Construction of an expression plasmid

Cloning and characterization of a cDNA encoding Pej-SGP-I, one of the CHHs from M. japonicus, were reported previously (Ohira et al., 1997). Two primers, 12-F and 12-R, were designed based on the nucleotide sequence of Pej-SGP-I cDNA, with the inclusion of three nucleotides (GGT in 12-F, and ACC in 12-R, underlined in Table 1) encoding a glycine residue to be inserted at position 12. Two primers, F1 and R1, which completely matched the untranslated region of the cDNA were designed as forward and reverse primers, respectively. Another set of primers, F2 and R2, were designed to contain 20 nucleotide residues encoding the Nand C-terminal regions, respectively, and restriction enzyme sites (an Nco I site in F2, and an EcoR I site in R2, indicated by italics in Table 1). The nucleotide sequences of these primers are shown in Table 1. Three rounds of PCR were conducted. First, two rounds of PCR were separately conducted with two sets of primers, F1 and 12-R, and 12-F and R1, using a plasmid containing the Pej-SGP-I cDNA as template. Each PCR product was separated on a 2.0% agarose gel, and extracted from the gel using a kit of Quantum Prep Freeze'N Squeeze DNA Gel Extraction Spin Columns (Bio-Rad, USA) according to instructions of the manufacturer. Two PCR products thus obtained were mixed with PCR buffer, dNTP mixture and Taq polymerase. The mixture was allowed to react under the following program: 5 cycles of 30 sec at 94°C, 30 sec at 55°C, and 30 sec at 72°C. To this tube, a set of primers, F2 and R2, were added, and PCR was performed with the following program: 20 cycles of 30 sec at 94°C, 30 sec at 55°C, and 30 sec at 72°C. The amplified PCR product was cloned with a pCR2.1 plasmid (Invitrogen, USA), and the nucleotide sequence of the PCR product was analyzed. Subsequently, the insert was released from the pCR2.1 plasmid by digestion with Nco I/EcoR I, and then ligated into the Nco I/EcoR I site of an expression plasmid pProEX HTa (Invitrogen, USA).

Table 1

Nucleotide sequences of primers used in this study

Preparation of CHH-Gly¹²

Expression, refolding reaction, cleavage of His-tag, amidating reaction and purification of CHH-Gly¹² were performed according to the method used for preparation of recombinant CHH (Katayama et al., 2002). The amidating enzyme was a kind gift from Drs. Ohsuye and Furukawa of Daiichi Suntory Pharma Co. Ltd., Japan.

Bioassay for hyperglycemic activity

Kuruma prawns, M. japonicus, were obtained at a fish market in Tokyo, Japan, and maintained in an aquarium at 20°C during the experiment. Hyperglycemic activity was assessed essentially according to the method described previously (Yang et al., 1995; Katayama et al., 2002).

Trypsin digestion

CHH-Gly¹² (15 μg) was dissolved in 300 μl of 25 mM HEPES buffer (pH 6.8). To this solution, 2 μl of TPCK-treated trypsin (Promega, USA) solution (1 mg/ml) was added, and the mixture was incubated at 37°C for 20 h. The digestion was stopped by the addition of 2 μl of trifluoroacetic acid (TFA). The tryptic digests were separated by reverse-phase HPLC (RP-HPLC) on a Capcell-Pak C₁₈ column (2.0×250 mm, Shiseido, Japan) with a 30-min linear gradient of 0–40% acetonitrile in 0.05% TFA, and a 10-min linear gradient of 40–80% acetonitrile in 0.05% TFA at a flow rate of 0.2 ml/min.

Amino acid sequence analysis

The N-terminal amino acid sequences of peptides were analyzed on an Applied Biosystems model 476A protein sequencer in the pulsed-liquid mode. The phenylthiohydantoin derivative of cystine (PTH-cystine) was detected in amino acid sequencing using an Applied Biosystems model 492HT protein sequencer with a modified HPLC program (Katayama et al., 2002).

Mass spectral analysis

Mass spectra were measured on a matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer (Voyager-DE™ STR, Applied Biosystems, USA) with 3,5-dimethoxy-4-hydroxycinnamic acid or α-cyano-4-hydroxycinnamic acid as a matrix in the positive ion mode.

Circular dichroism spectral analysis

The recombinant peptide (about 15 μg) was dissolved in 250 μl of PBS. The circular dichroism (CD) spectrum was recorded from 200 to 260 nm on a spectropolarimeter (Jasco J-720, Japan) at room temperature with a 1-mm path length cell.

