Four uric acid reducing peptides derived from mottled bamboo sharks

Abstract

The invention relates to rapid screening of uric acid reducing peptides in marine-derived mottled bamboo sharks, belongs to the technical field of marine biological medicines, and particularly relates to four uric acid reducing peptides from the mottled bamboo sharks. The number of the xanthine oxidase (XO) inhibitory peptides is four, and the amino acid sequences of the xanthine oxidase (XO) inhibitory peptides are respectively NCDEK, PGEAG, GDSGL and GDDGL. The XO inhibitory peptide is applied to serving as or preparing an XO inhibitor, and is applied to serving as or preparing a medicine with the effect of inhibiting the uric acid level. According to the invention, 8 potential uric acid-reducing peptides of Top8 are rapidly screened out from mottled bamboo shark protein by using enzymolysis and molecular docking technologies, the XO inhibitory activity is determined by high performance liquid chromatography, and the result shows that four small molecule peptides have obvious inhibitory activity on XO, and the four small molecule peptides have the potential of becoming XO inhibitors / uric acid-reducing drugs.

Description

Technical Field

[0001] The present invention relates to the rapid screening of uric acid-lowering polypeptides from the protein of Chiloscyllium plagiosum sourced from the ocean, and belongs to the technical field of marine biomedicine. Background Art

[0002] Uric acid is the end product of purine metabolism, and a significant increase in its level will lead to hyperuricemia. Xanthine oxidase (XO) is the key enzyme that catalyzes the formation of uric acid from hypoxanthine and xanthine, and is therefore considered an important target for lowering uric acid. Although traditional uric acid-lowering drugs have good uric acid-lowering effects, they often have serious side effects. Therefore, it is of great significance to develop a class of natural uric acid-lowering peptides with significant efficacy and no side effects.

[0003] XO inhibitory peptides are a class of natural active peptides that play a uric acid-lowering role by inhibiting the activity of XO and are widely present in a variety of foods. Currently, marine-derived proteins are considered high-quality sources for preparing XO inhibitory peptides. The muscle protein content of Chiloscyllium plagiosum is high, and it has both edible and medicinal values and is a good raw material for obtaining active peptides. However, currently, Chiloscyllium plagiosum is mostly processed into fish paste or fish balls, etc., and its economic added value is low. Therefore, obtaining XO inhibitory peptides from marine-derived Chiloscyllium plagiosum is an effective measure to improve its market utilization value.

[0004] The traditional preparation process of XO inhibitory peptides includes enzymatic hydrolysis and separation and purification. Among them, the separation and purification process is time-consuming and inefficient, and may also cause loss of the target polypeptide. In recent years, means such as molecular docking have been gradually applied to the separation and purification of active peptides. Through computer simulation docking, the separation and purification process of active peptides can be accelerated, providing the possibility for the rapid screening of active peptides.

[0005] The main idea of the present invention is to obtain polypeptide sequences from the protein of Chiloscyllium plagiosum through enzymatic hydrolysis, then quickly screen out XO inhibitory peptides with uric acid-lowering activity from the polypeptide sequences by means of molecular docking technology, and finally analyze and clarify the action mechanism of XO inhibitory peptides through molecular docking. Summary of the Invention

[0006] In view of the above-mentioned prior art, in order to develop XO inhibitory peptides, the present invention uses enzymatic hydrolysis and molecular docking to quickly screen out 4 small-molecule polypeptides with significant uric acid-lowering activity from Chiloscyllium plagiosum, and analyzes the action mechanism of XO inhibitory peptides through molecular docking.

[0007] The present invention is realized through the following technical solutions: XO inhibitory peptides, including the following 4 kinds, and their amino acid sequences are respectively: NCDEK, as shown in SEQ ID NO.1; PGEAG, as shown in SEQ ID NO.2; GDSGL, as shown in SEQ ID NO.3; GDDGL, as shown in SEQ ID NO.4.

[0008] Use of the above 4 kinds of XO inhibitory peptides in the manufacture of or as XO inhibitors; use in the manufacture of or as a drug with uric acid lowering efficacy.

[0009] The XO inhibitory peptide of the present invention is rapidly screened from Chiloscyllium plagiosum by enzymatic hydrolysis and molecular docking, and the process is as follows: (1) Preparation and ultrafiltration of the enzymatic hydrolysate of Chiloscyllium plagiosum The Chiloscyllium plagiosum meat was minced and stirred evenly with distilled water at a solid-liquid ratio of 1:5 (w / v), the initial pH was adjusted to 2.0, and pepsin was added at an enzyme addition amount of 1.5% (w / w), and the reaction was carried out at 37 °C for 1.5 h. Then the pH of the mixture was adjusted to 6.5, and 1.0% (w / w) of trypsin and α-chymotrypsin were added and the reaction was continued at 37 °C for 2.0 h. The enzymatic hydrolysate was further separated by 3 kDa and 10 kDa ultrafiltration tubes, the ultrafiltered solution was collected and freeze-dried to obtain the Chiloscyllium plagiosum polypeptide component with uric acid lowering activity.

[0010] (2) Liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis of the polypeptide component An appropriate amount of 0.1% trifluoroacetic acid was added to the polypeptide component, mixed evenly, desalted using a C18 StageTip, and vacuum dried. The dried sample was redissolved with 0.1% fluoroacetic acid, the sample concentration was measured, and LC-MS / MS analysis was carried out. The sample was chromatographically separated using a nano-flow rate Easy nLC1200 chromatographic system (Thermo Scientific). Buffer: Solution A was 0.1% formic acid aqueous solution, solution B was a mixed solution of 0.1% formic acid, acetonitrile and water (where acetonitrile was 80%), and the flow rate was 300 nL / min.

[0011] (3) Molecular docking to screen potential XO inhibitory peptides The polypeptides released during the enzymatic hydrolysis of Chiloscyllium plagiosum were analyzed by molecular docking technology, and these polypeptides were used as ligands for molecular docking, with XO as the receptor. The crystal structure of XO (PDB ID: 1N5X) was obtained from the RCSB Protein Data Bank ( https: / / www.rcsb.org / ). Autodock vina was used as the tool for molecular docking. The structure of the polypeptide was drawn by the software Chemdraw 19.0. Finally, potential XO inhibitory peptides were screened by the vina value.

[0012] (4) Synthesis and activity determination of XO inhibitory peptides Using the above method, the present invention screened out 8 (Top8) potential XO inhibitory peptides of marine origin. The XO inhibitory rate was determined by high performance liquid chromatography (HPLC). The results showed that 4 marine-derived polypeptides had significant inhibitory activity against XO. These marine-derived polypeptides have the potential to be used as xanthine oxidase inhibitors and can be used to prepare drugs with the efficacy of inhibiting uric acid levels. Among them, the polypeptides NCDEK, PGEAG, GDSGL and GDDGL had the best XO inhibitory activity and had the potential to be used as functional products / drugs for reducing uric acid.

[0013] Positive and beneficial effects of the present invention: The present invention is an innovation of the traditional screening method of marine-derived peptides for reducing uric acid. The use of molecular docking technology enables the rapid screening of XO inhibitory peptides and improves the screening efficiency of peptides for reducing uric acid. At the same time, the XO inhibitory peptides screened by the present invention have potential value for the treatment of hyperuricemia.

[0014] All terms and phrases used in the present invention have the general meanings known to those skilled in the art. Description of the drawings

[0015] Figure 1 : Inhibitory activity of Chiloscyllium plagiosum enzymatic hydrolysate and ultrafiltration fractions against XO.

[0016] Figure 2 : Mass spectrometry Basepeak diagram of the sample.

[0017] Figure 3 : XO inhibitory activity of Top8 polypeptides and vina docking energy values. Among them, A: vina docking energy values of active Top8 polypeptides; B: XO inhibitory rate of Top8 polypeptides.

[0018] Figure 4 : Molecular docking results of XO inhibitory peptide NCDEK and XO.

[0019] Figure 5 : Amino acid residues and interaction forces of XO inhibitory peptide NCDEK binding to XO; among them, A: main amino acid residues of XO inhibitory peptide NCDEK forming hydrogen bond interaction with XO; B: all interaction forces formed by XO inhibitory peptide NCDEK binding to XO.

[0020] Figure 6 : Molecular docking results of XO inhibitory peptide PGEAG and XO.

[0021] Figure 7 : Amino acid residues and interaction forces of XO inhibitory peptide PGEAG binding to XO; among them, A: main amino acid residues of XO inhibitory peptide PGEAG forming hydrogen bond interaction with XO; B: all interaction forces formed by XO inhibitory peptide PGEAG binding to XO.

[0022] Figure 8 : Molecular docking results of XO inhibitory peptide GDSGL and XO.

[0023] Figure 9 : Amino acid residues and interaction forces of XO inhibitory peptide GDSGL binding to XO; among them, A: Main amino acid residues of XO inhibitory peptide GDSGL forming hydrogen bonds with XO; B: All interaction forces formed by XO inhibitory peptide GDSGL binding to XO.

[0024] Figure 10 : Molecular docking results of XO inhibitory peptide GDDGL and XO.

[0025] Figure 11 : Amino acid residues and interaction forces of XO inhibitory peptide GDDGL binding to XO; among them, A: Main amino acid residues of XO inhibitory peptide GDDGL forming hydrogen bonds with XO; B: All interaction forces formed by XO inhibitory peptide GDDGL binding to XO. Specific embodiments

[0026] The following specific embodiments further illustrate the present invention. However, the scope of the present invention is not limited to the following examples. Those skilled in the art can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention.

[0027] For the instruments, reagents, materials, etc. involved in the following examples, unless otherwise specified, they are all conventional instruments, reagents, materials, etc. existing in the prior art and can be obtained through regular commercial channels. For the experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, they are all conventional experimental methods, detection methods, etc. existing in the prior art.

[0028] Example 1 Preparation and ultrafiltration of Chiloscyllium plagiosum enzymatic hydrolysate The Chiloscyllium plagiosum meat was minced and stirred evenly with distilled water at a solid-liquid ratio of 1:5 (w / v), the initial pH was adjusted to 2.0, and pepsin was added at an enzyme addition amount of 1.5% (w / w), and the reaction was carried out at 37 °C for 1.5 h. Then the pH of the mixture was adjusted to 6.5, and 1.0% (w / w) of trypsin and α-chymotrypsin were added and further reacted at 37 °C for 2.0 h. After the reaction, the mixture was boiled for 10 min to inactivate the enzyme, centrifuged at 4 °C and 8000 r / min for 20 min, and the supernatant was taken, and then its XO inhibitory activity was measured.

[0029] The enzymatic hydrolysate was ultrafiltered and separated using 3 kDa and 10 kDa ultrafiltration tubes respectively, and the solution was divided into three components with molecular weights > 10 kDa, molecular weights between 3 kDa and 10 kDa, and molecular weights < 3 kDa. The corresponding component solutions were collected, freeze-dried, and the XO inhibition rate was measured. The results are as Figure 1 shown. The enzymatic hydrolysate was ultrafiltered into three components with different molecular weights. The component with a molecular weight < 3 kDa had the best inhibitory activity against XO, and the inhibition rate was 54.47%. Therefore, this component was selected for the next LC-MS / MS analysis.

[0030] Example 2 LC-MS / MS analysis of polypeptide components Sample preparation: An appropriate amount of 0.1% trifluoroacetic acid was added to the polypeptide component, mixed well, desalted using a C18 StageTip, and vacuum dried. After drying, the sample was redissolved with 0.1% fluoroacetic acid, the sample concentration was measured, and LC-MS / MS analysis was performed.

[0031] LC-MS / MS analysis: The sample was chromatographically separated using a nano-flow rate Easy nLC1200 chromatographic system (Thermo Scientific). Buffer: Solution A was an aqueous solution of 0.1% formic acid, and solution B was a mixed solution of 0.1% formic acid, acetonitrile, and water (where acetonitrile was 80%). The chromatographic column was equilibrated with 100% solution A. After the sample was injected into the Trap Column (100 µm * 20 mm, 5 µm, C18, Dr. Maisch GmbH), it was gradient separated through the analytical column (75 µm * 150 mm, 3 µm, C18, Dr. Maisch GmbH) at a flow rate of 300 nL / min. The liquid phase separation gradient was as follows: from 0 min to 2 min, the linear gradient of solution B was from 2% to 5%; from 2 min to 44 min, the linear gradient of solution B was from 5% to 28%; from 44 min to 51 min, the linear gradient of solution B was from 28% to 40%; from 51 min to 53 min, the linear gradient of solution B was from 40% to 100%; from 53 min to 60 min, solution B was maintained at 100%. After peptide separation, DDA (data-dependent acquisition) mass spectrometry analysis was performed using a Q-Exactive HF mass spectrometer (Thermo Scientific). The analysis duration was 60 min, the detection mode: positive ion, the parent ion scan range: 400 - 1500 m / z, the first-stage mass spectrometry resolution: 120,000 @ m / z 200, AGC target: 3e6, the first-stage Maximum IT: 30 ms. The second-stage mass spectrometry analysis of peptides was acquired according to the following method: After each full scan, the second-stage mass spectrometry spectra (MS2 scan) of the 20 highest-intensity parent ions were triggered for acquisition. The second-stage mass spectrometry resolution: 15,000 @ m / z 200, AGC target: 1e5, the second-stage Maximum IT: 35 ms, MS2 Activation Type: HCD, Isolation window: 1.6 m / z, Normalized collision energy: 28.

[0032] Database search: The mass spectrometry database search software used was MaxQuant 2.4.14.0, and the analysis parameters of the MaxQuant database search software were set as shown in Table 1. A total of 64 polypeptide sequences were searched, and the mass spectrometry Basepeak map of the sample is as Figure 2 shown.

[0033] Table 1 Analysis parameter settings of MaxQuant database search software Item Value Enzyme Unspecific MaxMissed Cleavages 0 Precursor Tolerance(Main search) 4.5 ppm Precursor Tolerance(First search) 20 ppm MS / MS Tolerance 20 ppm Fixed modifications none Variable modifications Oxidation (M), Acetyl (Protein N-term) Database uniprotkb-Chiloscyllium plagiosum

[36176] -67-20250123.fasta Database pattern Target-Reverse PSM FDR 0.01 Protein FDR 0.01 Site FDR 0.01 Example 3 Molecular docking to screen potential XO inhibitory peptides ChemDraw 19.0 and Autodock vina were used as the software for polypeptide structure drawing and docking, respectively.

[0034] The polypeptides released during the enzymatic hydrolysis of Chiloscyllium plagiosum were analyzed by molecular docking technology. These polypeptides were used as ligands for molecular docking, and XO was used as the receptor. The crystal structure of XO (PDB ID: 1N5X) was obtained from the RCSB Protein Data Bank ( https: / / www.rcsb.org / ). Autodock vina was used as the tool for molecular docking. The structure of the polypeptide was drawn by the software Chemdraw 19.0. Finally, potential XO inhibitory peptides were screened based on the vina values.

[0035] The coordinates of the vina docking box were set as x = 96, y = 54, z = 39, the grid spacing was 0.375 Å, the box size was set to 40 Å × 40 Å × 40 Å, and other parameters were default values. Finally, the binding degree between the polypeptide and XO was determined according to the vina values, and the top 8 polypeptides with the highest vina values were screened. The results are shown in Figure 3 Figure A below.

[0036] Example 4 Determination of XO inhibitory activity of marine-derived polypeptides Sample treatment: A polypeptide solution with a concentration of 2 mg / mL was prepared using 100 mmol / L PBS buffer at pH 7.4. At the same time, xanthine with a final concentration of 0.7 mmol / L and XO solution with a concentration of 0.15 U / mL were added, and the reaction was carried out at 37 °C for 20 min. Immediately after the reaction, a hydrochloric acid solution with a concentration of 1.0 mol / L was added.

[0037] HPLC determination conditions: The chromatographic column used was Agilent XDB C18 (250×4.6 mm, 5μm), the mobile phase was 85% 10 mmol / L ammonium dihydrogen phosphate aqueous solution and 15% methanol solution, and the flow rate was 1.0 mL / min. The absorbance at 290 nm was measured. The calculation formula for the XO inhibition rate of the sample was: [(peak area of the blank group - peak area of the sample group) / peak area of the blank group] × 100%.

[0038] Example 5 Synthesis and activity verification of marine-derived XO inhibitory peptides The top 8 polypeptides were all synthesized by Sangon Biotech (Shanghai) Co., Ltd. The verification of the XO inhibitory activity of the polypeptides was carried out according to the method in Example 4. The results of the activity determination are shown in Figure 3As shown in B of [reference]. All 8 polypeptides showed varying degrees of uric acid-lowering activity. Among them, polypeptides NCDEK, PGEAG, GDSGL, and GDDGL showed good inhibitory activity against XO, with XO inhibition rates of 72.29%, 37.27%, 62.52%, and 36.44% respectively, indicating that these 4 polypeptides have the potential to become XO inhibitors. To further explore the reasons for the activity of marine-derived XO inhibitory peptides, we analyzed the molecular docking results of these 4 XO inhibitory peptides in order to clarify their mechanism of action.

[0039] Residues such as amino acids Ser876, Vall011, Glu1261, Glu802, Phe649, Asn768, Phe914, Leu873, Phe1009, Leu1014, Lys771, and Thr1010 are key amino acids in the XO active center. The docking results of XO inhibitory peptides NCDEK, PGEAG, GDSGL, and GDDGL with XO are shown respectively in Figure 4 , Figure 6 , Figure 8 and Figure 10 as shown, and these 4 XO inhibitory peptides can all bind well with XO.

[0040] As Figure 5 shown in A of [reference], the XO inhibitory peptide NCDEK binds to amino acids Lys771 and Thr1010 of XO through hydrogen bond interactions, and also forms carbon-hydrogen bonds with amino acids Vall011 and His875 ( Figure 5 in B of [reference]), indicating that the XO inhibitory peptide NCDEK can be well embedded in the active pocket of XO. As Figure 7 shown in A of [reference], the XO inhibitory peptide PGEAG is connected to amino acids Ser876 and Thr1010 of XO through hydrogen bond forces, and also forms UnfavorableDonor-Donor interaction forces with amino acids Vall011 and Thr1010 ( Figure 7 in B of [reference]), and these interaction forces may be the reasons for the XO inhibitory activity of the XO inhibitory peptide PGEAG. In addition, as Figure 9 shown in A of [reference], the XO inhibitory peptide GDSGL binds to amino acids Ser876 and Thr1010 in XO through hydrogen bond forces, and also forms carbon-hydrogen bond interaction forces with amino acids such as Glu802 ( Figure 9 in B of [reference]), and the binding of the XO inhibitory peptide GDSGL to these key amino acids of XO is the reason for its activity. In addition, the XO inhibitory peptide GDDGL also forms hydrogen bond interaction forces with amino acids Ser876, Thr1010, and Phe1009 in XO ( Figure 11 in A of [reference]), and at the same time also forms Pi-Sigma interaction forces with amino acids such as Phe914 (Figure 11 In B) above. The above experimental results show that traditional hydrogen bond interactions and hydrophobic interactions are the key forces for the binding of XO inhibitory peptides NCDEK, PGEAG, GDSGL, and GDDGL to XO. They bind to the amino acids in the active center of XO, thereby inhibiting the interaction between XO and the substrate, and finally exerting the uric acid-lowering activity.

[0041] The above embodiments are provided to those skilled in the art to fully disclose and describe how to implement and use the claimed embodiments, rather than to limit the scope disclosed herein. Modifications that are obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A Chiloscyllium plagiosum - derived XO inhibitory peptide with uric acid - lowering activity, characterized in that, Its amino acid sequence is: NCDEK, as shown in SEQ ID NO.

1.

2. A Chiloscyllium plagiosum - sourced XO inhibitory peptide with uric acid - lowering activity, characterized in that, Its amino acid sequence is: PGEAG, as shown in SEQ ID NO.

2.

3. A Chiloscyllium plagiosum - sourced XO inhibitory peptide with uric acid - lowering activity, characterized in that, Its amino acid sequence is: GDSG, as shown in SEQ ID NO.

3.

4. A Chiloscyllium plagiosum - derived XO inhibitory peptide with uric acid - lowering activity, characterized in that, Its amino acid sequence is: GDDGL, as shown in SEQ ID NO.

4.

5. Use of the XO inhibitory peptide according to any one of claims 1 to 4 as or in the preparation of an XO inhibitor.

6. Use of the XO inhibitory peptide according to any one of claims 1 to 4 as or in the preparation of a drug for reducing uric acid.

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