Construction method of jellyfish toxin metalloproteinase substrate peptide library
By constructing a jellyfish toxin metalloproteinase substrate peptide library, the problems of large workload and large error in existing jellyfish toxin research have been solved, and the peptide library has been efficiently applied to jellyfish sting research and therapeutic drug screening.
Patent Information
- Application Number
- CN202511007633.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-22
- Publication Date
- 2025-12-05
AI Technical Summary
Existing technologies cannot effectively construct jellyfish toxin metalloproteinase substrate peptide libraries, resulting in a large workload, large errors, and difficulty in applying them to jellyfish sting research.
Mice with varying degrees of dermatitis were induced by metalloproteinase inhibitors; potential substrate sequences located in the β chain of extracellular sequences were screened by bioinformatics analysis, and a peptide library with a length of no less than 8 amino acids was constructed.
The constructed peptide library contains only a small number of potential substrate sequences, and the hydrolysis products are easy to identify, reducing errors and ensuring that metalloproteinases play a dominant role. It can be applied to jellyfish sting research and therapeutic drug screening.
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Figure CN121075447A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of biological technology, in particular to a method for constructing a jellyfish toxin metalloprotease substrate peptide library. BACKGROUND
[0002] Jellyfish toxin is the material basis of jellyfish stings, containing a variety of polypeptides and proteins. Metalloprotease is one of the main components of jellyfish toxin, which has pro-inflammatory, edema, hemolysis and other biological activities, and plays a leading role in skin diseases caused by stings. Understanding the metalloprotease of jellyfish toxin is closely related to protecting the health of patients and improving the level of prevention and treatment of jellyfish stings.
[0003] Substrate peptide library is an important tool for studying the target of protease, which has important guiding role for in-depth study of related diseases and targeted drugs, and has been applied to drug research and development of cancer, AIDS, influenza and other diseases. The original protease substrate peptide library construction method is derived from a proteomics-driven protease cleavage site recognition technology. This technology was established by Christopher M Overall et al., which identifies the product of the target protease substrate peptide library by proteomics technology, thereby analyzing the specificity of the cleavage site and the substrate sequence, and the construction method of the substrate peptide library is to hydrolyze the whole proteome of the tissue cells (Nat Biotechnol, 2008, 26, 685-694). The original method relies on high-purity target protease monomers to construct a large peptide library derived from the whole proteome of tissue cells for high-throughput analysis of substrate sequences and cleavage sites. The substrate peptide library can be prepared according to the characteristics of the target protease to achieve personalized analysis of the target protease. Since the metalloprotease monomer of jellyfish toxin has not been obtained in the field at present, the substrate peptide library can only be hydrolyzed by crude jellyfish toxin. If the substrate peptide library contains the whole proteome of tissue cells, the workload is huge when identifying the product, and there are a large number of errors. Therefore, the original technology method has not been applied in the study of jellyfish toxin. In addition, the limitation of the original technology also lies in that the substrate of metalloprotease is relatively low in the peptide library derived from the proteome, so it is necessary to enrich and purify the hydrolysis product to improve the signal strength of mass spectrometry, and the purification process may cause the loss of part of the peptide segment and affect the subsequent analysis.
[0004] The present application intends to improve the original protease substrate peptide library construction method, break through the limitation of the original method, and realize the construction of the jellyfish toxin metalloprotease substrate peptide library, thereby providing strong technical support for the field of jellyfish sting research. SUMMARY
[0005] The application aims to provide a method for constructing a jellyfish toxin metalloprotease substrate peptide library to solve the problems in the prior art.
[0006] To achieve the above object, the application provides the following scheme:
[0007] The application provides a method for constructing a jellyfish toxin metalloprotease substrate peptide library, comprising the following steps:
[0008] A mouse with different degrees of dermatitis is induced by using a metalloprotease inhibitor and a jellyfish toxin;
[0009] Skin tissues with different degrees of dermatitis are collected for proteomic identification, and a substrate membrane protein of the jellyfish toxin metalloprotease is screened through bioinformatics analysis;
[0010] Potential substrate sequences in the substrate membrane protein are obtained through screening of a protein database;
[0011] According to application conditions, sequences are screened from the potential substrate sequences to construct a peptide library, and the jellyfish toxin metalloprotease substrate peptide library is synthesized.
