A Highly Active Transglutaminase Peptide Substrate and Screening Method
By designing highly active transglutaminase peptide substrates AFQSAY and FMKHKFV, and combining molecular docking and experimental optimization, the problem of insensitive enzyme activity detection in traditional methods has been solved, achieving efficient enzymatic reactions and disease diagnosis, which is applicable to food safety and biomedical fields.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- JILIN UNIVERSITY
- Filing Date
- 2023-09-27
- Publication Date
- 2026-07-03
AI Technical Summary
Existing technologies are insufficient for efficiently screening highly active microbial transglutaminase (mTG) substrates, resulting in insensitive enzyme activity detection. Furthermore, traditional methods are time-consuming and labor-intensive, making it difficult to identify a large number of structurally diverse compounds.
A highly active transglutaminase peptide substrate was designed, comprising an acyl donor AFQSAY and an acyl acceptor FMKHKFV. By combining molecular docking technology with traditional experiments, the enzyme reaction system conditions were optimized to improve the sensitivity of enzyme activity detection.
It improves the sensitivity of mTG enzyme activity detection, avoids non-specific coupling, reduces costs, and achieves efficient enzymatic reactions and disease diagnosis, making it suitable for food safety and biomedical fields.
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Figure CN117430661B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of peptide screening technology, and in particular to a highly active transglutaminase peptide substrate and screening method. Background Technology
[0002] Microbial transglutaminase (mTG, EC 2.3.2.13) is a transglutaminase that catalyzes the formation of isopeptide bonds through acyl transfer reactions between the ε-amino group of lysine and the γ-carboxamide group of glutamate, either between protein molecules or within the molecule. The acyl donor is glutamine, and the acyl acceptor is the ε-amino group of lysine. mTG can cross-link proteins, and this cross-linking reaction can alter the adhesive properties of proteins, thereby polymerizing small proteins into larger molecules. Compared to other TG sources, mTG is independent of calcium... 2+ With broader substrate specificity, better stability, and a "glue" label, mTG can be widely applied in fields such as food safety and emerging biomedical engineering. In tissue engineering, mTG can crosslink natural biomaterials such as collagen, hyaluronic acid, chitosan, alginate, and elastin to form large molecular polymers, improving their textural properties and biocompatibility, ensuring non-toxicity, and rapidly and tightly binding them to living tissues for cell growth and response. In food processing, mTGase can bind small pieces of meat and minced meat together without the need for sodium chloride or phosphates to produce "healthy" large pieces of meat, reducing waste. mTGase can also overcome the problem of serum separation caused by temperature changes or physical influences, producing low-fat dairy products such as yogurt, ice cream, and cheese. In the biopharmaceutical field, mTGase can catalyze the coupling of proteins with water-soluble polymers to reduce drug immunogenicity, and catalyze the coupling of proteins with oligonucleotides as molecular probes for drug delivery.
[0003] In recent years, the increasingly widespread application of mTG in tissue engineering, medical adhesives, pharmaceutical protein delivery, and antibody functionalization has led to a new surge in research on mTGase activity. Numerous studies have revealed certain limitations of mTGases; they readily undergo substrate over-coupling and non-specific binding, affecting enzyme activity detection. Therefore, identifying highly active TGase substrates to compensate for the lack of specific coupling sites between the enzyme and substrate has become crucial. However, based on domestic and international literature and patent reports, the structures of highly efficient catalytic substrates recognizable by mTG have not yet been fully elucidated.
[0004] mTG can recognize two substrates: acyl acceptors and acyl donors. mTG has less stringent requirements for substrates of acyl acceptors than for acyl donors and has broad tolerance. Generally speaking, lysine residues act as the main acyl acceptors because their ε-amino groups are recognized by mTGase. Currently, the better acyl acceptor substrates reported in experiments include KAYA, MRHKGS, MKHK(GS), etc. Better acyl donor substrates include: motifs composed of dipeptides LQ; tripeptides LQR, YQR, YQS; tetrapeptides LLQG, TQGA, LQSP, etc. ([1] DOTIN, CAPORALE A, MONTI A, et al. A recent update on the use of microbial transglutaminase for the generation of biotherapeutics[J]. World Journal of Microbiology and Biotechnology, 2020, 36(4):53).
[0005] Generally speaking, screening commercial peptide libraries is the most commonly used method for substrate optimization. However, this traditional screening method is both time-consuming and labor-intensive, and it is difficult to identify a large number of compounds with rich composition, diverse properties, and diverse structures.
[0006] Molecular docking is a computer simulation technique that mimics the interaction between receptors and ligands, predicting the binding strength based on their binding modes and affinities. With the rapid development of computer technology, many researchers have begun using simulations to reproduce the molecular recognition process, searching for the optimal conformation between the protein and ligand, and their interactions. Compared to high-throughput screening in traditional experiments, it offers advantages such as lower cost and shorter timeframes. Summary of the Invention
[0007] The purpose of this invention is to overcome the shortcomings of traditional methods by designing a pair of highly reactive microbial transglutaminase polypeptide substrates based on molecular docking. By optimizing the enzyme reaction system by changing conditions such as temperature, substrate concentration, and pH, a highly sensitive method for testing mTG enzyme activity is provided.
[0008] To achieve the above objectives, the present invention provides a highly active transglutaminase polypeptide substrate, the specific technical solution of which is as follows:
[0009] A highly active transglutaminase polypeptide substrate, the polypeptide substrate comprising an acyl donor and an acyl acceptor, wherein the acyl donor sequence is AFQSAY and the acyl acceptor sequence is FMKHKFV.
[0010] In addition, the present invention also provides a method for screening highly active transglutaminase peptide substrates, comprising the following steps:
[0011] (1) After washing the chromatographic column, dissolve the mTGase powder in the MES solution, filter it and load it into the chromatographic column, elute and purify it. All the eluted mTGase solution is collected in an Eppendorf tube. The purified mTGase is analyzed by polyacrylamide gel electrophoresis.
[0012] (2) Design acyl acceptors and acyl donor substrates according to the rules of substrate structure;
[0013] (3) Hydrophobicity and isoelectric point of acyl acceptor and donor primary sequences were tested on the ExPASy website, and steric hindrance energy of peptides was measured using chem3D to preliminarily screen peptide substrates.
[0014] (4) Using molecular docking technology, the recognition between enzymes and substrate peptides designed based on active sites and structural characteristics was carried out. Based on the highest docking score, 5 pairs of polypeptide substrates were screened out. By measuring the enzyme activity of the 5 groups of substrates against mTGase, the sensitivity of mTGase detection was determined, and the substrate peptide with the best sensitivity was identified.
[0015] Preferably, the chromatographic column in step (1) is a Superdex column. TM 75 pg column.
[0016] Beneficial effects of the present invention
[0017] Compared with the prior art, the present invention has the following beneficial effects:
[0018] (1) This invention provides a pair of highly active mTGase polypeptide substrates. Compared with collagen (the preferred natural substrate of mTGase), the sensitivity of the polypeptide to mTG is increased by 100 times. Using it to test enzyme activity can avoid substrate flooding caused by non-specific coupling in biological samples, improve the efficiency of enzymatic reaction, and realize high-sensitivity detection of mTGase in the field of food safety.
[0019] (2) This invention provides a method for finding highly active substrates, namely, combining molecular docking design with traditional enzyme experiments. Compared with traditional methods, this method reduces costs and improves the efficiency of cross-linking and protein-modified cations, enabling highly sensitive detection and disease diagnosis, which will bring great benefits to biomedical applications. Attached Figure Description
[0020] Figure 1 Statistical analysis of OD values after 30 min of reaction between 5 pairs of peptide substrates and mTGase;
[0021] Figure 2 A comparison of OD values after the reaction of PA3+PD14 peptide substrate and collagen substrate;
[0022] Figure 3 The graph shows the activity analysis of the PA3+PD14 peptide substrate under various pH conditions.
[0023] Figure 4 This is a graph showing the activity of the PA3+PD14 peptide substrate at various temperatures. Detailed Implementation
[0024] Example 1
[0025] This invention relates to the design and screening of highly active transglutaminase peptide substrates. Based on molecular docking, five pairs of highly active transglutaminase peptide substrates were obtained. After enzyme activity determination of the five pairs of peptide substrates, the pair of peptide substrates with the highest activity was finally determined.
[0026] This invention provides a highly active transglutaminase polypeptide substrate, the specific technical solution of which is as follows:
[0027] A highly active transglutaminase polypeptide substrate, the polypeptide substrate comprising an acyl donor and an acyl acceptor, wherein the acyl donor sequence is AFQSAY and the acyl acceptor sequence is FMKHKFV.
[0028] In addition, the present invention also provides a method for screening highly active transglutaminase peptide substrates, comprising the following steps:
[0029] (1) mTG purification:
[0030] The Superdex™ 75 pg column (GE Healthcare Life Sciences, UK) was washed with at least 5 column volumes of dH2O and then equilibrated with the same volume of 50 mM MES (pH 6.0) buffer. 30 mg of mTGase powder was dissolved in 30 mL of MES solution, filtered through a 0.22 μM microporous membrane, and then packed into the column. The column was purified by elution with 50 mM MES (pH 6.0) buffer using an AKTA purifier (GE Healthcare, UK). All eluted mTGase was collected in 2 mL Eppendorf tubes. The purified mTGase was analyzed using SDS-PAGE.
[0031] (2) Design of mTG peptide substrates:
[0032] Based on the rules of substrate structure, acyl acceptor and acyl donor substrates were designed independently. Since mTG has a wide range of acyl acceptor substrates, the number of designs was relatively small. For acyl donor substrates, tetrapeptides, pentapeptides and hexapeptides were designed according to the rules. Among them, the hexapeptide was designed by permutation and combination according to empirical rules.
[0033] A. Design of acyl acceptor substrates
[0034] Due to the broad specificity of acyl receptors, the design of acyl receptors is mainly based on the modification of receptor substrates with good specificity reported in the above literature, so as to make their hydrophobicity as suitable as possible for the donor substrate. As shown in Table 1.
[0035] Table 1
[0036]
[0037]
[0038] B. Design of acyl donor substrates
[0039] Tetrapeptides and pentapeptides mostly utilize the excellent substrates reported in the aforementioned literature. Hexapeptide design follows the patterns of amino acid characteristics near Gln as described in the literature, using Gln(Q) as a baseline, with the N-terminus being negative and the C-terminus positive. The characteristic patterns at different positions are shown in Table 2.
[0040] Table 2
[0041]
[0042] (3) Software simulation and substrate screening
[0043] A. Initial screening of peptide substrates
[0044] Hydrophobicity and isoelectric point of the acyl receptor and donor primary sequences were tested on the ExPASy website, and the steric hindrance energy of the peptide was measured using chem3D. Four acyl receptors and seventeen acyl donors were screened and obtained, as shown in Table 3.
[0045] Table 3
[0046]
[0047]
[0048] B. Secondary screening of acyl donors using molecular docking
[0049] Determine the range of the docking boxes, and the spacing between the docking grid boxes is... The container was designed as a cube with sides of 40 grid points to cover all residual sites on the mtase active site and was large enough for substrate docking. The centers of the active sites were set to -2.718, 22.138, and -4.549 (x, y, z), and Cys64, Asp255, and His274 were designated as flexible fragments. Flexible docking of 17 acyl donors was performed using Autodockvina. The average score of 10 conformations was calculated, and PyMOL was used to examine the results. The percentage of conformations that can interact with active sites Cys64, Asp255, and His274 within the specified range is shown in Table 4.
[0050] Table 4 shows that the top five average scores for conformational ratings are: 6, 8, 12, 14, and 10. The top five proportions of the conformations of Cys64, Asp255, and His277 within the range are: 15, 12, 10, 14 = 17, 9 = 8.
[0051] In summary, donors with the highest common ranking from both rankings, namely 12, 14, 10, and 8, were selected as substrates for the second round of screening of acyl donors. Based on the principle that the hydrophobicity of the acyl donor should be similar to that of the acceptor, acyl donor 8 was chosen for docking with acyl acceptors 2 and 4, and acyl donors 12, 14, and 10 were chosen for docking with acyl acceptor 3.
[0052] Table 4
[0053]
[0054]
[0055] (4) Determine the mTGase activity of the five selected peptide substrates.
[0056] 1 μM of peptide substrates (PD8+PA2, PD8+PA4, PD12+PA3, PD14+PA3, PD10+PA3) were dissolved in 50 mM MES buffer at pH 6 at a 1:1 ratio. The substrates were incubated with 26 μM mTGase at 40 °C for 30 minutes. Using CP (1 μM) as a negative control, the optical density (OD) at 600 nm was measured using a TECAN Infinite200 PRO multi-plate reader (TECAN). The results are as follows: Figure 1 As shown, the peptide sequence pair with the best reactivity was obtained through screening, namely PD14+PA3.
[0057] Comparative Example 1
[0058] The reactivity of peptide substrates and collagen with different concentrations of mTGase was determined.
[0059] 1 μM of the peptide substrate PA3+PD14 was reacted with mTGase at concentrations of 0.026, 0.13, 0.26, 1.3, 2.6, 13, 26, and 130 μM at 40 °C. 5 μM of collagen was reacted with mTGase at concentrations of 1.3, 2.6, 13, 26, 130, and 260 μM at 40 °C. CP (1 μM) was used as a negative control. After 60 min of reaction, the optical density (OD) at 600 nm was measured using a microplate reader. The results are as follows: Figure 2 As shown, when PA3+PD14 is used as a peptide substrate, mTGase activity can be detected at concentrations as low as 26 nM. Between 130 nM and 26 μM, the detection signal exhibits a linear relationship with the logarithm of the mTGase concentration. When collagen is used as a substrate, mTGase activity can only be detected at concentrations as low as 2.6 μM. Therefore, compared to collagen, the PA3+PD14 peptide substrate pair exhibits 100-fold increased mTGase activity. Such low concentrations of mTGase can prevent non-specific binding, improve the efficiency of enzymatic reactions, and enable highly sensitive detection of mTGase in the field of food safety.
[0060] Example 2
[0061] Optimization of pH conditions for mTG enzyme reaction.
[0062] The pH conditions for mTG enzymatic reaction were optimized by investigating the enzyme activity of mTG at pH values of 5, 6, 7, 8, and 9. The pH values were adjusted to 5, 6, 7, 8, and 9 using pre-prepared PBS buffer. 20 mM PBS buffer at different pH values was used as the reaction system. The concentrations of the peptide substrate were 1 μM, collagen 5 μM, and mTG 26 μM. CP (1 μM) was used as a negative control. After reacting at 37°C for 60 minutes, the absorbance at 600 nm was measured.
[0063] The results are as follows Figure 3 As shown, mTG exhibits catalytic activity for protein cross-linking across a wide pH range. Its enzymatic activity is highest between pH 6 and 8, and since pH 7 is physiologically relevant, this substrate also demonstrates high reactivity under physiological pH conditions, making it well-suited for research in the biomedical field. Furthermore, we observed that the enzyme activity of this substrate is higher than that of collagen-based enzymes under the same conditions at almost all pH levels.
[0064] Example 3
[0065] Optimization of mTG enzyme reaction temperature conditions.
[0066] The temperature conditions for mTG enzymatic reaction were optimized by investigating the enzyme activity of mTG at six temperatures: 25℃, 30℃, 37℃, 40℃, 50℃, and 60℃. In this experiment, PBS was used instead of MES buffer to dissolve the substrate because PBS provides a wider buffering range. The concentrations of the peptide substrate, collagen, and mTG in the system were 1 μM, 5 μM, and 26 μM, respectively. CP (1 μM) was used as a negative control. After reacting in 20 mM PBS buffer at pH 6 for 60 minutes, the absorbance at 600 nm was measured.
[0067] The results are as follows Figure 4 As shown, mTG exhibits catalytic activity for protein cross-linking over a wide temperature range. Its enzymatic activity is high between 30℃ and 45℃, with optimal activity at 37℃. Since 37℃ is approximately the physiological temperature, this substrate also demonstrates high reactivity under physiological conditions, making it well-suited for research in the biomedical field. Furthermore, we observed that the enzyme activity of this substrate is higher than that of collagen-based enzymes under the same conditions at almost all temperatures.
[0068] This application optimizes the enzyme reaction system by changing conditions such as temperature, substrate concentration, and pH, thereby providing a highly sensitive method for testing mTG enzyme activity. At the same time, the experimental results confirm the potential of combining molecular docking with traditional experiments under physiological conditions to design highly active substrates.
[0069] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A high activity glutaminase polypeptide substrate, characterized in that, The polypeptide substrate is an acyl donor and an acyl acceptor, respectively, the acyl donor sequence is AFQSAY, and the acyl acceptor sequence is FMKHKFV.