KR20110009451A - Calcitonin-like peptide having relaxing activity of smooth muscle - Google Patents

Abstract

PURPOSE: A novel calcitonin-relating peptide(SF-calcitonin) with relaxation activity is provided to ensure relaxation activity to DRM(dorsal retractor muscle). CONSTITUTION: A novel calcitonin-relating peptide(SF-CT) with relaxation activity of smooth muscle has an amino acid sequence of sequence number 1. SF-CT is isolated from Asterina pectinifera. SF-CT is obtained by preparing methanol-acid extract from Asterina pectinifera, purifying by CM52 anion exchange resin column chromatography, and isolating through HPLC system. SF-CT has homology with calcitonin.

Description

Calcitonin-related peptide having smooth muscle relaxation activity {Calcitonin-like Peptide Having Relaxing Activity of Smooth Muscle}

The present invention relates to a novel peptide having a relaxing activity isolated from the dermal animal, and more particularly to a novel calcitonin-related peptide exhibiting smooth muscle relaxation activity having the amino acid sequence of SEQ ID NO: 1.

Calcitonin is a thyroid hormone that regulates the amount of calcium in the blood in the endocrine system of vertebrates. It is a polypeptide consisting of 32 amino acids secreted from thyroid C cells. It is connected to each other by a disulfied bond, and C-terminal proline (Pro) is amide (-NH ₂ ). Calcitonin is a hormone that acts to lower the amount of calcium in the blood when it is higher than normal. It acts as opposed to parathyroid hormone, which raises calcium in the blood, but they are all hormones that regulate calcium metabolism.

Calcitonin has been found in various kinds of vertebrates, and there is a difference in the degree of physiological activity and amino acid sequence according to species. When humans and rats are tested to reduce calcium concentrations, it is found in salmon, eel and chicken. Calcitonin was found to be highly potent. In particular, since calcitonin of salmon exhibits strong activity against the human body, it is currently used more widely than human calcitonin for treating osteoporosis and medicine. Salmon calcitonin has 20-30 times stronger activity to lower calcium concentration than human calcitonin, but has a side effect that requires a continuous increase in dose due to antigenicity to the living body and its neutralizing effect upon administration. It also adversely affects the digestive tract, causing vomiting and nausea, leading to weight loss and avoidance of treatment. Human calcitonin, on the other hand, has no antigenicity and adverse effects on the digestive tract, but very low compared to salmon calcitonin but is unsuitable for use in therapy. In order to supplement the above limitations of salmon and human calcitonin, they are currently being used as a medicine by mixing them.

On the other hand, the muscle physiology of echinoderm, which is recognized as an evolutionary species more closely related to vertebrates than mollusks, is thought to be more similar to that of vertebrates than that of mollusks, but studies using them are insufficient. Recently, extracts from eukaryotic animals have been identified to induce relaxation in vitro.However , studies on neurotransmitters in eukaryotic animals have been conducted using basic neurotransmitters such as acetylcholine, catecholamine and GABA. Limited to active studies. In addition, most of the starfish used as experimental animals in the present invention have been disposed of, and studies on the verification of the active effects on physiologically active substances or their association with separation, purification and structural activity have been extremely limited.

Accordingly, the present inventors earnestly tried to isolate a new peptide having smooth muscle relaxation activity against starfish, a marine organism, and obtained methanol-acid extract from Asterina pectinifera , an epidermal animal, and extracted the CM- After adjusting by cellulose column chromatography, a novel calcitonin-related peptide having smooth muscle relaxation activity was isolated through an HPLC system to complete the present invention.

The main object of the present invention is to provide a novel calcitonin-related peptide having smooth muscle relaxation activity.

In order to achieve the above object, the present invention provides a peptide having a smooth muscle relaxation activity having the amino acid sequence of SEQ ID NO: 1.

The present invention has the effect of providing a novel calcitonin-related peptide having smooth muscle relaxation activity.

In one aspect, the present invention relates to a peptide having smooth muscle relaxation activity having the amino acid sequence of SEQ ID NO: 1.

In the present invention, the peptide may be characterized in that the star star ( Asterina pectinifera ) derived from.

As a result of the study on the association between calcitonin and muscle relaxation activity of the present invention, a study confirming that salmon calcitonin is associated with metabolism of skeletal muscle (Kenney MA et al., Biochem Biophys Res Commun., 30 ; 197 (1): 8-14, 1993, Nov), and studies confirming that calcitonin exhibits a strong relaxation effect on the gallbladder of humans (Jonderko K, J et al., Gastroenterol Hepatol., 4 (6) : 505-11, 1989 , Nov-Dec).

