CN108017711B - Anti-calcification short peptide and application thereof - Google Patents
Anti-calcification short peptide and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to an anti-calcification short peptide, in particular to an anti-calcification short peptide and application thereof, wherein the amino acid sequence of the anti-calcification short peptide is shown as SEQ ID NO. 1. Experiments prove that the short peptide provided by the invention can obviously inhibit warfarin and high-calcium and high-phosphorus induced valvular interstitial cell calcification and inhibit low-sugar culture induced valvular interstitial cell apoptosis. Therefore, the short peptide provided by the invention can be used as a good anti-calcification drug with low side effect.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to an anti-calcification short peptide and application thereof.
Background
Calcific diseases are clinically quite common diseases which are mainly characterized by non-bone tissues, which have osteogenesis similar processes and calcium salt deposition, and calcification is preferably generated in soft tissues with elasticity, such as heart valves, large blood vessels, synovium, tracheal cartilage and the like. In recent years, more and more research has shown that ectopic calcification, as is the most common heart valve and blood vessel, is an actively regulated process under the combined action of both external and internal causes. At present, the main causes of ectopic calcification lesions in clinic comprise chronic inflammatory diseases such as long-term atherosclerosis, calcium and phosphorus metabolic disorder caused by renal insufficiency and calcium and phosphorus metabolic disorder caused by endocrine diseases, and medicines which can cause calcification such as warfarin and the like are taken for a long time. Unfortunately, there is no effective means for preventing or treating calcification in clinic, and the current treatment is only to replace severely calcified tissue with artificial organs, such as a heart valve, a blood vessel, etc. For patients with renal insufficiency, no effective method for delaying long-term dialysis-related cardiovascular calcification exists, and development of effective treatment methods, particularly long-term treatment methods, for alleviating the risks of calcified diseases is urgently needed. The effect of peptidase-inhibiting protein PI16 on inhibiting aortic valve calcification is researched for the first time at the early stage of research groups of the applicant under the support of national science foundation project (81100162,81570247), and the protein encoded by over-expression human PI16 gene is proved to be capable of obviously inhibiting heart valve calcification induced by warfarin and high calcium and high phosphorus for the first time. Through analysis of PI16 protein structural domains and protein interaction research, I find for the first time that a section of amino acid sequence of PI16 can play a calcium-resistant role as the whole protein, and we invent a calcium-resistant short peptide with more stable physicochemical properties by mutating and modifying the section of amino acid.
Disclosure of Invention
The invention aims to provide a short peptide which is derived from human peptidase inhibitory protein PI16 and subjected to point mutation, and the short peptide has remarkable stability and anti-calcification effect.
The invention also aims to provide the medicinal application of the anti-calcification short peptide.
The amino acid sequence of the anti-calcification short peptide is shown in SEQ ID NO.1(Val-Ala-Leu-His-Asn-Leu-Tyr-Arg-Ala-Gln-Val-Ser-Pro-Thr), and the polypeptide consists of 14 amino acids.
The invention also includes a method for preparing the short peptide of the invention, wherein the method is selected from a conventional solid phase synthesis method, a solution synthesis method, a gene recombination method or a chemical synthesis method.
The medicinal application of the invention is the medicinal application for treating calcific diseases. The calcified diseases are calcification of heart valves, calcification of valve annuluses, and calcification of ectopic soft tissues caused by kidney diseases, including but not limited to calcification of heart valves, blood vessels, trachea and the like, and calcification of heart valves and blood vessels caused by atherosclerosis or calcium-phosphorus metabolic disorder.
The invention also includes pharmaceutical combinations comprising the short peptides of the invention. The pharmaceutical composition of the invention may contain various pharmaceutically acceptable carriers. The pharmaceutical combination of the present invention may be presented in any form of formulation, such as injection or oral formulation, preferably as freeze-dried injection.
The polypeptide having the amino acid sequence of the present invention can be used alone in the form of a polypeptide, or can be fused with other proteins or polypeptides, or can be used by being subjected to modification such as acetylation, methylation, or substitution or mutation of individual amino acids, or inserted into a specific site on the surface of a protein, or linked with other substances. In any form, polypeptides of this amino acid structure may also exhibit anticalcification properties.
The substance containing the amino acid sequence can be used in various application forms, including in vivo and in vitro anti-calcification activity, and can be used for treating diseases with calcium resistance by various administration modes.
Has the advantages that:
the short peptide can be specifically combined with p53 protein to inhibit cell and tissue calcification mediated by the short peptide, and can also act on other unknown targets to prepare medicines for treating calcific diseases.
The polypeptide of the invention has the characteristics of short length, easy synthesis and stability in acid-base environment.
Drawings
The invention is further illustrated with reference to the following figures and examples.
