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: 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.
Publication Date: 2004-06-12 PubMed ID: 15191359DOI: 10.1530/eje.0.1500877Google Scholar: Lookup
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- Journal Article
Summary
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The research identified specific amino acids within the alpha-subunit that play a crucial role in the variations of follicle-stimulating hormone (FSH) activity between horse (e) and donkey (dk) luteinizing hormone/choriogonadotropin (LH/CG), supporting the concept that even minute differences in hormone composition can lead to significant variations in biological activity.
Research Methodology
- The research investigated the FSH activity of eLH and dkLH. Notably, the FSH/LH ratio is ten times higher for eLH than for dkLH.
- The team created chimeric alpha-subunits containing the first 63 amino acids of the porcine (p) and the last 33 amino acids of the donkey alpha-subunit, and likewise the inverse.
- Porcine-specific amino acids were introduced by mutagenesis at set positions in donkey alpha-subunit.
- The different alpha-subunits were co-transfected in COS-7 cells with beta-eLH, beta-dkLH, and beta-eFSH.
- The bioactivity of the FSH and LH in the dimers were evaluated using two heterologous in vitro bioassays.
Research Findings
- The chimeric alpha-subunits showed FSH activity when co-expressed with beta-eLH while none was exhibited with beta-dkLH.
- The mutated hybrids displayed no FSH activity but high LH activity when expressed with beta-dkLH.
- A mutation at position 78 in alpha-dk did not affect FSH bioactivity when co-expressed with beta-eFSH.
Conclusions and Implications
- Amino acids in the first two-thirds and the last third of the equid LH alpha-subunits contribute significantly to their heterologous biospecificity. Ile alpha78 influences this to a large extent, equivalent to the beta102-103 residues; however, its effect is irrelevant to the FSH specificity of eFSH.
- The findings provide a valuable contribution to understanding the factors that govern the specific activity of these hormones and the fundamental biological differences between equid species.
Cite This Article
APA
Chopineau M, Martinat N, Gibrat JF, Galet C, Lecompte F, Foulon-Gauze F, Pourchet C, Guillou F, Combarnous Y.
(2004).
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.
Eur J Endocrinol, 150(6), 877-884.
https://doi.org/10.1530/eje.0.1500877 Publication
Researcher Affiliations
- Physiologie de la Reproduction et des Comportements (PRC), UMR INRA-CNRS 6073, 37380 Nouzilly, France. chopinea@tours.inra.fr
MeSH Terms
- Amino Acid Sequence
- Amino Acids / analysis
- Animals
- Biological Assay
- COS Cells
- Chlorocebus aethiops
- Chorionic Gonadotropin / chemistry
- Dimerization
- Equidae
- Follicle Stimulating Hormone / chemistry
- Follicle Stimulating Hormone / pharmacology
- Horses
- Luteinizing Hormone / chemistry
- Luteinizing Hormone / pharmacology
- Luteinizing Hormone, beta Subunit / chemistry
- Luteinizing Hormone, beta Subunit / genetics
- Models, Molecular
- Molecular Sequence Data
- Mutagenesis
- Mutagenesis, Site-Directed
- Recombinant Fusion Proteins / chemistry
- Recombinant Fusion Proteins / pharmacology
- Swine
- Testosterone / biosynthesis
- Transfection
Citations
This article has been cited 1 times.- Bhardwaj A, Nayan V, Sharma P, Kumar S, Pal Y, Singh J. Molecular characterization, modeling, in silico analysis of equine pituitary gonadotropin alpha subunit and docking interaction studies with ganirelix. In Silico Pharmacol 2016 Dec;5(1):5.
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