A lactadherin-derived calcium-chelating peptide and use thereof

Abstract

This invention discloses a lactobridged protein-derived calcium chelating peptide and its applications, belonging to the field of bioactive small molecule peptide technology. The calcium chelating peptide provided by this invention is safe and non-toxic, exhibiting better physicochemical activity compared to traditional calcium supplements. Its calcium chelating capacity reaches over 419.36 μg / mg, and it can promote calcium transport in the Caco-2 cell monolayer, thereby improving calcium absorption and bioavailability in the human body, making it suitable for use as a calcium supplement. This invention can develop a new protein source for calcium supplements and makes a significant contribution to the high-value utilization of dairy products.

Description

Technical Field

[0001] This invention relates to a lactoadenosine-derived calcium chelating peptide and its application, belonging to the field of bioactive small molecule peptide technology. Background Technology

[0002] Calcium is the main component of bone minerals. The human body primarily obtains calcium through food intake. Studies have found that calcium metabolism imbalance caused by insufficient calcium intake is a contributing factor to various diseases affecting people of all ages. Currently, calcium deficiency has become a global nutritional phenomenon.

[0003] There are currently three main types of calcium supplements on the market:

[0004] Inorganic calcium: First-generation calcium preparations are mainly composed of inorganic salts. The disadvantages are that they have low solubility and will form precipitates in the alkaline environment of the small intestine, resulting in low soluble calcium content. In addition, this type of calcium preparation will irritate the stomach after reacting with gastric acid and will also produce gas in the gastrointestinal tract, leading to side effects such as bloating.

[0005] Organic acid calcium: Second-generation calcium preparations are mainly composed of organic acid salts. Compared with first-generation calcium preparations, their solubility and absorption rate are improved, but their calcium content is generally lower.

[0006] Organic acid chelated calcium: Third-generation calcium supplements primarily use organic acid chelated calcium sources, mainly chelates formed from amino acids and calcium ions, and are compound calcium supplements made with vitamin C and vitamin D as excipients. This generation of calcium supplements has good solubility and can be absorbed without dissociating into calcium ions, resulting in a high calcium absorption rate. However, its transport is affected by food, so it is only suitable for calcium supplementation on an empty stomach.

[0007] In recent years, the development of small molecule peptides has propelled the research and development of fourth-generation calcium supplements. Fourth-generation calcium supplements refer to chelates formed by the chelation of calcium ions with small molecule peptides through coordination covalent bonds. Compared to third-generation calcium supplements, structurally, the presence of non-chelating oxygen peptide bonds in the coordination process makes the peptides... Calcium complexes are more stable than amino acid-chelated calcium. From an absorption perspective, both the terminal amino and carboxyl groups of amino acids participate in calcium chelation, forming a bicyclic chelate structure. Since there are no free carboxyl groups, the amino group itself cannot be completely absorbed. Peptides, on the other hand... Due to steric hindrance, calcium complexes contain free carboxyl groups and are absorbed in the small intestine in their entirety, releasing calcium ions when needed. Small molecule bioactive peptides, acting as carriers, possess characteristics such as rapid transport, low energy consumption, and low saturation, and have the ability to prevent calcium phosphate precipitation, thus enabling the peptides to... The calcium complex remains in a soluble calcium state and is directly decomposed into ionic form at the absorption site for absorption, thereby greatly improving the calcium absorption rate.

[0008] Literature reports (Liao W, Liu S, Liu X, et al. The purification, identification and bioactivity study of a novel calcium-binding peptide from casein hydrolysate. Food Funct. 2019;10(12):7724-7732. doi:10.1039 / c9fo01383k) that the heptapeptide VLPVPQK derived from casein hydrolysate has strong calcium-binding activity (129.46 mg / g) and can effectively enhance calcium transport and absorption in Caco-2 cell monolayers in a concentration-dependent manner.

[0009] Lactopontin (LPN) is a highly phosphorylated protein isolated from mammalian milk. LPN and its hydrolysates are rich in acidic amino acids and phosphoserine residues, exhibiting strong affinity for a large number of calcium ions. However, its binding sites and mechanism of action remain unclear, limiting its application as a calcium supplement. LPN can be enzymatically hydrolyzed into smaller peptides, thereby exposing its active site.

[0010] Therefore, the isolation and preparation of calcium chelate peptides from LPN hydrolysates is of great significance for the basic theoretical research on the regulation of biomineralization by phosphorylated proteins, can develop new protein sources for calcium supplements, and can make important contributions to the high-value utilization of dairy products. Summary of the Invention

[0011] To address the aforementioned issues, this invention provides a calcium chelating peptide DDDLDDDDDNSQDV or IE(pS)QEN(pS)KL(pS)QE, which exhibits extremely high biocompatibility, calcium chelation capacity, and calcium permeability.

[0012] The first objective of this invention is to provide a calcium chelating peptide, wherein the amino acid sequence of the calcium chelating peptide is DDLDDDDDNSQDV or IESQENSKLSQE.

[0013] In one embodiment, the serine in the calcium chelate peptide may also be a phosphate-modified serine.

[0014] In one embodiment, the calcium chelating peptide is DDLDDDDDNSQDV or DDLDDDDDN(pS)QDV or IE(pS)QEN(pS)KL(pS)QE; wherein (pS) is a phosphate-modified serine.

[0015] In one embodiment, the method for preparing the calcium chelate peptide includes solid-phase synthesis, enzymatic hydrolysis, and microbial expression.

[0016] A second object of the present invention is to provide a medicine comprising an effective dose of any of the above-mentioned calcium chelating peptides;

[0017] Optionally, the drug may also contain any of the above-mentioned calcium chelating peptides; the derivatives of the calcium chelating peptides refer to calcium chelating peptide derivatives obtained by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification or glycosylation of the amino acid side chain groups, amino terminus or carbonyl terminus of the calcium chelating peptides.

[0018] In one embodiment, the drug further contains pharmaceutically acceptable excipients; the excipients refer to conventional drug carriers in the pharmaceutical field.

[0019] In one embodiment, the excipients include one or more of the following: binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, and mannitol; fillers such as starch and sucrose; humectants such as glycerin; disintegrants such as sodium carboxymethyl starch, croscarmellose, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients such as flavoring agents and sweeteners may also be added to the composition.

[0020] Optionally, the dosage form of the drug includes, but is not limited to, oral dosage form, injectable dosage form, and inhaled dosage form;

[0021] Optionally, the oral dosage form includes, but is not limited to, tablets, capsules, granules, oral liquids, and oral suspensions;

[0022] Optionally, the injectable dosage form includes, but is not limited to, injection solution and injection powder for injection;

[0023] Optionally, the inhalation dosage form includes, but is not limited to, aerosols and powder inhalers.

[0024] A third object of the present invention is to provide the use of any of the above-described calcium chelating peptides in the preparation of a pharmaceutical product for promoting calcium absorption.

[0025] In one embodiment, the calcium chelate peptide can also be used in the preparation of products with antioxidant, anti-inflammatory, cartilage and tissue regeneration repair (e.g., promoting bone matrix mineralization, increasing bone density, accelerating bone tissue growth, and shortening fracture healing period), metabolic and immune function regulation (e.g., regulating enzyme activity and enhancing immunity), dental caries prevention, and digestive function improvement.

[0026] In one embodiment, the drug is used to promote calcium absorption.

[0027] In one embodiment, the drug further contains pharmaceutically acceptable excipients; the excipients refer to conventional drug carriers in the pharmaceutical field.

[0028] In one embodiment, the excipients include one or more of the following: binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, and mannitol; fillers such as starch and sucrose; humectants such as glycerin; disintegrants such as sodium carboxymethyl starch, croscarmellose, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients such as flavoring agents and sweeteners may also be added to the composition.

[0029] Optionally, the dosage form of the drug includes, but is not limited to, oral dosage form, injectable dosage form, and inhaled dosage form;

[0030] Optionally, the oral dosage form includes, but is not limited to, tablets, capsules, granules, oral liquids, and oral suspensions;

[0031] Optionally, the injectable dosage form includes, but is not limited to, injection solution and injection powder for injection;

[0032] Optionally, the inhalation dosage form includes, but is not limited to, aerosols and powder inhalers;

[0033] Beneficial effects of the present invention

[0034] The main reason why the peptide chain binds to calcium ions in this invention is that the amino acid sequence contains a large number of acidic amino acids. The free carboxyl groups on these amino acids can form coordinate bonds with calcium ions, while the free amino groups also participate in the reaction to form soluble complexes, which prevent calcium ion precipitation and increase the absorption of calcium in the small intestine and its accumulation in the body.

[0035] The calcium chelating peptides DDLDDDDDNSQDV, DDLDDDDDN(pS)QDV, and IE(pS)QEN(pS)KL(pS)QE provided by this invention are safe and non-toxic, and have better physicochemical activity compared with traditional calcium supplements. Their calcium chelating capacities are 638.78 μg / mg, 446.95 μg / mg, and 419.36 μg / mg, respectively, and all of them can promote calcium transport in the Caco-2 cell monolayer, thereby improving the absorption and bioavailability of calcium in the human body, and can be used as calcium supplements. Attached Figure Description

[0036] Figure 1 HPLC identification results for calcium chelate peptide DDLDDDDDNSQDV;

[0037] Figure 2 LC-MS identification results of calcium chelate peptide DDLDDDDDNSQDV;

[0038] Figure 3 The HPLC identification results of the calcium chelating peptide IE(pS)QEN(pS)KL(pS)QE;

[0039] Figure 4 LC-MS identification results of calcium chelating peptide IE(pS)QEN(pS)KL(pS)QE;

[0040] Figure 5 Effects of calcium chelating peptide DDLDDDDDNSQDV on Caco-2 cell viability;

[0041] Figure 6 The calcium chelating capacity of the calcium chelating peptides; among them, VK7 is VLPVPQK, DV13 is DDLDDDDDNSQDV, pDV13 is DDLDDDDDN(pS)QDV, and IE12 is IE(pS)QEN(pS)KL(pS)QE.

[0042] Figure 7 The effect of calcium chelating peptides on calcium transport in Caco-2 cell monolayers was investigated; among them, VK7 was VLPVPQK, DV13 was DDLDDDDDNSQDV, and pDV13DDLDDDDDN(pS)QDV. Detailed Implementation

[0043] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0044] Raw materials used in the examples:

[0045] LPN was isolated from fresh milk. For specific methods, please refer to the literature (Ma Ping, Sun Jie, Liu Ning. Isolation, purification and identification of osteopontin in milk [J]. Food and Fermentation Industries, 2008, (06): 135-139.).

[0046] Pepsin, pancreatin, and bile salts were purchased from Sigma-Aldrich, USA, while potassium chloride, sodium chloride, and sodium hydroxide were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0047] The calcium chelating peptides used in the examples were all synthesized by Shanghai Ketai Biotechnology Co., Ltd.

[0048] Test method:

[0049] 1. HPLC method

[0050] High-performance liquid chromatography (HPLC) was used with a Kromasil 100-5-C18 column (4.6 × 250 mm × 5 μm). The mobile phases were 0.1% trifluoroacetic acid aqueous solution (A) and 0.1% trifluoroacetic acid acetonitrile solution (B), with a total flow rate of 1 mL / min and a detection wavelength of 214 nm. Samples were dissolved in ultrapure water, with an injection volume of 7 μL. The gradient elution program was as follows:

[0051]

[0052] 2. LC MS method

[0053] An electrospray ionization (ESI) interface was used, with the detector voltage set to -0.2 kV, CDL temperature to 250 °C, CDL voltage to 0 V, block temperature to 200 °C, nebulizing gas flow rate to 1.5 L / min, preset bias to +4.5 kV, and T-Flow to 0.2 ml / min. Samples were dissolved in a mixed solvent of 15% acetonitrile (ACN) and 85% water (H₂O) at an injection volume of 0.2 μL.

[0054] Example 1: Preparation of calcium chelating peptides

[0055] A method for preparing calcium chelate peptides, using an infant model for in vitro simulated digestion, includes the following steps:

[0056] (1) Stomach digestion stage:

[0057] 10 mg / mL LPN solution was added to the enzyme reactor and preheated, and the pH was adjusted to 5.3. Then it was mixed with simulated gastric juice (pH 5.3, 724.3 U pepsin / mL, 13 mM potassium chloride, 94 mM sodium chloride) at a ratio of 63:37 (v / v). The reaction was carried out at 37 °C and pH 5.3 for 60 min. The pH was adjusted to 7.0 with 2 M sodium hydroxide to terminate this stage of digestion, and the gastric digestion stage product was obtained.

[0058] (2) Intestinal digestion stage:

[0059] The pH of the gastric digestion products was adjusted to 6.6, and then mixed with simulated intestinal fluid (pH 6.6, 42.1 U pancreatic enzyme / mL, 8.2 mM bile salts, 10 mM potassium chloride, 249 mM sodium chloride) at a ratio of 62:38 (v / v). Digestion was carried out at 37°C and pH 6.6 for 60 min, and intestinal digestion was stopped at 95°C for 10 min to obtain the final digestion products.

