High-glucuronic acid activity heparin 2-O-sulfate transferase mutant and application thereof

Abstract

The invention relates to a heparin 2-O-sulfate transferase mutant with high glucuronic acid activity and application of the heparin 2-O-sulfate transferase mutant. The amino acid sequence of the heparin 2-O-sulfate transferase mutant 2OST (R189G) with high glucuronic acid activity is as shown in SEQ ID NO. 4, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO. 3. According to the invention, a mutant 2OST (R189G) is obtained through protein engineering and mutation design of heparin 2-O-sulfate transferase 2OST. The mutant 2OST (R189G) has higher and stronger GlcA selectivity which is 9.51 times of that of 2OST, the GlcA2S synthesis efficiency in heparin synthesis is improved, the efficiency of preparing non-anticoagulant heparin by a chemical enzyme method is greatly promoted, and IdoA2S in a pentasaccharide sequence is replaced by the formed GlcA2S, so that a new thought is provided for synthesizing the non-anticoagulant heparin from the source, and the method is suitable for industrial production. And a new foundation is provided for research and development of glycosaminoglycan drugs.

Description

Technical Field

[0001] The present invention relates to a mutant of high glucuronic acid active heparin 2-O-sulfotransferase and its application, specifically to a heparin 2-O-sulfotransferase mutant and its application in the synthesis of non-anticoagulant heparin, belonging to the field of biotechnology. Background Art

[0002] As a natural glycosaminoglycan, heparin has rich physiological and pathophysiological functions. It is mainly composed of repeating glucosamine and glucuronic acid units, and forms a complex and diverse structure through modifications such as different degrees of sulfation and N-acetylation of glucosamine. Heparin is widely used as an anticoagulant in medicine for the prevention and treatment of thromboembolic diseases, myocardial infarction, and cardiovascular surgery, and also exhibits many other biological activities, such as applications in immunomodulation, anti-inflammation, anti-tumor, and inhibition of virus infection. In particular, the development of non-anticoagulant heparin has expanded its potential in the treatment of tumors, radiation therapy syndrome, and other inflammatory diseases.

[0003] Traditionally, heparin is derived from animal tissues (such as porcine intestinal mucosa and bovine lung) and obtained through chemical and physical treatments. In current technologies, the preparation of non-anticoagulant heparin usually adopts chemical selective treatment to remove or reduce its anticoagulant activity from heparin. For example, periodate is used to selectively oxidize the non-sulfated glucuronic acid (GlcA) sites in heparin to reduce its anticoagulant properties. However, this method requires the use of animal-derived heparin as raw material, which is prone to introducing impurities during the processing, has a potential risk of pathogen contamination, and has complex steps and high costs. In addition, due to the complex source and preparation process of heparin, and the structural heterogeneity of heparin increasing the difficulty of quality control, side reactions during the purification process may affect the purity and activity of the final product. Therefore, the development of more controllable and safe synthesis methods is of great significance for the development of non-anticoagulant heparin.

[0004] To overcome the deficiencies of traditional chemical methods, chemoenzymatic synthesis has become a promising strategy in recent years. This method combines the high stereoselectivity of enzymes and the flexibility of chemical synthesis, can efficiently synthesize heparin and its derivatives under mild reaction conditions, and can precisely control the sulfation sites, thus providing an innovative preparation approach for the development of non-anticoagulant heparin. The anticoagulant activity of heparin mainly depends on its specific pentasaccharide sequence, in which the skew-boat conformation of iduronic acid (IdoA) is crucial for anticoagulation. Therefore, the selectivity to distinguish IdoA from GlcA is a key challenge in the development of non-anticoagulant heparin.

[0005] 2-O-sulfotransferase (2OST) plays an important role in the sulfation modification process of glycosaminoglycans. It can transfer sulfate groups to the C2-OH position of glucuronic acid, which is closely related to the formation of anticoagulant structural units. However, 2OST usually has a much higher selectivity for iduronic acid (IdoA) than for glucuronic acid (GlcA). This preference limits its application potential in the synthesis of non-anticoagulant heparin. Therefore, searching for isoenzymes or mutants of this enzyme helps to efficiently synthesize heparin backbone glycan chains and to deeply study the reaction mechanism of this enzyme family. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the present invention provides a heparin 2-O-sulfotransferase mutant with high glucuronic acid activity and its application. The heparin 2-O-sulfotransferase mutant in the present invention is obtained by site-saturation mutagenesis of heparin 2-O-sulfotransferase derived from Gallus gallus. This mutant with high glucuronic acid activity can be applied to the synthesis of non-anticoagulant heparin oligosaccharides.

