Structure-activity relationship of crustacean molt-inhibiting hormone from the kuruma prawn Marsupenaeus japonicus.
In crustaceans, molt-inhibiting hormone (MIH) controls molting by suppressing the synthesis and/or secretion of molting hormone. In our previous study, which determined the solution structure of MIH by NMR, we hypothesized that the peptide's functional site spanned the region encompassing the N-terminal alpha-helix and a portion of the C-terminus, both of which are located sterically close to each other [Katayama et al. (2003) J. Biol. Chem. 278, 9620-9623]. To confirm this hypothesis, various mutants of MIH were prepared and their molt-inhibiting activities were assessed. All peptides mutated at the putative functional site exhibited circular dichroism spectra similar to the natural MIH, suggesting that the mutants retained their natural conformation regardless of the mutations. As expected, a majority of the mutants, except for Delta12 (a deletion mutant of Gly(12)) and Delta75-77 (a deletion mutant of the last three residues of the C-terminus), were less active than the natural MIH. In particular, I72G exhibited no molt-inhibiting activity even at 200 nM, while N13A and S71Y exhibited low activity at the same concentration. In contrast, the natural and recombinant MIHs exhibited full inhibitory activity at 20 nM. All these results indicate that the functional site of MIH is located in the region containing the C-terminal ends of the N- and C-terminal alpha-helices, and that Asn(13), Ser(71), and Ile(72) are especially significant for conferring molt-inhibiting activity. Furthermore, these findings agree with the results and the proposed hypothesis presented in previous studies on the structure-activity relationship of MIH and its related peptides.
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi, Bunkyo, Tokyo 113-8657, Japan.
Hidekazu
H
Katayama
Tsuyoshi
T
Ohira
Shinji
S
Nagata
Hiromichi
H
Nagasawa
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D060829:Arthropod Proteins; D003400:Astacoidea; D026461:Ecdysteroids / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D007447:Invertebrate Hormones / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D008969:Molecular Sequence Data; D018978:Molting / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D009924:Organ Culture Techniques; D033561:Penaeidae / Q000737:chemistry* / Q000502:physiology; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011487:Protein Conformation; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013329:Structure-Activity Relationship
D060829:Arthropod Proteins; C116855:CHH-like protein, shrimp; D026461:Ecdysteroids; D007447:Invertebrate Hormones; D010446:Peptide Fragments; D011994:Recombinant Proteins; C012445:molt-inhibiting hormone