High-affinity antigen of autoimmune disease antibody as well as synthesis method and application of high-affinity antigen

Abstract

The invention belongs to the technical field of biological medicines, and relates to a high-affinity antigen of an autoimmune disease antibody as well as a synthesis method and application of the high-affinity antigen. According to the method, a one-pot multi-enzyme system is utilized, a compound 1 is taken as a starting receptor, and N-acetylglucosamine is connected to a non-reducing terminal of the compound 1 through a beta1, 3-glucosidic bond to generate a trisaccharide compound; the method comprises the following steps: connecting galactose to a non-reducing terminal of a trisaccharide compound through a beta 1, 3-glucosidic bond by using a one-pot multi-enzyme system to generate a tetrasaccharide compound: connecting glucuronic acid to a non-reducing terminal of the tetrasaccharide compound through a beta 1, 3-glucosidic bond by using the one-pot multi-enzyme system to generate a pentasaccharide compound; a one-pot multi-enzyme system is utilized to obtain a sulfuric acid group from PAPS, and the sulfuric acid group is transferred to a C3 site of glucuronic acid at the tail end of a pentasaccharide compound to form an HNK-1 epitope. According to the method disclosed by the invention, the preparation of the HNK-1 can be realized without protection and deprotection strategies. And the prepared I-type HNK-1 antigen has relatively high affinity to an anti-MAG antibody.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and relates to an autoimmune disease antibody high-affinity antigen and a synthetic method and application thereof. BACKGROUND

[0002] Neurons are the basic units of the nervous system. They transmit electrical signals through axons and dendrites. Axons are wrapped in myelin, a membrane rich in lipids and proteins. Myelin provides insulation, structural stability, and metabolic support to ensure efficient signal transmission. Loss of myelin impairs neural function and leads to neurological diseases. Myelin-associated glycoprotein (MAG) is a protein essential for myelin formation and maintenance. Defects in MAG are associated with demyelinating diseases such as anti-MAG neuropathy.

[0003] Anti-MAG neuropathy is a rare, slowly progressive demyelinating peripheral neuropathy characterized by the presence of autoantibodies against MAG. Clinically, patients present with distal symmetrical sensory impairment, gait ataxia, and tremor. Although the disease usually progresses slowly, gradually worsening sensory impairment can lead to severe disability. Diagnosis relies on detecting anti-MAG IgM antibodies that specifically recognize the human natural killer cell-1 (HNK-1) epitope.

[0004] The HNK-1 epitope is a sulfated trisaccharide (HSO3–3GlcAβ1–3Galβ1–4GlcNAc) composed of a 3-O-sulfated glucuronic acid (GlcA) and a type II N-acetyllactosamine (LacNAc). It was initially identified as a differentiation antigen (CD57) in human natural killer (NK) cells. Subsequent studies have shown that it is highly expressed in myelin of the peripheral and central nervous systems.

[0005] Currently, there is no effective treatment for anti-MAG neuropathy. Clinical treatment focuses on immunosuppression or strategies targeting B cells to reduce the production of autoantibodies, including intravenous immunoglobulin, cytotoxic drugs, and anti-CD20 monoclonal antibodies. Among these therapies, rituximab is the preferred option in clinical practice, but randomized controlled trials have shown no significant difference compared to placebo.

[0006] Several novel therapeutic strategies have been explored, such as the use of tyrosine kinase inhibitors and autoantibody competitors. For example, a glycopolymer was designed to mimic the HNK-1 epitope by combining a sulfated disaccharide (HSO3–3GlcAβ1–3Galβ) with chloroacetylated poly-L-lysine. The glycopolymer showed good results in vivo by selectively binding and neutralizing anti-MAG IgM autoantibodies. These findings highlight the need for novel antigens with high affinity that can neutralize anti-MAG IgM antibodies.

[0007] The synthesis of HNK-1 epitope is relatively complex at present, mainly adopting chemical synthesis or chemical enzyme synthesis strategy, and frequent protection and deprotection operations are needed, so that the preparation is difficult. In addition, there is also a report on the enzyme synthesis of type II HNK-1 antigen, but GlcAT-P used in the report is expressed in mammals, and the expression cost is high, so it cannot be used for mass production of HNK-1 antigen. SUMMARY

[0008] In order to solve the problems in the prior art, the present application provides a human glucuronosyltransferase, and through heterologous expression conditions, the expression level of the synthesized HNK-1 antigen related enzyme element in Escherichia coli is realized, and the HNK-1 preparation related enzyme element can be obtained at low cost and high efficiency; based on the enzyme, the present application develops an effective enzymatic strategy to efficiently generate HNK-1 glycan, without protection and deprotection strategy, so that the preparation of HNK-1 can be realized, thereby providing a practical method for preparing HNK-1 glycan and sugar complex.

