Screening method of jellyfish toxin metalloproteinase substrate sequence

Abstract

The invention discloses a screening method of a jellyfish toxin metalloproteinase substrate sequence, and relates to the technical field of biology. The screening method comprises the following steps: regulating and controlling the metalloproteinase activity of the jellyfish toxin by using a compound protease inhibitor, inhibiting the activity of nonmetal protease in the toxin, retaining the metalloproteinase activity and causing significant difference of the metalloproteinase activity; the hydrolysis effect of the jellyfish toxin on a substrate peptide library is detected, and it is ensured that toxins with different enzyme activities have significant differences on the hydrolysis capacity of the substrate peptide library; and identifying the hydrolysate of the substrate peptide library, and screening the jellyfish toxin metalloproteinase substrate sequence in combination with the change trend of toxin enzyme activity and the change trend of hydrolysate abundance. By adopting the screening method, a toxin metalloproteinase substrate sequence and a cleavage site can be successfully analyzed by utilizing the jellyfish crude toxin, so that a technical support is provided for research and development of polypeptide drugs stung by jellyfish.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for screening substrate sequences of jellyfish toxin metalloproteinases. Background Technology

[0002] Jellyfish stings involve toxin metalloproteinases that play a significant pro-inflammatory role, making toxin metalloproteinase inhibitors a potential treatment for jellyfish stings. Compared to small molecule metal ion chelators, peptide inhibitors have more binding sites to proteases, theoretically offering better performance in terms of affinity, selectivity, and side effects. Peptides and peptide mimics are a crucial part of the protease inhibitor field.

[0003] Protease substrate sequences are the structural basis for the development of protease peptide inhibitors and can serve as backbone molecules for the design and modification of peptide drugs. Before the availability of protease substrate sequence profiles and the analysis of binding site preferences, the development of protease peptide inhibitors could only utilize a limited number of known substrate sequences, resulting in limitations such as narrow applicability, few modification sites, and poor selectivity.

[0004] Currently, the efficient acquisition of protease substrate sequences relies on proteomics-driven protease cleavage site identification technology. This technology mainly includes three steps: "constructing a peptide library," "protease-degraded peptide library," and "analysis of degradation products." By constructing different peptide libraries, research objectives such as analyzing different proteases and screening high-affinity substrate sequences can be achieved. Considering the complex composition of jellyfish toxins, this invention aims to develop a novel method for screening jellyfish toxin metalloproteinase substrate sequences, thereby providing technical support for the development of peptide drugs for jellyfish stings. Summary of the Invention

[0005] The purpose of this invention is to provide a method for screening the substrate sequences of jellyfish toxin metalloproteinases, thereby addressing the problems existing in the prior art. This screening method enables the successful analysis of toxin metalloproteinase substrate sequences and cleavage sites using crude jellyfish venom, thus providing technical support for the development of peptide drugs for jellyfish stings.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a method for screening substrate sequences of jellyfish toxin metalloproteinases, comprising the following steps:

[0008] By using a complex protease inhibitor to regulate the metalloproteinase activity of jellyfish toxins, the activity of non-metalloproteinases in the toxins was inhibited, while the activity of metalloproteinases was preserved and significant differences in metalloproteinase activity were caused.

[0009] The study investigated the hydrolytic effect of jellyfish toxins on a substrate peptide library, ensuring that toxins with different enzyme activities also exhibited significant differences in their hydrolytic capacity for the substrate peptide library.

[0010] We identified the hydrolysis products of the substrate peptide library and screened jellyfish toxin metalloproteinase substrate sequences by combining the trends of toxin enzyme activity and hydrolysis product abundance.

[0011] Furthermore, the complex protease inhibitor includes the following inhibitors: benzyl sulfonyl fluoride, ubenimex, pepsin A, E-64, aprotinin, and leucinogen.

[0012] Further, the contents of the benzyl sulfonyl fluoride, the ubenimex, the pepsin A, the E-64, the aprotinin and the leucinogen are 100mM, 1mM, 1.5mM, 1.4mM, 0.08mM and 1mM, respectively.

[0013] Furthermore, the azocasein substrate method was used to detect the metalloproteinase activity of jellyfish toxins.

[0014] Furthermore, the phthalaldehyde method was used to detect the hydrolytic effect of jellyfish toxins on the substrate peptide library.

[0015] Furthermore, the hydrolysis products of the substrate peptide library were identified using LC-MS / MS, and the peptide sequences and abundance values ​​generated in different reaction systems were obtained.

[0016] Furthermore, the reaction system includes a negative control group, a strong metalloproteinase activity group, and a weak metalloproteinase activity group;

[0017] A substrate peptide library without the addition of the jellyfish toxin was used as a negative control group;

[0018] A substrate peptide library incubated with the jellyfish toxin and the complex protease inhibitor at a volume ratio of 100:1 was used as a group with strong metalloproteinase activity.

[0019] A substrate peptide library that was incubated with the jellyfish toxin and the complex protease inhibitor at a volume ratio of 25:1 was used as the weak metalloproteinase activity group.

[0020] Furthermore, the affinity between the substrate sequence and the jellyfish toxin metalloproteinase was analyzed by abundance difference fold. The higher the abundance difference fold, the higher the sequence yield, that is, the stronger the affinity between the substrate sequence and the jellyfish toxin metalloproteinase.

[0021] The present invention discloses the following technical effects:

[0022] This invention provides a method for screening substrate sequences of metalloproteinases from jellyfish toxins. A complex protease inhibitor is used to eliminate non-metallic protease activity in the toxin, reducing interfering sequences in the degradation products and ensuring that the dominant products originate from the degradation of the peptide library by the metalloproteinases. To further reduce false-positive errors caused by other experimental conditions that might break the original sequences, this invention includes a weak metalloproteinase activity control group, allowing changes in the abundance values ​​of the substrate sequence degradation products. The substrate sequences of the metalloproteinases are determined by screening the trends in abundance value changes. Using this method, this invention, for the first time, successfully analyzes the substrate sequences and cleavage sites of metalloproteinases from crude jellyfish venom, thus providing technical support for the development of peptide drugs for jellyfish stings. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a graph showing the results of metalloproteinase activity detection;

[0025] Figure 2 This is a graph showing the results of free amino group concentration detection;

[0026] Figure 3 Total ion chromatogram obtained during sequence identification of experimental samples in the Peptide group;

[0027] Figure 4 Total ion chromatogram obtained during sequence identification of N1 experimental samples;

[0028] Figure 5 Total ion chromatogram obtained during sequence identification of N2 group experimental samples;

[0029] Figure 6 Total ion chromatogram obtained when identifying sequences in a peptide library after direct incubation with jellyfish toxin for 6 hours;

[0030] Figure 7 Total ion chromatogram obtained when identifying sequences in a peptide library after direct incubation with jellyfish toxin for 12 hours;

[0031] Figure 8 After incubating the peptide library with jellyfish toxin for 6 hours, we identified the cleavage products and abundance values ​​of the sequence EVLYTPTAMIRPE in the peptide library.

[0032] Figure 9The cleavage products and abundance values ​​of the sequence EVLYTPTAMIRPE in the peptide library were identified after 12 hours of direct incubation with jellyfish toxin. Detailed Implementation

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0035] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0036] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0037] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0038] The crude jellyfish toxin (NnNV) used in the following examples was derived from the tentacles of the sand jellyfish, and the preparation method is as follows:

[0039] Frozen jellyfish tentacle tissue was placed in a 4°C freezer and autolyzed for 2-5 days with 50% (v / v) filtered natural seawater. After complete autolysis, the lower precipitate and solution were collected and filtered through 60-mesh and 100-mesh sieves to remove residual impurities. The filtrate was collected, centrifuged at 3000g for 15 min, and the precipitate was collected and resuspended and washed with 20mM PBS (pH 7.4, containing 150mM NaCl). Centrifugation at 12000g, 4°C for 15 min yielded relatively pure nematocysts. The nematocysts were weighed on an analytical balance, and approximately 5 times their weight of 20mM PBS buffer (pH 7.4, containing 150mM NaCl) was added to fully resuspend the precipitate. The nematocysts were then homogenized using a Bertin biological sample homogenizer at 8000 rpm, 3 × 30 s, at 4-10°C. Collect 12,000g of the crushed homogenate, centrifuge at 4°C for 15 minutes, and the supernatant is the jellyfish toxin.

[0040] Example 1

[0041] Regulation of toxin protease activity:

[0042] The protein concentration of crude jellyfish toxin (NnNV) was adjusted to 500 μg / mL. The activity of the toxin protease was regulated using a complex protease inhibitor (Shanghai Sangon Biotech, C600386). The components and contents of the complex protease inhibitor were as follows: benzyl sulfonyl 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.

[0043] Jellyfish toxin NnNV was mixed with a complex protease inhibitor at different ratios to obtain three groups of jellyfish toxin samples. The v / v ratios of crude jellyfish toxin to complex protease inhibitor were N0 (100:0), N1 (100:1), and N2 (25:1), respectively. After mixing, the samples were incubated at room temperature for 30 min, and the changes in metalloproteinase activity of jellyfish toxin NnNV were detected using the azocasein substrate method. The specific experimental method is as follows:

[0044] First, weigh 50.0 mg of azocasein and dissolve it in PBS buffer to prepare a 5 mg / mL substrate solution. Transfer 25 μL of jellyfish toxin sample (12.5 μg) to a 1.5 mL centrifuge tube containing 100 μL of 5 mg / mL Azocasein. Set up three replicates for each toxin sample, using the buffer as a negative control. Incubate the reaction system at 37°C for 90 min, then add 200 μL of 5% trichloroacetic acid (w / v) to terminate the reaction. Incubate at room temperature for 30 min, then centrifuge at 1000 g, 4°C for 20 min. Transfer 150 μL of the supernatant to a 96-well plate and add an equal volume of 0.5 mol / L sodium hydroxide. Detect Azocasein using a microplate reader. 450nm .

[0045] Results of metalloproteinase activity assay are shown below Figure 1 The absorbance values ​​were as follows: negative control group (PBS) 0.07, N0 group (no inhibitor) 0.14, N1 group (100:1) 0.14, and N2 group (25:1) 0.10. These data indicate that NvNV, exhibiting strong metalloproteinase activity, showed no effect from the addition of a 100:1 (v / v) complex protease inhibitor, but its activity significantly decreased upon the addition of a 25:1 (v / v) complex protease inhibitor.

[0046] Degradation of the peptide library was detected using the o-phthalaldehyde (OPA) method. The specific steps were as follows: jellyfish toxin (N0) and mixed inhibitor jellyfish toxin samples (N1, N2) were mixed with the peptide library (Table 1), with a toxin / peptide pool sample (v / v) ratio of 1:50, and three replicates per group. The peptide library was used as a blank control. The mixture was incubated at 37°C for 12 hours. After 6 hours of reaction, the reaction was terminated by heating at 70°C for 30 minutes. 50 μL of the reaction solution was added to a 1.5 mL EP tube containing 300 μL of OPA reagent. Each group was reacted with the OPA reagent for the same time (2-3 minutes), and the absorbance was measured at 340 nm. The free amino concentration of each group was calculated using a standard curve of serine concentration-absorbance values. An increase in free amino concentration indicated that the peptide library sequence had broken down, releasing new ends.

[0047] Table 1 Substrate peptide library

[0048]

[0049]

[0050] The results of the free amino concentration test are shown in Figure 2The concentrations of peptides in the blank control group (NvNV) were 1.38 mmol / L, in the N0 group (NnNV) 4.89 mmol / L, in the N1 group (100:1) 4.25 mmol / L, and in the N2 group (25:1) 4.31 mmol / L. The data indicate that the toxin NvNV significantly degrades the peptide library, and the degradation effect significantly decreased after the addition of a 100:1 complex protease inhibitor. Even after the addition of a 25:1 complex protease inhibitor, the concentration of free amino acids still showed a significant decrease.

[0051] The lack of significant difference between groups N2 and N1 could be due to interference from free amino groups in the complex protease inhibitors, or the presence of uninhibited non-metallic proteases in the toxins. Calculations ruled out inhibitor interference. The four components of the complex protease inhibitors—Bestatin (1 mol of -NH2 per mole), E-64 (2 mol of -NH2 per mole), Aprotinin (21 mol of -NH2 per mole), and Leupeptin (2 mol of -NH2 per mole)—contain free amino groups. Calculations showed that the increase in free amino concentration at inhibitor ratios of 100:1 and 25:1 was 0.0014 mmol / L and 0.0056 mmol / L, respectively, within the error range. Therefore, the lack of further decrease in free amino concentration in group N2 may be due to uninhibited non-metallic proteases. These proteases partially overlap with the substrates of metalloproteinases, and their common substrates are primarily degraded by these non-metalloproteinases when metalloproteinase activity decreases, thus compensating for the decrease in free amino concentration.

[0052] The above analysis further highlights the necessity of this invention in screening metalloproteinase substrate sequences using differences in metalloproteinase activity and changes in product abundance values, which can further exclude non-selective substrates.

[0053] Peptide identification: Peptides in the samples were identified by LC-MS / MS analysis using an online nanojet ion source, providing a systematic and comprehensive study of endogenous peptides in the samples. The Q Exactive index of the tandem EASY-nanoLC 1200 was utilized. TM Mass spectrometry technology enables precise identification and separation of peptides, combined with annotation of mass spectrometry database information.

[0054] The total ion chromatograms of samples peptide, N1, and N2 are shown below. Figures 3-5 The peak shapes in the mass spectrometry are similar in samples N1 and N2, indicating that the main components of the three samples are the original substrate sequences from the peptide library, while the sequences generated by protease degradation are relatively low in content. Further analysis of the abundance changes of each sequence is needed.

[0055] Substrate sequence analysis: Fragments with high confidence (FDR ≤ 0.01) were screened from peptide identification results. Newly generated ends in the fragments were identified as cleavage sites of toxin proteases. In the experimental group that regulated toxin metalloproteinase activity, the abundance value of the fragments was included in the analysis. Fragments whose abundance value change trend was the same as that of enzyme activity regulation were considered as hydrolysis products of toxin metalloproteinases. The larger the fold change (Fc) of the fragment abundance value between the toxin group and the negative control group, the higher the fragment yield and the stronger the selectivity between the substrate and the toxin metalloproteinase.

[0056] A total of 67 high-confidence peptides (FDR≤0.01) were identified from three samples. Based on the abundance change trend of N1>N2>Peptide, hydrolysis products of toxin metalloproteinases were screened from these peptides. The screening results are shown in Table 2. In Table 2, sequences are sorted from high to low according to the fold change (Fc=Abundance N1 / AbundancePeptide). Sequences with a high ratio indicate high content and are likely to be dominant substrate sequences for toxin metalloproteinases. Among them, the peptides ranked 1st, 3rd, and 4th all originated from the substrate sequence EPRGQPIIQDV, indicating that this sequence may be a highly selective sequence for toxin metalloproteinases. The hydrolysis product of this sequence, [E].PRGQPIIQDV.[-], ranked 1st in the table, meets the condition that the abundance change is related to the activity of toxin metalloproteinases. It is also the main product of the direct hydrolysis peptide pool of the toxin sample, further increasing its possibility of high selectivity with toxin metalloproteinases. The newly generated amino acid ends in the above sequence are the cleavage sites, which include N-terminal (6), C-terminal (2) and internal cleavage sites (6).

[0057] Table 2. Hydrolysis products and abundance values ​​of toxin metalloproteinases

[0058]

[0059] Comparative Example 1

[0060] This comparative example is intended to illustrate that without using the method of the present invention, the product sequences of the peptide library degraded by jellyfish toxin cannot be analyzed to obtain the metalloproteinase substrate sequences.

[0061] Jellyfish toxins were incubated directly with the peptide library (Table 1) for 6 and 12 hours, followed by sequence identification of the peptide library. The incubation method and peptide sequence identification method were the same as in Example 1.

[0062] Total ion chromatograms of samples incubated for 6 hours and 12 hours are shown below. Figures 6-7It is evident that the sample incubated for 12 hours is weaker in both peak number and peak intensity than the sample incubated for 6 hours. This indicates that prolonged incubation reduces the number of sequences that can be identified by mass spectrometry, and that the toxin has an exonucleating effect on each peptide in the peptide library, resulting in unidentifiable free amino acids and residual short peptides.

[0063] The major sequences and abundance values ​​of the peptide library were identified after 6 hours and 12 hours of incubation, as shown in the figure. Figures 8-9 The products mainly originated from the sequences EVLYTPTAMIRPE and EPRGQPIIQDV. The N-terminus of EVLYTPTAMIRPE showed a clear sequential hydrolysis phenomenon, indicating that the toxin protease has an exonuclease activity at its N-terminus, and its activity is similar to that of aminopeptidases with a broad substrate spectrum. In terms of peptide abundance, [T].PTAMIRPE.[-] was the most abundant hydrolysis product, suggesting that the TP site in this sequence may be the cleavage site of another endopeptidase. The cleavage sites at the C-terminus of this sequence showed no obvious pattern. The EPRGQPIIQDV sequence also showed N-terminal and C-terminal breaks, and the products retained the [Q].PIIQ.[D] portion at sites 6-9, indicating that this sequence may be the binding site of the inactive site of the protease. The most abundant product was [E].PRGQPIIQDV.[-], but there was no obvious pattern of abundance variation among the products.

[0064] The analysis of the two substrate sequences indicates that the same substrate sequence can be acted upon by multiple proteases, and is more likely to be simultaneously hydrolyzed by aminopeptidase, carboxypeptidase, and endopeptidase. The complex composition of toxin proteases significantly interferes with the experimental results. Due to the lack of prior knowledge about the catalytic mechanism of toxin metalloproteinases, it is impossible to further determine which sites are generated by metalloproteinase hydrolysis in this case.

[0065] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for screening substrate sequences of jellyfish toxin metalloproteinases, characterized in that, Includes the following steps: By using a complex protease inhibitor to regulate the metalloproteinase activity of jellyfish toxins, the activity of non-metalloproteinases in the toxins was inhibited, while the activity of metalloproteinases was preserved and significant differences in metalloproteinase activity were caused. The hydrolytic effect of the jellyfish toxin on the substrate peptide library was detected to ensure that toxins with different enzyme activities also have significant differences in their hydrolytic ability on the substrate peptide library; We identified the hydrolysis products of the substrate peptide library and screened jellyfish toxin metalloproteinase substrate sequences by combining the trends of toxin enzyme activity and hydrolysis product abundance.

2. The screening method according to claim 1, characterized in that, The complex protease inhibitors include the following inhibitors: benzyl sulfonyl fluoride, ubenimex, pepsin A, E-64, aprotinin, and leucinogen.

3. The screening method according to claim 2, characterized in that, The contents of the benzyl sulfonyl fluoride, the ubenimex, the pepsin A, the E-64, the aprotinin and the leucinogen are 100 mM, 1 mM, 1.5 mM, 1.4 mM, 0.08 mM and 1 mM, respectively.

4. The screening method according to claim 1, characterized in that, The activity of metalloproteinases in jellyfish toxins was detected using the azocasein substrate method.

5. The screening method according to claim 1, characterized in that, The hydrolytic effect of jellyfish toxins on a substrate peptide library was detected using the phthalaldehyde method.

6. The screening method according to claim 1, characterized in that, The hydrolysis products of the substrate peptide library were identified using LC-MS / MS, and the peptide sequences and abundance values ​​generated in different reaction systems were obtained.

7. The screening method according to claim 6, characterized in that, The reaction system includes a negative control group, a strong metalloproteinase activity group, and a weak metalloproteinase activity group. A substrate peptide library without the addition of the jellyfish toxin was used as a negative control group; A substrate peptide library that was incubated with the jellyfish toxin and the complex protease inhibitor at a volume ratio of 100:1 was used as a strong metalloproteinase activity group. A substrate peptide library incubated with the jellyfish toxin and the complex protease inhibitor at a volume ratio of 25:1 was used as the weak metalloproteinase activity group.

8. The screening method according to claim 7, characterized in that, The affinity between substrate sequences and jellyfish toxin metalloproteinases was analyzed by abundance difference fold analysis. A higher abundance difference fold indicates a higher sequence yield, that is, a stronger affinity between the substrate sequence and jellyfish toxin metalloproteinases.

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