JPH0680696A - Novel peptide - Google Patents

Abstract

PURPOSE:To provide a novel peptide having a specific amino acid sequence comprising omega-conotoxin GVIA whose 2-lysine residue has been replaced with an alanine residue, and useful for the researches of Ca channel functions, etc., as a reagent for isolating N type Ca channels. CONSTITUTION:Amino acids whose alpha-amino groups and side chain functional groups are protected are successively condensed on a p-methylbenzhydrylamine resin as a solid phase carrier from the C terminal side in accordance with the amino acid sequence of a peptide by a peptide solid phase synthetic method, and the synthesized side chain-protected peptide resin is treated with hydrogen fluoride in the presence of anisole for the release of the peptide from the resin and for the removal of the protecting groups to produce a linear peptide. The linear peptide is diluted with distilled water, mixed with ammonia, oxidized with air, mixed with acetic acid, subjected to insolubles removal, and subsequently lyophilized to provide the novel peptide having an amino acid sequence of the formula comprising omega-conotoxin GVIA whose 2-lysine residue has been replaced with an alanine residue and useful for the isolation of N type Ca channels, etc.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel peptide. Specifically, it relates to a novel peptide effective for isolating N-type calcium channels.

[0002]

2. Description of the Related Art Calcium ions are involved in muscle contraction, neurotransmitter release, and the like, and there is a large concentration difference between inside and outside of cells. This difference in concentration changes when cells are activated, but a mechanism known as the calcium channel (Ca channel) for calcium ions to pass through the cell membrane is known.
[Annual Review of Neuroscience, 13, 337-356 (199
0)]. There are many unclear points about the calcium channel, and a substance that specifically binds to it is effective for isolating and purifying it and clarifying its function.

Conventionally, ω-conotoxin GVIA, which is a peptide consisting of 27 amino acid residues, is a substance that specifically binds to an N-type calcium channel found only in nerve cells.
(ω-conotoxin GVIA) is known [Annual Review of
Biochemistry, 57, 665-700 (1988)]. However, since this substance has a remarkably high affinity for calcium channels and its binding is irreversible, it was extremely difficult to isolate calcium channels by purification by ordinary affinity chromatography.

[0004]

DISCLOSURE OF THE INVENTION The object of the present invention is to provide an effective substance for isolating and purifying calcium channels.

[0005]

[Means for Solving the Problems] In order to achieve the above-mentioned object, the present inventors have searched for and investigated the analogues of ω-conotoxin GVIA, and as a result,
A novel peptide in which the lysine residue at the position is replaced with an alanine residue has a significantly weaker affinity for the calcium channel than ω-conotoxin GVIA, and is extremely effective for the isolation and purification of N-type calcium channel. The present invention has been achieved. That is, the gist of the present invention resides in a novel peptide having the amino acid sequence represented by the formula (1) in claim 1.

The present invention will be described in detail below. The novel peptide of the present invention has the following formula (1)

[Chemical 2] It consists of the 27 amino acid sequence shown by. However, Hyp in the above amino acid sequence means 4-trans-hydroxyproline.

The peptide of formula (1) can be easily produced by the method described below. The symbols used in the following description are as follows. MBHA resin: p-methylbenzhydrylamine resin; Boc: t-butyloxycarbonyl group; Br-Z: 2-bromobenzyloxycarbonyl group; C
1Z: 2-chlorobenzyloxycarbonyl group; MeBzl: 4-methylbenzyl group; Bzl: benzyl group; Tos: p-toluenesulfonyl group.

To produce the peptide of the present invention, a well-known solid phase method for peptide synthesis is applied. For example, MBHA
The resin was set in an automatic peptide synthesizer, and Boc-Tyr (Br-Z) -OH, Boc-Cys (MeBzl) -OH, Boc-Arg that had been manufactured in advance were set on the resin.
(Tos) -OH, Boc-Lys (Cl-Z) -OH, Boc-Thr (Bzl) -OH, Boc-T
yr (Br-Z) -OH, Boc-Hyp (Bzl) -OH, Boc-Asn-OH, Boc-Cys
(MeBzl) -OH, Boc-Ser (Bzl) -OH, Boc-Arg (Tos) -OH, Boc-
Cys (MeBzl) -OH, Boc-Cys (MeBzl) -OH, Boc-Asn-OH, Boc-
Tyr (Br-Z) -OH, Boc-Ser (Bzl) -OH, Boc-Thr (Bzl) -OH, Bo
c-Hyp (Bzl) -OH, Boc-Ser (Bzl) -OH, Boc-Cys (MeBzl) -OH,
Boc-Ser (Bzl) -OH, Boc-Ser (Bzl) -OH, Boc-Gly-OH, Boc
-Hyp (Bzl) -OH, Boc-Ser (Bzl) -OH, Boc-Ala-OH, Boc-Cys
(MeBzl) -OH is supplied to cause sequential condensation.

