CN103539834A - Chemerin derived peptide and expressed gene and application thereof - Google Patents

Abstract

The invention relates to a chemerin derived peptide. The chemerin derived peptide is polypeptide having the following structural formula: Xaa1-Leu-Gln-Val-Ala-Leu-Glu-Glu-Phe-His, wherein Xaa1 is any one of 20 L-amino acids. The chemical nature of the chemerin derived peptide is polypeptide, so the defects that the traditional natural chemerin and enzyme-digested products thereof have relatively large molecular weight, are difficult to prepare, and have poor antigenicity and poor stability are overcome, and the chemerin derived peptide can be produced in a large scale, and can be used in experimental study of large animals and human bodies and drug development. The invention also discloses an expressed gene of the chemerin derived peptide and the application of the chemerin derived peptide in curing cancers, obesity, fatty liver, diabetes, cardiovascular disease, nervous system, reproductive system and metabolic syndrome.

Description

Chemokine-derived peptides and their expression genes and applications

【Technical field】

The invention relates to the field of polypeptide medicine, in particular to a chemokine-derived peptide, its expression gene and its application.

【Background technique】

Protein is the most important class of biomacromolecules in the body, and is currently widely used as medicine for the treatment of diseases (Li Wei 2004). However, protein drugs also have disadvantages, such as large molecular weight, difficult preparation, antigenicity, and easy degradation in vivo. Fortunately, it has been found that some polypeptides with smaller molecular weights also have protein-like activities, and their functions are more significant. These biologically active polypeptides have very little (or even no) content in the body, but their effects are extremely strong. The occurrence and development of many diseases are related to their imbalance. The prospect of drug development is very good. Therefore, the research and development of polypeptide drugs has become a Internationally emerging high-tech field of biotechnology has great market potential.

By the end of the 1990s, scientists had discovered tens of thousands of natural polypeptide physiological substances, involving hormones, nerves, cell growth, reproduction, tumor lesions, neurohormone transmitters, and immune regulation. In 2006, , the total sales of protein/peptide drugs in the world have exceeded 60 billion US dollars, and it is expected to reach 75 billion to 80 billion US dollars in 2008, with an annual growth rate of more than 20%. Although peptides have been discovered a hundred years ago, they have only been developed as medicines for only 20 years. In the past 20 years, at least 100 kinds of peptide drugs have been developed and marketed by countries around the world, such as leuprolide, gorelin, buserelin, luteinizing hormone antagonists, Fuzeon and the use of Various drugs produced by bioengineering methods (such as human growth hormone, interleukin, human insulin, interferon and colony cell growth factor, etc.). According to related reports, in 2006, the total sales of global protein/polypeptide drugs exceeded 60 billion US dollars, and it is expected to reach 75 billion to 80 billion US dollars in 2008, with an annual growth rate of more than 20%. Peptide drugs have become an important category in the international market.

Compared with most organic small molecule drugs, peptide drugs have outstanding characteristics such as high activity, small dosage, low toxicity and side effects, and amino acids as the end product of metabolism; compared with proteins, smaller peptides have almost no immunogenicity; Chemical synthesis, high product purity and controllable quality. It is a pity that there are a considerable number of polypeptides in organisms, and only 1% or less of them have been discovered so far. Therefore, it is necessary to discover and develop new peptides with physiological activity, which may represent new therapeutic methods.

At present, peptide drugs on the market mainly come from three production methods: animal tissue extraction, chemical synthesis and gene recombinant expression. It is understood that due to the reduction of raw material costs, economies of scale and advancements in separation technology, the cost of chemical peptide synthesis, especially solid-phase peptide synthesis, has dropped significantly.

According to reports, compared with chemical synthesis, genetic recombination is more suitable for the preparation of long peptides; and with the advancement of technology, the cost of producing polypeptide drugs by genetic recombination is also decreasing.

Moneta believes in the research report that peptide drugs extracted from animal tissues will be gradually eliminated, and chemical synthesis and gene recombinant expression will become complementary peptide drug production methods for a long time.

Chemerin, also known as retinoic acid receptor responder 2 (RARRES2), encoded by tazarotene-induced gene 2 (TIG2), is a G protein-coupled receptor Chemerin R, also known as ChemR23 or the natural ligand of chemokine-like receptor 1 (CMKLR1) (Wittamer et al. 2003, Zabel et al. It is named after its role and is also a newly identified adipokine in 2007 (Bozaoglu et al.2007). Recently, it was also found that GPR1, a member of the GPCRs family, and CCRL2 on mast cells are also chemokine receptors, but they do not directly support chemotaxis (Barnea et al. 2008, Zabel et al. 2008).