[0009] The technical solution adopted by the present application to solve the technical problems is: an autoimmune disease antibody high-affinity antigen, the antigen is type I HNK-1 antigen, and has the following structural formula: ; Among them, R is a glycolipid or a glycoprotein.

[0010] Further, the present application provides an enzyme synthesis method of the autoimmune disease antibody high-affinity antigen, which utilizes a "one-pot multi-enzyme" system, uses compound 1 as a starting acceptor, and uses UDP-GlcNAc as a glycosyl donor to connect GlcNAc to compound 1 by a β1,3-glycosidic bond to generate a trisaccharide compound; utilizes a "one-pot multi-enzyme" system, uses UDP-Gal as a glycosyl donor to connect galactose to the non-reducing end of the trisaccharide compound by a β1,3-glycosidic bond to generate a tetrasaccharide compound; utilizes a "one-pot multi-enzyme" system, uses UDP-GlcA as a glycosyl donor to connect glucuronic acid to the non-reducing end of the tetrasaccharide compound by a β1,3-glycosidic bond to generate a pentasaccharide compound; utilizes a "one-pot multi-enzyme" system to obtain a sulfate group from PAPS and transfer it to the glucuronic acid at the end of the pentasaccharide compound to form an HNK-1 epitope, and synthesize type I sulfated pentasaccharide compound; wherein the structural formula of compound 1 is: ; In the reaction of synthesizing the trisaccharide compound, the "one-pot multi-enzyme" system used contains β1,3-N-acetylglucosamine transferase, N-acetylglucosamine kinase and UDP-sugar pyrophosphorylase; In the reaction of synthesizing the tetrasaccharide compound, the "one-pot multi-enzyme" system used contains β-1,3-galactosyltransferase, galactose kinase, UDP-sugar pyrophosphorylase; In the reaction for synthesizing the pentasaccharide compound, the "one-pot multi-enzyme" system used includes human glucuronyltransferase, glucuronyl kinase, and UDP-glucose pyrophosphorylase; the amino acid sequence of the human glucuronyltransferase is shown in SEQ ID NO.1 of the sequence listing; The "one-pot multi-enzyme" system used in the synthesis of type I sulfated pentasaccharide compounds includes HNK-1 sulfotransferase, adenosine triphosphate thioylase, and adenosine sulfate kinase.

[0011] Furthermore, the present invention provides the application of the high-affinity antigen of the autoimmune disease antibody, which is used to prepare a drug for treating MAG neuropathy.

[0012] Furthermore, the present invention also provides a human glucuronyl transferase, the amino acid sequence of which is shown in SEQ ID NO.1, and the enzyme is used for the addition of GlcA in the enzymatic synthesis of the high-affinity antigen of the autoimmune disease antibody.

[0013] Furthermore, the present invention also provides a DNA molecule encoding the aforementioned human glucuronyl transferase, the nucleotide sequence of which is shown in SEQ ID NO.2 or is a complementary sequence to the sequence shown in SEQ ID NO.2.

[0014] Furthermore, the present invention also provides the application of the human glucuronyl transferase or the DNA molecule in the enzymatic synthesis of high-affinity antigens of autoimmune disease antibodies; wherein the high-affinity antigen of autoimmune disease antibodies is type I HNK-1 antigen, type II HNK-1 antigen, or a carbohydrate compound containing type I HNK-1 antigen or type II HNK-1 antigen.

[0015] Furthermore, this invention also provides another enzymatic method for synthesizing high-affinity antigens of autoimmune disease antibodies. This method utilizes a "one-pot multi-enzyme" system, using compound 1 as the initiator acceptor and UDP-GlcNAc as the glycosyl donor, to link N-acetylglucosamine to the non-reducing end of compound 1 via a β1,3-glycosidic bond, generating a trisaccharide compound; using the same system, UDP-Gal as the glycosyl donor, galactose is linked to the non-reducing end of the trisaccharide compound via a β1,4-glycosidic bond, generating a tetrasaccharide compound; using the same system, UDP-GlcA as the glycosyl donor, glucuronic acid is linked to the non-reducing end of the tetrasaccharide compound via a β1,3-glycosidic bond, generating a pentasaccharide compound; using the same system, a sulfate group is obtained from PAPS and transferred to the glucuronic acid at the end of the pentasaccharide compound to form the HNK-1 epitope, synthesizing a type II sulfated pentasaccharide compound; wherein, the structural formula of compound 1 is: Where R represents glycolipids or glycoproteins; The "one-pot multi-enzyme" system used in the reaction to synthesize trisaccharide compounds includes β1,3-N-acetylglucosamine transferase, N-acetylglucosamine kinase and UDP-glucose pyrophosphorylase. The "one-pot multi-enzyme" system used in the reaction to synthesize tetrasaccharide compounds includes β-1,4-galactosyltransferase, galactokinase, and UDP-glucose pyrophosphorylase. In the reaction for synthesizing the pentasaccharide compound, the "one-pot multi-enzyme" system used includes human glucuronyltransferase, glucuronyl kinase, and UDP-glucose pyrophosphorylase; the amino acid sequence of the human glucuronyltransferase is shown in SEQ ID NO.1 of the sequence listing; The "one-pot multi-enzyme" system used in the synthesis of type I sulfated pentasaccharide compounds includes HNK-1 sulfotransferase, adenosine triphosphate thioylase, and adenosine sulfate kinase.

