title: Roles of aromatic residues in the structure and biological activity of the small cytokine, growth-blocking peptide (GBP).
abstract: Growth-blocking peptide (GBP) is a small (25 amino acids) insect cytokine with a variety of functions: controlling the larval development of lepidopteran insects, acting as a mitogen for various types of cultured cells, and stimulating insect blood cells. The aromatic residues of GBP (Phe-3, Tyr-11, and Phe-23) are highly conserved in the ENF peptide family found in lepidopteran insects. We investigated the relationship between the biological activities and structural properties of a series of GBP mutants, in which each of the three aromatic residues is replaced by a different residue. The results of the hemocytes-stimulating assays of GBP mutants indicated that Phe-3 is the key residue in this activity: Ala or Tyr replacement resulted in significant loss of the activity, but Leu replacement did not. The replacements of other aromatic residues hardly affected the activity. On the other hand, NMR analysis of the mutants suggested that Tyr-11 is a key residue for maintaining the core structure of GBP. Surprisingly, the Y11A mutant maintained its biological activity, although its native-like secondary structure was disordered. Detailed analyses of the (15)N-labeled Y11A mutant by heteronuclear NMR spectroscopy showed that the native-like beta-sheet structure of Y11A was induced by the addition of 2,2,2-trifluoroethanol. The results suggest that Y11A has a tendency to form a native-like structure, and this property may give the Y11A mutant native-like activity.
authors.affiliation: Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Japan.
authors.forename: Masahito
authors.initials: M
authors.lastname: Tada
authors.forename: Tomoyasu
authors.initials: T
authors.lastname: Aizawa
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Shinohara
authors.forename: Kimiaki
authors.initials: K
authors.lastname: Matsubara
authors.forename: Kazunori
authors.initials: K
authors.lastname: Miura
authors.forename: Masanobu
authors.initials: M
authors.lastname: Yoshida
authors.forename: Kunio
authors.initials: K
authors.lastname: Shitara
authors.forename: Takahide
authors.initials: T
authors.lastname: Kouno
authors.forename: Mineyuki
authors.initials: M
authors.lastname: Mizuguchi
authors.forename: Katsutoshi
authors.initials: K
authors.lastname: Nitta
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Hayakawa
authors.forename: Keiichi
authors.initials: K
authors.lastname: Kawano
mesh_terms: D000595:Amino Acid Sequence; D024322:Amino Acids, Aromatic / Q000737:chemistry*; D000818:Animals; D016207:Cytokines / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D019476:Insect Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D007814:Larva / Q000187:drug effects / Q000254:growth & development; D007915:Lepidoptera; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D017354:Point Mutation; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship
chemical_list: D024322:Amino Acids, Aromatic; D016207:Cytokines; D019476:Insect Proteins; C068690:growth blocking peptide, Apanteles kariyai