JP3162509B2 - Mollusc neuropeptides containing cystine - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION The present invention relates to novel neuropeptides, and more particularly to Helix Helix.
(Pomatia) ganglia.

[0002]

2. Description of the Related Art Since the nervous system of mollusks is generally simpler than higher animals, it can be used to elucidate the mechanism of information processing. It is believed that findings from molluscs can be generalized to the nervous system of higher animals, at least if they relate to subcellular mechanisms. At that time, a search for a neurotransmitter is indispensable, and in addition to a classical transmitter, a peptide-based transmitter or a neuromodulator has been energetically searched.

[0003] The present inventors have previously described the mussels (My
The following amino acid sequence formula (6) was obtained from the ganglia of S. tilus edulis, using as an index the effect on the strong contraction of the anterior retractor muscle of the mussel by electrical stimulation. -Val-N
H ₂ . . . . (6) (In the present specification, amino acid residues are IUPAC and IUB
X indicates a Ser residue or an Ala residue, and NH ₂ indicates that the C-terminal is amidated. ), A novel peptide (MI
P: MytilusInhibitory Pepti
de) was found to be an endogenous neuropeptide, and these peptides were synthesized and a patent application was filed (see JP-A-1-221392).

[0004]

Until now, molluscs have been obtained from Myomodulin in addition to the above-mentioned MIPs.
(Cropper, Tenenbaum, Kolks,
Kupffermann, Weiss, Pro. Nat
l. Acad. Sci. USA 84, 5483-54
86 1987), CARP (Catch-Relax).
ing Peptide: Hirata, Kubota, Takahata, Kuwahara,
Muneoka Brain Res. , 442 373-37
6), SCP (Small Cardioactive)
Peptide: Lloyd, Kupferman
n, Weiss, J.M. Comp. Physiol. , A
156, 959 to 667 1985), and the like, but it is necessary to find more neuropeptides for studying the structure-activity relationship, species specificity, etc. of neuropeptides.

[0005]

SUMMARY OF THE INVENTION In view of the above, the inventors of the present invention have found that from the ganglia of Hungarian Maimai, the anterior tractor muscle (Anterior Byssus) of mussels is used.
Based on the effect of RetractorMuscle (ABRM) on the strong contraction caused by electrical stimulation as an index, intensive studies were conducted to isolate endogenous neuropeptides by systematically combining high performance liquid chromatography (HPLC) as shown in FIG. An active substance was obtained.

[0006] These active substances were found to be novel peptides having the amino acid sequences shown in SEQ ID NOs: 1 to 5 by structural analysis, and the respective peptides were synthesized by the solid phase method to confirm their structures. Thus, the present invention has been completed.

According to the present invention, there is provided a novel neuropeptide having the amino acid sequence shown in SEQ ID NO: 1-5.

[0008] The peptide of the present invention can be isolated and purified by using the ganglion of Hungarian Maimai as a raw material by the following method. For example, ganglia of Hungarian Maimai are homogenized in acetone, the obtained homogenate is centrifuged, the supernatant obtained is adsorbed to a C18 cartridge, and the adsorbed fraction is treated with 0.1% trifluoroacetic acid (hereinafter TFA). ) / Methanol and subjected to reverse-phase chromatography, ion-exchange chromatography, etc., and finally to ultraviolet absorption at 220 nm (hereinafter referred to as U
By purified to show a single peak in V ₂₂₀ for short) monitor, can be a peptide of interest to isolate and purify.

Further, the peptide of the present invention has S-
Since the oligopeptide has an S bond, a commercially available peptide synthesizer (for example, a fully automatic peptide synthesizer 430A manufactured by Applied Biosystems) can be used to prepare the corresponding SS.
It can be obtained by synthesizing a peptide having no bond and then introducing an SS bond into the molecule. For purification of the peptide during the synthesis, as is known to those skilled in the art, C18
Reverse phase high performance liquid chromatography, ion exchange high performance liquid chromatography, and the like can be used.

[0010]

The peptide of the present invention has a physiological activity that modifies the strong contraction of the mussel anterior traction muscle by electrical stimulation, and can be used as a useful reagent in the study of the neurotransmission system.

[0011]

The present invention will be further described with reference to the following examples, but the scope of the present invention is not limited only to these examples.

