Targeted immunoregulation and anti-tumor morchella bifunctional glycopeptide

Abstract

The invention relates to a toadstool bifunctional glycopeptide for targeted immunoregulation and tumor resistance, and belongs to the technical field of biology. The molecular sequence of the morchella glycopeptide provided by the invention is LPS (Fuc) PMLLPQ, and the morchella glycopeptide molecule is obtained by connecting fucose to serine hydroxyl of a peptide chain through an alpha-glucosidic bond in an O-connection glycosylation mode. The morchella glycopeptide is identified from a morchella extract obtained by extracting morchella with pectinase by taking morchella as a substrate, is derived from edible mushrooms and is safe to use; the compound can be effectively combined with TLR4 (Toll-like receptor 4), block inflammatory response and effectively combine and inhibit VEGFR2 (vascular endothelial growth factor receptor-2), and has anti-tumor activity; meanwhile, release of LPS-induced macrophage cytokines can be inhibited, and it is further shown that the morchella glycopeptide has immunoregulation and anti-tumor activity.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a Morchella esculenta bifunctional glycopeptide targeting immune regulation and anti-tumor effects. Background Art

[0002] Morchella esculenta (L.) Pers., also known as Morchella esculenta, Morchella esculenta mushroom, and Phylloporus rhodoxanthus, belongs to the genus Morchella of the order Pezizales in the phylum Ascomycota, and is a rare medicinal and edible fungus. It is recorded in "Compendium of Materia Medica" that Morchella esculenta is flat in nature and sweet in taste, and has the effects of benefiting the stomach and intestines, promoting digestion, resolving phlegm and regulating qi, tonifying the kidney and receiving qi, and boosting the brain and refreshing the mind. Morchella esculenta is rich in various nutritional elements such as protein, polysaccharide, polypeptide, vitamin, and mineral, and shows broad application prospects in the fields of food, medicine, health products, etc.

[0003] Glycopeptides are composed of peptide molecules and sugar molecules connected by glycopeptide bonds, and their structures are more complex. The glycopeptides developed into drugs mainly focus on glycopeptide antibiotics (such as vancomycin, etc.), which are mainly used to treat Gram-positive bacterial infections, especially drug-resistant bacteria (such as methicillin-resistant Staphylococcus aureus, MRSA). The glycopeptide drugs that have been marketed or are in clinical use mainly come from natural products of microorganisms (such as actinomycetes, streptomyces, etc.), and some are improved in their properties through chemical modification (semi-synthesis).

[0004] TLR4 (Toll-like receptor 4) is a key pattern recognition receptor of the innate immune system, which mainly recognizes pathogen-associated molecular patterns (PAMPs, such as bacterial lipopolysaccharide LPS) and damage-associated molecular patterns (DAMPs). The molecular recognition of TLR4 mainly depends on its interaction with MD-2 protein. MD-2 is a cofactor of TLR4, and MD-2 has a hydrophobic pocket, which is the core region for molecular binding. If it binds to the acidic residues (such as Asp, Glu) of TLR4, it will promote the dimerization and signal activation of TLR4, and can play an agonist role. If it occupies the MD-2 pocket, it can competitively inhibit the binding of LPS, block the excessive inflammatory response, and play an antagonist role.

[0005] VEGFR2 (vascular endothelial growth factor receptor-2) is an important target for tumor treatment. VEGFR2 belongs to the tyrosine kinase receptor, and the ATP binding pocket in the kinase domain of VEGFR2 is the core region for catalyzing the phosphorylation transfer reaction. Its catalytic key residue Lys868 is involved in the positioning of the γ-phosphate group of ATP and stabilizes the transition state. Mutation of this residue will significantly reduce the kinase activity; Asp1046 is located in the catalytic loop and binds to Mg 2+Ionic coordination coordinates the three phosphate groups of ATP; Cys919 is located at the entrance of the ATP-binding pocket and is the target of covalent inhibitors (such as apatinib), which can form reversible / irreversible covalent bonds. The hinge region residues Glu885 and Val888 form hydrogen bonds with the adenine ring of ATP and are the binding sites of most ATP-competitive inhibitors. The DFG motif Asp1046-Phe1047-Gly1048 (D-F-G) determines the kinase activity state. After non-ATP-competitively binding to the hydrophobic pocket in the DFG-out conformation (such as regorafenib), it can block kinase activation. Therefore, the ATP-binding pocket of VEGFR2, as the core target for antitumor drug action, provides a structural basis for inhibitor optimization at key sites such as its hinge region (Glu885 / Val888), catalytic residues (Lys868 / Asp1046), and Cys919.

[0006] Macrophages play an important role in many diseases and are the main participants in the body's innate immune response. In in vitro cell experiments, an inflammatory model of macrophages was established by inducing with LPS. LPS promotes macrophage activation through the TLR4 pathway and stimulates macrophages to secrete various inflammatory factors and mediators. As an inflammatory mediator secreted by macrophages, the secretion level of NO is a common indicator for observing macrophage immune activity. Macrophages are divided into M1 and M2 types according to the type of immune response. TNF-α, IL-1β, IL-6, IL-12, and NO are the signature cytokines of M1-type macrophages. By treating RAW264.7 macrophages with LPS and analyzing the reduction in the secretion levels of the above signature cytokines after intervention with active substances, the role of active substances in immune regulation and anti-tumor can be clarified.

[0007] Currently, there are no relevant reports on food-derived glycopeptide molecules with immunomodulatory and anti-tumor activities. Summary of the Invention

[0008] In view of this, the purpose of the present invention is to provide morchella glycopeptide, morchella extract, its preparation method and application. The morchella glycopeptide identified in the morchella extract obtained by the preparation method can effectively bind to TLR4 and VEGFR2, block the inflammatory response, and has immunomodulatory and anti-tumor activities.

[0009] The present invention provides a morchella glycopeptide, and the molecular sequence of the morchella glycopeptide is LPS(Fuc)PM LLPQ; in the morchella glycopeptide molecule, fucose is connected to the serine hydroxyl group of the peptide chain through an O-linked glycosylation manner by an α-glycosidic bond.

[0010] The present invention also provides a morchella extract containing the morchella glycopeptide.

[0011] The present invention also provides a method for preparing the morel mushroom extract, comprising the following steps:

[0012] The morel mushroom and water are mixed at a ratio of 1 g∶(10 - 30) mL, and enzymatically hydrolyzed at 50°C - 70°C for 18 - 100 min using pectinase; the mass ratio of the pectinase to the morel mushroom is (3 - 8)∶100.

[0013] Preferably, the particle size of the morel mushroom is <100 mesh.

[0014] The present invention also provides the use of the morel mushroom glycopeptide, the morel mushroom extract or the preparation method in the preparation of a product for reducing the secretion levels of macrophage NO, IL-6, IL-12, IL-1β or TNF-α.

[0015] The present invention also provides the use of the morel mushroom glycopeptide, the morel mushroom extract or the preparation method in the preparation of an anti-inflammatory product.

[0016] The present invention also provides the use of the morel mushroom glycopeptide, the morel mushroom extract or the preparation method in the preparation of a TLR4 protein inhibitor.

[0017] The present invention also provides the use of the morel mushroom glycopeptide, the morel mushroom extract or the preparation method in the preparation of a VEGFR2 protein inhibitor.

[0018] The present invention also provides the use of the morel mushroom glycopeptide, the morel mushroom extract or the preparation method in the preparation of an immune regulation product.

[0019] The present invention also provides the use of the morel mushroom glycopeptide, the morel mushroom extract or the preparation method in the preparation of an anti-tumor product.

[0020] Advantages of the present invention:

[0021] By enzymatically hydrolyzing the morel mushroom, the morel mushroom glycopeptide of the present invention is identified in the obtained morel mushroom extract. The morel mushroom glycopeptide is derived from edible fungi and is safe to use; it can effectively bind to TLR4, block the inflammatory response, effectively bind to and inhibit VEGFR2, and has anti-tumor activity; at the same time, the morel mushroom glycopeptide of the present invention can inhibit the expression of macrophage cytokines induced by LPS, further indicating that the morel mushroom glycopeptide of the present invention has immune regulation and anti-tumor activities. Description of the Drawings

[0022] Figure 1 It is the molecular mass spectrum of the morel mushroom glycopeptide.

[0023] Figure 2It is the 3D structure diagram of the morchella glycopeptide molecule; among them, yellow is the peptide molecule, orange is fucose, red is the oxygen atom, blue is the carbon atom, and gray is the hydrogen atom.

[0024] Figure 3 It is the diagram of the effect of morchella glycopeptide on the secretion of cytokines by RAW264.7 macrophages; among them, **** indicates P < 0.0001.

[0025] Figure 4 It is the docking diagram of the morchella glycopeptide molecule LPS(Fuc)PMLLPQ and the TLR4 molecule. Among them, in the morchella glycopeptide molecule, red is the peptide molecule and green is the fucose molecule.

[0026] Figure 5 It is the docking diagram of the morchella glycopeptide molecule LPS(Fuc)PMLLPQ and the VEGFR2 molecule. Among them, in the morchella glycopeptide molecule, red is the peptide molecule and green is the fucose molecule. Detailed implementation mode

[0027] The present invention provides a morchella glycopeptide, and the molecular sequence of the morchella glycopeptide is LPS(Fuc)PMLLPQ; in the morchella glycopeptide molecule, fucose is connected to the serine hydroxyl group of the peptide chain through an O-linked glycosylation method by an α-glycosidic bond.

[0028] In the present invention, in the molecular sequence LPS(Fuc)PMLLPQ of the morchella glycopeptide, (Fuc) is located after serine (S), indicating that fucose O-linked glycosylation modification occurs at serine (S), resulting in an increase in molecular weight by 146.06 Da. The molecular weight of the morchella glycopeptide is preferably 1140 Da. The peptide sequence in the morchella glycopeptide molecule is preferably LPSPMLLPQ (SEQ ID NO.1).

[0029] Preferably, the structural formula of the morchella glycopeptide is as shown in Formula I:

[0030]

[0031] The present invention also provides a morchella extract containing the morchella glycopeptide. The morchella glycopeptide can be identified in the morchella extract.

[0032] The present invention also provides a preparation method of the morchella extract, including the following steps:

[0033] Morchella and water are mixed at a ratio of 1 g:(10 - 30) mL, pectinase is added, and enzymatic hydrolysis is carried out at 50°C - 70°C for 18 - 100 min; the mass ratio of the pectinase to the morchella is (3 - 8):100.

[0034] In the present invention, the morel is preferably a morel with a particle size < 100 mesh. The present invention has no special limitation on the method for controlling the particle size of the morel, and a conventional method for controlling the particle size in the art can be adopted. In one embodiment, the morel with a particle size < 100 mesh can be obtained by crushing and sieving; the morel with a particle size < 100 mesh is mixed with water at a ratio of 1 g : (10 - 30) mL, and the mixing ratio of the morel to water is preferably 1 g : 10 mL, 1 g : 15 mL, 1 g : 20 mL, 1 g : 25 mL or 1 g : 30 mL; after mixing, pectinase is added, and the mass ratio of the pectinase to the morel is (3 - 8) : 100, preferably 3 : 100, 4 : 100, 5 : 100, 6 : 100, 7 : 100 or 8 : 100; enzymatic hydrolysis is carried out at 50°C - 70°C for 18 - 100 min; in one embodiment, enzymatic hydrolysis can be carried out at 50°C, 55°C, 60°C, 65°C or 70°C for 18 min, 20 min, 40 min, 50 min, 60 min, 80 min or 100 min. In the present invention, the preparation method preferably further includes the steps of centrifugation and freeze-drying. The centrifugation is preferably carried out after enzymatic hydrolysis. The present invention has no special limitation on the conditions of the centrifugation, and conventional centrifugation conditions in the art can be adopted. In one embodiment, centrifugation can be carried out at a rotational speed of 6000 rpm - 8000 rpm for 10 min - 20 min, such as centrifugation at a rotational speed of 6000 rpm, 7000 rpm or 8000 rpm for 10 min, 15 min or 20 min; after centrifugation, the supernatant after centrifugation is preferably taken for freeze-drying. The freeze-drying is preferably carried out at -68°C - -80°C for 24 - 50 h. In one embodiment, freeze-drying can be carried out at -68°C, -70°C, -75°C or -80°C for 24 h, 30 h, 36 h, 40 h, 48 h or 50 h.

[0035] The present invention has no special limitation on the sources of the morel and the pectinase, and conventional commercially available products in the art can be adopted.

[0036] The morel extract containing the morel glycoprotein of the present invention is obtained for the first time by the preparation method of the present invention. The morel glycoprotein of the present invention can effectively bind to TLR4, block the inflammatory reaction, and effectively bind to VEGFR2, thereby inhibiting VEGFR2, and has antitumor activity; at the same time, the morel glycoprotein of the present invention can inhibit the expression of macrophage cytokines induced by LPS, further indicating that the morel glycoprotein of the present invention has immunomodulatory and antitumor activities.

[0037] The present invention also provides the application of the morel glycoprotein, the morel extract or the preparation method in the preparation of products for reducing the secretion levels of macrophage NO, IL-6, IL-12, IL-1β or TNF-α, anti-inflammatory products, immunomodulatory products or antitumor products. In the present invention, the products preferably include drugs.

[0038] The active ingredient of the product preferably contains the morchella glycoprotein as described above. The product preferably further includes carriers commonly used in the art, and the carriers include but are not limited to fillers, diluents, disintegrants, colorants, and / or flavoring agents.

[0039] The present invention also provides the application of the morchella glycoprotein, the morchella extract or the preparation method in the preparation of TLR4 protein inhibitor or VEGFR2 protein inhibitor. The active ingredient of the inhibitor preferably includes the morchella glycoprotein as described above. The inhibitor preferably further includes carriers commonly used in the art, and the carriers include but are not limited to fillers, diluents, disintegrants, colorants, and / or flavoring agents.

[0040] The technical solutions provided by the present invention will be described in detail below in conjunction with examples, but they cannot be construed as limiting the protection scope of the present invention.

[0041] In the following examples, unless otherwise specified, all are conventional methods.

[0042] In the following examples, the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0043] In the following examples, the pectinase used was purchased from Beijing Solarbio Science & Technology Co., Ltd., and the enzyme activity of the pectinase was 100,000 U / g.

[0044] Example 1

[0045] Preparation of a morchella extract containing morchella glycoprotein molecules

[0046] Select dry morchella as the raw material, and after pulverization and sieving, obtain morchella powder with a particle size less than 100 mesh. Mix the morchella powder with water at a solid-liquid ratio of 1:30 (g / mL), add 7% (w / w, enzyme / morchella powder) pectinase, and enzymatically hydrolyze at 50 °C for 50 min to obtain an enzymatic hydrolysate. Centrifuge the enzymatic hydrolysate at 8000 rpm for 15 min, collect the supernatant, and freeze-dry it at -70 °C for 48 h to obtain the morchella extract.

[0047] Example 2

[0048] Preparation of a morchella extract containing morchella glycoprotein molecules

[0049] Select dry morchella as the raw material, and after pulverization and sieving, obtain morchella powder with a particle size less than 100 mesh. Mix the morchella powder with water at a solid-liquid ratio of 1:10 (g / mL), add 8% (w / w, enzyme / morchella powder) pectinase, and enzymatically hydrolyze at 60 °C for 100 min to obtain an enzymatic hydrolysate. Centrifuge the enzymatic hydrolysate at 7000 rpm for 10 min, collect the supernatant, and freeze-dry it at -80 °C for 24 h to obtain the morchella extract.

[0050] Example 3

[0051] Preparation of Morchella extract containing Morchella glycoprotein molecules

[0052] Select dry Morchella as raw material, crush and sieve it to obtain Morchella powder with a particle size less than 100 mesh. Mix the Morchella powder with water at a solid-liquid ratio of 1:20 (g / mL), add 3% (w / w, enzyme / Morchella powder) pectinase, and enzymatically hydrolyze at 70 °C for 18 min to obtain an enzymatic hydrolysate. Centrifuge the enzymatic hydrolysate at 6000 rpm for 20 min, collect the supernatant, and freeze-dry it at -68 °C for 50 h to obtain the Morchella extract.

[0053] Comparative Example 1

[0054] Preparation of Morchella extract

[0055] Select dry Morchella as raw material, crush and sieve it to obtain Morchella powder with a particle size less than 100 mesh. Mix the Morchella powder with water at a solid-liquid ratio of 1:35 (g / mL), add 9% (w / w, enzyme / Morchella powder) pectinase, and enzymatically hydrolyze at 45 °C for 120 min to obtain an enzymatic hydrolysate. Centrifuge the enzymatic hydrolysate at 8000 rpm for 15 min, collect the supernatant, and freeze-dry it at -70 °C for 48 h to obtain the Morchella extract.

[0056] Test Example 1

[0057] Structure identification of Morchella glycoprotein

[0058] The peptide molecules in the Morchella extracts obtained in Example 1 and Comparative Example 1 were identified by mass spectrometry. A ZipTip C18 microchromatography column (Merck-Millipore, Shanghai Anpu Experimental Technology Co., Ltd.) was used to perform desalting pretreatment on the extracts. The desalting method was as follows: 1.0 mg of the Morchella extract was accurately weighed and dissolved in 10 μL of 0.1% (v / v) trifluoroacetic acid (TFA) to obtain a Morchella extract solution; the chromatography column was rinsed 10 times with 50 μL of a solution prepared with pure water containing 60% (v / v) acetonitrile (ACN) and 0.1% TFA; the chromatography column was rinsed 10 times with 10 μL of 0.1% TFA; the Morchella extract solution was aspirated and discharged through the chromatography column 20 times; the chromatography column was rinsed 5 times with 10 μL of 0.1% TFA; the chromatography column was eluted with 10 μL of a solution prepared with pure water containing 60% ACN and 0.1% TFA, and the eluate was collected and transferred to a polypropylene centrifuge tube and vacuum-dried to obtain a dry powder. The dry powder was dissolved in 20 μL of a dissolution solution (aqueous solution containing 0.1% (v / v) formic acid), vortexed, centrifuged at 17,000 rpm at 4 °C for 20 min, the supernatant was collected and transferred to a sample injection tube, and the injection volume was 3 μL for LC-MS / MS sequence analysis. The mobile phase A of the liquid chromatography was an aqueous solution of 0.1% formic acid, and the mobile phase B was an acetonitrile solution containing 0.1% formic acid; the LC-MS / MS set parameters are shown in Table 1. The PEAKS software was used for identification sequence database retrieval.

[0059] Table 1 LC-MS / MS Parameter Settings

[0060]

[0061] After LC-MS / MS identification, in the Morchella extract sample obtained in Example 1, a glycopeptide molecule with a high mass spectrometry abundance (peak area 1.22×10 5 ) was obtained. The mass spectrometry diagram of the glycopeptide molecule is shown in Figure 1 ; the glycopeptide sequence was LPS(Fuc)PMLLPQ, with a molecular weight of 1140 Da; the structural formula was as shown in Formula I; in the glycopeptide molecule, fucose was connected to the serine (Ser) hydroxyl group of the peptide chain by an O-linked glycosylation method through an α-glycosidic bond. The glycopeptide molecule was named Morchella glycopeptide, and the 3D structure diagram of Morchella glycopeptide is shown in Figure 2 . The same Morchella glycopeptide molecule was not identified in the Morchella extract sample obtained in Comparative Example 1.

[0062]

[0063] Experimental Example 2

[0064] Efficacy Analysis of Morchella Glycopeptide

[0065] The Morchella esculenta glycopeptide molecule LPS(Fuc)PMLLPQ used in this experimental example was synthesized by Gil Biochemical (Shanghai) Co., Ltd. according to the molecular structure of the Morchella esculenta glycopeptide identified in Experimental Example 1, and the purity of the synthesized sample was greater than 98%.

[0066] 1. Analysis of the activity of Morchella esculenta glycopeptide at the cellular level

[0067] Macrophages play an important role in many diseases and are the main participants in the body's innate immune response. In in vitro cell experiments, a macrophage inflammation model was established by inducing with LPS. LPS promotes macrophage activation through the TLR4 pathway, stimulating macrophages to secrete various inflammatory factors and mediators. As an inflammatory mediator secreted by macrophages, the secretion level of NO is a common indicator for observing macrophage immune activity. Macrophages are divided into M1 and M2 types according to the type of immune response. TNF-α, IL-1β, IL-6, NO, etc. are the signature cytokines of M1 macrophages. RAW264.7 macrophages were treated with LPS, and by analyzing the reduction of the secretion levels of the above signature cytokines after the intervention of Morchella esculenta glycopeptide, the role of Morchella esculenta glycopeptide in immune regulation and anti-tumor can be clarified.

[0068] RAW264.7 macrophages were cultured using complete culture medium, which consisted of DMEM high-glucose culture medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. Cells in the logarithmic growth phase were inoculated into 96-well plates (containing 1×10 6 cells / well). A blank group (RAW264.7 cells cultured normally), a group of RAW264.7 cells induced with LPS (LPS group), and a group of RAW264.7 cells cultured with Morchella esculenta glycopeptide LPS(Fuc)PMLLPQ (200 μM) after induction (glycopeptide group) were set up. In the LPS group, RAW264.7 cells were cultured in a culture medium containing LPS (2 μM) at 37 °C for 24 h for induction. In the glycopeptide group, the cell culture medium induced with LPS for 24 h was discarded, and complete culture medium containing 200 μM Morchella esculenta glycopeptide LPS(Fuc)PMLLPQ was added and cultured at 37 °C for 24 h. The cell culture supernatants obtained from each group were collected, and the supernatants were collected by centrifugation at 1500 r / min for 5 min. The Griess kit (Hangzhou Gosun Biotechnology Co., Ltd.) was used to detect the level of nitric oxide (NO) secreted by macrophages. The ELISA kit (Sangon Biotech (Shanghai) Co., Ltd.) was used to measure the contents of inflammatory factors IL-6, IL-12, IL-1β, and TNF-α secreted by cells.

[0069] The results of the effect of Morchella esculenta glycopeptide LPS(Fuc)PMLLPQ on the secretion of cytokines by RAW264.7 are as followsFigure 3 As shown in the figure, the results showed that the secretion levels of NO, IL-6, IL-12, IL-1β and TNF-α in the LPS group were significantly increased, indicating that LPS induced an inflammatory response in RAW264.7 macrophages. The release amount of NO and the secretion levels of IL-6, IL-12, IL-1β and TNF-α in the Morchella esculenta glycopeptide LPS(Fuc)PMLLPQ treatment group were significantly decreased. Among them, the release amount of NO decreased by 49.75%, and the secretion amounts of IL-6, IL-12, IL-1β and TNF-α decreased by 42.11%, 48.57%, 47.44% and 46.03% respectively. This shows that Morchella esculenta glycopeptide regulates macrophage function, inhibits inflammatory response, significantly down-regulates the levels of pro-inflammatory factors (significant analysis P value < 0.0001), and has immunomodulatory and anti-tumor activities.

[0070] 2. Molecular docking and activity analysis of Morchella esculenta glycopeptide

[0071] Download the crystal structures of the immune receptor protein TLR4 (PDB: 3FXI) and the tumor treatment target receptor protein VEGFR2 (PDB ID: 3VHE) from the RCSB database ( https: / / www.rcsb.org / ). Use the MOE 2019 molecular docking software to optimize the crystal structures of the receptor proteins, remove water molecules, and complete hydrogen atoms. Use the MOE software to construct the 3D structure of Morchella esculenta glycopeptide and perform molecular energy minimization. Use the Site Finder module of the MOE software to determine the amino acid residue active sites of the receptor proteins. Using the docking score, the number of bonds formed, and the binding bond energy between the glycopeptide and the receptor protein as screening indicators, select the complex in which the glycopeptide tightly binds to the receptor protein, and use the MOE software to analyze the binding sites and interaction modes between the glycopeptide molecule and the receptor protein in the complex.

[0072] There were 18 hydrogen bond interactions between the Morchella esculenta glycopeptide molecule LPS(Fuc)PMLLPQ and the TLR4 molecule, and the binding bond energy was -33.3 kcal / mol. Among them, the binding bond energy between the peptide molecule and TLR4 was -30.6 kcal / mol, and the binding bond energy between the sugar molecule and TLR4 was -2.7 kcal / mol. In the Morchella esculenta glycopeptide molecule, the peptide molecule formed a binding bond with the acidic residue GLU439 of TLR4. The oxygen atom in fucose binds to GLN505, which is also a key amino acid residue that can block the binding of LPS to TLR4. In summary, the binding of the Morchella esculenta glycopeptide molecule LPS(Fuc)PMLLPQ to TLR4 can block the inflammatory response (Table 2, Figure 4 ).

[0073] Table 2 Docking results of the Morchella esculenta glycopeptide molecule LPS(Fuc)PMLLPQ and the TLR4 molecule

[0074]

[0075]

[0076] Eleven hydrogen bonds and one H-pi interaction are formed between the morchella glycopeptide molecule LPS(Fuc)PMLLPQ and the VEGFR2 molecule, with a binding energy of -21.3 kcal / mol. Among them, the binding energy of the peptide molecule binding to VEGFR2 is -12.6 kcal / mol, and the binding energy of the sugar molecule binding to VEGFR2 is -8.7 kcal / mol. In the morchella glycopeptide molecule, the peptide molecule targets and binds to CYS919, forming a hydrogen bond interaction with a binding energy of -8.5 kcal / mol. This residue is also the binding site of the covalent inhibitor. Therefore, binding to this residue can enhance the binding persistence of the molecule. In the morchella glycopeptide molecule, the oxygen atom in fucose forms hydrogen bond interactions with the catalytic key amino acid residues ASP1046 and LYS868 and an H-pi interaction with PHE1047 in the form of an ATP competitive inhibitor, blocking kinase activation. In summary, the morchella glycopeptide molecule LPS(Fuc)PMLLPQ significantly inhibits VEGFR2 activity by targeting and binding to the ATP binding pocket of the tumor-related receptor protein VEGFR2 (Table 3, Figure 5 ).

[0077] Table 3 Docking results of the morchella glycopeptide molecule LPS(Fuc)PMLLPQ and the VEGFR2 molecule

[0078]

[0079]

[0080] The above results indicate that the morchella glycopeptide of the present invention can effectively bind to the immune receptor protein TLR4 and the tumor treatment target receptor protein VEGFR2, and has a good inhibitory effect on them, further indicating that the morchella glycopeptide of the present invention has immunomodulatory and anti-tumor activities.

[0081] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A morel glycopeptide, characterized in that, The molecular sequence of the morchella glycopeptide is LPS(Fuc)PMLLPQ; in the morchella glycopeptide molecule, fucose is linked to the serine hydroxyl group of the peptide chain by an O-glycosylation method through an α-glycosidic bond.

2. A morchella extract containing the morchella glycopeptide described in claim 1.

3. The preparation method of the morel extract according to claim 2, characterized in that, Comprising the following steps: Mix morchella with water at a ratio of 1 g:(10 - 30) mL, and enzymatically hydrolyze at 50°C - 70°C for 18 - 100 min using pectinase; the mass ratio of the pectinase to morchella is (3 - 8):

100.

4. The preparation method according to claim 3, characterized in that, The particle size of the morchella is <100 mesh.

5. Use of the morchella glycopeptide described in claim 1, the morchella extract described in claim 2, or the preparation method described in any one of claims 3 - 4 in the preparation of a product for reducing the secretion levels of macrophage NO, IL-6, IL-12, IL-1β or TNF-α.

6. Use of the morchella glycopeptide described in claim 1, the morchella extract described in claim 2, or the preparation method described in any one of claims 3 - 4 in the preparation of an anti-inflammatory product.

7. Use of the morchella glycopeptide described in claim 1, the morchella extract described in claim 2, or the preparation method described in any one of claims 3 - 4 in the preparation of a TLR4 protein inhibitor.

8. Use of the morchella glycopeptide described in claim 1, the morchella extract described in claim 2, or the preparation method described in any one of claims 3 - 4 in the preparation of a VEGFR2 protein inhibitor.

9. Use of the morchella glycopeptide described in claim 1, the morchella extract described in claim 2, or the preparation method described in any one of claims 3 - 4 in the preparation of an immunomodulatory product.

10. Use of the morchella glycopeptide described in claim 1, the morchella extract described in claim 2, or the preparation method described in any one of claims 3 - 4 in the preparation of an anti-tumor product.

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