[0012] Further, when the potential substrate sequences in the substrate membrane protein are obtained through screening of a protein database, a beta chain in an extracellular sequence is screened as a potential substrate sequence.
[0013] Further, sequences with a length of no less than 8 amino acids are screened from the potential substrate sequences to construct a peptide library.
[0014] The application further provides a jellyfish toxin metalloprotease substrate peptide library constructed according to the above method.
[0015] The application further provides application of the jellyfish toxin metalloprotease substrate peptide library in jellyfish stings research.
[0016] The application further provides application of the jellyfish toxin metalloprotease substrate peptide library in jellyfish sting treatment drug screening.
[0017] The application discloses the following technical effects:
[0018] The construction method provided by the application can break through the limitation of anhydrous toxin metalloprotease monomer, and a metalloprotease substrate peptide library applied to crude extraction of jellyfish toxin is constructed. The peptide library can avoid the interference of other components of the toxin when the jellyfish toxin is hydrolyzed, and ensure that the metalloprotease plays a dominant role. The peptide library only contains a small amount of potential substrate sequences, so the hydrolysis product is easy to identify, and there is no need for enrichment and purification when applied, which can reduce errors. The sequence contained in the peptide library constructed by the method of the application is clear, and the effect of polypeptide hydrolysis by the toxin is remarkable, which further ensures the application prospect of the peptide library in jellyfish sting research. BRIEF DESCRIPTION OF DRAWINGS
[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.
[0020] Figure 1 Design idea diagram for the construction method of the jellyfish toxin metalloprotease substrate peptide library of the present application;
[0021] Figure 2 BMT regulated jellyfish toxin metalloprotease activity detection result diagram;
[0022] Figure 3 Evaluation result diagram of the dermatitis level of each group of mice; A is the serum TNF-α content detection result; B is the skin thickness detection result; C is the tissue section diagram;
[0023] Figure 4 Adjacent phthalic aldehyde (OPA) method detection of the hydrolysis effect of jellyfish toxin on the peptide library result diagram; A is the detection result of free amino acid concentration at different times; B is the metalloprotease activity detection result; C is the detection result of A after adding OPA reagent reaction. 340nm DETAILED DESCRIPTION
[0024] Now, various exemplary embodiments of the present application will be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.
[0025] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of, for example, concentrations, amounts, and so forth, is to be understood as specifically encompassing every value falling within the range. Additionally, particular values within a stated range, and any other stated or inherent ranges, are intended to be encompassed even if not specifically stated.
[0026] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict, the content of the present specification will control.
[0027] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples presented herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.
[0028] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional, unrecited elements or method steps.
[0029] The NnNV used in the following examples was derived from the tentacle tissue of the jellyfish N. numida and was prepared as follows: The frozen tentacle tissue was placed in a 4°C refrigerator and autolysed in 50% (v / v) filtered natural seawater for 2-5 days. After the tissue was completely autolysed, the lower precipitate and solution were collected and passed through 60 mesh and 100 mesh sieves, respectively, to remove residual impurities. The filtrate was collected and centrifuged at 3000g for 15 min. The precipitate was collected and resuspended and washed with 20 mM PBS (pH 7.4, containing 150 mM NaCl). The precipitate was centrifuged at 12000g at 4°C for 15 min to obtain relatively pure nematocyst. The weight of the nematocyst was measured on an analytical balance, and about 5 times the mass of 20 mM PBS buffer (pH 7.4, containing 150 mM NaCl) was added to resuspend the precipitate. The nematocyst was broken using a bertin biological sample homogenizer at 8000 rpm for 3 x 30 s at 4-10°C. The homogenate was collected and centrifuged at 12000g at 4°C for 15 min, and the supernatant was the NnNV of N. numida.
[0030] Example 1
[0031] Construction method of chrysopeptide metalloprotease substrate peptide library (design idea see Figure 1 ) :
[0032] (1) Detection of batimastat (BMT) regulating metalloprotease activity of chrysopeptide
[0033] Inhibitor BMT (CAS: 130370-60-4) is a broad-spectrum inhibitor that selectively inhibits metalloproteases, has a peptidomimetic structure and active groups designed for metalloproteases, and has an inhibitory effect on jellyfish toxin metalloproteases without interfering with the biological activity of other toxin components. It is the basis for subsequent design of proteomic analysis methods and screening of substrate membrane proteins.
