. The Asn 82 oligosaccharide plays an important role in facilitating subunit folding (Strickland et al 1985, Thijssen-van Zuylen et al 1998, stabilizes the subunit (van Zuylen et al 1997) and, in some instances, modulates receptorbinding activity (Butnev et al 1998). The -subunit carbohydrates play a less important, but measurable role in signal transduction (Liu et al 1984, Matzuk et al 1989.…”

“…We have employed truncated, des(121-149)eLH (eLH t) preparations to prepare hybrid hormone preparations that enabled us to study the effects of hormonespecific changes in Asn 56 oligosaccharide structure on biological activity (Gotschall & Bousfield 1996, Butnev et al 1998. These derivatives were employed to solve two technical problems: elimination of non-dissociable eLH dimer and separation of hybrid heterodimers from nonassociated subunits .…”