A bioactive peptide derived from black truffle and its application

Abstract

The present invention discloses a bioactive peptide derived from black truffle, belonging to the technical field of food-derived bioactive peptides. The present invention uses polypeptideomics, computer virtual screening and molecular docking technologies to rapidly screen DPP-IV inhibitory peptides from the fruiting bodies of Yunnan black truffles. The bioactive peptides of the present invention are selected from APIKIPVGPGTL, IEDPDAPPPPPPREVIKN, KPPVSIPEDRLAGL, SLAKPPVSIPEDRLAGL, KPPVSIPEDRLAGLVN, EPKAPVQLDPPKDDPISL. The bioactive peptides obtained by screening in the present invention have high DPP-IV inhibitory activity, providing a new approach for the treatment of type 2 diabetes.

Description

Technical Field

[0001] The present invention relates to a bioactive peptide derived from black truffle and its application in the preparation of dipeptidyl peptidase-IV inhibitors, belonging to the technical field of food-derived bioactive peptides. Background Art

[0002] Diabetes Mellitus is one of the fastest-growing diseases globally. In 2021, the number of global diabetes patients was 537 million, which is expected to increase to 643 million by 2030 and to approximately 783 million by 2045. Diabetes and its complications are the main causes of death.

[0003] Type 2 diabetes is the most common type of diabetes. Currently, among the drugs for controlling blood glucose in type 2 diabetes patients, dipeptidyl peptidase-IV (DPP-IV) inhibitors have been well developed. Inhibiting DPP-IV can prevent 80-97% of glucagon-like peptide-1 (GLP-1) from being degraded and extend its half-life to 2-40 hours. GLP-1 directly acts on endocrine systems such as the pancreas, heart, stomach, and brain. In addition to promoting insulin secretion, it also has various physiological functions such as reducing gastric emptying and suppressing appetite, which can assist in controlling blood glucose homeostasis and regulating cardiovascular function.

[0004] Therefore, regulating blood glucose through the DPP-IV inhibition pathway is a multi-effective strategy, and it is necessary to develop more natural and safe DPP-IV inhibitors. Among them, DPP-IV inhibitory peptides from food sources have good application prospects. Traditional DPP-IV inhibitory peptides are usually screened using separation methods such as ultrafiltration, anion and cation chromatography, gel column chromatography, and high-performance liquid chromatography, combined with in vivo and in vitro activity evaluations during the separation process. With the development of science and technology, computer-aided design, online data, and virtual screening methods can be used to explore peptide activity, which can reduce labor intensity and labor costs, shorten the development cycle, and improve the success rate of active peptide identification.

[0005] Black truffle ( Tuber sinense ), a precious macrofungus, has a unique flavor and rich nutrition. It is known as one of the "world's three most delicious delicacies" together with foie gras and caviar. The fruiting body of black truffle is rich in protein. Truffle proteins are involved in translation, ribosome structure, energy metabolism, and environmental stress responses. During these processes, peptide bonds in proteins are broken, and some peptides with potential bioactive functions are released. So far, no reports have been found on small endogenous peptides in black truffle as DPP-IV enzyme inhibitory peptides. Summary of the Invention

[0006] In order to search for DPP-IV inhibitory peptides with higher safety and reduce the huge experimental costs during the separation and identification of traditional DPP-IV inhibitory peptides, the present invention uses the methods of peptidomics, network virtual screening, and molecular docking to screen and identify 6 bioactive peptides from black truffle ( Tuber sinense ), and the bioactive peptides are selected from the amino acid sequences shown in any one of the following (1) to (6):

[0007] (1) Ala-Pro-Ile-Lys-Ile-Pro-Val-Gly-Pro-Gly-Thr-Leu;

[0008] (2) Ile-Glu-Asp-Pro-Asp-Ala-Pro-Pro-Pro-Pro-Pro-Pro-Arg-Glu-Val-Ile-Lys-Asn;

[0009] (3) Lys-Pro-Pro-Val-Ser-Ile-Pro-Glu-Asp-Arg-Leu-Ala-Gly-Leu;

[0010] (4) Ser-Leu-Ala-Lys-Pro-Pro-Val-Ser-Ile-Pro-Glu-Asp-Arg-Leu-Ala-Gly-Leu;

[0011] (5) Lys-Pro-Pro-Val-Ser-Ile-Pro-Glu-Asp-Arg-Leu-Ala-Gly-Leu-Val-Asn;

[0012] (6) Glu-Pro-Lys-Ala-Pro-Val-Gln-Leu-Asp-Pro-Pro-Lys-Asp-Asp-Pro-Ile-Ser-Leu; and the small molecule peptides are applied in the preparation of dipeptidyl peptidase-IV inhibitors. The said bioactive peptides have good inhibitory activity and are safe and reliable for reducing blood sugar in diabetic patients.

