Four uricosuric acid-lowering peptides from the striped bamboo shark

Abstract

The present invention relates to the rapid screening of uric acid-lowering peptides from the marine-derived striped bamboo shark (Araneus striata), belonging to the field of marine biopharmaceuticals. Specifically, it relates to four uric acid-lowering peptides derived from the striped bamboo shark. The four xanthine oxidase (XO) inhibitory peptides have amino acid sequences of NCDEK, PGEAG, GDSGL, and GDDGL, respectively. These XO inhibitory peptides are used as or in the preparation of XO inhibitors, and as or in the preparation of drugs that inhibit uric acid levels. The present invention utilizes enzymatic hydrolysis and molecular docking techniques to rapidly screen the top eight potential uric acid-lowering peptides from striped bamboo shark proteins. High-performance liquid chromatography assays for XO inhibitory activity revealed that four small-molecule peptides exhibited significant XO inhibitory activity, indicating potential as XO inhibitors / uric acid-lowering drugs.

Description

Technical Field

[0001] The present invention relates to the rapid screening of uric acid-lowering polypeptides in marine-derived striped bamboo shark proteins, and belongs to the technical field of marine biomedicine. Background Art

[0002] Uric acid is the end product of purine metabolism, and significantly elevated levels can lead to hyperuricemia. Xanthine oxidase (XO) is a key enzyme that catalyzes the conversion of hypoxanthine and xanthine to uric acid, and is therefore considered an important target for uric acid lowering. While traditional uric acid-lowering drugs have demonstrated promising uric acid-lowering effects, they are often associated with severe side effects. Therefore, the development of a class of naturally occurring uric acid-lowering peptides with significant efficacy and no side effects is of great significance.

[0003] XO inhibitory peptides are a class of naturally occurring active peptides that lower uric acid levels by inhibiting XO activity. They are widely found in a variety of foods, and marine proteins are currently considered a high-quality source for their production. The muscle protein content of the striped bamboo shark is high, offering both edible and medicinal value, making it a promising source for active peptides. However, currently, the striped bamboo shark is primarily processed into fish paste or fish balls, resulting in low economic value. Therefore, obtaining XO inhibitory peptides from marine sources is an effective measure to increase their market value.

[0004] The traditional preparation process for XO inhibitory peptides involves enzymatic hydrolysis and separation and purification. This separation and purification process is time-consuming and inefficient, and may also result in loss of the target peptide. In recent years, molecular docking and other methods have been gradually applied to the separation and purification of active peptides. Computer-simulated docking can accelerate the separation and purification process of active peptides, providing the possibility of rapid screening of active peptides.

[0005] The present invention mainly aims to obtain polypeptide sequences from striped bamboo shark proteins through enzymatic hydrolysis, and then use molecular docking technology to quickly screen XO inhibitory peptides with uric acid-lowering activity from the polypeptide sequences, and finally analyze and clarify the mechanism of action of XO inhibitory peptides through molecular docking. Summary of the Invention

[0006] In response to the above-mentioned existing technologies, in order to develop XO inhibitory peptides, the present invention uses enzymatic hydrolysis and molecular docking to quickly screen out four small molecule peptides with significant uric acid-lowering activity from the striped bamboo shark, and analyzes the mechanism of action of the XO inhibitory peptides through molecular docking.

[0007] The present invention is achieved through the following technical solutions:

[0008] XO inhibitory peptides include the following four types, with amino acid sequences:

[0009] NCDEK, as shown in SEQ ID NO. 1;

[0010] PGEAG, as shown in SEQ ID NO. 2;

[0011] GDSGL, as shown in SEQ ID NO. 3;

[0012] GDDGL, as shown in SEQ ID NO.4.

[0013] The above four XO inhibitory peptides are used as or in the preparation of XO inhibitors; and are used as or in the preparation of drugs with uric acid-lowering effects.

[0014] The XO inhibitory peptide of the present invention was rapidly screened from the striped bamboo shark using enzymatic hydrolysis and molecular docking. The process is as follows:

[0015] (1) Preparation and ultrafiltration of bamboo shark enzymatic hydrolysate

[0016] The minced bamboo shark meat was mixed with distilled water at a material-liquid ratio of 1:5 (w / v). The initial pH was adjusted to 2.0, and pepsin was added at 1.5% (w / w) enzyme dosage. The reaction was incubated at 37°C for 1.5 hours. The pH of the mixture was then adjusted to 6.5, and 1.0% (w / w) trypsin and α-chymotrypsin were added, and the reaction was continued at 37°C for 2.0 hours. The enzymatic hydrolyzate was further separated by 3 kDa and 10 kDa ultrafiltration tubes. The ultrafiltered solution was collected and lyophilized to obtain the bamboo shark polypeptide fraction with uric acid-lowering activity.

