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

Objective: To identify amino-acids in the α-subunit important for expression of heterospecific FSH activity of horse (e) LH/choriogonadotropin (CG) (eLH) and donkey (dk) LH/CG (dkLH) IFSH/LH ratio ten times higher for eLH than for dkLH); this FSH activity absolutely requires an equid (donkey or horse) α-subunit combined with an equid β-LH subunit. Design: Chimeric α-subunits possessing the first 63 amino-acids of the porcine (p) and the last 33 amino-acids of the donkey α-subunit (ap-dk) and the inverse (αdk-p) were constructed. Porcine-specific amino-acids were introduced by mutagenesis in donkey α-subunit at positions 70, 85, 89, 93 and 96 (adk5xmut). 18 (αdk K18E ) or 78 (αdk 178A ). Methods: These different α-subunits were co-transfected in COS-7 cells with β-eLH, β-dkLH and β-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 β-eLH but not with β-dkLH. αdk K18E or αdk 17A gave hybrids with no FSH activity and important LH activity when expressed with β-dkLH. αdk 178A /βeLH displayed an FSH/LH ratio as low as that of dkLH. However. mutation at 78 in α-dk had no effect on FSH bioactivity when co-expressed with β-eFSH. Conclusions: Amino-acids present in both the first two-thirds and the last third of the α-subunit of equid LHs are involved in their heterologous biospecificity. He α78 exerts as strong an influence on it as the β102-103 residues. By contrast, this residue plays no role in the FSH specificitu of eFSH.