JP3160013B2 - New peptide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel peptide. More specifically, the present invention relates to a novel peptide having a neurotrophic factor-like activity.

[0002]

2. Description of the Related Art The brain is composed of many neurons and glial cells, which interact to express complex functions.
Have maintained. One of the important factors involved in such neuron differentiation and its function and survival is neurotrophic factor.
(neurotrophic factor). Recently, in the central nervous system, nerve growth factor (NGF)
And several neurotrophic factors constituting the family have been discovered (Nerve Advancement, Vol. 34, No. 4, 515-52).
3 pages, August 1990). However, the neurons that act on them are still limited, and it is expected that many unknown factors exist and act when considering the function of the brain composed of various types of neurons. .

[0003]

DISCLOSURE OF THE INVENTION The present invention aims to clarify the mechanism of the function development, maintenance and repair of the brain, and as a part thereof, seeks for a new neurotrophic factor and examines its structure and physiological role. And to provide a novel substance expected to be used for the treatment of cranial nerve diseases.

[0004]

Means for Solving the Problems In order to solve the above problems, the present inventors have collected a protein exhibiting a neurotrophic factor-like activity from an injured site in a rat brain, and further decomposed the protein to obtain a strong nerve. The present inventors have found a novel peptide having trophic factor-like activity and have achieved the present invention. That is, the gist of the present invention is:
A novel polypeptide having the amino acid sequence represented by the formula (1) according to claim 1 and having a neurotrophic factor-like activity.
A further gist is that the amino represented by the above formula (1)
A novel polypeptide consisting of an acid sequence .

Hereinafter, the present invention will be described in detail. The novel peptide of the present invention comprises the amino acid sequence of 13 represented by the formula (1) in claim 1 and can be obtained by the method described below. For example, secretions exuding from the damaged site of the rat frontal cortex in a newborn rat are collected, DF medium (tissue culture medium) is added thereto, homogenized, centrifuged, and the supernatant is fractionated with ammonium sulfate. An active fraction exhibiting a neurotrophic factor-like activity is collected. This active fraction was subjected to gel filtration,
Subsequently, by purifying with an ion-exchange resin, a single band having a molecular weight of about 1.1 showing a single band by slab gel electrophoresis of sodium dodecyl sulfate (SDS) -polyacrylamide.
50,000 proteins with neurotrophic factor-like activity are obtained.

[0006] The protein obtained by the above method is decomposed with a protease, and reverse-phase high performance liquid chromatography (H
The peak of the peptide which is separated by PLC) and has a high absorbance at 215 nm is analyzed for its amino acid sequence using a protein sequencer. Formula (1) showing strong neurotrophic factor-like activity from the peptide thus obtained

[0007]

Embedded image

A peptide of the present invention having the amino acid sequence represented by The above peptide can be easily produced by the method described below. The symbols used in the following description indicate the following, respectively. MBHA resin: p-methylbenzhydrylamine resin; Boc: t-butyloxycarbonyl group; Bzl: benzyl group; Tos: p-toluenesulfonyl group.

To produce the peptide of the present invention, a well-known synthesis method using a solid phase method is applied. For example, an MBHA resin is set in an automatic peptide synthesizer, and Boc-Ala-OH, Boc-Gln-OH, Boc-Phe-OH, Boc-Ile-OH, Boc-
Ala-OH, Boc-Gly-OH, Boc-Arg (Tos) -OH, Boc-Ala-OH, Boc-L
eu-OH, Boc-Glu (OBzl) -OH, Boc-Leu-OH, Boc-Ala-OH, Boc-G
Lu (OBzl) -OH is supplied to sequentially condense. Then, the resulting side chain is protected H-Glu (OBzl) -Ala-Leu-Glu (OB
zl) -Leu-Ala-Arg (Tos) -Gly-Ala-Ile-Phe-Gln-Ala-MBHA
The resin may be reacted with hydrogen fluoride to remove all side chain protecting groups and the peptide may be cleaved from the MBHA resin.

[0010] The peptide of the present invention exhibits extremely excellent neurotrophic factor-like activity, as specifically described in the Examples below. For example, using a primary culture system of the septum corinergic neuron in the postnatal rat brain, the survival maintenance effect of neuron by the addition of the peptide of the present invention, the choline acetyl transferase (ChAT) activity. When measured as an indicator, the activity was significantly higher than when no peptide was added.
Ron is confirmed to be present. In addition, the corina
Zick neuron is a neuron using acetylcholine as a neurotransmitter, and ChAT activity is specific to corina-gic neuron.