[0017] (2) Liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis of peptide components

[0018] An appropriate amount of 0.1% trifluoroacetic acid was added to the peptide fraction, mixed thoroughly, desalted using a C18 StageTip, and dried under vacuum. After drying, the sample was reconstituted with 0.1% trifluoroacetic acid, the sample concentration was determined, and LC-MS / MS analysis was performed. The sample was chromatographed using a nanoflow Easy nLC1200 system (Thermo Scientific). Buffers: Solution A was 0.1% formic acid in water, and Solution B was a mixture of 0.1% formic acid, acetonitrile, and water (80% acetonitrile). The flow rate was 300 nL / min.

[0019] (3) Molecular docking screening of potential XO inhibitory peptides

[0020] The peptides released by the bamboo shark during enzymatic hydrolysis were analyzed by molecular docking technology, and these peptides 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 Database ( https: / / www.rcsb.org / ). Autodock vina was used as a molecular docking tool. The peptide structures were drawn using Chemdraw 19.0. Potential XO inhibitory peptides were screened based on vina values.

[0021] (4) Synthesis and activity determination of XO inhibitory peptides

[0022] Using the above method, the present invention screened eight potential marine-derived XO-inhibiting peptides (Top 8). High-performance liquid chromatography (HPLC) assays for XO inhibition revealed that four marine-derived peptides exhibited significant XO-inhibiting activity. These marine-derived peptides have the potential to act as xanthine oxidase inhibitors and could be used to prepare drugs for suppressing uric acid levels. Among these, the peptides NCDEK, PGEAG, GDSGL, and GDDGL exhibited the highest XO-inhibiting activity, suggesting potential as uric acid-lowering functional products or drugs.

[0023] The positive beneficial effects of the present invention are:

[0024] This invention innovates traditional methods for screening marine-derived uric acid-lowering peptides. It utilizes molecular docking technology to rapidly screen for XO inhibitory peptides, improving screening efficiency. Furthermore, the XO inhibitory peptides screened by this invention have the potential to treat hyperuricemia.

[0025] Various terms and phrases used herein have the general meanings that are well known to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : Inhibitory activity of the enzymatic hydrolysate and ultrafiltration fractions of the striped bamboo shark against XO.

[0027] Figure 2 : Basepeak diagram of mass spectrum of the sample.

[0028] Figure 3 : XO inhibitory activity and vina docking energy values ​​of Top8 peptides. A: vina docking energy value of active Top8 peptides; B: XO inhibition rate of Top8 peptides.

[0029] Figure 4 : Molecular docking results of XO inhibitory peptide NCDEK and XO.

[0030] Figure 5 : Amino acid residues and interaction forces between XO inhibitory peptide NCDEK and XO; A: main amino acid residues that form hydrogen bonds between XO inhibitory peptide NCDEK and XO; B: total interaction forces formed by the binding of XO inhibitory peptide NCDEK and XO.

[0031] Figure 6 : Molecular docking results of XO inhibitory peptide PGEAG and XO.

[0032] Figure 7 : Amino acid residues and interaction forces between XO inhibitory peptide PGEAG and XO; A: main amino acid residues that form hydrogen bonds between XO inhibitory peptide PGEAG and XO; B: total interaction forces formed by the binding of XO inhibitory peptide PGEAG and XO.

[0033] Figure 8 : Molecular docking results of XO inhibitory peptide GDSGL and XO.

[0034] Figure 9 : Amino acid residues and interaction forces between XO inhibitory peptide GDSGL and XO; A: main amino acid residues that form hydrogen bonds between XO inhibitory peptide GDSGL and XO; B: total interaction forces formed by the binding of XO inhibitory peptide GDSGL and XO.

[0035] Figure 10 : Molecular docking results of XO inhibitory peptide GDDGL and XO.

[0036] Figure 11 : Amino acid residues and interaction forces between XO inhibitory peptide GDDGL and XO; A: main amino acid residues that form hydrogen bonds between XO inhibitory peptide GDDGL and XO; B: total interaction forces formed by the binding of XO inhibitory peptide GDDGL and XO. DETAILED DESCRIPTION

[0037] 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 will appreciate that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0038] Unless otherwise specified, the instruments, reagents, and materials used in the following examples are all conventional instruments, reagents, and materials available in the prior art and can be obtained through regular commercial channels. The experimental methods and detection methods used in the following examples are all conventional experimental methods and detection methods available in the prior art, unless otherwise specified.