[0013] The object of the present invention is achieved through the following technical solutions:

[0014] (1) Extraction of endogenous polypeptides from black truffle

[0015] Grind the fresh fruiting body of black truffle into powder in liquid nitrogen, add the extraction solution, homogenize on ice, take the supernatant after centrifugation, ultrafilter to collect the filtered liquid, discard macromolecules such as residual proteins with a molecular weight greater than 10 kDa in the ultrafiltration tube, and perform desalting treatment on the filtered liquid after freeze-drying;

[0016] The desalting treatment is to dissolve the freeze-dried sample with a solution containing TFA and acetonitrile, add it into the pre-treated desalting column, and let the sample slowly flow through the desalting column. The polypeptide is trapped by the desalting column, and other non-hydrophobic small molecules such as salts flow out and are discarded. Then, add a solution containing TFA and acetonitrile to wash the desalting column to wash away the residual salts. Continue to slowly flow the solution containing TFA and acetonitrile through the desalting column to elute the polypeptide. Use a new EP tube to collect the elution solution, and freeze-dry the eluate;

[0017] (2)Perform amino acid sequencing on the extract obtained in step (1) through proteomics;

[0018] Specifically, the extract is separated by an EASY-nLC 1000 ultra-high pressure nano-liquid chromatography system. Then, peptide sequence identification is performed by an Orbitrap Fusion mass spectrometer, and the endogenous peptides of black truffle are determined by comparing with the Uniprot database;

[0019] (3)Use an online tool to screen for potential DPP-IV inhibitory peptides among the top 200 peptides with the highest content identified in step (2);

[0020] (4)Use an online database to screen for peptides containing more DPP-IV inhibitory active sites in step (3);

[0021] The online database is selected from Peptide Ranker, BIOPEP, and admetSAR;

[0022] (5)Use an online database to screen for active peptides with good ADMET properties in step (4);

[0023] (6)Use molecular docking technology to screen for highly active DPP-IV inhibitory peptides from step (5);

[0024] Finally, 6 small peptides are obtained by the above method. The peptide with the amino acid sequence APIKIPVGPGTL has a molecular weight of 1162.43; the peptide with the amino acid sequence IEDPDAPPPPPPREVIKN has a molecular weight of 1981.22; the peptide with the amino acid sequence KPPVSIPEDRLAGL has a molecular weight of 1491.74; the peptide with the amino acid sequence SLAKPPVSIPEDRLAGL has a molecular weight of 1763.05; the peptide with the amino acid sequence KPPVSIPEDRLAGLVN has a molecular weight of 1704.97; the peptide with the amino acid sequence EPKAPVQLDPPKDDPISL has a molecular weight of 1958.03.

[0025] Advantages of the present invention:

[0026] The present invention rapidly and effectively identifies highly active DPP-IV inhibitory peptides from the fruiting bodies of Tuber melanosporum through virtual screening methods of peptidomics, databases, and molecular docking.

[0027] The present invention combines peptidomics, database screening, and molecular docking to construct a method for rapidly screening DPP-IV inhibitory peptides, avoiding the time-consuming and laborious drawbacks caused by step-by-step separation and purification; First, high-throughput sequencing is performed on the peptide segments contained in the fruiting bodies of Tuber melanosporum through peptidomics to obtain the sequences of all peptide segments; Secondly, databases are used to screen potential DPP-IV inhibitory peptides, and then the screened polypeptides are docked with DPP-IV in turn using molecular docking technology, and their Vina Score (binding energy) is sorted. Theoretically, the lower the Vina Score, the stronger the DPP-IV inhibitory activity of the polypeptide. Finally, polypeptides with lower Vina Scores are selected for in vitro synthesis and their activities are verified.

[0028] The present invention effectively screens 6 bioactive peptides from the fruiting bodies of Tuber melanosporum, and the bioactive peptides have the effect of inhibiting the activity of dipeptidyl peptidase-IV. The bioactive peptides of the present invention can be applied to the treatment or adjuvant treatment of diabetes, can be prepared into drugs or foods, adding pharmaceutically or food-acceptable excipients to form a pharmaceutically suitable dosage form for use, or a suitable edible form in the food field. The present invention provides a new approach for the treatment of type 2 diabetes. Description of the Drawings

[0029] Figure 1 It is the liquid phase diagram of APIKIPVGPGTL;

[0030] Figure 2 It is the molecular structure diagram of APIKIPVGPGTL;

[0031] Figure 3 It is the three-dimensional diagram of the molecular docking result of the DPP-IV inhibitory peptide APIKIPVGPGTL and DPP-IV;

[0032] Figure 4 It is the analysis diagram of the interaction between the DPP-IV inhibitory peptide APIKIPVGPGTL and DPP-IV molecules. Detailed Embodiments

[0033] The present invention will be further described in detail below with reference to the drawings and embodiments. However, the protection scope of the present invention is not limited to the described content. The reagents used in the embodiments are all conventional commercially available reagents or reagents prepared according to conventional methods unless otherwise specified. The methods used in the embodiments are all conventional experimental methods unless otherwise specified. The fruiting bodies of Tuber melanosporum used in the embodiments ( Tuber sinense ) were purchased from the Wuding Farmers' Market in Yunnan.

[0034] Example 1: Isolation and Identification of DPP-IV Inhibitory Peptides from the Fruiting Bodies of Tuber melanosporum

[0035] 1. Extraction and Desalting of Endogenous Peptides from the Fruiting Bodies of Tuber melanosporum

[0036] Take 0.5 g of Tuber melanosporum fruiting body sample, grind it into powder in liquid nitrogen, add 2 mL of extraction solution (0.1% TFA aqueous solution) to the powder, homogenize on ice for 10 min, then centrifuge at 4 °C and 17000 g for 30 min. Take the supernatant, ultrafilter it using a 10 kDa ultrafiltration tube at 10000 g, collect the filtered liquid, discard the macromolecules such as residual proteins with molecular weight greater than 10 kDa in the ultrafiltration tube, and freeze-dry the filtered liquid into dry powder;

[0037] Activate the desalting column with 200 µL of a solution containing 0.1% TFA and 80% acetonitrile; balance the desalting column with 600 µL of a solution containing 0.1% TFA and 1% acetonitrile; dissolve the freeze-dried sample with 400 µL of a solution containing 0.1% TFA and 1% acetonitrile, add it into the desalting column, and let the sample slowly flow through the desalting column. The polypeptide is trapped by the desalting column, and other non-hydrophobic small molecules such as salts flow out and are discarded. Then add 200 µL of a solution containing 0.1% TFA and 0.5% acetonitrile to wash the desalting column to remove the residual salts; finally, add 300 µL of a solution containing 0.1% TFA and 80% acetonitrile, let the liquid slowly flow through the desalting column, elute the polypeptide, collect the elution solution using a new EP tube, and freeze-dry the eluate;

[0038] 2. Sequencing of Endogenous Peptides from the Fruiting Bodies of Tuber melanosporum by LC-MS-based Peptidomics

[0039] Use the EASY-nLC 1000 ultra-high pressure nanoLC system for endogenous peptide separation. The liquid phase A is 0.1% formic acid-aqueous solution, and the liquid phase B is 0.1% formic acid-acetonitrile solution; in step (1), dissolve the freeze-dried sample with 20 µL of liquid phase A. After the automatic sampler aspirates 2 µL of the sample, load it onto the analytical column at a flow rate of 500 nL / min; the sample is chromatographically separated on the analytical column at a flow rate of 300 nL / min. The relevant liquid phase gradient is as follows: from 0 min to 105 min, the linear gradient of liquid phase B is from 5% to 30%; from 105 min to 110 min, the linear gradient of liquid phase B is from 30% to 90%; from 110 min to 112 min, liquid phase B is maintained at 90%; from 112 min to 113 min, the linear gradient of liquid phase B is from 90% to 5%; liquid phase B at 5% is maintained for 7 min;

[0040] The spray voltage of the ion source was 2.4 kV, the temperature of the heated capillary of the Fusion mass spectrometer was set at 320 °C, and data-dependent mode was used to automatically switch between MS and MS / MS acquisitions. Full-scan MS was performed using Orbitrap for the first-stage scan, with a scan range of m / z 400 - 1600 and a resolution set at 120,000 (at m / z 200). The maximum ion injection time was 50 ms, and the automatic gain control (AGC) was set at 1×10 6 , and then within 3 seconds, higher energy C-trap dissociation (HCD) was used to fragment the precursor ions that met the tandem (MS / MS) fragmentation conditions, and the scan was performed using Orbitrap with a scan resolution set at 15,000. The scan range was automatically controlled according to the precursor ion mass-to-charge ratio, with the lowest scan range fixed at m / z = 110 and the highest up to 2000. The minimum ion intensity value for MS / MS was set at 50,000, the maximum ion injection time for MS / MS was 80 ms, and the AGC control was set at 1.0×10 5 , and the precursor ion selection window was set at 1.6 Da. MS / MS acquisitions were performed for ions with 2 - 6 charges, and dynamic exclusion was set to perform MS / MS on each precursor ion once within 10 seconds, followed by exclusion for 18 seconds with 30% collision energy.

[0041] The tandem mass spectra were analyzed using MaxQuant (version 1.6.10.43), searching against the Uniprot-Tuber (truffles) database (version 202106, 39058 entries), and setting no enzyme digestion. The search parameters were as follows: fragment ion mass tolerance: 0.02 Da, precursor ion mass tolerance: 4.5 ppm, maximum missed cleavage: 2, variable modifications: Oxidation(M) 15.99, Deamidation(NQ) 0.98. The protein cut-off value was: -10lgP≥0, with at least 1 unique peptide; the peptide cut-off value was: -10lgP≥20. After sequencing the endogenous peptides in the truffle fruiting body by peptidomics, a total of 4038 peptides were identified.

[0042] 3. Prediction of the biological activity of endogenous peptides

[0043] The possibility of biological activity of the top 200 most abundant peptides was analyzed using the Peptide Ranker program. Peptides with a biological activity score of greater than or equal to 0.5 were selected as potential bioactive peptides in the experiment. The "profiles of potential biological activity" function in the BIOPEP program was used for database search to define their potential biological activities and possible bioactive peptide segments; as a result, 68.75% of the segments may have DPP-IV inhibitor function, and the physicochemical properties and absorption of bioactive peptides were predicted using AdmetSAR and PepDraw.

[0044] 4. Screening of highly active DPP-IV inhibitory peptides from the polypeptides identified in (3) using molecular docking technology, mainly including the following steps:

[0045] ① Preparation of DPP-IV enzyme PDB file and small molecule file: Retrieve the protein crystal structure of DPP-IV enzyme (code: 4PNZ) from the Protein Data Bank, remove the redundant ligands and H2O from 4PNZ, and use the WHAT IF server to repair the missing residues as a Dimer system for subsequent docking experiments; select peptides with a Peptide Ranker score greater than 0.5 and good ADMET properties for molecular docking, convert the peptide sequence into a SMILES number using PepSMI, and then convert the peptide SMILES number into a 3D structure model using NovoPro, save it as a PDB file, and minimize the internal forces of the peptide model using Pymol;

[0046] Determine the polar hydrogen atoms and Kollman charges of the polypeptide and DPP-IV monomer, and merge the non-polar hydrogen atoms through the AutoDock tool and save as pdbqt Format;

[0047] ② During the docking process, set the polypeptide to be flexible in the grid box and the DPP-IV monomer to be rigid, and all torsion angles of the polypeptide are in a free state. Use AutoGrid to generate a grid box with dimensions of 90 × 96 × 86 Å, a grid spacing of 0.375 Å, and coordinates X = +11.500, Y = -4.556, Z = +4.889;

[0048] ③ Extract the original ligand peptide from the protein crystal structure and redock it, calculate the RMSD value. If RMSD < 2 Å, the docking program is reliable. Screen for polypeptides with lower binding energy according to the Vina Score, exclude the DPP-IV inhibitory peptides with publicly available sequences, and the remaining polypeptides are determined as potential DPP-IV inhibitory peptides, and their DPP-IV inhibitory activity is verified through in vitro solid-phase synthesis.

[0049] The lower the value of the binding energy, the stronger the DPP-IV inhibitory activity of the corresponding polypeptide; the in vitro solid-phase synthesis is specifically a polypeptide chemical synthesis method (Fmoc solid-phase synthesis method); the verification of the DPP-IV inhibitory activity is specifically that after the screened polypeptide is synthesized, the substrate chemical method is used to determine the strength of its DPP-IV inhibitory activity to verify the screening result;

[0050] Specifically, the Vina Score of 10 polypeptides is less than -7.0 Kcal / mol (Table 1); considering the ATMET property, 6 polypeptides are selected for verification of their DPP-IV inhibitory activity after in vitro solid-phase synthesis (Table 2);

[0051] Table 1

[0052] 。

[0053] Example 2: Fluorometric determination of the in vitro inhibitory activity of 6 polypeptides against DPP-IV

[0054] The DPP-IV activity assay kit was provided by Cayman Chemical Co. (Ann Arbor, MI, USA), and the experiment was carried out according to the following steps:

[0055] 1. Prepare the buffer, DPP-IV enzyme solution, and substrate solution according to the operation instructions in the kit; the 6 obtained polypeptides were configured into solutions with a concentration range of 0-3 mg / mL using the buffer;

[0056] 2. DPP-IV inhibitory activity assay

[0057] Set the initial activity group, blank group, and peptide assay group on a 96-well plate; in the initial activity group, add 30 μL of buffer, 10 μL of DPP-IV enzyme solution, and 10 μL of blank solvent (buffer) in sequence; in the blank group, add 40 μL of buffer and 10 μL of blank solvent (buffer) in sequence; in the peptide assay group, add 30 μL of buffer, 10 μL of DPP-IV enzyme solution, and 10 μL of different concentration polypeptide solutions in sequence; each group was replicated 3 times;

[0058] After adding the reagents as described above, add 50 μL of substrate solution to start the reaction in all wells. The reaction temperature was 37 °C, the excitation wavelength was set at 255 nm, the emission wavelength was 455 nm, the fluorescence value of each well was detected every 60 s, and the reaction time was 30 min; the DPP-IV inhibitory activity was calculated according to the following formula:

[0059] DPP-IV inhibition rate % = [1 - A 样品 / A 初始 × DF × 100%; where A 样品It is the slope of the fluorescence value curve made from the difference between the fluorescence value measured by the 0 - 30 min peptide determination group and the fluorescence value of the blank group, A 初始 is the slope of the fluorescence value curve made from the difference between the fluorescence value measured by the 0 - 30 min initial activity group and the fluorescence value of the blank group; DF is the dilution factor;

[0060] As shown in Table 2, 6 polypeptides have DPP - Ⅳ inhibitory activity

[0061] Table 2

[0062] .

[0063] Example 3: Structural analysis of DPP - Ⅳ inhibitory peptides

[0064] 1. Structural analysis of APIKIPVGPGTL

[0065] As Figure 1 and Figure 2 shown, the liquid phase diagram and molecular structural formula of APIKIPVGPGTL;

[0066] 2. Molecular docking of APIKIPVGPGTL with DPP - Ⅳ

[0067] As Figure 3 、 Figure 4 shown, APIKIPVGPGTL forms 11 hydrogen bonds with Tyr48, Arg125, Tyr547, Lys554, Asn562, Ser630, Tyr662 in the active pocket of DPP - Ⅳ, and forms 20 hydrophobic interactions with Leu49, Glu206, Phe357, Asp545, Val546, Leu561, Trp563, Trp627, Gly628, Trp629, Tyr631, Val656, Trp659, Tyr662, Tyr666, Val711, His740, Gly741, Ala743, Try752 residues. Among them, the amino acid residues of APIKIPVGPGTL form hydrogen bond interactions with Tyr547, Tyr630, Try662 in the S1 pocket, and form hydrophobic interactions with Tyr631, Val656, Trp659, Tyr662, Tyr666, Val711, His740 in the S1 pocket, Glu206 in the S2 pocket and Phe357 in the S3 pocket, which is the key to its inhibitory ability. Subsequently, when selecting this method for virtual screening, the strength of the hydrogen bond interactions and hydrophobic interactions formed by the polypeptide with the S1, S2 and S3 pockets in the DPP - Ⅳ structure can be focused on to predict its DPP - Ⅳ inhibitory activity.

[0068] 3. Docking of the other 5 polypeptides with the DPP-IV molecule

[0069] As shown in Table 3, after the other 5 polypeptides were analyzed by the above method, they could all form complexes with the DPP-IV enzyme through different numbers of hydrogen bond interactions and hydrophobic interactions, and inhibit the activity of DPP-IV;

[0070] Table 3

[0071]

[0072] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A bioactive peptide derived from black truffle, wherein the bioactive peptide is selected from the amino acid sequences shown in any one of the following (1) to (6): (1) Ala-Pro-Ile-Lys-Ile-Pro-Val-Gly-Pro-Gly-Thr-Leu; (2) Ile-Glu-Asp-Pro-Asp-Ala-Pro-Pro-Pro-Pro-Pro-Pro-Arg-Glu-Val-Ile-Lys-Asn; (3) Lys-Pro-Pro-Val-Ser-Ile-Pro-Glu-Asp-Arg-Leu-Ala-Gly-Leu; (4) Ser-Leu-Ala-Lys-Pro-Pro-Val-Ser-Ile-Pro-Glu-Asp-Arg-Leu-Ala-Gly-Leu; (5) Lys-Pro-Pro-Val-Ser-Ile-Pro-Glu-Asp-Arg-Leu-Ala-Gly-Leu-Val-Asn; (6) Glu-Pro-Lys-Ala-Pro-Val-Gln-Leu-Asp-Pro-Pro-Lys-Asp-Asp-Pro-Ile-Ser-Leu.

2. Use of the bioactive peptide according to claim 1 in the preparation of a dipeptidyl peptidase-IV inhibitor.

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