[0034] Chrysopeptide nematocyst venom (NnNV) and metalloprotease inhibitor BMT were prepared as high-concentration solutions for standby. NnNV and BMT were mixed to prepare an inhibitory active toxin sample NnNV-B, with a final concentration of NnNV of 500 μg / mL and a final concentration of BMT of 1 mM.
[0035] Using PBS buffer (20 mM, pH 7.4, containing 0.15 M NaCl) as a negative control and NnNV (500 μg / mL) as a positive control, BMT (1 mM) was set up to exclude interference (named group B), and the metalloprotease activity of NnNV-B was detected by the azocasein substrate method. The specific experimental method is as follows:
[0036] 50.0 mg of azocasein was weighed and dissolved in 10 mL of 50 mM Tris-HCl buffer (pH 8.8, containing 100 mM NaCl and 5 mM CaCl2) to prepare a substrate solution of 5 mg / mL. The toxin samples were uniformly diluted to 500 μg / mL using 20 mM PBS (pH 7.4, containing 150 mM NaCl). 25 μL of toxin sample (12.5 μg) was transferred to a 1.5 mL centrifuge tube containing 100 μL of 5 mg / mL azocasein, and 3 parallel samples were set up for each toxin sample. The negative control group was replaced with Tris-HCl buffer instead of toxin sample. The reaction system was placed in a 37°C electric heating constant temperature drying oven for reaction for 90 min. 200 μL of 5% trichloroacetic acid (w / v) was added to terminate the reaction, and the reaction system was placed at room temperature for 30 min, and then centrifuged at 1000 g and 4°C for 20 min. 150 μL of supernatant was transferred to a 96-well plate and an equal volume of 0.5 mol / L sodium hydroxide was added to neutralize the excess trichloroacetic acid. The A 450nm .
[0037] The results of enzyme activity detection are shown in Figure 2NnNV-B can inhibit 40.3% of the enzymatic activity of NnNV, and the inhibitor itself does not affect the detection results.
[0038] (2) Inducing mice with different degrees of dermatitis
[0039] NnNV and NnNV-B were used to induce dermatitis in mice. The PBS group was the negative control for administering PBS buffer, the B group was the negative control for administering BMT, and the Blank group was the blank control without drug treatment. The specific experimental method is as follows:
[0040] ICR male mice, SPF level, body weight 31±1g, divided into 5 groups, 3 cages per group, 5 mice per cage. The mice were depilated on the back with 8% sodium sulfide and an electric depilator, and then injected subcutaneously on the depilated part. The injection site and needle direction of each mouse were kept consistent. The five groups of mice were injected with corresponding NnNV (500 μg / mL NnNV), NnNV-B (500 μg / mL NnNV+1 mM BMT), BMT (1 mM), PBS or no injection (Blank), with a dose of 60 μL per mouse. After injecting the sample, the timing started, and tissue samples were collected after 6 hours. First, the eyeball was removed to collect blood, and the whole blood was allowed to clot for more than 30 minutes. Centrifugation was performed at 4°C and 3000 rpm for 15 minutes, and the serum sample without hemolysis was collected. The serum of each mouse was divided into 50 μL per part. After completion of the division, the samples were immediately quenched with liquid nitrogen for 15 minutes and stored at -80°C. The skin tissue at the injection site of the mouse was cut. Four mice from each cage were taken for skin, which was stored in a centrifuge tube at -80°C; one mouse was taken for skin, which was pinned flat on a thick tin foil and immersed in formalin fixing solution. Then the level of dermatitis in the three groups of mice was evaluated by serum TNF-α, skin thickness and skin tissue section.
[0041] The experimental results are shown in Figure 3 . In the serum TNF-α detection Figure 3 and skin thickness detection Figure 3 , the NnNV and NnNV-B groups were significantly higher than the B, PBS and Blank groups, and the addition of inhibitor BMT in the toxin had a significant effect on reducing the content of serum TNF-α and reducing the skin thickness. In the tissue section Figure 3 , the difference in tissue thickness and inflammatory cell infiltration further proved that the dermatitis levels of the three groups of mice were significantly different.