Embodiment 1 FIG. Peace from Hungarian Maimai
Purification of peptide a. Crude extraction The cerebral ganglia and sub-esophageal ganglia of 1,075 heads of Himalayan maimai were extracted at 0 ° C in 180 ml of 100% acetone.
Homogenized under, centrifuged at 4 ° C., 16,000 × g for 40 minutes, and the supernatant was collected. The precipitate is 170 ml of 80
After extraction with% acetone, the mixture was centrifuged under the same conditions, and the supernatant was collected. The obtained precipitate was further extracted with 100 ml of 80% acetone, centrifuged under the same conditions, and the supernatant was collected. The supernatant obtained by these operations was collected, concentrated under reduced pressure to 1 ml,
30 ml of 0.1N hydrochloric acid was added thereto, and the mixture was centrifuged again under the same conditions, and the supernatant was used as an extract.

B. Adsorption to C18 cartridge a. The extract obtained in Step 3 was connected in series with three Sep-
The solution was passed through a Pak C18 cartridge (manufactured by Nippon Millipore Limited). After washing the cartridge with 0.1% TFA, the retentate was eluted with 7 ml of 0.1% TFA / methanol and concentrated to dryness under reduced pressure.

C. Reverse phase column chromatography b. 0.5 ml of 0.1% TF
A-1 dissolved in A, and C-1 using Asahipak ODP-50 (φ7.6 × 250 mm, manufactured by Asahi Kasei Corporation)
8 Reversed-phase high performance liquid column chromatography was performed at a flow rate of 1 ml / min in 0.1% TFA (pH 2.2).
Elution was performed with a linear gradient of 0% to 60% acetonitrile over 120 minutes. Fractionation was performed in 2 ml portions, and five fractions (fraction I in the order of shorter retention time,
To V).

D. Cation exchange column chromatography
-C . The fraction I obtained in the above was used for TSKgel SP-5PW
(Tosoh Corp., φ7.5 × 75 mm) by cation exchange column chromatography, and the flow rate was 0.5
10 mM phosphate buffer (pH 6.9) at ml / min
The mixture was eluted with a linear gradient of 0 M to 0.6 M NaCl over 60 minutes, and fractionated in 1 ml portions. By bioassay,
From fraction I, four active fractions (in the order of shorter retention time, referred to as Fl-4) were obtained. Similarly, fraction II
From 8 active fractions (also referred to as F5 to 12) and 5 active fractions from Fraction III (also referred to as F13 to 17).

For fractions IV and V, the elution conditions were changed to a linear gradient of NaCl from 0 M to 0.7 M for 70 minutes, and cation exchange column chromatography was performed to obtain two active fractions from the fractions. Minutes (also F18
~ 19), and two active fractions (also referred to as F20-21) from fraction V.

E. Purification of compound 1 d. The F7 fraction obtained in the above was used for TSKgel ODS 80
The mixture was subjected to C18 reversed-phase high-performance liquid column chromatography using T _M (manufactured by Tosoh Corporation, φ4.6 × 150 mm) at a flow rate of 0.5 ml / min and 0.1% TFA (pH
In 2.2), elution was performed with a linear gradient of 12% to 22% acetonitrile in 50 minutes. Compound 1 has the first peak of the four activity peaks obtained by the bioassay in acetonitrile concentration of 0.5% to 1% higher than the elution concentration in 0.1% TFA (pH 2.2). In an isocratic mode by final phase chromatography until finally a single peak was observed on the _UV220 monitor.

F. Purification of compound 2 d. The F8 fraction obtained in the above was subjected to TSKgel ODS 80
The mixture was subjected to C18 reversed-phase high-performance liquid column chromatography using T _M (manufactured by Tosoh Corporation, φ4.6 × 150 mm) at a flow rate of 0.5 ml / min and 0.1% TFA (pH
In 2.2), elution was performed with a linear gradient of 12% to 22% acetonitrile in 50 minutes. Compound 2 has the first peak of the four active peaks obtained by the bioassay in acetonitrile concentration of 0.5% to 1% higher than the elution concentration in 0.1% TFA (pH 2.2). In an isocratic mode by final phase chromatography until finally a single peak was observed on the _UV220 monitor.

G. Purification of compound 3 d. The F19 fraction obtained in the above was subjected to TSKgel ODS 8
0T _M (Tosoh Corp., φ4.6 × 150mm) subjected to C18 reverse phase high performance liquid column chromatography using a flow rate of 0.5ml / min, 0.1% TFA ( pH
In 2.2), a linear gradient of 19% to 29% acetonitrile was eluted in 50 minutes. Compound 3 has a second peak among the six active peaks obtained by the bioassay in acetonitrile concentration 0.5% to 1% higher than the elution concentration in 0.1% TFA (pH 2.2). In an isocratic mode by final phase chromatography until finally a single peak was observed on the _UV220 monitor.