[0007] Term Explanation:

[0008] GlcA-GlcNS-GlcA-GlcNS-GlcA-pNP: The full Chinese name is 4-nitrophenyl-glucuronic acid-N-sulfo-glucosamine-glucuronic acid-N-sulfo-glucosamine-glucuronic acid, and its function is to serve as the starting substrate for the synthesis of non-anticoagulant heparin oligosaccharides.

[0009] PAPS: The full Chinese name is 3′-phosphoadenosine-5′-phosphosulfate, and its function is to be a sulfate donor.

[0010] The technical solution of the present invention is as follows:

[0011] A heparin 2-O-sulfotransferase mutant 2OST (R189G) with high glucuronic acid activity is obtained by site-directed mutagenesis of arginine at position 189 to glycine on the basis of heparin 2-O-sulfotransferase 2OST; the amino acid sequence of the heparin 2-O-sulfotransferase mutant 2OST is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0012] Preferably according to the present invention, the amino acid sequence of the heparin 2-O-sulfotransferase mutant 2OST (R189G) with high glucuronic acid activity is shown in SEQ ID NO.4, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.3.

[0013] A recombinant vector is obtained by inserting the encoding gene of the heparin 2-O-sulfotransferase mutant 2OST (R189G) into a plasmid vector.

[0014] Preferably according to the present invention, the plasmid vector is pMAL-c5x.

[0015] In the present invention, the recombinant vector containing the target gene is synthesized by Nanjing GenScript Corporation.

[0016] A recombinant strain is obtained by transforming the above recombinant vector into a host cell.

[0017] Preferably according to the present invention, the host cell is Escherichia coli Origami B(DE3).

[0018] The application of the above heparin 2-O-sulfotransferase mutant 2OST(R189G) in heparin oligosaccharide synthesis.

[0019] Preferably according to the present invention, in the application, a heparin backbone pentasaccharide of GlcA-GlcNS-GlcA-GlcNS-GlcA-pNP is used as the starting acceptor, PAPS is used as the donor, and through the catalytic reaction of the heparin 2-O-sulfotransferase mutant 2OST(R189G), a heparin backbone pentasaccharide with the structure of GlcA-GlcNS-GlcA2S-GlcNS-GlcA-pNP is generated.

[0020] In the present invention, the experimental operations not described in detail can be carried out according to the conventional experimental operations in this technical field.

[0021] Beneficial effects

[0022] 1. Through protein engineering and mutation design of heparin 2-O-sulfotransferase 2OST, the present invention obtains the heparin 2-O-sulfotransferase mutant 2OST(R189G). This heparin 2-O-sulfotransferase mutant 2OST(R189G) has higher and stronger selectivity for glucuronic acid, reaching 9.51 times that of heparin 2-O-sulfotransferase 2OST, improving the synthesis efficiency of GlcA2S in heparin synthesis, greatly promoting the efficiency of chemoenzymatic preparation of non-anticoagulant heparin, and providing a new basis for the research and development of glycosaminoglycan drugs.

[0023] 2. Through protein engineering transformation of 2OST, the present invention creates a heparin 2-O-sulfotransferase mutant 2OST(R189G) with stronger selectivity for glucuronic acid, which can prepare a heparin backbone pentasaccharide with the structure of GlcA-GlcNS-GlcA2S-GlcNS-GlcA-pNP to replace IdoA2S in the pentasaccharide sequence, thus providing a new idea for the way of synthesizing non-anticoagulant heparin from the source. Brief description of the drawings

[0024] Figure 1SDS-PAGE detection results of the soluble expression and purification of heparin 2-O-sulfotransferase 2OST and its mutant heparin 2-O-sulfotransferase 2OST(R189G) in Escherichia coli;

[0025] In the figure: M is the Marker; the remaining bands are the recombinant proteins of heparin 2-O-sulfotransferase 2OST and its mutant heparin 2-O-sulfotransferase 2OST(R189G) obtained by purification.

[0026] Figure 2 Protein quantitative standard curve of heparin 2-O-sulfotransferase 2OST.

[0027] Figure 3 Histogram of the soluble expression levels of heparin 2-O-sulfotransferase 2OST and its mutant heparin 2-O-sulfotransferase 2OST(R189G) in Escherichia coli.