In the present invention, the peptide described in SEQ ID NO: 1 obtains a methanol-acid extract from Asterina pectinifera , which is an dermal animal, and adjusts the extract by CM 52 cation exchange resin column chromatography, followed by an HPLC system. Separated. The isolated peptide is a novel calcitonin-related peptide having a relaxation activity, and after passing through the HPLC system, a 'MALDI-TOF mass spectrometer (Voyager-DE ^TM STR spectrometer, Perseptive Biosystems, USA)' and 'automated protein sequencer PPSQ-21A (Shimadzu, Ltd., Japan) 'amino acid sequence was determined (SEQ ID NO: 1). In addition, the dorsal retractor muscle (DRM) of the starfish was used to measure activity. The primary structure of the peptide of SEQ ID NO: 1 is homologous to the protein sequence previously identified using GenBankTM / EMBL Data Bank, and has homology with calcitonin, a substance that plays a role in calcium regulation in vivo. It was confirmed.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following Examples only describe the present invention in detail and do not limit the present invention by these Examples.

Example 1 Extraction of Relaxing Peptide Fraction from Starfish

The starfish used in this example were starfish ( Asterina pectinifera ), which was collected from Cape Homi Pohang, Gyeongbuk, and collected 450 starfish with a length of 8-15 cm and immediately used for extraction.

450 live starfish were placed in a 70% methanol solution and bathed at 65 ° C for 5 minutes. After 5% acetic acid was added to crush the tissue, the supernatant was concentrated by centrifugation (4 ° C., 40 minutes, 10,000 × g), and ethanol was added so that the ratio of sample to ethanol was 2: 9 (v / v). The precipitate was removed by freezing at -20 ° C. and then centrifuged (4 ° C., 40 min, 10,000 × g). The separated supernatant was concentrated again and ethanol was added so that the ratio of sample to ethanol was 1:10 (v / v), followed by centrifugation (4 ° C, 40 minutes, 10,000 xg) to remove the precipitate once more. . Supernatant obtained by adding ethanol and NaCl in the ratio of sample (1 L): ethanol (8 L): NaCl (11 g) to this supernatant after centrifugation (4 ° C., 40 minutes, 10,000 × g). After the solution was concentrated, 1 N HCl was added so that the total concentration was 0.1 N HCl, followed by centrifugation (4 ° C., 50 minutes, 20,000 × g).

The supernatant was partially injected into Sep-Pak C ₁₈ cartridge (20 mL, Waters) to separate the material, followed by H ₂ O (DW), 10% methanol (retained materials; RM10) and 60 containing 0.1% TFA. The materials were eluted with% methanol (RM60) and 100% methanol (RM100) to obtain DW fraction, RM10 fraction, RM 60 fraction and RM 100 fraction, respectively.

Example 2. Determination of the Relaxation Activity of Starfish Fraction on Starfish DRM

The relaxation activity of the RM10, RM60, and RM 100 fractions extracted in Example 1 was measured using a dorsal retractor muscle (DRM) of Asterina pectinifera .

In order to sample the starfish DRM, the starfish was removed from the eye spot, the DRM across the center was separated with a scalpel, and cut into 20 mm fragments. Each of the prepared fragments was fixed to a support in the reactor, and the upper part was connected to an isometric transducer. After 1 g of tension was applied to the connected tissue, equilibration was performed by replacing artificial seawater (55 mM Mg ²⁺ artificial sea water) every 15 minutes at room temperature. Thereafter, 10 ^-6 M acetylcholine was activated to activate, and after 15 minutes, ^10-6 M acetylcholine was administered to contract the muscles, and the fractions obtained in each HPLC purification step were administered. All procedures were performed under artificial seawater (NaCl 445 mM, KCl 10 mM, CaCl ₂ · 2H ₂ O 10 mM, MgCl ₂ · 6H ₂ O 55 mM, Glucose 10 mM and Tris-HCl (pH 7.8) 20 mM). As a result, smooth muscle relaxation activity was confirmed in RM60 aliquots (FIG. 1).

Example 3. Isolation and Purification of Relaxing Peptides from RM60 Fractions

Starfish extract RM60 fractions confirmed in Example 2 were separated through six consecutive HPLC steps using different columns. The fractions obtained through each HPLC purification were measured for the relaxation activity against the starfish DRM in the same manner as in Example 2, and the fractions showing the activity were concentrated and used for the next step HPLC purification.

① CM52 column : Concentration gradient using CM52 (2.5 × 30 cm) column, 20 mM → 1.5 M ammonium acetate (pH 5.0, 6 hours); Isolate under flow rate, 2.75 mL / min. Relaxation activity was observed in fractions near retention time 40-45 minutes (FIG. 2).

