Identification of the hydrophobic strand in the A–B loop of leptin as major binding site III: implications for large-scale preparation of potent recombinant human and ovine leptin antagonists
| Autor: | Krishnan V. Ramanujan, Eugene E. Gussakovsky, Dana Gonen-Berger, Jean Djiane, Eva Biener, Leonora Niv-Spector, Yackir Benomar, Arieh Gertler, Brian Herman, Isabelle Gourdou, Mohammed Taouis, Isabelle Callebaut |
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| Přispěvatelé: | Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University, The Hebrew University of Jerusalem (HUJ), Institut National de la Recherche Agronomique, Neuroendocrinologie Moleculaire de la Prise Alimentaire, Institut National de la Recherche Agronomique (INRA), Department of Life Sciences, Bar Ilan University, Bar-Ilan University [Israël], Institute of Horticulture, The Volcani Center, Agricultural Research Organization, Agricultural Research Organization, Laboratoire d'Endocrinologie, University of Paris XI, Université Paris-Sud - Paris 11 (UP11), Department of Cellular and Structural Biology, University of Texas Health Science Center, The University of Texas Health Science Center at Houston (UTHealth), Institut de minéralogie et de physique des milieux condensés (IMPMC), Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Diderot - Paris 7 (UPD7)-Institut de Physique du Globe de Paris (IPG Paris)-Centre National de la Recherche Scientifique (CNRS), Neurobiologie de l'Olfaction et de la Prise Alimentaire (NOPA), Neurobiologie de l'apprentissage, de la mémoire et de la communication (NAMC), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Institut de minéralogie, de physique des matériaux et de cosmochimie (IMPMC), Muséum national d'Histoire naturelle (MNHN)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de recherche pour le développement [IRD] : UR206-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-IPG PARIS-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS) |
| Jazyk: | angličtina |
| Rok vydání: | 2005 |
| Předmět: |
Leptin
Protein Conformation Molecular Sequence Data Receptors Cell Surface Biochemistry hydrophobic strand 03 medical and health sciences Mice Cell Line Tumor Animals Humans [SDV.BBM]Life Sciences [q-bio]/Biochemistry Molecular Biology Amino Acid Sequence Binding site Site-directed mutagenesis Receptor Molecular Biology 030304 developmental biology Alanine chemistry.chemical_classification A–B loop of leptin antagonists 0303 health sciences Leptin receptor Binding Sites Sheep Chemistry 030302 biochemistry & molecular biology digestive oral and skin physiology Cell Biology Ligand (biochemistry) Recombinant Proteins Amino acid Gene Expression Regulation Mutation fluorescence resonance energy transfer (FRET) Receptors Leptin immunoglobulin-like domain (IGD) site-directed mutagenesis Hydrophobic and Hydrophilic Interactions Research Article Protein Binding |
| Zdroj: | Biochemical Journal Biochemical Journal, 2005, 391, pp.221-230. ⟨10.1042/BJ20050457⟩ Biochemical Journal, 2005, 391 (2), pp.221-230. ⟨10.1042/BJ20050457⟩ Biochemical Journal, Portland Press, 2005, 391, pp.221-230. ⟨10.1042/BJ20050457⟩ Biochemical Journal, Portland Press, 2005, 391 (2), pp.221-230. ⟨10.1042/BJ20050457⟩ |
| ISSN: | 0264-6021 1470-8728 |
| DOI: | 10.1042/BJ20050457⟩ |
| Popis: | International audience; Interaction of leptin with its receptors resembles that of interleukin-6 and granulocyte colony-stimulating factor, which interact with their receptors through binding sites I–III. Site III plays a pivotal role in receptors' dimerization or tetramerization and subsequent activation. Leptin's site III also mediates the formation of an active multimeric complex through its interaction with the IGD (immunoglobulin-like domain) of LEPRs (leptin receptors). Using a sensitive hydrophobic cluster analysis of leptin's and LEPR's sequences, we identified hydrophobic stretches in leptin's A–B loop (amino acids 39–42) and in the N-terminal end of LEPR's IGD (amino acids 325–328) that are predicted to participate in site III and to interact with each other in a β-sheet-like configuration. To verify this hypothesis, we prepared and purified to homogeneity (as verified by SDS/PAGE, gel filtration and reverse-phase chromatography) several alanine muteins of amino acids 39–42 in human and ovine leptins. CD analyses revealed that those mutations hardly affect the secondary structure. All muteins acted as true antagonists, i.e. they bound LEPR with an affinity similar to the wild-type hormone, had no agonistic activity and specifically inhibited leptin action in several leptin-responsive in vitro bioassays. Alanine mutagenesis of LEPR's IGD (amino acids 325–328) drastically reduced its biological but not binding activity, indicating the importance of this region for interaction with leptin's site III. FRET (fluorescence resonance energy transfer) microscopy experiments have documented that the transient FRET signalling occurring upon exposure to leptin results not from binding of the ligand, but from ligand-induced oligomerization of LEPRs mediated by leptin's site III. |
| Databáze: | OpenAIRE |
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