FIG. 1 alizarin Red staining for inhibition of warfarin-induced valvular stromal cell calcification by short peptides
FIG. 2 intracellular calcium concentration assay of short peptides to inhibit warfarin-induced valvular stromal cell calcification
FIG. 3 alizarin red staining for short peptide inhibition of high calcium and high phosphorus induced calcification of valve stromal cells
FIG. 4 intracellular calcium concentration assay of short peptides to inhibit high calcium and high phosphorus induced valvular stromal cell calcification
FIG. 5 is a schematic diagram of a flow cytometer for inhibiting serum-free induction of valve interstitial cell apoptosis by short peptides
FIG. 6 statistical analysis of flow cytometry assays
FIG. 7 Co-immunoprecipitation and Western blotting method showed that the short peptide specifically binds to p53 protein
FIG. 8 degradation rates of short peptides in ph2.5 and ph8.0 aqueous solutions
Detailed Description
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention.
Example 1, procedure for the preparation of short peptides:
the main operation steps of the solid-phase short peptide are as follows:
(1) 0.5mmol of Fmoc-Thr (tBu) wang resin with loading of 0.35mmol/g is weighed out and put into a reaction column, and an appropriate amount of DCM is added for soaking for 30min, DCM is extracted and washed 3 times with DMF.
(2) Deprotection, adding appropriate amount of deprotection solution (20% piperidine in DMF) to react for 20min, pumping off deprotection solution, washing with DMF for 6 times.
(3) And (2) coupling amino acid, weighing 3eq of Fmoc-Pro-OH and TBTU, adding the mixture into a reaction column, adding 3eq of DIEA by taking DMF as a solvent, reacting for 1h, detecting whether the reaction is complete or not by ninhydrin, after the reaction is finished, pumping out the reaction solution, and washing with DMF for three times.
(4) Repeating the steps (2) and (3) until the last amino acid reaction is finished.
(5) And (3) carrying out deprotection shrinkage, adding a proper amount of deprotection solution (20% piperidine solution in DMF) to react for 20min, pumping out the deprotection solution, washing with DMF, DCM and methanol for three times respectively, and pumping out the resin.
(6) Adding an appropriate amount of lysate (TFA 95%/2.5% water/2.5% Tis), shaking in a shaking table at a constant temperature of 30 ℃ for 2h, filtering to remove resin, adding an appropriate amount of anhydrous ethyl glacial ether into the filtrate, separating out a solid, centrifuging to precipitate the solid, pouring out a supernatant, washing with the anhydrous ethyl glacial ether for three times, and pumping to dry to obtain a crude product.
(7) And detecting the amino acid sequence of the short peptide by adopting a mass spectrometry on the purified sample, and determining the short peptide product with the correct amino acid sequence.
Example 2 experiment of inhibition of warfarin-induced valvular stromal cell calcification by short peptides
Culturing primary porcine aortic valve interstitial cells in DMEM medium containing 10% fetal bovine serum at a cell density of about 1 x 105Adding the cell amount of each cell/hole into a 12-hole plate, adding 1.6mM warfarin solution respectively, culturing for 7 days, adding the short peptide and the negative control peptide (SEQ ID NO.3) with different concentrations respectively, fixing the cells by 4% paraformaldehyde on the 7 th day, staining by 1% alizarin red to show the calcium deposition condition, dissolving the calcium in the cells by 0.6M dilute hydrochloric acid, and detecting the level of the calcium content in the cells by a colorimetric calcium concentration determination kit.
The result shows that the oligopeptide can obviously inhibit the calcium ion deposition of valve interstitial cells induced by warfarin, the inhibition effect has a dose-effect relationship, and the negative control peptide has no obvious effect. Alizarin red staining (shown in figure 1) and calcium content determination (shown in figure 2) results show that 200-400ug/ml short peptide can significantly inhibit warfarin-induced valvular interstitial cell calcification.
Example 3 experiment of short peptides to inhibit high calcium and high phosphorus induced calcification of valve stromal cells
Culturing primary porcine aortic valve interstitial cells in DMEM medium containing 10% fetal bovine serum at a cell density of about 1 x 105Adding each cell/well cell amount into a 12-well plate, adding 2.0mM calcium chloride and 2.0mM (sodium hydrogen phosphate + disodium hydrogen phosphate) solution respectively, culturing for 5 days, adding the short peptide and the negative control peptide (SEQ ID NO.3) with different concentrations respectively, fixing the cells by 4% paraformaldehyde on the 5 th day, staining by 1% alizarin red to show calcium deposition, dissolving the calcium in the cells by 0.6M dilute hydrochloric acid, and detecting the level of the calcium content in the cells by using a colorimetric calcium concentration determination kit. The results show that the short peptide of the invention can be shownThe inhibition effect has a dose-effect relationship while the negative control peptide has no obvious effect on the calcium ion deposition of valve interstitial cells induced by high calcium and high phosphorus. Alizarin red staining (shown in figure 3) and calcium content determination (shown in figure 4) results show that 400ug/ml concentration of short peptide can significantly inhibit high-calcium high-phosphorus induced calcification of valve interstitial cells.
