Identification of amino-acids in the alpha-subunit first and third loops that are crucial for the heterospecific follicle-stimulating hormone activity of equid luteinizing hormone/choriogonadotropin

Abstract

Identification of amino-acids in the a-subunit first and third loops that are crucial for the heterospecific follicle-stimulating hormone activity of equid luteinizing hormone/choriogonadotropin M Chopineau, N Martinat, J F Gibrat 1 , C Galet, F Lecompte, F Foulon-Gauze, C Pourchet, F Guillou and Y Combarnous AbstractObjective: To identify amino-acids in the a-subunit important for expression of heterospecific FSH activity of horse (e) LH/choriogonadotropin (CG) (eLH) and donkey (dk) LH/CG (dkLH) (FSH/LH ratio ten times higher for eLH than for dkLH); this FSH activity absolutely requires an equid (donkey or horse) a-subunit combined with an equid b-LH subunit. Design: Chimeric a-subunits possessing the first 63 amino-acids of the porcine (p) and the last 33 amino-acids of the donkey a-subunit (ap-dk) and the inverse (adk-p) were constructed. Porcinespecific amino-acids were introduced by mutagenesis in donkey a-subunit at positions 70, 85, 89, 93 and 96 (adk5xmut), 18 (adk K18E ) or 78 (adk I78A ). Methods: These different a-subunits were co-transfected in COS-7 cells with b-eLH, b-dkLH and b-eFSH. The LH and FSH bioactivities of the dimers were then assessed in two heterologous in vitro bioassays. Results: ap-dk or adk-p exhibited FSH activity when co-expressed with b-eLH but not with b-dkLH. adk K18E or adk I78A gave hybrids with no FSH activity and important LH activity when expressed with b-dkLH. adk I78A /beLH displayed an FSH/LH ratio as low as that of dkLH. However, mutation at 78 in a-dk had no effect on FSH bioactivity when co-expressed with b-eFSH. Conclusions: Amino-acids present in both the first two-thirds and the last third of the a-subunit of equid LHs are involved in their heterologous biospecificity. Ile a78 exerts as strong an influence on it as the b102-103 residues. By contrast, this residue plays no role in the FSH specificity of eFSH.

Experimental and computational study of inter- and intra- species specificity of gonadotropins for various gonadotropin receptors

“…rat FSHR and LHR, as summarized in the SSFA-GPHR and GRIS servers (Kleinau et al, 2007;Van Durme et al, 2006), combinations of a and b subunits for horse and donkey CG and FSH (Chopineau et al, 2004), duck FSH (Shen et al, 2006), horse TSHR (Royer et al, 2008) and the LH and FSH b subunits of snakehead fish (Chatterjee et al, 2005).…”

Molecular characterization, modeling, in silico analysis of equine pituitary gonadotropin alpha subunit and docking interaction studies with ganirelix

“…Many workers across the globe reported the studies on alpha-subunit of pituitary glycoprotein hormones. Chopineau et al (2004), reported the identification of aminoacids in the alpha-subunit first and third loops, that are crucial for the hetero-specific follicle-stimulating hormone activity of equid luteinizing hormone/gonadotropin. It was precisely shown that which amino-acids of the alpha-subunit are implicated in the interaction with gonadotropin receptors.…”

Transcription of equine chorionic gonadotropin/luteinizing hormone b subunit gene in the endometrium of non-pregnant mares

“…Although many studies, including the presented one, focus on the b subunit of equine gonadotropic hormones, the a subunit has been shown to play a crucial role for the biological activity of the dimeric molecule (Papkoff 1974, Chopineau et al 1997, 2004. Wolkersdörfer et al (1998) suggested a circulating pool of free a subunits in women, being systemically available due to an excessive secretion into the blood stream by the pituitary gland (Hagen et al 1976).…”