[0028] Figure 4 HPLC chromatogram of the reaction products of heparin 2-O-sulfotransferase 2OST and its mutant heparin 2-O-sulfotransferase 2OST(R189G).

[0029] Figure 5 Enzyme activity comparison of heparin 2-O-sulfotransferase mutant 2OST(R189G) in the synthesis of GlcA-GlcNS-GlcA2S-GlcNS-GlcA-pNP.

[0030] Figure 6 Enzyme activity comparison of heparin 2-O-sulfotransferase 2OST and its mutant heparin 2-O-sulfotransferase 2OST(R189G) against GlcA in the synthesis of GlcA-GlcNS-GlcA2S-GlcNS-GlcA-pNP. Detailed implementation mode

[0031] The technical solutions of the present invention will be further described below in combination with the embodiments and the accompanying drawings of the specification, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the present invention are all methods well known to those skilled in the art.

[0032] Through literature research, the inventors found that heparin 2-O-sulfotransferase 2OST derived from Gallus Gallus has been reported. However, due to the weak activity of this heparin 2-O-sulfotransferase 2OST and its weak selectivity for GlcA, it is difficult to be used for the biosynthesis of non-anticoagulant heparin oligosaccharides with a defined structure. Therefore, the inventors expect to carry out directed evolution of the enzyme in various ways to improve the enzyme activity and selectivity.

[0033] The inventors performed sequence homology analysis on the amino acid sequence of heparin 2-O-sulfotransferase 2OST, carried out multiple sequence alignment using the EMBL Clustal Omega tool, and analyzed the highly conserved regions of the amino acid sequence using the Jalview software. At the same time, the Swiss-Model tool was used to perform protein simulation modeling on heparin 2-O-sulfotransferase 2OST, and the HotSpot Wizard 2.0 was used to predict the active center of this enzyme. It was found that position 189 of the amino acid sequence of heparin 2-O-sulfotransferase 2OST is near the active center and the highly conserved region, and site-directed mutagenesis of it is very likely to improve the catalytic activity of 2OST and the selectivity for glucuronic acid. Therefore, site-directed saturation mutagenesis was selected for position 189 of the amino acid sequence, and ten mutants, namely 2OST(R189N), 2OST(R189C), 2OST(R189Q), 2OST(R189G), 2OST(R189I), 2OST(R189F), 2OST(R189S), 2OST(R189T), 2OST(R189W), and 2OST(R189Y), were designed.

[0034] Among them, the nucleotide sequence of 2OST(R189G) is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

[0035] Unless otherwise specified, the carbohydrate substrates used in the present invention are all purchased from Sigma. The mutant plasmids of the heparin 2-O-sulfotransferase 2OST used were all commissioned by Nanjing Genscript Biotech Co., Ltd. to be produced according to the sequence information. The Escherichia coli Origami B(DE3) competent cells used were purchased from Nanjing Novozymes Biotech Co., Ltd. The amino column of YMC was used for HPLC detection. The liquid phase system was produced by Shimadzu Corporation of Japan, and the ultraviolet detection system was SPD-20A. The ultraviolet absorption at 310 nm of each component after the catalytic products of 2OST and its mutants were separated by the chromatographic column was detected. The HPLC mobile phase conditions are shown in Table 1.

[0036] Table 1. HPLC analysis method for detecting heparin oligosaccharides

[0037]

[0038] Example 1. Expression and purification of the protein of heparin 2-O-sulfotransferase 2OST and its mutants of heparin 2-O-sulfotransferase

[0039] 1. Construction of the expression strain

[0040] The recombinant vector plasmid pMAL-c5x-2OST (containing the 2OST gene) produced by Nanjing Genscript Biotech Corporation was transformed into Escherichia coli Origami B (DE3) competent cells, spread on LB solid medium containing carbenicillin (50 μg / mL), and cultured in an incubator at 37 °C for 12 h. After screening, 2OST positive transformants were obtained; at the same time, the empty plasmid was transformed and cultured under the same conditions as a negative control;

[0041] Using the same method as above, positive transformants of ten mutants 2OST(R189N), 2OST(R189C), 2OST(R189Q), 2OST(R189G), 2OST(R189I), 2OST(R189F), 2OST(R189S), 2OST(R189T), 2OST(R189W), 2OST(R189Y) were obtained.