[0016] Furthermore, the present invention also provides a drug for treating MAG neuropathy, the drug comprising the aforementioned type I HNK-1 antigen.

[0017] The beneficial effects of this invention are as follows: 1. This invention provides a human β-1,3-glucuronyl transferase (GlcAT-S), and by optimizing the expression of key enzyme elements, heterologous soluble expression of GlcAT-P and GlcAT-S is achieved. 2. This invention develops an effective enzymatic strategy that can efficiently generate HNK-1 glycans without the need for protection and deprotection strategies, thus providing a practical method for preparing HNK-1 glycans and glycan complexes. 3. The type I HNK-1 antigen provided by this invention has a high affinity for anti-MAG antibodies, which provides new possibilities for the development of anti-MAG neuropathy treatment. Attached Figure Description

[0018] Figure 1 It is a purified human glucuronyl transferase (GlcAT-S) protein with an MBP tag; Figure 2 This is a schematic diagram of the synthesis pathway of HNK-1, a high-affinity antigen for autoimmune diseases, in an embodiment of the present invention. Figure 3 For the trisaccharide compound Lc3-pro-N3 1 H-NMR spectrum (600 MHz, D2O, 25°C); Figure 4 For the trisaccharide compound Lc3-pro-N3 13C-NMR spectrum (150 MHz, D2O, 25°C); Figure 5 For type II tetrasaccharide compound LNnT-pro-N3 1 H-NMR spectrum (600 MHz, D2O, 25°C); Figure 6 For type II tetrasaccharide compound LNnT-pro-N3 13 C-NMR spectrum (150 MHz, D2O, 25°C); Figure 7 For type II pentasaccharide compound LNnTA-pro-N3 1 H-NMR spectrum (600 MHz, D2O, 25°C); Figure 8 For type II pentasaccharide compound LNnTA-pro-N3 13 C-NMR spectrum (150 MHz, D2O, 25°C); Figure 9 For type II sulfated pentasaccharide compound LNnTAS-pro-N3 1 H-NMR spectrum (600 MHz, D2O, 25°C); Figure 10 For type II sulfated pentasaccharide compound LNnTAS-pro-N3 13 C-NMR spectrum (150 MHz, D2O, 25°C); Figure 11 For type I tetrasaccharide compound LNT-pro-N3 1 H-NMR spectrum (600 MHz, D2O, 25°C); Figure 12 For type I tetrasaccharide compound LNT-pro-N3 13 C-NMR spectrum (150 MHz, D2O, 25°C); Figure 13 For type I pentasaccharide compound LNTA-pro-N3 1 H-NMR spectrum (600 MHz, D2O, 25°C); Figure 14 For type I pentasaccharide compound LNTA-pro-N3 13 C-NMR spectrum (150 MHz, D2O, 25°C); Figure 15 LNTAS-pro-N3 is a type I sulfated pentasaccharide compound. 1H-NMR spectrum (600 MHz, D2O, 25°C); Figure 16 LNTAS-pro-N3 is a type I sulfated pentasaccharide compound. 13 C-NMR spectrum (150 MHz, D2O, 25°C); Figure 17 The results of surface plasmon resonance (SPR) experiments for two types of HNK-1 antigens synthesized in the embodiments of the present invention are shown; wherein, (a) is type II HNK-1 antigen; and (b) is type I HNK-1 antigen. Detailed Implementation

[0019] To facilitate understanding of the present invention, the specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and specific examples. The following examples or drawings are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0020] In the human body, two glucuronyl transferases (GlcAT-P and GlcAT-S) are primarily responsible for the biosynthesis of the HNK-1 epitope. GlcAT-P is mainly expressed in the brain, while GlcAT-S is expressed in the kidneys. Previous studies have reported significant differences in the receptor-substrate specificity of the two enzymes. GlcAT-P catalyzes the glucuronidation of type II LacNAc (Galβ1-4GlcNAc), while GlcAT-S exhibits broader substrate specificity, recognizing both type I LacNAc (Galβ1-3GlcNAc) and type II LacNAc.

