Leech polypeptide and use thereof

Abstract

A leech polypeptide with activity to inhibit macrophage migration in vitro, which is useful in the prevention of atherosclerosis. The preparation method of the leech peptide includes: firstly, a leech zymolyte HE is prepared from Whitmania pigra Whitman, and the amino acid sequence of leech polypeptide HE 4-1 is obtained and synthesized artificially after analysis and sequencing.

Claims

1. A leech polypeptide, having an amino acid sequence as shown in SEQ ID No: 1.

2. The leech polypeptide according to claim 1, wherein the polypeptide is produced by solid-phase polypeptide synthesis.

3. A method for preventing or treating a disease caused by macrophage migration, comprising: administering a leech polypeptide to a subject in need thereof, wherein the leech polypeptide has an amino acid sequence as shown in SEQ ID NO: 1.

4. The method according to claim 3, wherein the polypeptide is produced by solid-phase polypeptide synthesis.

5. The method according to claim 3, wherein the disease is atherosclerosis.

6. A method of preparing a drug by synthesizing the peptide of SEQ ID NO: 1 by solid-phase synthesis, and isolating the peptide and adding a pharmaceutically acceptable excipient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The accompanying drawings constituting a part of the present invention are used for providing further understanding for the present invention. Exemplary embodiments of the present invention and descriptions thereof are used for explaining the present invention and do not constitute an improper limitation to the present invention.

(2) FIG. 1 shows an elution curve by chromatography on Q Sepharose FF strong basic anion column of HE in Example 1 of the present invention.

(3) FIG. 2 shows the effect of HE1-HE6 on the migration activity of macrophages RAW264.7 in Example 1 of the present invention, in which FIG. 2(A) shows the migration result of macrophages RAW264.7 (100×); and FIG. 2(B) shows the statistical result of cell count in the lower chamber. **p<0.01 VS controlgroup, #p<0.05 VS LPS group, and ##p<0.01 VS LPS group.

(4) FIG. 3 shows an elution curve from Superdex30 gel filtration chromatography of HE4 in Example 1 of the present invention.

(5) FIG. 4 shows the effect of RE1-HE6 on the survival rate of macrophages RAW264.7 in Example 1 of the present invention.

(6) FIG. 4(A) shows the migration result of macrophages RAW264.7 after treatment with HE4-1 (100×); FIG. 4(B) shows the statistical result of cell count in the lower chamber after treatment with HE4-1; FIG. 4(C) shows the migration result of macrophages RAW264.7 after treatment with HE4-2 (100×); and FIG. 4(D) shows the statistical result of cell count in the lower chamber after treatment with HE4-2. *p<0.05 VS control group, **p<0.01 VS control group, #p<0.05 VS LPS group.

(7) FIG. 5 shows an elution curve from Superdexpeptide gel filtration chromatography of HE4-1 in Example 1 of the present invention.

(8) FIG. 6 shows an elution curve from G10 gel filtration chromatography of HE4-1 in Example 1 of the present invention.

(9) FIG. 7 shows the effect of HE4-1 on the migration activity of macrophages RAW264.7 in Example 1 of the present invention, in which FIG. 7(A) shows the migration result of macrophages RAW264.7 (100×); and FIG. 7(B) shows the statistical result of cell count in the lower chamber. **p<0.01 VS control group, #p<0.05 VS LPS group.

(10) FIG. 8 is an MALDI-TOF mass spectrum of HE4-1 in Example 2 of the present invention.

(11) FIG. 9 is an LC-MS/MS mass spectrum of HE4-1 in Example 2 of the present invention.

(12) FIG. 10 shows the effect of HE-D on the migration activity of macrophages RAW264.7 in Example 4 of the present invention, in which FIG. 10(A) is a photograph showing the effect of HE-D on the migration activity of macrophages RAW264.7 based on Transwell assay; and FIG. 10(B) is a histogram of statistical data.

(13) FIG. 11 shows the effect of HE-D on JNK and p38 signaling pathways in RAW264.7 cells induced with LPS in Example 5 of the present invention, in which FIG. 11(A) shows the result of Western blot of JNK and p38 signaling pathways; and FIG. 11(B) shows the result of data processing by Image J. *p<0.05 VS LPS group.

(14) FIG. 12 shows the effect of HE-D on the phagocytosis ability and lysozyme secretion ability of RAW264.7 cells in Example 6 of the present invention, in which FIG. 12(A) shows that HE-D has no significant effect on the phagocytosis ability of macrophages; and 12(B) shows that HE-D has no significant effect on the lysozyme secretion ability of macrophages. ***p<0.001 VS control group.

