The Gly-Gly linker region of the insect cytokine growth-blocking peptide is essential for activity.
Growth-blocking peptide (GBP) is a 25-amino acid cytokine isolated from the lepidopteran insect Pseudaletia separata. GBP exhibits various biological activities such as regulation of larval growth of insects, proliferation of a few kinds of cultured cells, and stimulation of a class of insect immune cells called plasmatocytes. The tertiary structure of GBP consists of a well structured core domain and disordered N and C termini. Our previous studies revealed that, in addition to the structured core, specific residues in the unstructured N-terminal region (Glu1 and Phe3) are also essential for the plasmatocyte-stimulating activity. In this study, a number of deletion, insertion, and site-directed mutants targeting the unstructured N-terminal residues of GBP were constructed to gain more detailed insight into the mode of interaction between the N-terminal region and GBP receptor. Alteration of the backbone length of the linker region between the core structure and N-terminal domain reduced plasmatocyte-stimulating activity. The substitutions of Gly5 or Gly6 in this linker region with more bulky residues, such as Phe and Pro, also remarkably reduced this activity. We conclude that the interaction of GBP with its receptor depends on the relative position of the N-terminal domain to the core structure, and therefore the backbone flexibility of Gly residues in the linker region is necessary for adoption of a proper conformation suited to receptor binding. Additionally, antagonistic experiments using deletion mutants confirmed that not only the core domain but also the N-terminal region of GBP are required for "receptor-binding," and furthermore Phe3 is a binding determinant of the N-terminal domain.
Yoshida
Masanobu
M
Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama 930-0194, Japan.
Aizawa
Tomoyasu
T
Nakamura
Takashi
T
Shitara
Kunio
K
Hayakawa
Yoichi
Y
Matsubara
Kimiaki
K
Miura
Kazunori
K
Kouno
Takahide
T
Clark
Kevin D
KD
Strand
Michael R
MR
Mizuguchi
Mineyuki
M
Demura
Makoto
M
Nitta
Katsutoshi
K
Kawano
Keiichi
K
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001681:Biological Assay; D049109:Cell Proliferation; D016207:Cytokines / Q000737:chemistry* / Q000378:metabolism; D017353:Gene Deletion; D005998:Glycine / Q000737:chemistry*; D019476:Insect Proteins / Q000737:chemistry* / Q000378:metabolism; D007313:Insecta; D007915:Lepidoptera; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D016207:Cytokines; D019476:Insect Proteins; D010455:Peptides; C068690:growth blocking peptide, Apanteles kariyai; D005998:Glycine