[0042] 2. Expression and purification of heparin 2-O-sulfotransferase 2OST and its recombinant proteins of heparin 2-O-sulfotransferase mutants

[0043] Pick a single colony of 2OST positive transformant into 20 mL of sterilized LB liquid medium (50 μg / mL carbenicillin), and culture it overnight at 37 °C and 225 r / min; inoculate the activated bacterial solution into 1 L of LB liquid medium (50 μg / mL carbenicillin) at an inoculation amount of 1%, and culture it at 37 °C and 225 r / min for 3 h until the OD 600 is about 0.6 - 0.8, add IPTG with a final concentration of 0.2 mM, and induce it at 22 °C and 225 r / min for 16 - 18 h, then collect the bacteria; resuspend with equilibration buffer (1×PBS, pH = 8.00), ultrasonically disrupt on ice (work for 15 s, intermittent for 45 s, amplitude 33%, energy 1500 KJ, 4 °C) for 30 min, centrifuge the disrupted bacteria at 12000 rpm for 20 min, and collect the supernatant; filter the supernatant with a 0.22 μm filter membrane, purify the protein using an MBP column, wash it with equilibration buffer after loading, then wash away the miscellaneous proteins with washing buffer (1×PBS pH = 8.00), and finally elute with elution buffer (1×PBS, 20 mM maltose, pH = 8.00) to obtain the target protein. Store the purified 2OST protein in 20% glycerol and aliquot it in a -80 °C refrigerator.

[0044] Recombinant proteins of ten mutants 2OST(R189N), 2OST(R189C), 2OST(R189Q), 2OST(R189G), 2OST(R189I), 2OST(R189F), 2OST(R189S), 2OST(R189T), 2OST(R189W), and 2OST(R189Y) were purified by the same method as described above.

[0045] 3. The purified heparin 2-O-sulfotransferase 2OST and its mutant proteins were identified by SDS-PAGE as Figure 1 shown.

[0046] As can be seen from Figure 1 this, recombinant proteins of heparin 2-O-sulfotransferase 2OST and its ten mutants 2OST(R189N), 2OST(R189C), 2OST(R189Q), 2OST(R189G), 2OST(R189I), 2OST(R189F), 2OST(R189S), 2OST(R189T), 2OST(R189W), and 2OST(R189Y) were successfully purified by this method, and the relative purity of all reached over 50%.

[0047] 4. The Bradford protein concentration assay kit (Beyotime P0006) was used to quantitatively analyze the purified proteins. The specific method was as follows: Take the BSA protein standard solution (5 mg / mL) in the kit and prepare protein standard solutions of 0, 0.3, 0.6, 0.9, 1.2, and 1.5 mg / mL respectively. Take 5 μL of protein standards with different concentrations and add them to the protein standard wells of a 96-well plate, and take 5 μL of the sample and add it to the sample wells of the 96-well plate. Add 250 μL of G250 staining solution to each well, and use a microplate reader to measure the absorbance at 595 nm of each well. Calculate the protein concentration in the sample according to the standard curve and the volume of the sample used. The standard curve for the quantification of 2OST protein is as Figure 2 shown.

[0048] After calculation, the recombinant expression level of 2OST in each liter of LB liquid medium can reach 25.1 mg / L. At the same time, the recombinant expression levels of the ten mutants 2OST(R189N), 2OST(R189C), 2OST(R189Q), 2OST(R189G), 2OST(R189I), 2OST(R189F), 2OST(R189S), 2OST(R189T), 2OST(R189W), and 2OST(R189Y) in each liter of LB liquid medium were measured by the same method as described above, and the results are as Figure 3 shown.

[0049] As can be seen fromFigure 3 It can be seen that among the obtained ten mutants, the expression levels of mutants R189G, R189N, and R189Q are relatively high.

[0050] Example 2. Comparison of the catalytic activities of heparin 2-O-sulfotransferase 2OST and its heparin 2-O-sulfotransferase mutants

[0051] Using the heparin backbone pentasaccharide GlcA-GlcNS-GlcA-GlcNS-GlcA-pNP (final concentration 0.2 mM) as the acceptor substrate, PAPS (final concentration 0.6 mM) as the donor substrate, and 2OST as the catalytic enzyme for the reaction, the reaction system is shown in Table 2. The reaction system was placed in a 37 °C water bath overnight, heated in boiling water for 5 min to inactivate the enzyme and terminate the reaction, and the reaction solution was filtered through a 0.22 μm filter membrane and then detected by HPLC. The detection method is shown in Table 1, the mobile phase flow rate is 0.5 mL / min, and the obtained chromatographic results are as Figure 4 shown.