[0039] Example 1 Preparation and ultrafiltration of bamboo shark hydrolysate

[0040] Minced striped bamboo shark meat was mixed with distilled water at a material-to-liquid ratio of 1:5 (w / v). The initial pH was adjusted to 2.0, and pepsin was added at 1.5% (w / w) enzyme dosage. The mixture was reacted at 37°C for 1.5 h. The pH of the mixture was then adjusted to 6.5, and 1.0% (w / w) trypsin and α-chymotrypsin were added, followed by a further reaction at 37°C for 2.0 h. After the reaction, the mixture was boiled for 10 min to inactivate the enzymes. The supernatant was then centrifuged at 4°C and 8000 rpm for 20 min, and its XO inhibitory activity was determined.

[0041] The enzymatic hydrolysate was subjected to ultrafiltration separation using 3 kDa and 10 kDa ultrafiltration tubes, respectively, to separate the solution into three components: molecular weight >10 kDa, molecular weight between 3 kDa and 10 kDa, and molecular weight <3 kDa. The corresponding component solutions were collected, freeze-dried, and then the XO inhibition rate was determined. The results are as follows: Figure 1 As shown in the figure, the enzymatic hydrolysate was ultrafiltrated into three fractions with different molecular weights. The fraction with a molecular weight <3 kDa had the best XO inhibitory activity, with an inhibition rate of 54.47%. Therefore, this fraction was selected for the next LC-MS / MS analysis.

[0042] Example 2 LC-MS / MS analysis of polypeptide components

[0043] Sample Preparation: Add an appropriate amount of 0.1% trifluoroacetic acid to the peptide fraction, mix thoroughly, desalt using a C18 StageTip, and vacuum dry. After drying, reconstitute the sample with 0.1% trifluoroacetic acid, determine the sample concentration, and perform LC-MS / MS analysis.

[0044] LC-MS / MS analysis: Samples were separated using a nanoflow Easy nLC1200 chromatography system (Thermo Scientific). Buffer A consisted of 0.1% formic acid in water and buffer B consisted of a mixture of 0.1% formic acid, acetonitrile, and water (80% acetonitrile). The columns were equilibrated with 100% buffer A. Samples were injected into a trap column (100µm x 20mm, 5µm, C18, Dr. Maisch GmbH) and then passed through an analytical column (75µm x 150mm, 3µm, C18, Dr. Maisch GmbH) for gradient separation at a flow rate of 300 nL / min. The liquid phase separation gradient was as follows: from 0 min to 2 min, a linear gradient of 2% to 5% buffer B; from 2 min to 44 min, a linear gradient of 5% to 28% buffer B; from 44 min to 51 min, a linear gradient of 28% to 40% buffer B; from 51 min to 53 min, a linear gradient of 40% to 100% buffer B; and from 53 min to 60 min, a constant buffer B of 100%. After peptide separation, DDA (data-dependent acquisition) mass spectrometry analysis was performed on a Q-Exactive HF mass spectrometer (Thermo Scientific). The analysis lasted 60 min, with positive ion detection mode, precursor ion scan range: 400–1500 m / z, primary mass spectrometer resolution: 120,000 at m / z 200, AGC target: 3e6, and primary maximum time interval: 30 ms. The peptide secondary mass spectrometry analysis was acquired according to the following method: after each full scan, the 20 highest intensity precursor ion secondary mass spectra (MS2scan) were triggered to acquire, the secondary mass spectrometry resolution was 15,000@m / z200, AGCtarget:1e5, secondary MaximumIT:35ms, MS2Activation Type:HCD, Isolation window:1.6m / z, Normalized collision energy:28.

[0045] Database search: The mass spectrometry database search software used was MaxQuant 2.4.14.0, and the analysis parameter settings of the MaxQuant database search software are shown in Table 1. A total of 64 peptide sequences were searched, and the mass spectrum Basepeak diagram of the sample is shown in Figure 2 shown.

[0046] Table 1 Analysis parameter settings of MaxQuant library search software

[0047] 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

[0048] Example 3 Molecular docking screening of potential XO inhibitory peptides

[0049] ChemDraw 19.0 and Autodock vina were used as peptide structure drawing and docking software, respectively.

[0050] The peptides released by the bamboo shark during enzymatic hydrolysis were analyzed by molecular docking technology, and these peptides 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 Database ( https: / / www.rcsb.org / ). Autodock vina was used as a molecular docking tool. The peptide structures were drawn using Chemdraw 19.0. Potential XO inhibitory peptides were screened based on vina values.

