Effect of modification of the β-hairpin and long loops simultaneously in both α- and β-subunits on the function of human choriogonadotropin: part II

Shao, Kui; Bahl, Om P.

doi:10.1016/s0303-7207(97)04007-0

Cited by 8 publications

(3 citation statements)

References 31 publications

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“…It has been shown by peptide mapping, site-directed mutagenesis studies and epitope mapping of the hCG-receptor complex that residues at the αβ interface around glycosylation site αAsn-52 and the seat-belt residues are important for strong receptor binding [35][36][37][38]. The residues at the C-terminus of the αsubunit, comprising the residues 88-91, and αPhe-18 on the opposite face of the hCG molecule, are involved in cAMP production [39,40]. Furthermore, there is experimental evidence that upon binding to the receptor the conformation of hCG changes and that consequently the receptor is activated [39,[41][42][43].…”

Section: Figure 8 Enlarged X-ray Structure Of Hcg Around the Glycosylmentioning

confidence: 99%

Studies on the relevance of the glycan at Asn-52 of the α-subunit of human chorionic gonadotropin in the αβ dimer

Erbel

Haseley

Kamerling

et al. 2002

Biochemical Journal

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Glycosylation of Asn-52 of the alpha-subunit (alphaAsn-52) is required for bioactivity of the alphabeta-dimeric human chorionic gonadotropin (hCG), although at a molecular level the effect of the glycan at alphaAsn-52 is not yet understood. To study the role of this glycan for heterodimer stability, the beta-subunit was recombined in solution with either the alpha-subunit or the alpha-subunit enzymically deglycosylated at alphaAsn-52. Enzymic deglycosylation avoids modification of the glycans at alphaAsn-78 and disturbing the protein folding. The efficiency of recombination after 16 h is 80%, independent of whether alphaAsn-52 is glycosylated or not. The dissociation constant of the hCG complex, with or without the glycan at alphaAsn-52, is less than 1 x 10(-5) s(-1), indicating that the glycan at alphaAsn-52 does not contribute significantly to the stability of the dimer. CD and NMR spectra indicate a local conformational difference between both alphabeta-dimeric hCG variants, most probably involving amino acids of the hCG beta-subunit close to the glycan at alphaAsn-52. These data explain the native-like receptor-binding abilities of hCG lacking the glycan at alphaAsn-52. It is proposed that for bioactivity the glycan at alphaAsn-52 is necessary for inducing and stabilizing a conformational change in hCG upon binding to the receptor, resulting in activation of the signal-transduction pathway.

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Section: Figure 8 Enlarged X-ray Structure Of Hcg Around the Glycosylmentioning

confidence: 99%

Studies on the relevance of the glycan at Asn-52 of the α-subunit of human chorionic gonadotropin in the αβ dimer

Erbel

Haseley

Kamerling

et al. 2002

Biochemical Journal

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“…The contradictory view on the hCGα L1 of E12 epitope adds to this apparent dichotomy. On one hand, the hCG αL1 has been shown to be exposed in hCG–hLHR complex52 while on the other, it was shown to be involved in hormone binding,53 suggesting that mechanism of E12 inhibition might not be through binding to the receptor binding residues of the hormone. Superimposition of E12–hCG model on the hCG–LHR 51–266 model [Fig.…”

Section: Resultsmentioning

confidence: 99%

Docking and free energy simulations to predict conformational domains involved in hCG–LH receptor interactions using recombinant antibodies

Majumdar

Railkar

Dighe³

2011

Proteins

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Single chain fragment variables (ScFvs) have been extensively employed in studying the protein-protein interactions. ScFvs derived from phage display libraries have an additional advantage of being generated against a native antigen, circumventing loss of information on conformational epitopes. In the present study, an attempt has been made to elucidate human chorionic gonadotropin (hCG)-luteinizing hormone (LH) receptor interactions by using a neutral and two inhibitory ScFvs against hCG. The objective was to dock a computationally derived model of these ScFvs onto the crystal structure of hCG and understand the differential roles of the mapped epitopes in hCG-LH receptor interactions. An anti-hCG ScFv, whose epitope was mapped previously using biochemical tools, served as the positive control for assessing the quality of docking analysis. To evaluate the role of specific side chains at the hCG-ScFv interface, binding free energy as well as residue interaction energies of complexes in solution were calculated using molecular mechanics Poisson-Boltzmann/surface area method after performing the molecular dynamic simulations on the selected hCG-ScFv models and validated using biochemical and SPR analysis. The robustness of these calculations was demonstrated by comparing the theoretically determined binding energies with the experimentally obtained kinetic parameters for hCG-ScFv complexes. Superimposition of hCG-ScFv model onto a model of hCG complexed with the 51-266 residues of LH receptor revealed importance of the residues previously thought to be unimportant for hormone binding and response. This analysis provides an alternate tool for understanding the structure-function analysis of ligand-receptor interactions.

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“…3), and it points outside the molecule on its convex surface. It has been suggested that Phe 22 in human aL1 (the numbering of the human a-subunit has been adjusted to the numbering of all a-subunit species) may interact with the LH receptor, as replacement of this amino-acid by a hydrophilic residue (Thr) gave a hybrid hCG which was twice as active as hCG in the stimulation of progesterone (25,26). The authors hypothesize that this replacement probably results in weakening the interaction between the two subunits and that this short conformational change affects the receptor binding site probably located in close proximity to aPhe 22 .…”

Section: Discussionmentioning

confidence: 99%

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

Chopineau¹,

Martinat²,

Gibrat³

et al. 2004

European Journal of Endocrinology

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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.

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Effect of modification of the β-hairpin and long loops simultaneously in both α- and β-subunits on the function of human choriogonadotropin: part II

Cited by 8 publications

References 31 publications

Studies on the relevance of the glycan at Asn-52 of the α-subunit of human chorionic gonadotropin in the αβ dimer

Studies on the relevance of the glycan at Asn-52 of the α-subunit of human chorionic gonadotropin in the αβ dimer

Docking and free energy simulations to predict conformational domains involved in hCG–LH receptor interactions using recombinant antibodies

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

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