Next, the resulting side chain is protected with H-
Cys (MeBzl) -Ala-Ser (Bzl) -Hyp (Bzl) -Gly-Ser (Bzl) -Ser
(Bzl) -Cys (MeBzl) -Ser (Bzl) -Hyp (Bzl) -Thr (Bzl) -Ser (Bz
l) -Tyr (Br-Z) -Asn-Cys (MeBzl) -Cys (MeBzl) -Arg (Tos) -Se
r (Bzl) -Cys (MeBzl) -Asn-Hyp (Bzl) -Tyr (Br-Z) -Thr (Bzl)-
Lys (Cl-Z) -Arg (Tos) -Cys (MeBzl) -Tyr (Br-Z) -MBHA resin was reacted with fluorine to remove all side chain protecting groups and the peptide was treated with MBHA resin. It can be separated and air-oxidized.

The peptide of the present invention is compared with ω-conotoxin GVIA, as will be specifically described in Examples below,
It has a distinctly weak binding activity for calcium channels. For example, ω-conotoxin GVIA suppresses the binding of ¹²⁵ I-labeled ω-conotoxin GVIA to calcium channels contained in chicken brain synaptic membranes at a concentration of 1 nM or less, whereas the peptide of the present invention has a similar inhibitory effect. About 40 times higher concentration is needed to show.

[0011]

EXAMPLES Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples as long as the gist thereof is not exceeded.

[Production of peptide] (b) H-Cys (MeBzl) -Ala-Ser (Bzl) -Hyp (Bzl) -Gly-Ser (Bz
l) -Ser (Bzl) -Cys (MeBzl) -Ser (Bzl) -Hyp (Bzl) -Thr (Bzl)-
Ser (Bzl) -Tyr (Br-Z) -Asn-Cys (MeBzl) -Cys (MeBzl) -Arg (T
os) -Ser (Bzl) -Cys (MeBzl) -Asn-Hyp (Bzl) -Tyr (Br-Z) -Thr
(Bzl) -Lys (Cl-Z) -Arg (Tos) -Cys (MeBzl) -Tyr (Br-Z) -MBHA
Resin production:

MBHA resin 0.79 g (amine content 0.76 mmol / g
Resin) was set in a Biosearch 9500 type automatic peptide synthesizer, and Boc-Tyr (Br-Z) -OH, Boc-Cys (MeBzl) -O.
H, Boc-Arg (Tos) -OH, Boc-Lys (Cl-Z) -OH, Boc-Thr (Bzl)
-OH, Boc-Tyr (Br-Z) -OH, Boc-Hyp (Bzl) -OH, Boc-Asn-O
H, Boc-Cys (MeBzl) -OH, Boc-Ser (Bzl) -OH, Boc-Arg (To
s) -OH, Boc-Cys (MeBzl) -OH, Boc-Cys (MeBzl) -OH, Boc-A
sn-OH, Boc-Tyr (Br-Z) -OH, Boc-Ser (Bzl) -OH, Boc-Thr (B
zl) -OH, Boc-Hyp (Bzl) -OH, Boc-Ser (Bzl) -OH, Boc-Cys
(MeBzl) -OH, Boc-Ser (Bzl) -OH, Boc-Ser (Bzl) -OH, Boc-
Gly-OH, Boc-Hyp (Bzl) -OH, Boc-Ser (Bzl) -OH, Boc-Ala-
OH and Boc-Cys (MeBzl) -OH were supplied and sequentially condensed to obtain 4.24 g of the above side chain protected peptide-MBHA resin.

(B) Fluorine fluoride treatment: 2.12 g of the side chain protected peptide-MBHA resin obtained in (a) above was sampled and used as a fluorine fluoride reactor manufactured by Protein Research Institute Peptide Research Institute. The reaction mixture was set and reacted with 21 ml of fluorine fluoride in the presence of 3.2 ml of anisole at 0 ° C. for 1 hour. After the reaction was completed, the fluorine fluoride was distilled off under reduced pressure, the residue was washed with ethyl acetate, and then 2M acetic acid was added.
Extraction with 150 ml gave a chain peptide solution.

(C) Disulfide bond formation by air oxidation The solution of the chain peptide obtained in (b) above was diluted to 550 ml with distilled water. Ammonia water is added to this to adjust the pH of the solution to 7.8.
And distilled water was added so that the total volume became 600 ml. This solution was stirred at 5 ° C for 48 hours, pH was adjusted to 4 by adding acetic acid, insoluble matter was filtered off, and freeze-drying was repeated to obtain the cyclic crude compound represented by the above formula (1). 437 mg of peptide was obtained.