② C ₁₈ column : The concentrated active fraction was applied to a reversed phase HPLC column (Vydac protein & peptide C ₁₈ , 9.2 × 250 mm), containing 0.1% aqueous TFA solution (AH 2.2) as solvent A and 0.1% TFA as solvent B. Concentration was gradient with 0% to 100% B solvent (200 min) using 100% acetonitrile (pH 2.2), and the flow rate was separated at 40 ° C at 3.0 mL / min. Relaxation activity was observed in fractions near retention time 70 minutes (FIG. 3).

③ anion exchange column : purified using TSK-gel DEAE-5PW (7.5 × 75 mm) column, solvent A, 10 mM Tris-HCl buffer (pH 8.8); 10 mM Tris-HCl buffer (pH 8.8) containing B solvent, 1.0 M NaCl; Concentration gradient, 0 → 0.5 MB solvent (100 min), flow rate; 0.5 mL / min, temperature; 40 ° C .; Fraction volume (fraction volume), was carried out under the condition of 1.0 mL. Relaxation activity was observed in fractions near retention time 5 minutes (FIG. 4).

④ C ₁₈ column : Hypersil-BDS C ₁₈ (2 × 125 mm) was used, which is different from the previous phase reversed column. At this time, the concentration gradient of the solvent B, 20 → 40% was separated at 40 ℃ at a flow rate of 0.5 mL / min for 60 minutes, showed activity in the portion corresponding to 26% acetonitrile (acetonitrile) (Fig. 5).

Cation exchange column : purified using TSK-gel SP-5PW (7.5 × 75 mm) column, solvent A, 10 mM phosphate buffer solution (pH 6.0); 10 mM phosphate buffer solution (pH 6.0) containing B solvent, 1.0 M NaCl; Concentration gradient, 0 → 0.5 MB solvent (100 min), flow rate; 0.5 mL / min; Temperature; 40 ° C .; Fraction volume (fraction volume), was carried out under the condition of 1.0 mL. Relaxation activity was observed in fractions near retention time 20-25 minutes (FIG. 6).

⑥ Hypersil-BDS C ₁₈ column : As a sixth process, Hypersil-BDS C ₁₈ (2 × 125 mm) was used. At this time, the concentration gradient of solvent B, 25 → 30%, was separated at 40 ° C. at a flow rate of 0.5 mL / min for 50 minutes (Data not shown), and finally under an isocratic condition of 25% acetonitrile. Purification was done using a Hypersil-BDS C ₁₈ (2 × 125 mm) column. As a result, one pure material with a retention time of 22.93 minutes was finally purified (FIG. 7A).

Example 4 Determination of Molecular Weight and Amino Acid Sequence of Natural Peptides

The molecular weight of the peptide finally purified from the starfish and having relaxation activity in DRM was measured using a MALDI-TOF mass spectrometer (Voyager-DETM STR spectrometer, Perseptive Biosystems, U.S.A). The amino acid sequence was determined by Edman digestion using an Automated protein sequencer PPSQ-21A (Shimadzu, Ltd., Japan).

The molecular weight of the final purified peptide was 3655 Da (Fig. 7b), and the primary sequence was analyzed by Edman decomposition to determine the primary structure of the substance. As a result, two Met and two aromatic amino acids, Phe and (+) It was identified as a peptide consisting of 20 amino acids, including 1 Lys with charge, 1 Arg, 2 Asp with negative charge and 1 Glu. In particular, this material is characterized by having Lys-Asp and Arg-Glu, which can form salt bridges at the N-terminus and C-terminus, respectively, whose primary sequence is as follows; Ser-Gly-Thr-Gly-Cys-Thr-Gln-Phe-Ser-Gly-Cys-Ala-Gln-Leu-Lys-Val-Gly-Gln-Asp-Ala-Leu-Ser-Arg-Val-Leu- Ala-Asp-Ser-Asn-Ser-Arg-Phe-Gly-Ser-Gly-Gly-Pro (SEQ ID NO: 3).

On the other hand, according to the Edman decomposition method, the 5th and 11th residues of the N-terminal side were not detected, but based on the amino acid sequence analysis and the molecular weight value, it was found that the two Cys match the molecular weight of the form of SS bond. Could know. That is, the molecular weight of the purified material was 3655 Da, but the sum of the molecular weight of the amino acid except the two undetected Xaa was 3453.70 Da, it was found that the molecular weight difference between them is 201.3 Da. Here, since one Cys amino acid residue amount is 103.1 Da, two Cys becomes 206.2 Da, and SS bond is formed between them, so when two hydrogens are released, it becomes 204.2 Da, which finally corresponds to the molecular weight of amino acid not detected at 3453.70 Da If we add 204.2 Da, it is equivalent to 3657.90 Da. Thus, it could be estimated that the fifth and eleventh residues, two undetected Xaa, were Cys.

