JPH0517500A - New peptide - Google Patents

Abstract

PURPOSE:To obtain a new peptide useful for study on action mechanism of nerve transmitter substance and for research and development of agricultural chemicals for exterminating Achandina fulica, having a specific amino acid sequence containing a D type amino acid, prepared from a nerve node of Achandina fulica. CONSTITUTION:Cerebral nerve nodes in heads and nerve modes of esophagi of 1,000 Achandina fulica are separated and collected, immediately frozen, immersed in acetone, homogenized, and centrifuged, the supernatant liquid is collected and passed through a Sep-Pak C18-cartridge. The adsorbed substance is eluted with methanol and the eluted fraction is made into dryness under reduced pressure to give a crude substance. The crude substance is dissolved in 0.1 M acetic acid, subjected to gel filtration, developed with 0.1 M acetic acide, an active fraction is purified by a reversed-phase high-performance liquid chromatography to give fulicin which is the objective peptide having an amino acid sequence shown by the formula.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a novel peptide,
More specifically, it relates to a novel peptide obtained from the ganglion of African snail.

[0002]

[Prior Art] Achatina fulica Fer
(Ussac) is known to cause great damage to agricultural products, and effective control methods are required. The neurotransmitters of African snails are being researched in order to lead to the development of effective pesticides.

[0003] Regarding the molluscs African snail, the neuropeptides that have been discovered to date are achatin-I (HL-Gly-D-Phe-L-Ala-L-Asp isolated from ganglia. -
OH) (Kamatani, Y. et al., Biochemical and
Biophysical Research Communications, vol.160, p10
15,1989) and ACEP-1 (Fujimoto
(Fujimoto), K. et al., Biochemical and Biophysical
Research Communications, vol.167, p777, 1990), among which, Acatin-I is the first example reported as an endogenous neuropeptide containing a D-amino acid.

[0004]

DISCLOSURE OF THE INVENTION Peptides containing D-type amino acids that are not derived from microorganisms include Dermorphin, Dermenkephalin, and Dertrophi, which are present in the skin of frogs.
n) has been discovered since 1981 and is being actively studied from the aspects of pharmacology and biosynthesis. However, since the contents of these peptides are extremely small and it is difficult to purify them and determine their structures including stereoisomerism, a great deal of effort is required to newly find these substances.

It is an object of the present invention to isolate a new peptide containing D-type amino acid from African snail, determine its structure including stereoisomerism, and elucidate its usefulness.

[0006]

SUMMARY OF THE INVENTION The present inventors have found that about 100
A novel peptide was purified and isolated from the ganglion of 0 African snail, using its action on the penile traction muscle as a means of bioassay, to conduct an intensive study to isolate the endogenous neuropeptide of snail snail. Then, the amino acid sequence was determined to be represented by the formula (2) H-L-Phe -D-Asn-L-Glu-L-Phe-L-Val-NH 2 .... (2) ( Hereinafter, this compound is referred to as furicin). In the process leading to this determination, various synthetic peptides in which the D and L forms of each amino acid were replaced,
We found that they also had similar activity, although weaker than furicin.

Accordingly, the present invention relates to an optically active and inactive peptide represented by the amino acid sequence formula (1) H-Phe-Asn-Glu-Phe-Val-NH ₂ ... (1).

In the present specification, the amino acid residue is IUP.
Indicated by the three-letter code defined by AC and IUB, when optical isomers exist, L-form is L-, D-form is D-, and L-form, D-form, or racemic, unless otherwise specified. It shall indicate the body.

In the sequence formula (1), one having one D-type amino acid residue is preferable from the viewpoint of activity,
The most preferable one is represented by the sequence formula (2).

[0010] Of the novel peptides of the present invention, furicin can be isolated and purified from the cerebral ganglion and the subesophageal ganglion of African snail. For example,
Fresh or cryopreserved African locust ganglia are immersed in acetone and immediately homogenized. A supernatant is obtained from the homogenate by a treatment such as centrifugation, concentrated and dried if necessary, dissolved in an appropriate solvent, for example, a 0.1 N hydrochloric acid solution, and if necessary, centrifuged to remove insoluble matter. After removal, the target peptide can be separated and purified by subjecting to various ion exchange chromatography, gel filtration chromatography, reverse phase chromatography and the like.

Since the peptides of the present invention, including furicin, are oligopeptides, they can be easily synthesized by a commercially available peptide synthesizer. For example, using a peptide synthesizer 430A manufactured by Applied Biosystems,
Solid phase synthesis is performed using protected amino acids. Then, the peptide is cleaved from the resin and the protecting group is removed by the HF method. The obtained crude peptide can be subjected to various ion exchange chromatography, gel filtration chromatography, and high performance liquid chromatography using a reverse phase column to separate and purify the target peptide.