(D) Purification of peptide: The crude peptide obtained in (c) above was dissolved in 20 ml of 30% acetic acid to give Sephadex G-50F.
The column (internal diameter 5 cm, length 110 cm) was applied to the column and eluted with the same solvent, and fractions containing the target substance were collected. The yield of this partially purified product was 96 mg. 40 mg of it was dissolved in 5 ml of distilled water, and HPS using a reversed-phase column (inner diameter 2 cm, length 25 cm) in which ODS (octadecylsilane) was bound to silica was used.
Purified by C. Elution was performed by increasing the acetonitrile concentration in 0.1% trifluoroacetic acid from 13% to 16% over 30 minutes.

By the above operation, the final target formula
28 mg of the cyclic purified peptide represented by (1) was obtained. The structure of this substance was confirmed by FAB-MS and amino acid analysis. FAB-MS [M + H] ⁺ 2980, calculated (C ₁₁₇ H ₁₇₅ N ₃₇ O ₄₃ S ₆ + H) 298
1; Asp 1.68 (2), Ser 5.79 (6), Thr1.90 (2), Arg 1.85 (2), G
ly 1.00 (1), Ala 1.05 (1), (Cys) ₂ 2.27 (3), Lys 1.04 (1),
Tyr 3.00 (3)

[Measurement of Binding Activity to Calcium Channel] The binding activity of the peptide of the present invention and ω-conotoxin GVIA to the calcium channel was measured by binding ω-conotoxin GVIA labeled with ¹²⁵ I to the calcium channel contained in the chicken synapse membrane. Were compared by examining the inhibitory effect against Synaptic membranes prepared from chicken brain were suspended in 20 mM Hepes / Tris buffer (pH 7.3) at a protein concentration of 0.8 mg / ml, and various concentrations of ω
-Conotoxin GVIA and the peptide of the present invention were added separately and left at 4 ° C for 15 minutes.

Then, ¹²⁵ I-labeled ω-conotoxin GVIA was added to each buffer to a concentration of 0.2 nM, and the mixture was allowed to stand at 4 ° C. for 90 minutes. Then, using a filter paper made of glass fibers to separate synaptic membranes from the buffer solution, bound to calcium channels contained in the synaptic membrane ¹²⁵
The amount of I-labeled ω-conotoxin GVIA was measured by a γ counter. The result is shown in FIG. The vertical axis of Fig. 1 is ¹²⁵
The binding amount [count / min (cpm)] of the ω-conotoxin GVIA labeled with I to the calcium channel is shown, and the horizontal axis shows the peptide concentration [mol (Mol)].

In FIG. 1,  marks indicate ω-conotoxin labeled with ¹²⁵ I in the peptide solutions of the present invention at various concentrations.
The amount of GVIA bound is shown, and the circle indicates the same concentration of ω-conotoxin G.
Ω-conotoxin G labeled with ¹²⁵ I in VIA solution
The amount of VIA bound is shown. As shown in FIG. 1, both ω-conotoxin GVIA and the peptide of the present invention inhibited the binding of ¹²⁵ I-labeled ω-conotoxin GVIA to the calcium channel in a concentration-dependent manner. A difference was observed.

That is, ω-conotoxin GV labeled with ¹²⁵ I
Ω-conotoxin GVIA to suppress half the binding of IA
For example, a concentration of 0.146 nM was sufficient, whereas the peptide of the present invention required a concentration of 5.49 nM, which was about 40 times higher. This fact shows that the affinity of the peptide of the present invention for the calcium channel is clearly weaker than that of the ω-conotoxin GVIA and is extremely effective for the isolation and purification of N-type calcium channel.

[0022]

INDUSTRIAL APPLICABILITY The peptide of the present invention has an appropriate affinity for calcium channels, and is therefore extremely useful as a reagent for isolation and purification of calcium channels, which greatly contributes to the elucidation of calcium channel functions. It is expected.

[Brief description of drawings]

FIG. 1 shows the concentrations of solutions of the peptide of the present invention and a known peptide (ω-conotoxin GVIA), and the concentrations in these solutions.
FIG. 3 is a diagram showing the relationship with the binding amount of ω-conotoxin GVIA labeled with ¹²⁵ I to calcium channels.

[Explanation of symbols]

●: ^{125 in} solution of various concentrations of the peptide of the present invention
Binding amount of ω-conotoxin GVIA labeled with I. ○: ω-conotoxin GVIA at the same concentration as the peptide of the present invention
Ω-conotoxin GVIA labeled with ¹²⁵ I in solution
Binding amount of.

[Sequence list]

SEQ ID NO: 1 Sequence length: 27 Sequence type: Amino acid Topology-: Cyclic Sequence type: Peptide Sequence: Cys Ala Ser Hyp Gly Ser Ser Cys Ser
Hyp Thr Ser Tyr Asn Cys Cys Arg Ser 15
10 15 Cys Asn Hyp Tyr Thr Lys Arg Cys Tyr 20 25

Claims (1)

[Claims] 1. The following formula (1): A novel peptide having an amino acid sequence represented by: