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 alpha-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) alpha-subunit combined with an equid beta-LH subunit.


METHODS
Chimeric alpha-subunits possessing the first 63 amino-acids of the porcine (p) and the last 33 amino-acids of the donkey alpha-subunit (alphap-dk) and the inverse (alphadk-p) were constructed. Porcine-specific amino-acids were introduced by mutagenesis in donkey alpha-subunit at positions 70, 85, 89, 93 and 96 (alphadk5xmut), 18 (alphadkK18E) or 78 (alphadkI78A).


METHODS
These different alpha-subunits were co-transfected in COS-7 cells with beta-eLH, beta-dkLH and beta-eFSH. The LH and FSH bioactivities of the dimers were then assessed in two heterologous in vitro bioassays.


RESULTS
alphap-dk or alphadk-p exhibited FSH activity when co-expressed with beta-eLH but not with beta-dkLH. alphadkK18E or alphadkI78A gave hybrids with no FSH activity and important LH activity when expressed with beta-dkLH. alphadkI78A/betaeLH displayed an FSH/LH ratio as low as that of dkLH. However, mutation at 78 in alpha-dk had no effect on FSH bioactivity when co-expressed with beta-eFSH.


CONCLUSIONS
Amino-acids present in both the first two-thirds and the last third of the alpha-subunit of equid LHs are involved in their heterologous biospecificity. Ile alpha78 exerts as strong an influence on it as the beta102-103 residues. By contrast, this residue plays no role in the FSH specificity of eFSH.
Chopineau
M
M
Physiologie de la Reproduction et des Comportements (PRC), UMR INRA-CNRS 6073, 37380 Nouzilly, France. chopinea@tours.inra.fr
Martinat
N
N
Gibrat
J F
JF
Galet
C
C
Lecompte
F
F
Foulon-Gauze
F
F
Pourchet
C
C
Guillou
F
F
Combarnous
Y
Y
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D000818:Animals; D001681:Biological Assay; D019556:COS Cells; D002522:Chlorocebus aethiops; D006063:Chorionic Gonadotropin / Q000737:chemistry*; D019281:Dimerization; D018397:Equidae; D005640:Follicle Stimulating Hormone / Q000737:chemistry* / Q000494:pharmacology; D006736:Horses; D007986:Luteinizing Hormone / Q000737:chemistry* / Q000494:pharmacology; D037101:Luteinizing Hormone, beta Subunit / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000494:pharmacology; D013552:Swine; D013739:Testosterone / Q000096:biosynthesis; D014162:Transfection
D000596:Amino Acids; D006063:Chorionic Gonadotropin; D037101:Luteinizing Hormone, beta Subunit; D011993:Recombinant Fusion Proteins; D013739:Testosterone; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone