N-terminal mutational analysis of the interaction between growth-blocking peptide (GBP) and receptor of insect immune cells.
GBP, a small insect cytokine isolated from lepidopterans, has a variety of functions. We constructed a series of mutants focusing on the unstructured N-terminal residues of GBP by acetylation, deletion, and elongation in order to investigate the interaction between GBP and its receptor in plasmatocytes. The 1H NMR spectra showed no significant changes in the tertiary structures of these peptides, which indicated that all the mutants maintained their core beta-sheet structures. The deletion and acetylated mutants, 2-25GBP, Ac2-25GBP, and AcGBP, lost their activity. 2-25GBP was the strongest antagonist, while Ac2-25GBP and AcGBP were moderate. In contrast, the elongated mutants, (-1R)GBP, (-1A)GBP, and (-2G,-1R)GBP maintained their plasmatocyte-spreading activity. These results demonstrate the importance of the GBP N-terminal charged amine and length of N-terminal GBP-peptide backbone for plasmatocyte-spreading activity. Next, we analyzed other mutant peptides, 1-25(N2A)GBP and 2-25(N2A)GBP, focusing on Asn2. Surprisingly, 2-25(N2A)GBP had slight plasmatocyte-spreading activity, whereas 2-25GBP lost its activity. Finally, substituted mutant, F3AGBP, had neither plasmatocyte-spreading activity nor antagonistic activity. These results demonstrate the function of each N-terminal residue in the interaction between GBP and its receptor in plasmatocytes.
Watanabe
Satoshi
S
Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama 930-0194, Japan.
Tada
Masahito
M
Aizawa
Tomoyasu
T
Yoshida
Masanobu
M
Sugaya
Tadamasa
T
Taguchi
Makoto
M
Kouno
Takahide
T
Nakamura
Takashi
T
Mizuguchi
Mineyuki
M
Demura
Makoto
M
Hayakawa
Yoichi
Y
Kawano
Keiichi
K
D000595:Amino Acid Sequence; D000818:Animals; D016207:Cytokines / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006434:Hemocytes / Q000166:cytology / Q000378:metabolism; D019476:Insect Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007313:Insecta / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D018121:Receptors, Cytokine / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D016207:Cytokines; D019476:Insect Proteins; D018121:Receptors, Cytokine; C068690:growth blocking peptide, Apanteles kariyai