Chemokines are widely expressed in various tissues of the human body, including adipose tissue, adrenal gland, liver, lung, pancreas, placenta, ovary, skin, etc., and mainly expressed in white adipose tissue, liver and lung (review: Wang Xiaojuan and Yan Jianying, 2010). Chemokines have a wide range of functions, such as promoting the chemotaxis of dendritic cells, macrophages and NK cells to inflammatory sites, inhibiting the synthesis of pro-inflammatory mediators TNFα and IL-6, increasing the production of adiponectin, and promoting the formation of adipocytes. Differentiation and maturation, improving the sensitivity of adipocytes to insulin and glucose uptake, regulating lipolysis, increasing the synthesis of TNFβ, increasing the activity of NFκβ, increasing the synthesis of VEGF and MMPs and regulating angiogenesis and angiogenesis, etc. (Kukla et al. 2011). Therefore, chemokines play an important role in immune response, inflammatory response, lipogenesis and lipid metabolism (involving obesity, fatty liver, diabetes and metabolic syndrome), and have a good application prospect.

Chemokines are highly conserved in organisms such as humans, mice, and rats. As shown in Figure 1, the protein translated from human TIG2 gene expression is a chemokine preproprotein, called "Pre-prochemerin", which consists of 163 amino acids and has a signal peptide of 20 amino acids at the N-terminal (Table 1, Du and Leung. 2009, Ernst and Sinal. 2010). The signal peptide is cleaved to form prochemerin. In the normal state of the body, the chemokines produced by various tissues (except peripheral blood leukocytes) are secreted out of the cells in the form of proChemerin, which is its precursor. The activity of proChemerin is very low, and different forms of chemokines are produced under the action of different proteolytic enzymes. It has different degrees of immune chemotaxis (Table 1, Du and Leung.2009, Ernst and Sinal.2010). As can be seen from Table 1, the natural chemokines discovered so far (Chem21-157, Chem21-155 and Chem21-154) and the chemokines formed by enzymatic digestion (Chem21-158, Chem21-156, Chem21-152 ) are all proteins, and there are no polypeptides less than 50 amino acids. In terms of application, others have invented human recombinant chemerin protein, recombinant vector, transformant and preparation method thereof.

However, combined with Figure 1, it can be seen that various natural chemokines (chemerins) and their enzyme-cleaved products found in the body are all proteins, which have disadvantages such as relatively large molecular weight, difficult preparation, antigenicity, and poor stability. It is difficult to produce in large quantities and carry out experimental research and drug development on large animals and humans.

Table 1. Different forms of chemokines and their chemotactic activities

references:

Barnea et al. The genetic design of signaling cascades to record receptor activation. Proc. Natl. Acad. Sci. USA2008.105:64–69.

Bozaoglu et al. Chemerin is a novel adipokine associated with obesity and metabolic syndrome［J］. Endocrinology, 2007, 148 (10): 4687-4694.

Cash et al.Synthetic chemerin-derived peptides suppress inflammation through ChemR23.J Exp Med.2008;205(4):767-75.

Du and Leung.Proteolytic regulatory mechanism of chemerin bioactivity.ActaBiochim Biophys Sin(Shanghai).2009;41(12):973-9.

Ernst and Sinal. Chemerin: at the crossroads of inflammation and obesity. Trends Endocrinol Metab. 2010;21(11):660-7.

Goralski et al. Chemerin: a novel adipokine that regulates adipogenesis and adipocyte metabolism［J］. J Biol Chem,2007,282(38):28175-28188.

Kukla et al. Potential role of leptin, adiponectin and the noveladipokines-visfatin, chemerin and vaspin-in chronic hepatitis. Mol Med.2011.doi:10.2119/molmed.2010.00105.

Wittamer et al.Specific recruitment of antigen-presenting cells by chemerin,novel processed ligand from human inflammatory fluids.J.Exp.Med.2003.198:977-985.

Wittamer et al.The C-terminal nonapeptide of mature chemerin activates the chemerin receptor with low nanomolar potency［J］.J Biol Chem,2004,279(11):9956-9962.

Zabel et al. Chemoattractants, extracellular proteases, and the integrated hostdefense response. Exp. Hematol. 2006.34:1021-1032.