The peptide of the present invention enhances the transient contraction of the penis traction muscle of African snail at a low concentration, suppresses the transient contraction of the lingual tongue traction muscle, and increases the heart rate to the ventricles. It has been found to enhance contractility without change. In the central nervous system, B1 and B4 nerves, which are recognized as nerves that promote contraction of the oral muscles, cause periodic firing. The peptides of the present invention are believed to act on the muscular system and nervous system of other organisms including molluscs, and are not only expected to be used for exterminating pests of molluscs, but also useful in pharmacological experiments. It can be used as a reagent.

[0013]

The present invention will be further described with reference to examples, but the scope of the present invention is not limited to these. In addition, comparing each fraction of the purification process in Example 1 with the physiological activities of the natural product and the synthetic product in Examples 3 and 4, the effect on the transient contraction of the penis traction muscle of African snail, Muneoka and Twalog method (Muneoka, Y. and Twarog, BM,
Journal of Pharmacological & Experimental Therape
utics, vol.202, p601, 1977). That is, the penis traction muscle of African snail was taken out, and 1.5 ml of Hepes buffer solution (sodium chloride 6
1.0 mM, potassium chloride 3.3 mM, calcium chloride 10.7 mM, magnesium chloride 13.0 mM, glucose 5.0 mM, N- [2-hydroxyethyl] piperazine-N ′-[2-ethanesulfonic acid] (abbreviation Hepes) )
The ends were tied with a string to an experimental container filled with 10.0 mM of sodium hydroxide solution to adjust the pH to 7.5) to stimulate the muscle, and the effect on the transient contraction that occurred at this time was recorded.

Example 1 Purification of furicin [0014] Cerebral ganglia and subesophageal ganglia of 1,000 African snails (collected in Okinawa) were separated, and these were collectively frozen immediately on dry ice. In use, this was immersed in acetone, immediately homogenized, and centrifuged at 25,000 for 30 minutes at 15,000 × g. The obtained precipitate was reextracted with 80% acetone and centrifuged in the same manner.
The obtained supernatants were collected and dried under reduced pressure. The dried product was dissolved in 0.1 N hydrochloric acid and centrifuged in the same manner to obtain a supernatant. The obtained supernatant was used for two Sep-Pak Cs connected in series.
It was passed through a -18 cartridge (manufactured by Waters), and the adsorbed substance was eluted with 5 ml of methanol. The elution fraction was dried under reduced pressure to obtain a crude product.

This crudely purified product was dissolved in 0.1 M acetic acid, and then Sephadex G-15 column (φ26 × 400 mm).
I went to When developed with 0.1 M acetic acid and each fraction is subjected to a bioactivity assay test using the penis traction muscle of African snail, the activity is eluted first (the first fraction) and later eluted (the first fraction). The second fraction). Furicin was obtained from this second fraction.

The second fraction was collected and concentrated to obtain ODS-8.
C1 using 0TM (Tosoh, φ4.6 × 150mm)
8 Reversed-phase high performance liquid chromatography was performed to obtain a 0.1% trifluoroacetic acid (hereinafter abbreviated as TFA) solution (pH 2.
2) was eluted with a linear gradient of 0% to 60% acetonitrile in 60 minutes.

The active fractions were collected and collected as DEAE-5PW.
(Tosoh, φ7.5 × 75 mm) was subjected to anion exchange high performance liquid chromatography, and 10 mM Tris-
0M to 0.7M Na in hydrochloric acid buffer (pH 9.6)
Elution was performed with a linear concentration gradient of Cl for 70 minutes.

The active fractions were collected and again collected by ODS-80T.
C18 reverse phase high performance liquid chromatography using M (Tosoh, φ4.6 × 150 mm), 0.1% TF
Elution was performed with a linear concentration gradient of 10% to 30% acetonitrile in Solution A (pH 2.2) for 60 minutes. For the final purification, this active fraction was again treated with ODS-80TM (Tosoh,
C18 reverse phase high performance liquid chromatography using φ4.6 × 150 mm) containing 0.1% TFA 17
The purified peptide was obtained by eluting with a single solvent of% acetonitrile (Fig. 1).