H. Purification of compound 4 d. The F20 fraction obtained in the above was subjected to TSKgel ODS 8
0T _M (Tosoh Corp., φ4.6 × 150mm) subjected to C18 reverse phase high performance liquid column chromatography using a flow rate of 0.5ml / min, 0.1% TFA ( pH
In 2.2), elution was performed with a linear gradient of 21% to 31% acetonitrile in 50 minutes. Compound 4 has a ninth peak among the 13 active peaks obtained by the bioassay in acetonitrile concentration of 0.5% to 1% higher than the elution concentration in 0.1% TFA (pH 2.2). In an isocratic mode by final phase chromatography until finally a single peak was observed on the _UV220 monitor.

I. Compound 5 was obtained by purifying Compound 12 with the twelfth peak among the 13 active peaks obtained in h, using 0.1% TFA (pH 2.2).
Medium, in the isocratic mode with acetonitrile concentration of about 0.5% to 1% higher than the elution concentration, obtained by repeating reversed-phase high performance liquid column chromatography until finally showing a single peak on the _UV220 monitor. Was done.

Embodiment 2 FIG. Identification of peptides The amino acid composition of these peptides was determined by Tosoh CCP-8.
It was measured by a 000 type amino acid analysis system. The results are shown in [Table 1]. In addition, the amino acid sequences of these peptides were identified by Shimadzu PSQ-1 type fully automatic Edman degradation gas phase sequencer as shown in Table 2 except for cystine.

[0023]

[Table 1]

[0024]

[Table 2]

In general, in the amino acid sequence analysis of a peptide containing cystine, an N-terminal half cystine residue cannot be detected in the cycle, but as the Edman degradation proceeds, the SS is reduced by dithiothreitol contained in the reaction system.
It is reported that the bond is reduced and detected as a mixture of phenylthiohydantoyl cysteine and its degradation product phenylthiohydantoyl dehydroalanine dithiothreitol adduct in the cycle corresponding to the C-terminal half cystine residue. (PeptideChem
Istry 1990 pp. 159-164 1991
Therefore, in the amino acid sequence analysis of the compound of the present invention, a cycle in which no amino acid residue is detected is a N-terminal half cystine residue, and a cycle in which an unidentified amino acid residue is detected is a C-terminal half cystine residue. It is estimated to correspond to

Among these peptides, for the compounds 1, 2 and 5 obtained in relatively large amounts, the SS bond was determined by the method of Ekenbara et al. (Ekenbara, Morita, Yamaguchi, Watanabe: Peptide).
Chemistry 1990 pp. 159-164
1991), and the resulting cystine was converted into S-pyridylethyl cysteine. Similarly, the amino acid sequence was measured using a Shimadzu PSQ-1 fully automatic Edman degradation gas phase sequencer. [Table 3]
Shown in

[0027]

[Table 3]

Further, the mass spectra of these peptides were obtained from JEOL JMS-HX-110 / 110A FA
It was measured by a B mass spectrometer. The results are shown in [Table 4].

[0029]

[Table 4]

From these data, among these peptides, compounds 1, 2 and 5 were obtained from Sequence Listing 1 and 2, respectively.
It was presumed to be a peptide having the amino acid sequence shown in 2 and 5.

For Compounds 3 and 4, the amino acid sequences shown in Sequence Listings 3 and 4 were obtained from the amino acid analysis values, mass analysis values, and all amino acid sequences except for cystine shown in [Table 2]. Was presumed to be a peptide having

Example 3 Synthesis of Peptide by Solid-Phase Method
P-resin (manufactured by Applied Biosystems), whose C-terminus is amidated is Fmoc-aminoethyl
Using l-SAL resin (manufactured by Watanabe Chemical Industry) as a carrier, Fmo
c-Ala, Fmoc-Asn (Trt), Fmoc-
Asp (OBu ^t ), Fmoc-Cys (Trt),
Fmoc-Glu (OBu ^t ), Fmoc-Gln (T
rt), Fmoc-Gly, Fmoc-His (Tr
t), Fmoc-Ile, Fmoc-Leu, Fmoc
-Lys (Boc), Fmoc-Met, Fmoc-P
he, Fmoc-Pro, Fmoc-Ser (B
u ^t ), Fmoc-Trp, Fmoc-Tyr (B
u ^t ) using an Applied Biosystems fully automated peptide synthesizer, Model 430A, with FastMoc ^™
Was synthesized by the solid phase method. (However, HMP = 4- (Hy
(doxymethyl) -phenoxymethyl
1, Fmoc = 9-Fluorenylmethod
carbonyl, SAL = Super Acid L
abile, Trt = Trityl, Boc = t-Bu
^{tyloxycarbonyl,} Bu t = t-Buty
l. )