(15) FIG. 13 shows the effect of HE-D on the secretion of IL-1, IL-6, IL-12, and TNF-α by RAW264.7 cells in Example 6 of the present invention, in which FIG. 13(A) shows that HE-D has no significant effect on IL-1 secretion by macrophages; FIG. 13(B) shows that HE-D has no significant effect on IL-6 secretion by macrophages; FIG. 13(C) shows that HE-D has no significant effect on IL-12 secretion by macrophages; and FIG. 13(D) shows that 400 and 800 μg/mL HE-D can promote the secretion of TNF-α. *p<0.05 VS control group, ***p<0.001 VS control group.

DETAILED DESCRIPTION

(16) It should be noted that the following detailed description is exemplary and is intended to provide a further description of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

(17) It should be noted that terms used herein are only for describing specific implementations and are not intended to limit exemplary implementations according to the present invention. As used herein, the singular form is intended to include the plural form, unless the context clearly indicates otherwise. In addition, it should further be understood that terms “comprise” and/or “include” used in this specification indicate that there are features, steps, operations, devices, components, and/or combinations thereof.

(18) As described above, due to the complex composition in Whitmania pigra Whitman, it is difficult to control the quality of medicinal materials, and there are many adverse reactions. The most severe adverse reaction is bleeding, so regular monitoring of blood clotting is needed for patients who have been taking Whitmania pigra Whitman preparations for a long time.

(19) In view of this, in a typical embodiment of the present invention, a leech polypeptide is provided. The main amino acid sequence of the leech polypeptide is EAGSAKELEGDPVAG (SEQ ID NO: 1). The leech polypeptide has the effect of inhibiting the migration of macrophages.

(20) The synthesized leech polypeptide (designated as HE-D) in the present invention has a sequence abbreviated as EAGSAKELEGDPVAG (SEQ ID NO: 1), a molecular weight of 1429.294 Da, and a sequence of Glu-Ala-Gly-Ser-Ala-Lys-Glu-Leu-Glu-Gly-Asp-Pro-Val-Ala-Gly (SEQ ID NO: 1), where:

(21) Glu represents glutamic acid, and is a residue of the amino acid glutamic acid;

(22) Ala represents alanine, and is a residue of the amino acid alanine;

(23) Gly represents glicine, and is a residue of the amino acid glicine;

(24) Ser represents serine, and is a residue of the amino acid serine;

(25) Lys represents lysine, and is a residue of the amino acid lysine;

(26) Leu represents leucine, and is a residue of the amino acid leucine;

(27) Asp represents aspartic acid, and is a residue of the amino acid aspartic acid;

(28) Pro represents proline, and is a residue of the amino acid proline; and

(29) Val represents valine, and is a residue of the amino acid valine.

(30) The polypeptide of the present invention can be artificially synthesized by solid-phase polypeptide synthesis. Specifically, the amino acid sequence is obtained following a standard Fmoc protocol, screening by a resin, and a reasonable method for polypeptide synthesis. The C-terminal carboxyl group of the target polypeptide is covalently linked to an insoluble polymer resin, and then the amino group of this amino acid is used as a starting point to interact with the carboxyl group of another molecule of amino acid to form a peptide bond. This process is repeated continuously to obtain the target polypeptide product. After the synthesis reaction is completed, the protecting group is removed, and the polypeptide chain is separated from the resin to obtain the target product. Polypeptide synthesis is a process of repeatedly adding amino acids, and the solid-phase synthesis is synthesis from the C-terminus to the N-terminus.

(31) It has been proved by experiments that the leech polypeptide HE-D provided in the present invention can exert an inhibitory effect on macrophage migration by regulating the levels of JNK and p38 phosphorylation. HE-D has basically no effect on the biological functions of macrophages at the same time of inhibiting the migration of macrophages, which can effectively reduce the adverse reactions caused by HE-D. Therefore, synthetic leech polypeptide HE-D can be used for the early prevention of atherosclerosis and has the potential to prevent the further development of AS.

(32) In a further specific embodiment of the present invention, a leech polypeptide fragment is provided, which has a main amino acid sequence that is ≥70% identical and ≥90% similar to the main amino acid sequence of above-mentioned leech polypeptide, and is a leech polypeptide comprising 3 or 4 or 5 amino acids truncated from any position of the 15-amino-acid sequence and having the same biological activity as the leech polypeptide.

(33) In a further specific embodiment of the present invention, a leech polypeptide analogue inhibiting macrophage migration is provided, which has the same biological activity as the leech polypeptide. The analogue is a biologically active polypeptide sequence or protein formed by fusing the leech polypeptide to another compound or fusing the amino acid sequence of the leech polypeptide to another polypeptide or protein.

(34) In a further specific embodiment of the present invention, a leech polypeptide derivative inhibiting macrophage migration is provided, which has an amino acid sequence that is ≥70% identical and ≥90% similar to the main amino acid sequence of the leech polypeptide. The derivative is a leech polypeptide obtained by substituting a group of one or more amino acids in the amino acid sequence with an additional group and having the same biological activity as the leech polypeptide.