Preparation of CHH-Gly¹²

An expression plasmid containing the CHH-Gly¹² insert was constructed. Using this construct, CHH-Gly¹² was expressed as a fusion protein with an N-terminal histidine tag (His-tag). After breakage of the E. coli cells that expressed CHH-Gly¹², the lysate was centrifuged to obtain soluble and insoluble fractions. These fractions were subjected to SDS-polyacrylamide gel electrophoresis, and a heavily stained band (≈11.5 kDa) likely representing CHHGly¹² was detected only in the insoluble fraction (Fig. 1), indicating that this peptide was expressed as inclusion bodies in E. coli cells. Since a refolding reaction was considered necessary for obtaining the mutant peptide in its natural conformation, it was performed according to the method used for preparation of recombinant CHH (Katayama et al., 2002). Subsequently, a His-tag attached at the N-terminus of CHH-Gly¹² was removed by digestion with TEV protease. CHH-Gly¹² was amidated with peptidylglycine α-amidating enzyme, and the resulting peptide was purified by RPHPLC. The peptide showed an expected single molecular ion peak at m/z 8751.3 and an expected N-terminal sequence, Gly-Ala-Met-Gly-Ser-Leu-Phe-Asp-Pro-Ser-, which was identical to that of natural CHH except for the four N-terminal residues derived from the nucleotide sequence of the expression plasmid. Since the recombinant CHH having four additional residues at the N-terminus showed hyperglycemic activity as high as the natural peptide (Katayama et al., 2002), it was unlikely that these four residues in CHHGly¹² affected hormonal activity.

Fig. 1

SDS-polyacrylamide gel electrophoresis (15% acrylamide) of soluble and insoluble fractions after cell breakage from a 0.1-ml culture. Lanes 1 and 2 are the soluble and insoluble fractions of cells carrying an expression vector containing the CHH-Gly¹² insert, respectively. The positions of molecular weight markers are indicated.

In order to examine whether the recombinant peptide possessed natural conformation, we analyzed the arrangement of disulfide bonds in CHH-Gly¹² by identification of its tryptic digests, as was done for the recombinant CHH (Katayama et al., 2002). Two major peaks were observed in the elution profile of tryptic digests of CHH-Gly¹² on RPHPLC. Table 2 shows the results of mass spectral and amino acid sequence analyses. Fragment 2 contained one disulfide bond between Cys⁷ and Cys⁴⁴. In the amino acid sequence analysis of fragment 1, PTH-cystine was detected at both cycles 5 and 8, unambiguously establishing the two disulfide bonds between Cys²⁴ and Cys⁴⁰, and between Cys²⁷ and Cys⁵³. This disulfide bond arrangement was identical to that of the recombinant CHH (Katayama et al., 2002). The circular dichroism (CD) spectrum of CHH-Gly¹² exhibited a pattern typical for α-helix-rich proteins (data not shown), and it was calculated that the α-helix content in CHH-Gly¹² was 44.7%. These results were similar to those of the natural and recombinant CHHs (Katayama et al., 2002). All these results indicate that CHH-Gly¹² possesses a conformation similar to the conformations of natural and recombinant CHHs.

Table 2

Amino acid sequence and TOF-MS analyses of tryptic fragments of CHH-Gly¹².

Biological activity of CHH-Gly¹²

The dose-response relationship of CHH-Gly¹² in hyper-glycemic activity is shown in Fig. 2. Injection of 100 pmol of CHH-Gly¹² increased the hemolymph glucose levels slightly, and its hyperglycemic activity reached a maximum at 300 pmol. It was demonstrated that the recombinant CHH showed low hyperglycemic activity at 10 pmol and high activity at 100 pmol (Katayama et al., 2002). These results indicated that the hyperglycemic activity of CHH-Gly¹² was lower than that of the recombinant CHH by about one order of magnitude.

Fig. 2

(A) Dose-response relationship of CHH-Gly¹². (B) Negative control, injection of PBS; positive control, injection of sinus gland extract (one sinus gland equivalent) of M. japonicus. Results are expressed as the mean±S.E. (N=7–14). Asterisks indicate values significantly different from the negative control. Symbols, (*) and (**), indicate P<0.01 and P<0.001, respectively, which were calculated with the program of Fishers PLSD.

In the tertiary structure of M. japonicus MIH, the Gly¹² residue is positioned in the short N-terminal helix, and it was presumed that this helical structure might be lost by the absence of the Gly¹² residue (Katayama et al., 2003). Comparison of the tertiary and surface structures of the MIH with those of the modeled CHH suggests that this N-terminal helix is one of the significant regions for conferring MIH activity, and therefore the glycine residue at position 12 is likely to be one of the indices for diversification of the functions of the CHH family peptides. Thus, the present results are consistent with the hypothesis on the structure-activity relationship of the CHH family peptides.

The mutational analysis of M. japonicus MIH demonstrated that the residues at positions 13, 14, 71 and 72 were significant for conferring molt-inhibiting activity (Katayama et al., 2004). It was presumed that these residues were some of indices of dividing the functions of CHH-family peptides, and were not conserved between CHHs and MIHs. Therefore, it is likely that CHH-Gly¹² could not show the molt-inhibiting activity, and additional mutations at these positions would be required for exhibiting the molt-inhibiting activity.

Recently, it was proposed that molecular evolution of the CHH family peptides was similar to that of the growth hormone (GH) family proteins including GH and prolactin (PRL) (Chan et al., 2003); GH and PRL are structurally similar but functionally completely different. This is also true for the CHH family, but there are some exceptions; CHH of the lobster Homarus americanus showed both CHH and MIH activity (Chang et al., 1990). Therefore, it is likely that the CHH family peptides may be still on their way to attaining complete functional diversity. Studies on structural differences between CHH and MIH receptors and molecular mechanism of hormone-receptor interaction are required for revealing the significance of the glycine residue insertion or deletion in the functional and structural diversion.