Example 4 experiment of short peptides to inhibit serum-free Induction of apoptosis of valvular stromal cells
Culturing primary porcine aortic valve interstitial cells in DMEM medium containing 10% fetal bovine serum at a cell density of about 1 x 105Adding the cell amount of each cell/hole into a 12-hole plate, replacing a low-sugar culture medium after the cells are completely attached to the wall, simultaneously adding the short peptide and the negative control peptide (SEQ ID NO.3) with different concentrations respectively, culturing for 12 hours, digesting the cells by 0.25 percent of trypsin, then carrying out immunostaining on the cells by using a fluorescent antibody of an apoptosis marker Annexin V, detecting the proportion of Annexin V positive cells by a flow cytometer, and detecting by the flow cytometer shows that 200-dose 400ug/ml short peptide can obviously inhibit the valve interstitial apoptosis induced by the low-sugar culture medium. While the negative control peptide had no significant effect. As shown in fig. 5-6.
Example 5 short peptide binding specifically to p53 protein
293T cells were transfected with plasmids containing the short peptide and the negative control peptide (SEQ ID NO.3), respectively, and after 48 hours, proteins were extracted by lysis. Respectively incubating with anti-Flag antibody and anti-p 53 antibody, precipitating by magnetic bead method, and respectively hybridizing with p53 and Flag antibody for the protein after the membrane is converted by conventional western blot method. The results show that the short peptide of the invention binds to p53, whereas the negative control peptide does not. As shown in fig. 7.
Example 6 acid-base stability of short peptides
The original short peptide sequence (SEQ ID NO.2) and the mutant short peptide of the present invention (SEQ ID NO.1) were prepared as 1mg/ml aqueous solutions, and adjusted to pH2.5 and 8.0 with dilute hydrochloric acid and hydrogen peroxide, respectively. And (3) under the condition of 37-degree water area, carrying out concentration measurement on samples at different time points by adopting high performance liquid chromatography. The result shows that the mutant short peptide has better acid-base stability. As shown in fig. 8.
The parts not involved in the present invention are the same as or can be implemented using the prior art.
The invention is subsidized by national projects.
Sequence listing
<110> Sun Wei
<120> anti-calcification short peptide and application thereof
<160>3
<170>SIPOSequenceListing 1.0
<210>1
<211>14
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>1
Val Ala Leu His Asn Leu Tyr Arg Ala Gln Val Ser Pro Thr
1 5 10
<210>2
<211>14
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
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Val Glu Leu His Asn Leu Tyr Arg Ala Gln Val Ser Pro Thr
1 5 10
<210>3
<211>14
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>3
Val Ala Leu Ala Asn Leu Ala Ala Ala Ala Val Ala Pro Ala
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Claims (8)
1. An anti-calcification short peptide is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.
2. The process for producing an anticalcification oligopeptide according to claim 1, wherein: selected from conventional solid phase synthesis methods or gene recombination methods.
3. A pharmaceutical composition comprising the anti-calcification short peptide as claimed in claim 1.
4. The pharmaceutical composition of claim 3, in the form of a pharmaceutical formulation of any form.
5. The pharmaceutical composition of claim 3, wherein the pharmaceutical composition is a lyophilized injectable formulation or any other form of oral formulation.
6. The pharmaceutical composition of claim 3, further comprising other pharmaceutically acceptable excipients.
7. Use of the anti-calcification oligopeptide of claim 1 in the preparation of a medicament for treating valvular interstitial cell calcification.
8. The use according to claim 7, wherein the anticalcification short peptide is used alone in the form of polypeptide or fused with other proteins and polypeptides, or is acetylated and methylated in the preparation of a medicament.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102119224A (en) * | 2008-05-30 | 2011-07-06 | 不列颠哥伦比亚大学 | Methods of diagnosing rejection of a kidney allograft using genomic or proteomic expression profiling |
| WO2011127543A1 (en) * | 2010-04-16 | 2011-10-20 | Transbio Ltd | Proteins that bind pi16 and uses thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102119224A (en) * | 2008-05-30 | 2011-07-06 | 不列颠哥伦比亚大学 | Methods of diagnosing rejection of a kidney allograft using genomic or proteomic expression profiling |
| WO2011127543A1 (en) * | 2010-04-16 | 2011-10-20 | Transbio Ltd | Proteins that bind pi16 and uses thereof |
Non-Patent Citations (3)
| Title |
|---|
| Accession number：EAX03923.1;NCBI Database;《GenBank》;20150523;EAX03923 * |
| Peptidase inhibitor 16 identifies a unique subset of memory T helper cells with hyperproliferative and proinflammatory properties (IRC8P.477);Arunesh Mohandas等;《J Immunol》;20140501;第192卷;57-64 * |
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