Zabeletal.Chemokine-likereceptor 1 expression and chemerin-directedchemotaxis distinguishing plasmacytoid from myeloid dendritic cells in human blood.J.Immunol.2005.174:244251.

Zabel et al.Mast cell-expressed orphan receptor CCRL2 binds chemerin and is required for optimal induction of IgE-mediated passive cutaneous anaphylaxis.J.Exp.Med.2008.205:2207–2220.

Li Wei Shanghai Medicine 200425 (3): 119-121.

Research progress of Wang Xiaojuan and Yan Jianying's adipocyte factor chemerin International Journal of Obstetrics and Gynecology (JInt Obstet Gynecol) 201037 (1): 43-46.

【Content of invention】

Based on this, it is necessary to provide a chemokine-derived peptide and its expression gene and application.

A chemokine-derived peptide is a polypeptide having the following structural formula:

Xaa ₁ -Leu-Gln-Val-Ala-Leu-Glu-Glu-Phe-His;

Wherein, Xaa ₁ is any one of 20 L-amino acids.

Preferably, it is a polypeptide having the following sequence:

A polypeptide as shown in SEQ ID No.1, or

The polypeptide as shown in SEQ ID No.2.

An expressed gene whose expression product includes the above-mentioned chemokine-derived peptide.

Application of the above-mentioned chemokine-derived peptides in the treatment of cancer, obesity, fatty liver, diabetes, cardiovascular disease and metabolic syndrome.

Due to the chemical nature of the above-mentioned chemokine-derived peptides as polypeptides, it overcomes the shortcomings of traditional natural chemokines and their enzyme-cleaved products, such as relatively large molecular weight, difficult preparation, antigenicity, and poor stability, and can be mass-produced And carry out experimental research and drug development on large animals and humans.

【Description of drawings】

Figure 1 shows the sequences of chemokine pre-precursors in humans, mice, and rats; the boxes indicate the signal peptide, human Chem ^149-157 with chemotactic effect, and small protein with anti-inflammatory effect Sequence of murine Chem ^140-154 .

Figure 2 is a sequence alignment of the amino acids of pre-prochemerin in different species.

Figure 3 is an isotope binding experiment between chemokine-derived peptide N10 and CCRL2 receptor cells;

Fig. 4 is the mass spectrometric analysis result graph after thrombin and plasmin combined treatment mouse recombinant chemokine protein;

Figure 5 is a graph showing the expression results of CCRL2mRNA detected by quantitative PCR in cerebral cortex, caudate putamen, cerebellum, spinal cord, hypothalamus and pituitary;

Figure 6 is a graph showing the expression results of CCRL2mRNA in testis, epididymis, vas deferens, seminal vesicle, prostate, heart, kidney and epididymis by quantitative PCR method;

Fig. 7 is a diagram showing the results of quantitative PCR detection of CCRL2 mRNA expression in cancer cells.

【Detailed ways】

In recent years, in order to study the active peptides of chemokines, many peptides derived from the carboxy-terminal region of chemokine precursors have been artificially synthesized. It was found that the shortest bioactive peptide of chemokines is human Chem ^149-157 , also known as human The chemokine C9 peptide (chemerin9), which has strong chemotactic activity (Wittamer et al.2004); in mice, the 15 amino acid chemokine C15 peptide (Chem ^140-154 ), has Anti-inflammatory effect (Cash et al. 2008). This shows that the polypeptides derived from chemokines are active. This discovery reminds us that the discovery of natural chemokine polypeptides that exist and are active in the body obviously provides a form with low cost, simple process and better stability for the application of chemokines, and provides a new way for the development of related new drugs. convenient and effective way.

Although both human Chem ^149-157 and mouse Chem ^140-154 bind to CMKLR1 and/or GPR1 and have certain biological activities, there are certain problems:

(1) It is a synthetic polypeptide, and the enzyme cleavage site is not conserved among different species, suggesting that these two polypeptides are not in the form of endogenous existence;

(2) There is no detailed information showing the relationship between these two polypeptides and the three receptors of chemokine chemerin (CMKLR1, CCRL2, GPR1). Because different receptors have different biological functions, if it is not clarified which receptor it binds to, it is difficult to determine whether the two polypeptides are the ligands of the three receptors or just the ligands of a certain receptor. Therefore, it is difficult to determine the exact biological function and medicinal value of the two polypeptides.