In addition, using a primary culture system of midbrain catecholamine neuron of postnatal rats, the survival maintaining effect of the neuron by the addition of the peptide of the present invention is measured using the content of catecholamine as an index. As a result, a much larger amount of catecholamine was measured as compared with the case where no peptide was added, and it was confirmed that many catecholamine neurons were alive. Note that catecholamine neuron is a neuron using catecholamine as a neurotransmitter.

[0012]

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 unless it exceeds the gist.

Example 1 [Peptide Isolation] A 2 mm square hole was made in the frontal cortex of a 3 day old rat, and gelform (insoluble gelatin spongel, manufactured by Yamanouchi Pharmaceutical Co., Ltd.) was implanted for one week. Thereafter, the gel foam was taken out together with the peripheral tissues, immediately frozen on dry ice and stored at -80 ° C. Next, 10 times (weight) of ice-cooled DF medium [DME: F12 = 1: 1 by Gibco] was added to the gel form.
And homogenized at 4 ° C at 350,000 rpm
The mixture was ultracentrifuged at 60 m for 60 minutes, and the supernatant was subjected to 30-60% saturated ammonium sulfate fractionation.

The 30-60% saturated precipitate, which is the most active fraction, is dissolved in a sodium phosphate buffer, and the FPLC system (Pharmacia) using a Superrose 12 column is used. ), The obtained active fraction was applied to an ion exchange resin (Pharmacia Mono S) equilibrated with an acetate buffer (pH 5.0), and the non-adsorbed fraction was collected. A protein having a molecular weight of about 15,000 and exhibiting neurotrophic factor-like activity by electrophoresis was obtained. This protein is digested with trypsin and
A peptide peak that was separated by PLC and showed high absorbance at 215 nm was collected from Applied Biosystems Co., Ltd. 4
The amino acid sequence was analyzed using a 70A type gas phase protein sequencer. From the peptide thus obtained, a peptide of the present invention having an amino acid sequence represented by the above formula (1) exhibiting a strong neurotrophic factor-like activity was obtained.

[Production of peptide] (a) H-Glu (OBzl) -Ala-Leu-Glu (OBzl) -Leu-Ala-Arg (To
s) Production of -Gly-Ala-Ile-Phe-Gln-Ala-MBHA resin: 0.94 g of MBHA resin (amine content: 0.64 mmol / g resin) was set in a 9500 type automatic peptide synthesizer manufactured by Biosearch. Boc-Ala-OH, Boc-Gln-OH, Boc-Phe-OH, Boc
-Ile-OH, Boc-Ala, Boc-Gly-OH, Boc-Arg (Tos) -OH, Boc
-Ala-OH, Boc-Leu-OH, Boc-Glu (OBzl) -OH, Boc-Leu-OH,
Boc-Ala-OH and Boc-Glu (OBzl) -OH were supplied and sequentially condensed to obtain 2.15 g of the side chain-protected peptide-MBHA resin described above.

(B) Hydrofluoric acid treatment: The side chain-protected peptide obtained in the above (a) —1 in the MBHA resin.
07 g was collected, and this was set in a hydrogen fluoride reaction apparatus manufactured by Peptide Research Institute, Protein Research Promotion Association, and reacted with 11 ml of hydrogen fluoride at 0 ° C. for 1 hour in the presence of 1.6 ml of anisole. After the completion of the reaction, hydrogen fluoride was distilled off under reduced pressure, and the residue was washed with ethyl acetate.
H-Glu-Ala-Leu-Glu-Leu-Ala-Arg-
Crude peptide 3 represented by Gly-Ala-Ile-Phe-Gln-Ala-NH ₂
66 mg were obtained.

(C) Purification of peptide: 30% acetic acid 2
The residue was dissolved in 0 ml and applied to a column of Sephadex G-25 (inner diameter 5 cm, length 100 cm), and eluted with the same solvent to collect a fraction containing the target compound. The yield of this partially purified product was 194 mg. 72mg in it is 5ml
Of 10% acetic acid, and ODS (octadecylsilane) bonded to silica.
Purified by HPLC using 5 cm). Elution is 0.
Performed with 26% acetonitrile in 1% trifluoroacetic acid. The yield of the purified product was 39 mg.