As is well known to those skilled in the art, cleavage of the peptide from the peptide resin and deprotection of the crude peptide depend on the type of the protecting group of the amino acid, and thus the reagent A (6.25) is used.
% Phenol / 2.1% 1,2-ethanedithiol /
4.2% thioanisole / 4.1% water / 83.3% TF
A) and reagent B (2.5% 1,2-ethanedithiol / 2.5% water / 95.0% TFA) were used properly. However, with respect to Compound 5, 2 mg of 2-methylindole was added to 1 ml of TFA to Reagent B in order to prevent a side reaction with Trp. After completion of the reaction, the peptide resin was treated with either A or B reagent at room temperature for 1.5 to 3 hours to perform deprotection and cleavage, and ether was added to precipitate the peptide. The precipitate was washed three times with ether. Later, it was dissolved in TFA and precipitated again with ether to obtain a crude peptide.

The obtained crude peptide was prepared by the method of Hope et al. (Hope, Murti, du Vigneau.
J. Biol. Chem. , 237, 1563-15
66, 1962), and by oxidizing with potassium ferricyanide to introduce an SS bond into the molecule according to a modified Nantake method (Nantake, doctoral dissertation, Kyoto University, 1991) to introduce C18 reverse phase. Purification was performed by a combination of high performance liquid chromatography and strongly acidic or weakly basic ion exchange high performance liquid chromatography.

The synthetic peptide is TSKgel ODS8
Reverse phase chromatography and TSKg used 0T _M
In cation exchange chromatography using el SP-5PW, the retention time was exactly the same as that of the natural product. In addition, in the modification of strong contraction by electrical stimulation of the mussel anterior retractor muscle, the same as in natural products, Compound 1
5 was identified as a peptide having the amino acid sequence shown in Sequence Listings 1 to 5.

Example 4 Mussel Muscles
The bioactivity assay test using the muscle puller muscle of the anterior muscle of the mussel was performed according to the method of Muneoka et al. (Muneoka et al .: J. Pharmac.ex).
p. Ther. , Vol. 202, p601-609,
1977). That is, the upper and lower ends of the separated mussel muscular foreshortening muscle were ligated with cotton thread and
ml. The sample to be assayed is N
aCl: 445 mM, KCl: 10 mM, CaCl ₂ :
The solution was used by dissolving in 10 mM Tris-HCl buffer (pH 7.8) containing 10 mM and MgCl ₂ : 55 mM. Strong contraction was induced by applying a 15 V, 3 msec, 10 Hz, 50 pulse electrical stimulus every 10 minutes to a pair of silver electrodes in contact with the upper and lower cotton threads. The sample was allowed to act 8 minutes before the electrical stimulation, and immediately after the contraction was recorded, the sample was washed with the above buffer.

The effects of the peptide of the present invention on the strong contraction of the mussel anterior traction muscle are shown in FIGS. 2 to 6. As shown in the figure, the peptide of the present invention modifies the strong contraction of the anterior traction muscle of the mussel by electrical stimulation.

[0038]

INDUSTRIAL APPLICABILITY The peptide of the present invention is a neuropeptide that modifies the strong contraction of the mussel anterior tractor muscle, and is useful as a biochemical reagent for elucidating the neurotransmission system. It can provide a new clue to pharmaceutical and pesticide research.

[Sequence list]

SEQ ID NO: 1 Sequence length: 11 Sequence type: Amino acid Sequence type: Peptide Origin: Organism name: Helix Pomacia Sequence characteristics: Symbol indicating characteristics: disulfide-bonds Location: 3, 8 Characteristic Method determined: E Sequence characteristics: Other characteristics: C-terminal amidation Location: 11 Method determined: E sequence:

SEQ ID NO: 2 Sequence length: 11 Sequence type: Amino acid Sequence type: Peptide Origin: Organism name: Helix Pomacia Sequence features: Symbol indicating feature: disulfide-bonds Location: 2, 10 Features Method determined: E Sequence characteristics: Other characteristics: C-terminal amidation Location: 11 Method determined: E sequence:

SEQ ID NO: 3 Sequence length: 12 Sequence type: amino acid Sequence type: Peptide Origin: Organism name: Helix Pomacia Sequence characteristics: Symbol indicating characteristics: disulfide-bonds Location: 3, 9 Characteristic Method determined: E Sequence characteristics: Other characteristics: C-terminal amidation Location: 12 Method determined: E sequence:

SEQ ID NO: 4 Sequence length: 14 Sequence type: Amino acid Sequence type: Peptide Origin: Organism name: Helix Pomacia Sequence characteristics: Characterizing symbol: disulfide-bonds Location: 6, 13 Characteristic Determined method: E sequence:

SEQ ID NO: 5 Sequence length: 15 Sequence type: amino acid Sequence type: Peptide Origin: Organism name: Helix Pomacia Sequence characteristics: Symbol indicating characteristics: disulfide-bonds Location: 7, 13 Characteristic Method determined: E Sequence characteristics: Other characteristics: C-terminal amidation Location: 14 Method determined: E sequence:

[Brief description of the drawings]

FIG. 1 is a drawing showing a scheme for purifying a peptide of the present invention.

FIG. 2 (a) shows the results before compound addition, and FIG.
When adding an amount equivalent to 6 animals to the chamber,
(C), (d) and (e) are drawings in which the strong contraction of the mussel anterior traction muscle after washing with the buffer was recorded with the passage of time. Compound 1 enhances strong shrinkage and its effect persists for a while after washing.

FIG. 3 (a) shows a sample before addition of a sample, and FIG.
When adding an amount equivalent to 6 animals to the chamber,
(C) is a drawing in which the strong contraction of the mussel anterior retractor muscle after washing with the buffer was recorded with the passage of time. Compound 2 suppresses strong contraction.

4 (a) shows the results before compound addition, and FIG. 4 (b) shows compound 3
When adding an amount equivalent to 6 animals to the chamber,
(C), (d) and (e) are drawings in which the strong contraction of the mussel anterior traction muscle after washing with the buffer was recorded with the passage of time. Compound 3 enhances strong shrinkage, and its effect persists for a while after washing.

FIG. 5 (a) shows the results before compound addition, and FIG. 5 (b) shows the compound 4
When adding an amount equivalent to 6 animals to the chamber,
(C) is a drawing in which the strong contraction of the mussel anterior retractor muscle after washing with the buffer was recorded with the passage of time. Compound 4 suppresses strong contraction.

6 (a) shows the results before compound addition, and FIG. 6 (b) shows compound 5
When adding an amount equivalent to 6 animals to the chamber,
(C) is a drawing in which the strong contraction of the mussel anterior retractor muscle after washing with the buffer was recorded with the passage of time. Compound 5 suppresses strong contraction.

Continuation of the front page (58) Fields investigated (Int. Cl. ⁷ , DB name) C07K 7/06 C07K 7/08 BIOSIS (DIALOG) WPI (DIALOG) CA (STN) REGISTRY (STN) SwissProt / PIR / GeneSeq

Claims (5)

(57) [Claims] 1. The following amino acid sequence formula (1): H-Pro-Phe-Cys-Asn-Ser-Tyr
-Gly-Cys-Tyr-Asn-Ser-NH
₂ (1) (wherein, amino acid residues are represented by three-letter codes defined by IUPAC and IUB, and NH ₂ indicates that the C-terminal is amidated.) A peptide having an SS bond between Cys. 2. The following amino acid sequence formula (2): H-Met-Cys-Ser-Gly-Tyr-Asn
-Pro-Ala-Met-Cys-Lys-NH
₂ (2) (wherein, amino acid residues are represented by three-letter codes defined by IUPAC and IUB, and NH ₂ indicates that the C-terminus is amidated.) A peptide having an SS bond between Cys. 3. The following amino acid sequence formula (3): H-Leu-Phe-Cys-Asn-Gly-Tyr
-Gly-Gly-Cys-Gln-Asn-Leu-
NH ₂ (3) (wherein, amino acid residues are represented by three-letter codes defined by IUPAC and IUB, and NH ₂ indicates that the C-terminus is amidated). A peptide having an SS bond between Cys of the above. 4. The following amino acid sequence formula (4): H-Asn-Tyr-Ile-Gln-Ala-Cys
-Tyr-Phe-Gln-Ala-Ile-Ser-
Cys-Tyr-OH (4) (wherein, amino acid residues are represented by three-letter codes defined by IUPAC and IUB),
A peptide having an SS bond between the third Cys. 5. The following amino acid sequence formula (5): H-Tyr-His-Ala-Asp-Leu-Asn
-Cys-Leu-Asp-Pro-Glu-Trp-
Cys-Phe-Gly-NH ₂ (5) (wherein, amino acid residues are represented by three-letter codes defined by IUPAC and IUB, and NH ₂ indicates that the C-terminal is amidated). A peptide having an SS bond between Cys 7 and 13;