Example 2 Estimation of amino acid sequence of furicin The amino acid composition of furicin was analyzed by Hitachi L-8500 type amino acid analyzer, and Asp (aspartic acid) 1.0, Glu (glutamic acid) 0.9, Val was found.
(Valine) 1.0, and Phe (phenylalanine)
It was 1.7. Also, in the automated Edman degradation by Applied Biosystems' 477A gas phase sequencer, the amino acid sequence of furicin was determined as H-Phe-Asn-Glu-Phe -... (3) up to the 4th amino acid residue. However, the 5th amino acid residue was not revealed. However, since this molecule contains one valine or its derivative, the amino acid sequence of furicin is H-Phe-Asn-Glu-Phe-Val ... (4) or its C It was determined to be an amide derivative on the terminal side.

Further, from the FAB-MS spectrum,
Since ((M + H) ⁺ = 654.2 m / z] was observed,
The molecular weight of furicin was determined to be 653. From this, it was estimated that furicin was H-Phe-Asn-Glu-Phe-Val-NH ₂ .... (1) in which the C-terminal side was amidated.

Example 3 Synthesis of furisines Compound (5) in which all amino acids are in L-form in this sequence formula (1): HL-Phe-L-Asn-L-Glu-L-Phe-L-Val -NH ₂ .... (5) was used as a carrier with p-methylbenzhydrylamine resin as a carrier, and using an automated peptide synthesizer model 430A manufactured by Applied Biosystems, by the NMP-HOBt / t-BOC method, t- Boc-L-Phe, t-Boc-L-Asn, t-Boc-L-Glu (OBz
l), t-Boc-L-Val was used for synthesis (however, NMP = N-methy
lpyrrolidine, HOBt = hydroxybenztriazole, t-Boc = t-but
yloxy-carbonyl, Bzl = Benzyl)). The peptide resin was treated with 10% p-cresol-hydrogen fluoride at 0 ° C. for deprotection and cleavage, ether was added to precipitate the peptide, and the precipitate was washed with ether, then dissolved in TFA, and again. The crude peptide was obtained by precipitation with ether. The crude peptide was purified by C-18 reverse phase high performance liquid chromatography. The behavior of this synthetic pentapeptide in high performance liquid chromatography on a C-18 column is shown in Example 1.
Although it was similar to the furicin obtained in 1. (hereinafter referred to as a natural product), the behavior in anion exchange high performance liquid chromatography was different from that of the natural product. In addition, the activity of the synthetic pentapeptide on penile traction muscle was about 10 times that of natural products.
It showed only 0-fold less activity.

Taking into consideration the above results and the fact that Achatin-I obtained from the same ganglion of African snail contains one D-type amino acid, furicin contains one D-type amino acid. It was expected to include. Therefore, (1)
The following 5 compounds each containing D-type amino acid at each position in the amino acid sequence of were synthesized using the same method. That is, by using t-Boc-D-Phe in addition to t-Boc-L-Phe, (6) and (9) were converted into HD-Phe-L-Asn-L-Glu-L-Phe- L-Val-NH ₂ .... (6) H-L-Phe-L-Asn-L-Glu-D-Phe-L-Val-NH ₂ .... (9) t-Boc-L- the use of t-Boc-D-Asn in place of Asn to (7), H-L- Phe-D-Asn-L-Glu-L-Phe-L-Val-NH 2 .... (7 ) t-Boc-L-Glu instead of by the (8) the use of t-Boc-D-Glu- H-L-Phe L-Asn-D-Glu-L-Phe-L-Val-NH 2 .... (8) By substituting t-Boc-D-Val for t-Boc-D-Val, (10) was replaced with HL-Phe-L-Asn-L-Glu-L-Phe. _{-D-Val-NH 2 .... (} 10) were respectively synthesized.

Example 4 Structure determination of furicin A natural product and five kinds of pentapeptides containing one D amino acid residue synthesized in Example 3 were treated with DEAE-5PW (Tosoh φ7.5 × 75 mm) at 30 mM. NaCl
When analyzed under the conditions of 10 mM Tris-hydrochloric acid buffer (pH 9.6) containing 0.5 mL / min, the retention time (7) was consistent with that of the natural product. Further ODS-80T
When using M (Tosoh φ4.6 × 150 mm) under the conditions of 0.1% TFA in 18% acetonitrile (pH 2.2) and a flow rate of 0.5 ml / min, the retention time of (7) is a natural product. Matched with. In addition, the mixture of (7) and the natural product was eluted as a single peak at the same position as the natural product. [Table 1] Retention time of natural product and five kinds of pentapeptides ──────────────────────────────────── Compound DEAE-5PW ODS-80TM ─────────────────────────────────── Natural product 13 minutes 29 seconds 17 minutes 41 seconds (6) 1-D-Phe body 13 minutes 41 seconds 20 minutes 10 seconds (7) 2-D-Asn body 13 minutes 29 seconds 17 minutes 41 seconds (8) 3-D-Glu body 13 minutes 48 seconds Measurement No (9) 4-D-Phe body 14 minutes 04 seconds Not measured (10) 5-D-Val body 15 minutes 54 seconds Not measured ───────────────── ────────────────────

Both natural product and (7) enhanced transient penile traction muscle contraction in the same manner with a threshold concentration of about 10 ^-11 M (Fig. 2). Further, when the concentration exceeded 10 ⁻⁹ M, both of them repeatedly caused contraction immediately after administration to muscle (FIG. 3). Further, the other synthetic products had weaker activity than both of them, and exhibited only about 1 / 10,000 activity.

From the above facts, the structure of furicin was presumed to be the same as that of the synthetic pentapeptide (7). Next, in order to confirm this, after hydrolyzing furicin obtained from African snail for 24 hours with constant boiling hydrochloric acid, a highly sensitive amino acid enantiomer separation method (Buck, RH et a
l., Journal of Chromatography, 315, 279, 1984; Aswad, DW Analytical Biochemistry, 137, 4
05, 1984), orthophthalaldehyde as a fluorescent reagent and N-acetylcysteine as an optically active thiol were added to the hydrolyzate and reacted, and then the column of ODS (CAPCELL PAK C ₁₈ AG120 angstrom (φ4.6 × 250 mm) was used to perform high performance liquid chromatography, and as a result, the presence of D-aspartic acid was confirmed, and the amino acid sequence of furicin was:
Synthetic pentapeptide (7) H-L-Phe -D-Asn-L-Glu-L-Phe-L-Val-NH 2 .... is determined to be the same as (7) (Naoshiki (7 ) Is the same as the formula (2), but two numbers are used for convenience of description).

Example 5 Investigation of Physiological Activity of Furicin Furicin showed physiological activity not only on the penis traction muscle of African snail but also on several muscles and nerve cells of the same animal. To the tongue traction muscle, administration of more than 10 ^-8 M concentration of furicin suppresses transient contraction in response to electrical stimulation (Fig. 4), and washing out of furicin enhances contraction. There was a tendency. Also, in the one ventricle,
Furicin at a concentration of 10 ^-6 M enhanced the contraction degree without obviously changing the heart rate (Fig. 5). Furthermore, in the central nervous system, B1 and B4, which have been identified as nerve cells that cause contraction of the oral muscles, caused periodic firing (Fig. 6).

[0027]

The peptide of the present invention, furicin, is
The frog delmorphin attracts attention as a potent and highly selective agonist for each opiator receptor
(Dermorphin) and Acatin-I obtained from the same African snail, similar to Achatin-I
It is characterized by being a peptide containing type amino acids. Therefore, it serves as a new clue for elucidating the significance of D-type amino acids as neurotransmitters and the biosynthetic mechanism of active peptides.

Furthermore, the peptide furicin of the present invention and its optical isomers are useful as biochemical reagents that give important clues to the study of the structure-activity relationship with their receptors, and various species including African snails. It will contribute greatly to the development of new medicines and pesticides by elucidating the neurotransmission mechanism of animals.

[Brief description of drawings]

FIG. 1 is a graph showing the final active ingredient elution pattern in reverse phase high performance liquid chromatography.

FIG. 2 shows natural furicin (nP) and synthetic HL-
Phe-D-Asn-L-Glu-L-Phe-L-Val-N
Comparing the effect on the penis traction muscle H ₂ (sP), a graph showing the transient contraction enhancement and concentration relationships.

FIG. 3 shows natural furicin (nP) and synthetic HL-
Phe-D-Asn-L-Glu-L-Phe-L-Val-N
Comparing the effect on the penis traction muscle H ₂ (sP), a graph showing the transient contraction enhancement, a state causing the periodic contractions.

FIG. 4 is a graph showing the action of furicin other than the action on the penis traction muscle, and is a graph showing the action of furicin on transient contraction of the lingual traction muscle.

FIG. 5 is a graph showing the action of furicin other than the action on penile traction muscles, and is a graph showing the action of furicin on ventricle.

FIG. 6 shows actions other than the action of furicin on penile traction muscles, namely B1 and B in the orb ganglia.
4 is a graph showing the action of furicin on 4 nerve cells.

Claims (2)

[Claims] 1. The following amino acid sequence formula (1) H-Phe-Asn-Glu-Phe-Val-NH ₂ ........ (1) (wherein the amino acid residue is defined by IUPAC and IUB) Peptide represented by three-letter code). 2. The following amino acid sequence formula (2) H-L-Phe-D-Asn-L-Glu-L-Phe-L-Val-NH ₂ ... (2) (wherein the amino acid The residues are represented by IUPAC and IUB in a three-letter code, L- is the L-form and D- is the D-form)