(35) In a further specific embodiment of the present invention, a leech polypeptide variant inhibiting macrophage migration is provided, which has an amino acid sequence that is ≥70% identical and ≥90% similar to the main amino acid sequence of the leech polypeptide. The variant is an amino acid sequence or an encoding nucleotide sequence thereof having one or several amino acid or nucleotide changes including deletion, insertion or replacement of amino acid(s) or nucleotide(s) at any position(s) in the amino acid sequence or nucleotide sequence, or addition of amino acid(s) or nucleotide(s) at two ends of the sequence.

(36) In a further specific embodiment of the present invention, a nucleotide encoding the leech polypeptide, the leech polypeptide fragment, the leech polypeptide analogue, the leech polypeptide derivative, or the leech polypeptide variant is provided, which comprises any one of: (a) a nucleotide encoding a polypeptide having the amino acid sequence or a fragment, an analogue, a derivative or a variant thereof; (b) a nucleotide complementary to the nucleotide in (a); and (c) a nucleotide that is ≥75% identical to the nucleotide in (a) or (b).

(37) The nucleotide is produced by an artificial synthesis.

(38) In a further specific embodiment of the present invention, use of the leech polypeptide, the leech polypeptide fragment, the leech polypeptide analogue, the leech polypeptide derivative, and the leech polypeptide variant in the preparation of drugs for preventing or treating related diseases caused by macrophage migration such as atherosclerosis, and diagnosing and detecting the occurrence and severity of these related diseases. The disease includes atherosclerosis.

(39) In a further specific embodiment of the present invention, the leech polypeptide is used for preparing a drug useful as a protease inhibitor, promoter or affinity reagent, or a detection reagent.

(40) The present invention is further illustrated through examples below; however the present invention is not limited thereto. It is to be understood that these examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention.

EXAMPLE 1

Separation and Purification of Leech Polypeptide

(41) In this example, a leech zymolyte HE was firstly prepared from Whitmania pigra Whitman by enzymolysis. After establishing a model of LPS-induced macrophage RAW264.7 migration, Transwell migration assay showed that HE can inhibit the macrophage migration in a concentration-dependent manner. Then, HE was separated and purified by chromatography on QSepharose FF strong basic anion exchange column, and chromatography on Superdex30, Superdexpeptide and G10 gel column, and Transwell assay was done to track the activity. Finally leech polypeptide HE4-1 having activity to inhibit the migration of macrophages RAW264.7 was obtained.

(42) The QSepharoseFF strong basic anion column was used for the first separation of HE. As shown in FIG. 1, HE was separated into 6 peaks. The products of each eluted peak were collected, and lyophilized after alcohol precipitation and desalting to obtain HE1-HE6.

(43) Transwell migration assay was used to detect the activity of HE1-HE6 in inhibiting the macrophage migration. The results are shown in FIG. 2. After induction with LPS, the macrophage migration activity is increased significantly. After adding the six eluted products, HE3 and HE4 can significantly inhibit the migration of macrophages RAW264.7 induced by LPS. Considering that the yield of HE3 eluate was low and it had migration inhibitory activity only at a high concentration, the next step was mainly to isolate and purify HE4.

(44) HE4 was separated by chromatography on a Superdex30 gel column. As shown in FIG. 3, HE4 is separated into two peaks. The products of each eluted peak were collected, and lyophilized after alcohol precipitation and desalting to obtain the products HE4-1 and HE4-2.

(45) Transwell assay was used to detect the activity of HE4-1 and HE4-2 in inhibiting the migration of macrophages. The results are shown in FIG. 4. 800 μg/mL of HE4-1 can significantly inhibit the LPS-induced migration of macrophages RAW264.7, with an inhibition rate reaching 15.2%.

(46) HE4-1 was passed through Superdexpeptide gel filtration chromatography. The result is shown in FIG. 5. Only a single peak is eluted, namely HE4-1. Then HE4-1 was desalted by passing through a Sephadex G10 gel column (FIG. 6), and the HE4-1 peak before the salt peak was collected as the finally isolated and purified product.

(47) Transwell assay was used to test the activity of the finally eluted product HE4-1 to inhibit the migration of macrophages RAW264.7. The results are shown in FIG. 7. 1 μg/mL LPS can significantly promote the macrophage migration, while 800 μg/mL HE4-1 can inhibit the LPS-induced migration of macrophage RAW264.7 with an inhibition rate reaching 15.2%.