[0052] Table 2. Comparison reaction system of the catalytic activities of 2OST and its mutants

[0053]

[0054] It can be seen from Figure 4 that using the heparin backbone pentasaccharide GlcA-GlcNS-GlcA-GlcNS-GlcA-pNP as the starting acceptor, PAPS as the donor, and 2OST as the catalytic enzyme for the reaction, a heparin backbone pentasaccharide with the structure GlcA-GlcNS-GlcA2S-GlcNS-GlcA-pNP can be generated.

[0055] Using the same method, mutants 2OST(R189N), 2OST(R189C), 2OST(R189Q), 2OST(R189G), 2OST(R189I), 2OST(R189F), 2OST(R189S), 2OST(R189T), 2OST(R189W), and 2OST(R189Y) were used as the catalytic enzymes for the reaction, and the transferase activities of each mutant in

[0056] the synthesis of GlcA-GlcNS-GlcA2S-GlcNS-GlcA-pNP were calculated, and the enzyme activities of 2OST and its mutants on glucuronic acid in the synthesis of GlcA-GlcNS-GlcA2S-GlcNS-GlcA-pNP were calculated from the reaction conversion rate and the amount of protein used.

[0057] Among them, the chromatographic results of the heparin backbone pentasaccharide with the structure of GlcA-GlcNS-GlcA2S-GlcNS-GlcA-pNP generated by the mutant 2OST(R189G) are as Figure 4 shown; the results of the transferase activity of the 2OST mutant are as Figure 5 shown; the results of the enzyme activity of 2OST and its mutants against glucuronic acid are as Figure 6 shown.

[0058] It can be seen from Figure 5 that the total enzyme activity of the heparin 2-O-sulfotransferase mutant 2OST(R189G) is 0.51772 μmol / mg, which is significantly higher than that of other heparin 2-O-sulfotransferase mutants 2OST(R189G).

[0059] It can be seen from Figure 6 that the selectivity of 2OST(R189G) for glucuronic acid is 0.20406 μmol / mg, the selectivity of 2OST for glucuronic acid is 0.02146 μmol / mg, and the selectivity of 2OST(R189G) for glucuronic acid is significantly improved compared with 2OST, which is 9.51 times that of the original. Moreover, the enzyme activity of the heparin 2-O-sulfotransferase mutant 2OST(R189G) is also much higher than that of other heparin 2-O-sulfotransferase mutants, indicating that the heparin 2-O-sulfotransferase mutant 2OST(R189G) provided by the present invention is an effective tool enzyme for efficiently catalyzing the synthesis of non-anticoagulant heparin sugar chains.

Claims

1. A heparin 2-O-sulfotransferase mutant 2OST(R189G) with high glucuronic acid activity, characterized in that, Based on heparin 2-O-sulfotransferase 2OST, the arginine at position 189 was site-directed mutated to glycine; the amino acid sequence of the heparin 2-O-sulfotransferase mutant 2OST is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

2. The heparin 2-O-sulfotransferase mutant 2OST(R189G) with high glucuronic acid activity as claimed in claim 1, characterized in that, The amino acid sequence of the heparin 2-O-sulfotransferase mutant 2OST (R189G) with high glucuronic acid activity is shown in SEQ ID NO.4, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.

3.

3. A recombinant vector, characterized in that, It is inserting the encoding gene of the heparin 2-O-sulfotransferase mutant 2OST (R189G) described in claim 2 into a plasmid vector.

4. The recombinant vector according to claim 3, characterized in that, The plasmid vector is pMAL-c5x.

5. A recombinant strain, characterized in that, It is obtained by transforming the recombinant vector described in claim 3 into a host cell.

6. The recombinant strain according to claim 5, characterized in that, The host cell is Escherichia coli Origami B(DE3).

7. Use of the heparin 2-O-sulfotransferase mutant 2OST (R189G) described in claim 1 in heparan sulfate chain synthesis.

8. The application according to claim 7, wherein The use uses the heparan sulfate backbone pentasaccharide of GlcA-GlcNS-GlcA-GlcNS-GlcA-pNP as the starting acceptor, PAPS as the donor, and through the catalytic reaction of the heparin 2-O-sulfotransferase mutant 2OST (R189G), a heparan sulfate backbone pentasaccharide with the structure of GlcA-GlcNS-GlcA2S-GlcNS-GlcA-pNP is generated.

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