[0051] The coordinates of the vina docking box were set to x=96, y=54, and 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 kept to default values. Finally, the binding degree between the peptide and XO was determined based on the vina value, and the peptides with the top 8 vina values ​​were screened. The results are shown below. Figure 3 As shown in A.

[0052] Example 4 Determination of XO Inhibitory Activity of Marine-derived Peptides

[0053] Sample Preparation: A 2 mg / mL peptide solution was prepared in 100 mmol / L PBS buffer (pH 7.4). Xanthine (0.7 mmol / L) and 0.15 U / mL XO solution were added simultaneously. The mixture was reacted at 37°C for 20 min. Immediately after the reaction, 1.0 mol / L hydrochloric acid solution was added.

[0054] HPLC conditions: An Agilent XDB C18 column (250 × 4.6 mm, 5 μm) was used, and the mobile phase consisted of 85% 10 mmol / L ammonium dihydrogen phosphate aqueous solution and 15% methanol at a flow rate of 1.0 mL / min. The absorbance at 290 nm was measured, and the XO inhibition rate of the sample was calculated as: [(peak area of ​​blank group - peak area of ​​sample group) / peak area of ​​blank group] × 100%.

[0055] Example 5 Synthesis and Activity Verification of Marine-derived XO Inhibitory Peptides

[0056] The Top8 peptides were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The XO inhibitory activity of the peptides was verified by the method in Example 4. The activity assay results are shown in Figure 4. Figure 3As shown in Figure B. All eight peptides exhibited varying degrees of uric acid-lowering activity. Among them, peptides NCDEK, PGEAG, GDSGL, and GDDGL exhibited strong inhibitory activity against XO, with XO inhibition rates of 72.29%, 37.27%, 62.52%, and 36.44%, respectively, indicating that these four peptides have the potential to be XO inhibitors. To further explore the mechanisms underlying the activity of these marine-derived XO inhibitory peptides, we analyzed the molecular docking results of these four XO inhibitory peptides to elucidate their mechanisms of action.

[0057] Amino acids Ser876, Val1011, Glu1261, Glu802, Phe649, Asn768, Phe914, Leu873, Phe1009, Leu1014, Lys771 and Thr1010 are key amino acids in the active center of XO. The docking results of XO inhibitory peptides NCDEK, PGEAG, GDSGL and GDDGL with XO are shown in Figure 2. Figure 4 、 Figure 6 、 Figure 8 and Figure 10 As shown, these four XO inhibitory peptides can bind to XO very well.

[0058] like Figure 5 As shown in Figure A, the XO inhibitory peptide NCDEK binds to the amino acids Lys771 and Thr1010 of XO through hydrogen bonds, and also forms carbon-hydrogen bonds with amino acids Val1011 and His875 ( Figure 5 (B) shows that the XO inhibitory peptide NCDEK can be well embedded in the active pocket of XO. Figure 7 As shown in Figure A, the XO inhibitory peptide PGEAG is connected to the amino acids Ser876 and Thr1010 of XO through hydrogen bonding forces, and also forms UnfavorableDonor-Donor interactions with amino acids Val1011 and Thr1010 ( Figure 7 (B) These interactions may be the reason why the XO inhibitory peptide PGEAG has XO inhibitory activity. Figure 9 As shown in Figure A, the XO inhibitory peptide GDSGL binds to amino acids Ser876 and Thr1010 in XO through hydrogen bonds, and also forms carbon-hydrogen bonds with amino acids Glu802 ( Figure 9 In Figure B), 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 interactions with the amino acids Ser876, Thr1010 and Phe1009 in XO ( Figure 11 A), and also formed Pi-Sigma interaction with amino acid Phe914 and others ( Figure 11 These experimental results indicate that traditional hydrogen bonding and hydrophobic interactions are key forces for the binding of XO inhibitory peptides NCDEK, PGEAG, GDSGL, and GDDGL to XO. These interactions inhibit the interaction between XO and its substrate by binding to the amino acids in the active center of XO, ultimately exerting its uric acid-lowering activity.

[0059] The above examples 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 of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. A XO inhibitory peptide derived from a striped bamboo shark having uric acid-lowering activity, characterized in that: Its amino acid sequence is: NCDEK, as shown in SEQ ID NO.

1.

2. Use of the XO inhibitory peptide according to claim 1 in the preparation of uric acid-lowering drugs.

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