Using bioinformatics tools, according to the characteristics of polypeptide/protein hormones, combined with the inventor's accumulation of knowledge and experience in discovering new polypeptide/protein hormones for many years, the endogenous and endogenous hormones that exist in the body and have Biologically active, natural, novel chemokine-derived peptides, denoted as N10. This polypeptide has higher safety, more accurate binding receptors and biological activities, low cost, simple process, and better stability. It is a substitute for natural chemokines, which can replace natural chemokines for in-depth functional, Mechanism research can also replace natural chemokines for in-depth pharmacological research; it provides a new tool for the search for new drug targets of the three known receptors, and provides a new way for the development of related new drugs.

It can be seen from Figure 2 that the amino acid sequence of pre-prochemerin in different species is conserved in different species. Among them, the gray background part is the newly discovered sequence of N10.

Taking human chemokine-derived peptide N10 as an example for a brief description, its sequence is as follows: Gly ²⁹ -Leu ³⁰ -Gln ³¹ -Val ³² -Ala ³³ -Leu 34 -Glu ³⁵ -Glu ^{36 -Phe 37} -His ³⁸ .

For human chemokine-derived peptide N10, the sequence of the 29th amino acid can be replaced with a similar amino acid sequence, which will not have a great impact on the biological activity of chemokine-derived peptide N10; but Leu ³⁰ -Gln ³¹ -Val ³² -Ala ³³ The sequence of -Leu ³⁴ -Glu ³⁵ -Glu ³⁶ -Phe ³⁷ -His ³⁸ is very conservative and cannot be replaced. If the amino acid sequence is replaced, its biological activity may be affected.

Based on this, a chemokine-derived peptide N10 is provided, which is a polypeptide having the following structural formula:

Xaa ₁ -Leu-Gln-Val-Ala-Leu-Glu-Glu-Phe-His;

Wherein, Xaa ₁ is any one of 10 L-amino acids.

The sequence of this chemokine-derived peptide N10 in various species is shown below:

Human chemokine-derived peptide N10 (G ²⁹ -H ³⁸ ): GLQVALEEFH, which is the polypeptide shown in SEQ ID No.1;

Orangutan chemokine-derived peptide N10 (G ²⁹ -H ³⁸ ): GLQVALEEFH, which is the polypeptide shown in SEQ ID No.1;

Monkey chemoattractant-derived peptide N10 (G ²⁹ -H ³⁸ ): GLQVALEEFH, which is the polypeptide shown in SEQ ID No.1;

Rabbit chemokine-derived peptide N10 (G ²⁹ -H ³⁸ ): GLQVALEEFH, which is the polypeptide shown in SEQ ID No.1;

Dog chemokine-derived peptide N10 (G ²⁹ -H ³⁸ ): GLQVALEEFH, which is the polypeptide shown in SEQ ID No.1;

Porcine chemokine-derived peptide N10 (G ²⁹ -H ³⁸ ): GLQVALEEFH, which is the polypeptide shown in SEQ ID No.1;

Bovine chemokine-derived peptide N10 (G ²⁹ -H ³⁸ ): GLQVALEEFH, which is the polypeptide shown in SEQ ID No.1;

Rat chemokine-derived peptide N10 (G ³¹ -H ⁴⁰ ): GLQVALEEFH, which is the polypeptide shown in SEQ ID No.1;

Hamster chemokine-derived peptide N10 (G ³¹ -H ⁴⁰ ): GLQVALEEFH, which is the polypeptide shown in SEQ ID No.1;

Mouse chemokine-derived peptide N10 (S ³¹ -H ⁴⁰ ): SLQVALEEFH, which is the polypeptide shown in SEQ ID No.2.

The chemokine-derived peptide N10 can be synthesized in vitro according to its sequence. It can also be obtained by synthesizing the expressed gene and purifying it after expression in other organisms.

At the same time, it also provides an expression gene whose expression product includes chemokine-derived peptide N10.

Existing studies have shown that chemokines and three receptors CMKLR1, CCRL2 and GPR1 play roles in inflammatory response, chemotaxis, cancer, obesity, fatty liver, diabetes, cardiovascular disease and metabolic syndrome. In the future, more and more studies will discover more and more precise biological functions of the three receptors, CMKLR1, CCRL2 and GPR1.

Chemokine-derived peptide N10 competes with iodine-labeled human chemokine for binding to recipient cells transfected with human CCRL2, as shown in Figure 3.

It can be seen from Fig. 3 that human chemokine-derived peptide N10 binds to CCRL2 receptor.

It can thus be seen that the chemokine-derived peptide N10 is a ligand for the CCRL2 receptor. Therefore, the chemokine-derived peptide N10 acts as either an agonist or an antagonist of the CCRL2 receptor, and its biological function is related to the function of the CCRL2 receptor. The chemokine-derived peptide N10 will be a ligand for in-depth study of the biological function of CCRL2.

Figure 4 shows the results of mass spectrometry analysis of the mouse recombinant chemokine protein after combined treatment with thrombin (Thrombin) and plasmin (Plasmin). It can be seen from Fig. 4 that the chemokine-derived peptide N10 with a molecular weight of 1171 was obtained through joint digestion with thrombin (Thrombin) and plasmin (Plasmin).

In addition, carboxypeptidase B (Carboxypeptidase B) and thrombin (Thrombin) can also be used in combination to digest the chemokine protein and obtain the chemokine-derived peptide N10.

The above results indicate that in the human or animal body, the active endogenous chemokine-derived peptide N10 can be digested in tissues and organs with the above proteases.

The real-time quantitative PCR method was used for detection, and the detection results are shown in Figures 5 to 7. It can be seen from Figure 5 that CCRL2mRNA is expressed in the cerebral cortex, caudate putamen, cerebellum, spinal cord, hypothalamus and pituitary gland in the central nervous system, and the expression in the pituitary gland is the highest. It can be seen from Fig. 6 that CCRL2mRNA is also expressed in adipose tissue of testis, epididymis, vas deferens, seminal vesicle, prostate, heart, kidney and epididymis. It can be seen from Figure 7 that it is highly expressed in some cancer cells, especially human breast cancer cells, human endometrial cancer cells, mouse breast cancer cells and mouse prostate cancer cells.

The above results indicate that, as a ligand of CCRL2, chemokine-derived peptide N10 can act on the above tissues or organs expressing CCRL2 mRNA and play an important role. In the pituitary gland, there are somatotroph cells and prolactin cells, which secrete growth hormone (GH) and prolactin hormone (PRL), respectively, thyrotroph cells, gonadotroph cells, and adrenotroph cells, which secrete thyrotropin Hormone (TSH), adrenotroph cells secrete adrenotropic hormone (ACTH), mainly glucocorticoids. Gonadotrophs secrete luteinizing hormone (LH) and follicle stimulating hormone (FSH). Also in the pituitary gland are melanocytes that secrete melanocyte-stimulating hormone (MSH). As a ligand for CCRL2, the chemokine-derived peptide N10 will have an effect on the regulation of secretion of growth hormone, prolactin, thyrotropin, adrenotropin, luteinizing hormone, and follicle-stimulating hormone and melanocyte-stimulating hormone from pituitary cells.

The chemokine-derived peptide N10 can be used for the preparation of drugs for the treatment of cancer, obesity, fatty liver, diabetes, cardiovascular disease and metabolic syndrome, and will be used in combination with CCRL2 in inflammatory response, chemotaxis, cancer, obesity, fatty liver, New drug candidates related to diabetes, cardiovascular disease, and metabolic syndrome.

Chemokine-derived peptide N10, due to its chemical nature as a polypeptide, overcomes the shortcomings of traditional natural chemokines and their enzyme-cleaved products, such as relatively large molecular weight, difficult preparation, antigenicity, and poor stability, and can be mass-produced And carry out experimental research and drug development on large animals and humans. At the same time, the chemokine-derived peptide N10 has clear and specific receptors and specific physiological activities, so it has the advantages of higher safety, good activity, low cost, simple process, and better stability.

The above-mentioned embodiments only represent one or several implementations of the present invention, and the description thereof is relatively specific and detailed, but should not be construed as limiting the patent scope of the present invention. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims (4)

1. a chemotactic element derived peptide, is characterized in that, for thering is the polypeptide of following structural formula:

Xaa ₁-Leu-Gln-Val-Ala-Leu-Glu-Glu-Phe-His；

Wherein, Xaa ₁be any one in 20 L-amino acid.

2. chemotactic element derived peptide as claimed in claim 1, is characterized in that, for thering is the polypeptide of following sequence:

Polypeptide as shown in SEQ ID No.1, or

Polypeptide as shown in SEQ ID No.2.

3. an expressing gene, is characterized in that, its expression product comprises chemotactic element derived peptide as claimed in claim 1 or 2.

4. chemotactic element derived peptide as claimed in claim 1 or 2 is in the application for the treatment of cancer, obesity, fatty liver, diabetes, cardiovascular disorder, nerve, reproduction and metabolic syndrome.

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