EXAMPLE 2

Sequencing of Leech Polypeptide

(48) The leech polypeptide HE4-1 was shipped to Beijing Huada Protein Research And Development Center Co. Ltd for sequencing by LC-ESI-QUAD-TOF-MS/MS detection. Chromatographic column: Trap column: AcclaimPePmap100 (75 μm×2 cm, 3 μm, 100A, Thermoscientific); analytical column: needle-type column manufactured by Beijing Huada Protein Research And Development Center Co. Ltd, packing material: Venusil×BPC, C18 (L), 5 μm, 150A (AgelaTechnologies). Chromatographic conditions: mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: 0.1% formic acid in acetonitrile, gradient elution conditions: 0-48 min, A: 95%-20%, B: 5%-80%; 48-56 min, A: 20%-20%, B: 80%-80%; flow rate: 0.4 μL/min.

(49) As determined by high-resolution mass spectrometry, the molecular weight of leech polypeptide HE4-1 is 1429.294 Da (see FIG. 8) and the amino acid sequence is EAGSAKELEGDPVAG (SEQ ID: 1) (see FIG. 9).

EXAMPLE 3

Full Synthesis of Leech Polypeptide HE-D

(50) The determined amino acid sequence_Glu-Ala-Gly-Ser-Ala-Lys-Glu-Leu-Glu-Gly-Asp-Pro-Val-Ala-Gly (SEQ ID NO: 1) was shipped to Sangon Bioengineering (Shanghai) Co., Ltd. for peptide synthesis. The synthesized peptide was designated as HE-D, which has a purity of 95% or more detected by HPLC. The structure was identified by ESI-MS.

EXAMPLE 4

Activity of Synthetic Leech Polypeptide HE-D to Inhibit Macrophage Migration

(51) Transwell assay was used to detect the inhibition of HE-D on the migration activity of macrophages RAW264.7. The experiments were divided into five groups, including a blank control group, an LPS model group and 200, 400, 800 μg/mL HE-D groups. The results are shown in FIG. 10. 1 μg/mL LPS can significantly promote the macrophage migration, while 800 μg/mL HE-D can inhibit the LPS-induced migration of macrophage RAW264.7 with an inhibition rate reaching 14.9%.

EXAMPLE 5

Study on Signal Pathways of Synthetic Leech Polypeptide HE-D Inhibiting Macrophage Migration

(52) The mechanism of HE-D inhibiting macrophages RAW264.7 was tested by Western-blot. The results are shown in FIG. 11. HE-D has no significant effect on the expression of JNK and total p38 protein, but 800 μg/mL HE-D can significantly inhibit the increase in expression levels of INK and phosphorylated p38 proteins induced by LPS, which indicates that HE-D can inhibit the migration of macrophages by regulating the levels of JNK and p38 phosphorylation.

EXAMPLE 6

Effect of Synthetic Leech Polypeptide HE-D on Biological Functions of Macrophage

(53) The effects of HE-D on the phagocytosis ability and lysozyme activity of macrophage RAW264.7 were measured by neutral red phagocytosis test and lysozyme activity assay. The results are shown in FIG. 12. 1 μg/mL LPS can significantly increase the phagocytosis ability and lysozyme activity of macrophages, and 0-800 μg/mL HE-D has no obvious effect on the phagocytosis ability and lysozyme activity of macrophages RAW264.7.

(54) The effect of HE-D on the secretion of IL-1, IL-6, IL-12, and TNF-α by macrophages RAW264.7 was detected by ELISA. The results are shown in FIG. 13. 1 μg/mL LPS can significantly promote the release of the four inflammatory factors; and 0-800 μg/mL HE-D has no significant effect on the ability of macrophages to secrete the proinflammatory factors IL-1, IL-6, and IL-12, and 400 and 800 μg/mL HE-D can promote the secretion of TNF-α, but the promotion effect is small and is 11.4%.

(55) In summary, the mechanism of HE-D inhibiting macrophages RAW264.7 was tested by Western-blot. The results in FIG. 11 show that HE-D has no significant effect on the expression of INK and total p38 protein, but 800 μg/mL HE-D can significantly inhibit the increase in expression levels of JNK and phosphorylated p38 proteins induced by LPS, which indicates that HE-D can inhibit the migration of macrophages by regulating the levels of JNK and p38 phosphorylation. HE-D has basically no effect on the biological functions of macrophages at the same time of inhibiting the migration of macrophages, which can effectively reduce the adverse reactions caused by HE-D. Therefore, synthetic leech polypeptide HE-D can be used for the early prevention of atherosclerosis and has the potential to prevent the further development of AS.

(56) It should be finally noted that the foregoing descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Although the present invention is described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still make modifications to the technical solutions described in the foregoing embodiments or make equivalent substitutions to some of the technical solutions. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention. The specific implementations of the present invention are described above, but are not intended to limit the protection scope of the present invention. Those skilled in the art should understand that various modifications or deformations may be made without creative efforts based on the technical solutions of the present invention, and such modifications or deformations shall fall within the protection scope of the present invention.