Hidekazu Katayama

Significance of a carboxyl-terminal amide moiety in the folding and biological activity of crustacean hyperglycemic hormone

Recombinant peptides related to Pej-SGP-I, one of several crustacean hyperglycemic hormones (CHHs) existing in the kuruma prawn Penaeus japonicus, were expressed in bacterial cells, and then purified after being allowed to refold. Their circular dichroism spectra suggested that the recombinant Pej-SGP-I having a free carboxyl-terminus (rPej-SGP-I-OH) differed slightly in secondary structure from the recombinant Pej-SGP-I having an amidated C-terminus (rPej-SGP-I-amide). The hyperglycemic activity of rPej-SGP-I-amide was comparable to that of natural Pej-SGP-I, whereas rPej-SGP-I-OH showed weaker hyperglycemic activity by approximately one order of magnitude. These results indicate that the C-terminal amide of CHH affects secondary structure and is significant in conferring hyperglycemic activity.

Cloning and characterization of a molt-inhibiting hormone-like peptide from the prawn Marsupenaeus japonicus

Recently, it was demonstrated by PCR amplification that an additional molt-inhibiting hormone (MIH)-like peptide was present in the kuruma prawn Marsupenaeus japonicus. In this study, a cDNA encoding this peptide designated Pej-MIH-B was cloned. The Pej-MIH-B gene was expressed strongly in the nerve cord, and weakly in the eyestalk. It was possible to isolate Pej-MIH-B from the sinus glands in the eyestalks. The recombinant Pej-MIH-B expressed in Escherichia coli showed low molt-inhibiting activity, but did not exhibit hyperglycemic activity. These results suggest that Pej-MIH-B does not function as MIH or CHH intrinsically, but may have some unknown functions.

Characterization of a Molt‐Inhibiting Hormone (MIH) Receptor in the Y‐Organ of the Kuruma Prawn, Marsupenaeus japonicus

Abstract: To characterize a receptor for molt‐inhibiting hormone (MIH) in the kuruma prawn, Marsupenaeus japonicus, we performed a binding assay and chemical cross‐linking experiments using radiolabeled recombinant MIH ([125I]rMIH). The specific binding of [125I]rMIH was found in the membrane fraction of the Y‐organ (Kd= 4.76 × 10−10 M and Bmax= 5.51 × 10−12 M). Chemical cross‐linking between [125I]rMIH and the membrane fraction of the Y‐organ revealed that the MIH receptor protein has a molecular size of approximately 70 kDa.

In vivo Effects of a Recombinant Molt-Inhibiting Hormone on Molt Interval and Hemolymph Ecdysteroid Level in the Kuruma Prawn, Marsupenaeus japonicus

Abstract In order to determine the function of molt-inhibiting hormone (MIH) in vivo, we examined the effects of injecting of a recombinant MIH on the molt interval and hemolymph ecdysteroid level in the kuruma prawn, Marsupenaeus japonicus. The injection of recombinant MIH significantly prolonged the molt interval (9.0±0.4 days in the control group, 9.5±0.5 days in the 2500 ng/g-body weight/injection-group, mean±SD), and significantly decreased the hemolymph ecdysteroid level (ratio of levels between after and before injection: 1.94±1.09 in the control and 1.28±0.39 in the 3000 ng/g-body weight/injection-group, mean±SD). These results conclusively show the inhibitory effects of MIH on molting in vivo.

A recombinant molt-inhibiting hormone of the kuruma prawn has a similar secondary structure to a native hormone: determination of disulfide bond arrangement and measurements of circular dichroism spectra.

In crustaceans, molt-inhibiting hormone (MIH) is presumed to regulate molting through suppressing synthesis and/or secretion of ecdysteroids by the Y-organ. Recently, a recombinant MIH of the kuruma prawn Penaeus japonicus was produced in E. coli. To approximate the secondary structure of native and recombinant MIH of P. japonicus containing six cysteine residues, the arrangements of disulfide bridges in both MIHs were determined by characterizing their enzymatic digests, and their circular dichroism spectra were measured. The arrangements of disulfide bonds in both MIHs were determined to be identical, and they were linked between Cys7 and Cys44,Cys24 and Cys40, and Cys27 and Cys53. The circular dichroism spectra of both MIHs were very close, and demonstrated that they were rich in α-helix. α-Helix contents in native and recombinant MIHs were calculated to be 49.3% and 46.0%, respectively. All these results strongly suggested that the recombinant MIH was folded in the same manner as the native MIH.

Effect of a Glycine Residue Insertion into Crustacean Hyperglycemic Hormone on Hormonal Activity

Abstract Crustacean hyperglycemic hormone (CHH) and molt-inhibiting hormone (MIH) have similar amino acid sequences and therefore comprise a peptide family referred to as the CHH family. All MIHs unexceptionally have an additional glycine residue at position 12, which is lacking in all CHHs. In order to understand the relevance of the absence of the glycine residue for hyperglycemic activity, a mutant CHH having a glycine residue insertion was prepared, and its hyperglycemic activity was assessed. This mutant CHH had the same disulfide bond arrangement as the recombinant CHH produced in Escherichia coli cells, and exhibited a similar circular dichroism spectrum to the recombinant CHH, indicating that the two CHHs possessed similar conformations. The mutant CHH showed a hyperglycemic effect weaker than the recombinant CHH by about one order of magnitude. These results suggest that the insertion of a glycine residue is one of the indices for structural and functional divergence of the CHH family peptides.

The solution structure of molt-inhibiting hormone from the kuruma prawn

Molting in crustaceans is controlled by molt-inhibiting hormone (MIH) and ecdysteroids. It is presumed that MIH inhibits the synthesis and the secretion of ecdysteroids by the Y-organ, resulting in molt suppression. The amino acid sequence of MIH is similar to that of crustacean hyperglycemic hormone (CHH), and therefore, they form a peptide family referred to as the CHH family. Most of the CHH family peptides show no cross-activity, whereas a few peptides show multiple hormonal activities. To reveal the structural basis of this functional specificity, we determined the solution structure of MIH from the Kuruma prawn Marsupenaeus japonicus and compared the solution structure of MIH with a homology-modeled structure of M. japonicus CHH. The solution structure of MIH consisted of five alpha-helices and no beta-structures, constituting a novel structural motif. The homology-modeled structure of M. japonicus CHH was very similar to the solution structure of MIH with the exception of the absence of the N-terminal alpha-helix and the C-terminal tail, which were sterically close to each other. The surface properties of MIH around this region were quite different from those of CHH. These results strongly suggest that this region is a functionally important site for conferring molt-inhibiting activity.