Next, in order to remove the trifluoroacetic acid in the product, the whole amount thereof was dissolved in a small amount of 2M acetic acid, and the column was separated by Sephadex G-10 (inner diameter 1.8 cm, length 100 cm).
And eluted with the same solvent, collecting the fractions containing the peptide, H-Glu-Ala-Leu-Glu-Leu-Ala-Arg-
Peptide 29 represented by Gly-Ala-Ile-Phe-Gln-Ala-NH ₂
mg was obtained. The structure of this substance was confirmed by FAB-MS, amino acid analysis and the like. FAB-MS [M + H] ⁺
1387, calcd _{(C 62 H 102 N 18 O} 18 + H) 1387; G
lu 3.03 (3), Arg 0.98 (1), Gly 1.02
(1), Ala 3.94 (4), Ile 0.98 (1), Leu
2.02 (2), Phe 1.03 (1)

[Measurement of neurotrophic factor-like activity] The neurotrophic factor-like activity of the peptide represented by the formula (1) obtained by the above method was measured by the following two methods. (1) Method using septum: An index of survival of corina-gic neuron in culture using the primary culture system using the septum of rat brain about 2 weeks after birth by the method described below. As a choline acetyl transferase (Ch
AT) Fluctuations in activity were measured.

The septum is removed under a stereoscopic microscope and papain is removed.
(Protease), and this was digested with 5% quasi-fetal bovine serum,
The cells were seeded and cultured on a medium consisting of 5% horse serum, 1% rat serum and 89% DF medium so as to have a density of about 2 × 10 ⁵ cells / cm ² . One day later, the above-mentioned peptide of the present invention was separately added to this medium in an amount shown in the following Table 1, and cultured for 7 days.
M Tris-HCl buffer (pH 7.5) was added to disrupt the cells, and the ChAT activity of the obtained cell extract was determined by phonnam.
(Fonnum) method. For comparison, ChAT activity of a cell extract obtained by culturing using only serum without adding a peptide and treating in the same manner was measured. Each C
The hAT activity was as shown in Table 1.

[0021]

[Table 1]

For culture, 48 well plates (Costar) were used. As is clear from Table 1, ChAT activity when cultured in the presence of the peptide of the present invention is:
It increased almost dose-dependently compared to that when cultured in the absence of peptide, indicating that more corina-gic neurons are alive.

Also, the number of corina-gic neurons in the culture wells was counted by acetylcholine esterase (AchE) staining. The number of corina-gic neurons (AChE) when cultured in the presence of the peptide of the present invention was also determined. As shown in Table 2, the number of active neurons was much higher than that when cultured in the absence of the peptide.

[0024]

[Table 2]

(2) Measurement using the midbrain: Using a primary culture system using the lower midbrain of the rat brain about 2 weeks after birth, the survival of catecholamine neurons in culture was determined by the following method. As an index, the amount of catecholamine in the cell extract was measured.

The lower part of the midbrain was digested with papain in the same manner as in (1) above, and the peptides of the present invention were separately incubated for 7 days in the same serum medium as in (1) in the amounts shown in Table 3 below. After culturing, the cells were disrupted with a 0.2 M perchloric acid solution, and the proteins were removed by centrifugation. When the amount of catecholamine in the cell extract thus obtained was measured using HPLC-ECD (electrochemical detector), a particularly remarkable difference was found in the amount of dopamine. Table 3 shows the amount of dopamine in each case.

[0027]

[Table 3]

Also, the number of dopamine neurons in the culture wells was measured by staining with an anti-tyrosine hydroxylase (TH) antibody. The results also show that the number of catecholamine neurons when cultured in the presence of the peptide ( (Number of TH positive neurons)
Was clearly higher than when cultured in the absence of peptide, as shown in Table 4.

[0029]

[Table 4]

[0030]

As described above, the novel peptide of the present invention acts almost in a dose-dependent manner on the maintenance of the survival of corina-gic neuron, and reduces the ChAT activity of corina-gic neuron in culture. Maintain at a significantly higher level.
It also acts on the maintenance of catecholamine neuron survival in a substantially dose-dependent manner, and maintains a high level of catecholamine neuron in culture at a high level. From the above, the novel peptide of the present invention is expected to be used as a therapeutic drug for cranial nerve dysfunctions such as senile dementia, Alzheimer's dementia, and cranial nerve injury.

[Sequence list]

SEQ ID NO: 1 Sequence length: 13 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Sequence: Glu Ala Leu Glu Leu Ala Arg Gly Ala
Ile Phe Gln Ala 15
10

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) C07K 7/08 BIOSIS (DIALOG) CA (STN) GenBank / EMBL / DDBJ REGISTRY (STN) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. An amino acid sequence represented by the following formula (1):
Novel Polypep Having Novel and Neurotrophic Factor-Like Activity
Chid. Glu-Ala-Leu-Glu-Leu-Ala-Arg-Gly-Ala-Ile-Phe-Gln-Ala (1) Or wherein the amino acid sequence represented by the following formula (1)
A novel polypeptide comprising: Glu-Ala-Leu-Glu-Leu-Ala-Arg-Gly-Ala-Ile-Phe-Gln-Ala (1)