[0042] (3) Proteomic analysis of dermatitis tissue in NnNV, NnNV-B and PBS groups
[0043] The proteome of mouse skin tissue was detected by Lable free technology. The specific experimental method is as follows:
[0044] The protein in the mouse skin tissue was extracted by SDT lysis method, and the protein was quantified by BCA method. The appropriate amount of protein was subjected to trypsin digestion by Filter aided proteome preparation (FASP) method. The peptides were desalted and freeze-dried. 40 μL of 0.1% formic acid solution was added to the freeze-dried peptides for reconstitution. OD 280nm Peptide quantification was performed. The sample was separated by Easy nLC system. 0.1% formic acid aqueous solution was used as A liquid, and 0.1% formic acid aqueous solution containing 84% acetonitrile was used as B liquid. The chromatographic column was equilibrated with 95% A liquid. The sample was loaded by automatic sampler. The loading column was ThermoScientific Acclaim PepMap 100, 100 μm x 2 cm, nano Viper C18. The analysis column was ThermoScientific EASY column, 10 cm, ID 75 μm, 3 μm, C18-A2. The flow rate was set to 300 nL / min. Mass spectrometry was performed by Q-Exactive mass spectrometer, and the detection mode was positive ion. MaxQuant software (version number 1.5.3.17) was used to identify and quantify the protein of the RAW file of the original data. Bioinformatics analysis was performed on the protein identification and quantification data, including gene ontology annotation (GO) analysis and subcellular localization. The present application first identifies differential proteins through the comparison group "NnNV_VS_PBS", and filters out differential proteins (plasma membrane and extracellular matrix) in the main action site of toxins through bioinformatics analysis. Then, the content regulation trend of the differential proteins screened by the comparison group "NnNV_VS_NnNV-B_VS_PBS" is analyzed to determine the substrate protein.
[0045] Experimental results: the differential protein identification results of the comparison group "NnNV_VS_PBS" are shown in Table 1. The relative abundance difference data shows that NnNV up-regulates the content of 25 proteins and down-regulates the content of 99 proteins. The absolute abundance difference data shows that NnNV adds 49 proteins and loses 105 proteins in the tissue.
[0046] Table 1 Differential protein identification results of the comparison group "NnNV_VS_PBS"
[0047]
[0048] The key differential proteins related to the symptoms of dermatitis and distributed in the plasma membrane and extracellular matrix were screened from the 278 differential proteins by GO analysis and subcellular localization analysis, including Cell adhesion molecule 3, H-2class I histocompatibility antigen, Receptor-type tyrosine-protein phosphatase C, Receptor-type tyrosine-protein phosphatase F, Integrin alpha-1, and Integrin alpha-2. The abundance of the key differential proteins in the NnNV, NnNV-B and PBS groups is shown in Table 2. It can be seen that each protein meets the condition that the content is the highest in the PBS group and the lowest in the NnNV group, which can be regarded as the substrate protein of the toxic metalloproteinase. Among them, the content of Cell adhesion molecule 3 in the NnNV-B group is lower than the detection limit of the instrument, and its correlation with the activity of metalloproteinase cannot be directly determined. However, it is distributed in the extracellular matrix, and NnNV-B still has high metalloproteinase activity, so it is most likely to be a highly selective target protein of the toxic metalloproteinase.
[0049] Table 2 Comparison of the abundance of key differential proteins in NnNV, NnNV-B and PBS groups
[0050]
[0051]
[0052] Note: "n" represents that the abundance value is lower than the detection limit of the instrument.
[0053] (4) Potential substrate sequence screening and construction of peptide library
[0054] The substrate proteins in Table 2 were searched in the Uniprot protein database, and the key information of the potential substrate sequence in the extracellular domain was extracted, including the source of secondary structure, the location of beta chain, the length of beta chain and the sequence of beta chain. The sequences with a length of not less than 8 amino acids were used to construct the peptide library, and the sequence information is shown in Table 3.
[0055] Table 3 Peptide library constructed by 41 potential substrate sequences screened
[0056]
[0057]
[0058] (5) Peptide library synthesis and toxin hydrolysis
[0059] The HPLC purity of each polypeptide in the 41 polypeptide sequences in the synthetic peptide library was greater than 98%, and the synthesized polypeptide was indeed the target sequence by MS analysis. The 41 polypeptides were mixed in equal amounts to obtain a peptide pool sample, which was stored as a freeze-dried powder at -80°C. The hydrolysis of the peptide pool sample by palytoxin was detected by o-phthaldehyde (OPA) reaction. The specific experimental method was as follows:
[0060] The peptide pool sample was dissolved in PBS, and NnNV and the peptide pool were mixed at a mass ratio of w / w (NnNV / peptide pool) = 1:50. The reaction was incubated in a 37°C constant temperature oven. After reaching the reaction time, the reaction was terminated by heating at 70°C for 30 min. Then 50 μL of the reaction solution was added to 300 μL of OPA reagent. After 2 min of accurate reaction, the A 340nm In the analysis of the hydrolysis of the peptide pool by palytoxin, the peptide pool solution was used as a negative control. In the analysis of the hydrolysis of the peptide pool by toxin metalloproteinase, the reaction solution with a w / w (NnNV / peptide pool) = 1:50 was used as a positive control, and the peptide pool solution was used as a negative control. In the experimental group, the NnNV solution in the reaction system was replaced by NnNV with added complex protease inhibitors. The group without added inhibitors was labeled as N0, the group with a volume ratio (toxin / inhibitor) of 200:1 was labeled as N1, and the group with a volume ratio of 25:1 was labeled as N2. The complex protease inhibitor was a commercial protease inhibitor complex (Shanghai Sangon, C600386) with the following components and contents: phenylmethylsulfonyl fluoride (PMSF; CAS: 329-98-6), 100 mM; bestatin (CAS: 58970-76-6), 1 mM; pepstatin A (CAS: 26305-03-3), 1.5 mM; E-64 (CAS: 66701-25-5), 1.4 mM; aprotinin (CAS: 9087-70-1), 0.08 mM; and leupeptin (CAS: 103476-89-7), 1 mM.
[0061] The experimental results are shown in Figure 4 . Figure 4 As shown in Fig. A, the concentration of free amino groups increased with the increase of reaction time, indicating that palytoxin had hydrolysis effect on the peptide pool sample. Figure 4 As shown in Figs. B-C, under the action of complex protease inhibitors, the hydrolysis ability of the toxin sample on the peptide pool did not change when the activity of the toxin metalloproteinase was unchanged, and the degree of hydrolysis of the peptide pool also decreased significantly when the activity of the toxin metalloproteinase decreased significantly. This indicates that the toxin metalloproteinase component plays a dominant role in the hydrolysis of the peptide pool.
[0062] The above described embodiments are only to illustrate the preferred modes of the present application, and are not intended to limit the scope of the present application. Any modification and improvement made by those skilled in the art to the technical solutions of the present application without departing from the design spirit of the present application shall fall within the protection scope of the present application as defined by the claims.
Claims
1. A method for constructing a library of jellyfish toxin metalloprotease substrate peptides, characterized by, The method comprises the following steps: inducing different degrees of dermatitis in mice by using metalloproteinase inhibitors and jellyfish toxin; collecting skin tissues of different degrees of dermatitis for proteomic identification, screening the substrate membrane proteins of jellyfish toxin metalloproteinase by bioinformatics analysis; screening potential substrate sequences in the substrate membrane proteins by using protein database; constructing a peptide library from the potential substrate sequences according to application conditions, and synthesizing the jellyfish toxin metalloproteinase substrate peptide library.
2. The construction method according to claim 1, characterized in that, When screening potential substrate sequences in the substrate membrane proteins by using protein database, the beta chain in the extracellular sequence is screened as a potential substrate sequence.
3. The construction method of claim 1, wherein, A peptide library is constructed from sequences with a length of no less than 8 amino acids.
4. A jellyfish toxin metalloproteinase substrate peptide library constructed by the construction method according to any one of claims 1-3.
5. Application of the jellyfish toxin metalloproteinase substrate peptide library according to claim 4 in jellyfish sting research.
6. Application of the jellyfish toxin metalloproteinase substrate peptide library according to claim 4 in jellyfish sting treatment drug screening.