[0060] (3) Identification of polypeptide composition:

[0061] The final digestion products were analyzed by LC-MS / MS equipped with an online nanospray ion source. The entire system was an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, MA, USA) with an EASY-nanoLC 1200 in series. A total of 5 μL of sample was loaded (analytical column: Acclaim PepMap C18, 75 μm x 25 cm), and the sample was separated by a gradient at a rate of 60 min. The column flow rate was controlled at 300 nL / min, the column temperature was 40 °C, the electrospray voltage was 2 kV, and the gradient started from 4% B phase, increased nonlinearly to 50% within 53 min 40 sec, increased to 95% within 40 sec, and was maintained for 5 min 40 sec.

[0062] The mass spectrometer operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. The mass spectrometry parameters were set as follows: (1) MS: Scan range (m / z): 100-1500; resolution: 120,000; Normalized AGC target: 200%; maximum injection time: 100ms; (2) HCD-MS / MS: resolution: 50,000; Normalized AGC target: 200%; maximum injection time: 86ms; collision energy: 25%, 30%, 35%; dynamic exclusion time: 30s. The tandem mass spectra were analyzed using PEAKS Studio version 10.6 (BioinformaticsSolutions Inc., Waterloo, Canada). The database was Uniprot-Bostaurus (version 2024, 26635 entries). Library search parameters: Fragment ion mass tolerance: 0.02 Da, precursor ion mass tolerance: 10 ppm, maximum missed cuts: 2, fixed modifications: Carbamidomethylation 57.02, variable modifications: Oxidation (M) 15.99, Damidation (NQ) 0.98, Acetylation (Protein N-term) 42.01, Phosphorylation (STY) 79.97. Protein card value is 1% FDR, containing at least 1 unique peptide; peptide card value is 1% FDR.

[0063] 242 peptides derived from LPN were identified. Based on the proportion of acidic amino acids and the degree of phosphorylation, three potential calcium chelating peptides, DDLDDDDDNSQDV, DDLDDDDDN(pS)QDV, and IE(pS)QEN(pS)KL(pS)QE, were screened.

[0064] Shanghai Ketai Biotechnology Co., Ltd. was commissioned to synthesize calcium chelate peptides DDLDDDDDNSQDV, DDLDDDDDN(pS)QDV and IE(pS)QEN(pS)KL(pS)QE.

[0065] Example 2: Detection of calcium chelating peptides

[0066] The calcium chelating peptides DDLDDDDDNSQDV, DDLDDDDDN(pS)QDV, and IE(pS)QEN(pS)KL(pS)QE prepared in Example 1 were tested, and the results are as follows:

[0067] (1) HPLC detection results

[0068] HPLC detection results are as follows Figure 1 and Figure 3 As shown.

[0069] (2) LC-MS detection results

[0070] LC-MS detection results are as follows Figure 2 and Figure 4 As shown.

[0071] (3) Safety testing

[0072] The biosafety of DDLDDDDDNSQDV and IE(pS)QEN(pS)KL(pS)QE is tested using the following steps:

[0073] Take Caco-2 cells in the logarithmic growth phase and divide them into 1.0 × 10⁶ cells per well. 5 Cells were seeded into 96-well plates at 100 μL per well and cultured at 37 ℃, 5% CO2 and saturated humidity for 24 h. The culture medium was then removed, and complete culture medium containing calcium chelate peptide at final concentrations of 0.1, 1 and 10 μg / mL was added, respectively, and the cells were cultured for another 24 h. Cell viability was determined using the CCK-8 reagent.

[0074] The results are as follows Figure 5 As shown, the results indicate that 0.1–10 μg / mL of DDLDDDDDNSQDV, DDLDDDDDN(pS)QDV, and IE(pS)QEN(pS)KL(pS)QE do not affect cell viability, meaning that DDLDDDDDNSQDV, DDLDDDDDN(pS)QDV, and IE(pS)QEN(pS)KL(pS)QE have high biosafety.

[0075] (4) Calcium chelating ability

[0076] The calcium chelating capacity of DDLDDDDDNSQDV, DDLDDDDDN(pS)QDV, and IE(pS)QEN(pS)KL(pS)QE was determined by the following steps:

[0077] 2 mg of lyophilized powders DDLDDDDDNSQDV, DDLDDDDDN(pS)QDV, and IE(pS)QEN(pS)KL(pS)QE were mixed with 1 mL of 5 mM CaCl2 solution and 2 mL of phosphate buffer (pH 7.8, 20 mM) and shaken until completely dissolved. The mixture was stirred at 37 °C for 120 min, and the reaction solution was centrifuged at 4000 g for 20 min to precipitate insoluble calcium phosphate.

[0078] The calcium content in the supernatant was determined by flame atomic absorption spectrometry. Deionized water was used as a blank control instead of the sample. The calcium content was calculated as follows: soluble calcium in the supernatant of the peptide-containing sample minus soluble calcium in the blank supernatant. Each experiment was performed three times, and the results were expressed as mean ± standard deviation. The peptide VLPVPQK, as disclosed in existing literature, was used as a control.

[0079] The results are as follows Figure 6 As shown, the results indicate that the calcium chelating capacity of DDLDDDDDNSQDV reached 638.78 μg / mg, that of DDLDDDDDN(pS)QDV reached 446.95 μg / mg, and that of IE(pS)QEN(pS)KL(pS)QE reached 419.36 μg / mg, all of which were significantly higher than that of VK7 (342.81 μg / mg).

[0080] (5) Effects on calcium transport

[0081] The effect of calcium chelating peptides on calcium transport in Caco-2 cell monolayers was investigated using the following steps:

[0082] Adjust the concentration of Caco-2 cell suspension to 5 × 10⁻⁶ 4 Cells were seeded at a density of 1 / mL onto the filter membrane of a 12-well Transwell plate. 0.5 mL of cell suspension and 1.5 mL of complete DMEM culture medium were added to the apical side (AP) and basal side (BL) of the filter membrane, respectively. The culture medium was changed after 24 h. For the first 7 days, the medium was changed every other day, and then daily thereafter, until a tight monolayer of cells was formed.

[0083] Transwell plates inoculated with Caco-2 cells were removed from the cell culture incubator and equilibrated at room temperature for 30 min in a clean bench. Transepithelial electrical resistance (TEER) was then measured using a Millipore Millicell-ERS transepithelial electrical resistance meter. The test electrodes were sterilized by immersing in 70% ethanol for 15 min, air-dried for 15 s, rinsed with sterile PBS, and then rinsed with complete culture medium. During transepithelial resistance measurement, the short end of the electrode was immersed in the AP side, and the long end in the BL side. When TEER > 500 Ω·cm... 2 It can be used in small intestinal transport experiments.

[0084] After the Caco-2 cell monolayer formed, the Caco-2 cells in the Transwell plate were washed twice with HBSS buffer. During the third addition of HBSS, the plate was incubated at 37 ℃ for 30 min. The HBSS was then gently aspirated from the wells. 0.5 mL of working solution (HBSS containing 10 μg / mL calcium chelate peptide and 5 mM CaCl2) was added to the AP side, and 0.5 mL of HBSS containing 5 mM CaCl2 was added to the control group. 1.5 mL of HBSS buffer was added to the BL side. After transporting for 2 h, the sample from the BL side was removed. The calcium content of the samples was determined using flame atomic absorption spectrometry. Calcium permeability (%) = (calcium content on the BL side / initial calcium content on the AP side) * 100%.

[0085] The results are as follows Figure 7 As shown, the results indicate that the total calcium permeability of the DDLDDDDDNSQDV group was 36.53%, the DDLDDDDDN(pS)QDV group was 39.12%, and the IE(pS)QEN(pS)KL(pS)QE group was 40.79%, all of which were significantly higher than the 11.20% of the control group and the 34.55% of the VK7 group.

[0086] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A calcium chelating peptide, characterized in that, The amino acid sequence of the calcium chelate peptide is DDLDDDDDNSQDV, DDLDDDDDNpSQDV, or IEpSQENpSKLpSQE.

2. The calcium chelating peptide according to claim 1, characterized in that, The calcium chelate peptide is prepared by solid-phase synthesis or microbial expression.

3. A calcium supplement, characterized in that, The calcium supplement contains an effective dose of the calcium chelating peptide of claim 1.

4. The application of the calcium chelating peptide according to claim 1 in the preparation of calcium supplements, characterized in that, The calcium supplement is used to promote calcium absorption.

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