In addition, the primary structure of this peptide was examined for homology with previously known protein sequences using the GenBankTM / EMBL Data Bank, and showed homology with calcitonin, a substance that plays a role in calcium regulation in vivo. It was confirmed. These calcitonins have a common region in the molecule, that is, the first and seventh residues are Cys-linked to SS and the last residue is Pro-NH ₂ , but the peptides of the present invention are also composed of the same amino acids. In addition, the peptide of the present invention is characterized in that it has four more amino acids at the N-terminal region than other calcitonins so far known (Table 1, human: SEQ ID NO: 1, salmon: SEQ ID NO: 2, starfish calcitonin: SEQ ID NO: 3). Primary structure comparison), named as SF-CT (SF-Calcitonin) for convenience.

Human C G N L S T C M L G T Y T Q D - F N K F H T F P Q T A I G V G A P NH ₂ Salmon C S N L S T C V L G K L S Q E - L H K L Q T Y P R T N T G S G T P NH ₂ Starfish S G T G C T Q F S G C A Q L K V G Q D A L S R V L A D S N S R F G S G G P NH ₂

Example 5 Measurement of Relaxation Activity on Starfish DRM of SF-CT

Relaxation activity of SF-CT purified from starfish was measured using DRM of starfish in the same manner as in Example 2, SF-CT was cumulatively administered from 10 ^-12 M to 10 ^-5 M Activity is expressed as relative relaxation% to maximal contraction of ^10-6 M acetylcholine. On the other hand, the magnitude of the response was compared as ED ₅₀ (concentration when representing 50% of the highest relaxation) and E _max (% relaxation at 10 ⁻⁵ M). ED ₅₀ values were calculated using the least square method, and statistical processing was done by student's t-test. All response values are expressed as means ± se.

As a result, as shown in FIG. 8, SF-CT had a threshold value of 4.5% at 10 ⁻¹¹ M and showed a relaxation activity of 87.4 ± 6.4% at 10 ⁻⁵ M. FIG.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Figure 1 shows the results of the relaxation activity measurement for the starfish DRM (dorsal retractor muscle) of the four fractions separated from the starfish using a Sep-Pak C ₁₈ column.

Figure 2 shows the relaxation activity for CM52 cation exchange resin chromatogram and starfish DRM (■ indicates the active fraction).

Figure 3 shows the relaxation activity against reverse phase HPLC chromatogram and starfish DRM of the active fraction of Figure 2 (■ represents the active fraction).

Figure 4 shows the relaxation activity for the anion exchange resin chromatogram and starfish DRM of the active fraction of Figure 3 (■ represents the active fraction).

Figure 5 shows the relaxation activity against the reverse phase HPLC chromatogram and starfish DRM of the active fraction of Figure 4 (■ represents the active fraction).

Figure 6 shows the relaxation activity against reverse phase HPLC chromatogram and starfish DRM of the active fraction of Figure 5 (■ indicates the active fraction).

Figure 7a shows the relaxation activity for the starfish DRM of the last purification step of the active fraction of Figure 6, Figure 7b shows the MALDI-TOF Mass results of the active peak.

8 is a graph showing the concentration-dependent relaxation activity measurement results for starfish DRM of SF-CT (●) human-calcitonin (H-CT) and salmon-calcitonin (S-CT).

<110> Pukyong National University Industry-University Cooperation Foundation <120> Calcitonin-like Peptide Having Relaxing Activity of Smooth Muscle <130> P09-B113 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 32 <212> PRT <213> Human <400> 1 Cys Gly Asn Leu Ser Thr Cys Met Leu Gly Thr Tyr Thr Gln Asp Phe   1 5 10 15 Asn Lys Phe His Thr Phe Pro Gln Thr Ala Ile Gly Val Gly Ala Pro              20 25 30 <210> 2 <211> 32 <212> PRT <213> Salmon <400> 2 Cys Ser Asn Leu Ser Thr Cys Val Leu Gly Lys Leu Ser Gln Glu Leu   1 5 10 15 His Lys Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Gly Thr Pro              20 25 30 <210> 3 <211> 37 <212> PRT <213> Starfish <400> 3 Ser Gly Thr Gly Cys Thr Gln Phe Ser Gly Cys Ala Gln Leu Lys Val   1 5 10 15 Gly Gln Asp Ala Leu Ser Arg Val Leu Ala Asp Ser Asn Ser Arg Phe              20 25 30 Gly Ser Gly Gly Pro          35  

Claims (2)

Peptide having a smooth muscle relaxation activity having the amino acid sequence of SEQ ID NO: 1. The peptide of claim 1, wherein the peptide is derived from Asterina pectinifera .

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