Selective modification of recombinant bovine placental lactogen by site-directed mutagenesis at its C terminus
| Autor: | Gwen G. Krivi, Edna Sakal, N.R. Staten, Arieh Gertler, Jean Djiane, Russell E. McKinnie, Dorit Vashdi-Elberg |
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| Přispěvatelé: | The Hebrew University of Jerusalem (HUJ), Monsanto Company, Unité de biologie cellulaire et moléculaire, Institut National de la Recherche Agronomique (INRA) |
| Jazyk: | angličtina |
| Rok vydání: | 1996 |
| Předmět: |
Circular dichroism
bovin Lymphoma Protein Conformation récepteur hormonal Biochemistry law.invention 0302 clinical medicine law Tumor Cells Cultured Cloning Molecular Placental lactogen Receptor Site-directed mutagenesis ComputingMilieux_MISCELLANEOUS hormone lactogène placentaire 0303 health sciences Chemistry Circular Dichroism Biological activity Recombinant Proteins [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry Molecular Biology/Biomolecules [q-bio.BM] mutagénèse dirigée essai biologique Microsomes Liver Recombinant DNA Rabbits hormone recombinante Receptors Prolactin Molecular Sequence Data 030209 endocrinology & metabolism liaison Cell Line 03 medical and health sciences analogue Escherichia coli Animals Humans Point Mutation Amino Acid Sequence Molecular Biology 030304 developmental biology Sequence Homology Amino Acid Point mutation C-terminus Receptors Somatotropin Cell Biology Placental Lactogen Molecular biology chromatographie d'exclusion diffusion Rats Kinetics Growth Hormone Mutagenesis Site-Directed Cattle |
| Zdroj: | Journal of Biological Chemistry Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 1996, 271 (10), pp.5558-5564. ⟨10.1074/jbc.271.10.5558⟩ Journal of Biological Chemistry 10 (271), 5558-5564. (1996) |
| ISSN: | 0021-9258 1083-351X |
| DOI: | 10.1074/jbc.271.10.5558⟩ |
| Popis: | Five recombinant analogues of bovine placental lactogen (bPL) ((bPL(S184H), bPL(S187A), bPL(S187F), bPL(T188F), bPL(T188F,I190F)) were prepared, expressed in Escherichia coli, and purified to homogeneity. Circular dichroism analysis revealed no or minor structural changes, except in bPL(T188F,I190F). Binding and biological activities of bPL(T188F,I190F) were almost completely abolished, whereas bPL analogues mutated at position 187 retained their full activity. Point mutation T188F resulted in selective modification; binding to somatogenic receptors, their extracellular domains (ECDs), and to bPLR in the endometrium as well as somatogenic receptor-mediated biological activities were reduced or abolished, whereas binding to lactogenic receptors, their ECDs, and subsequent biological activity was fully or almost fully retained. This selective modification most likely results from a steric hindrance induced by a bulky Phe-188 chain of bPL which interacts with the Arg-43 of the human or Leu-43 of the non-human GHRs. Point mutation S184H abolished the interaction with hGHR, most likely due to the unfavorable charge-charge interaction, possibly accompanied by steric hindrance between Arg-43 of the receptor and the newly introduced His-184 and possible interference with the putative interaction between the alkyl portion of Thr-188 and Lys-185 of bPL with Trp-104 of hGHR. In contrast, bPL(S184H) retained its capacity to interact with nonhuman GHRs. Decrease in the biological activity of bPL(S184H) was also observed in two lactogenic receptor-mediated bioassays most likely due to the elimination of the intermolecular hydrogen bond of Ser-184 with a side chain of Tyr-127, which appears in all lactogenic receptors. |
| Databáze: | OpenAIRE |
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