[0021] GlcAT-S is a transmembrane protein composed of 323 amino acids, featuring an 8-amino acid tail, a 14-amino acid transmembrane segment, and a C-terminal catalytic domain. The crystal structure of GlcAT-S has been elucidated using a construct lacking the first 78 amino acids at the N-terminus (GlcAT-SΔ78). However, the expression level of GlcAT-SΔ78 is only 0.5–1.2 mg / L, which is unfavorable for large-scale enzymatic synthesis. Since the function of the N-terminal disordered region (residues 23–78) is unclear, this invention expressed two truncated GlcAT-S variants (Δ23 and Δ78) in an *E. coli* expression system. Both recombinant proteins were expressed as inclusion bodies and exhibited poor solubility, consistent with previous reports.

[0022] Example 1: Optimization of GlcAT-S Expression The GlcAT-S protein gene was truncated by removing the first 23 and 78 amino acids from the N-terminus, respectively, to obtain the GlcAT-SΔ23 and GlcAT-SΔ78 gene fragments. These fragments were then cloned into the pMAL-c5X vector tagged with maltose-binding protein (MBP) to enhance protein solubility. The amino acid sequence of GlcAT-SΔ23 with MBP is shown in SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID NO.2. The two recombinant plasmids were transformed into the *E. coli* host strain *Origami B* and cultured at 37 °C until the OD 600 reached 0.6-0.8. IPTG (final concentration 2 mM) was added to induce expression for 12-16 hours. After cell collection, the cells were sonicated and purified, and the results were analyzed by SDS-PAGE. The results are shown below. Figure 1 As shown, both GlcAT-SΔ23 and GlcAT-SΔ78 fused with the MBP tag can be expressed in a soluble form. GlcAT-SΔ23 exhibits a higher soluble protein yield (up to 41 mg / L) and better activity, with a conversion rate of 92%. Compared to the 78-amino acid truncated GlcAT-S protein in existing literature, the 23-amino acid truncated GlcAT-S protein provided by this invention shows significant improvements in soluble expression and enzyme activity.

[0023] From Examples 2 to 9, two different types of HNK-1 antigens were synthesized, and the overall synthetic routes are as follows: Figure 2 As shown, the bolded black lines represent specific catalytic enzymes. Except for GlcAT-S, all other enzymes are existing enzymes that have been reported.

[0024] Example 2: Synthesis of the trisaccharide compound Lc3-pro-N3 (GlcNAcβl-3Galβl-4GlcβPro-N3), the synthetic route is shown in the following formula: .

[0025] The receptor compound Lac-pro-N3 (372 mg), N-acetylglucosamine (387 mg), ATP (643 mg), UTP (642 mg), Tris-HCl buffer (100 mM, pH 7.5), and MgCl2 (50 mM, the amounts of Tris-HCl and MgCl2 added were calculated based on the final reaction volume) were dissolved in 50 mL centrifuge tubes. NahK (1.0–3.0 mg), AGX1 (1.0–3.0 mg), and Nmβ3GlcNAcT (0.5–2.0 mg) were then added, and double-distilled water was added to a total volume of 50 mL. The reaction system was incubated in a shaker at 37 °C and 140 r / min for 5 h. The reaction progress was monitored by thin-layer chromatography (EtOAc:MeOH:H2O:EtOH = 4:2:1:0.5). After the reaction was complete, the reaction solution was boiled in boiling water for 10 min to terminate the reaction. Subsequently, it was centrifuged at 4 ℃ and 11000 r / min for 10 min, and the supernatant was collected and concentrated by rotary evaporation. The resulting concentrate was purified by polyacrylamide gel column chromatography to obtain a white powder, namely the trisaccharide compound Lc3-pro-N3 (338 mg, yield 91%). 1 H-NMR and 13 The C-NMR spectra are shown in Figure 3 and 4 respectively. Figure 4 The specific parameters are as follows: 1 H NMR (600 MHz, Deuterium Oxide) δ 4.76 (d, J = 8.4 Hz, 1H), 4.55(d, J = 8.0 Hz, 1H), 4.51 (d, J = 7.9 Hz, 1H), 4.21 (d, J = 3.3 Hz, 1H), 4.08- 4.01 (m, 2H), 3.95 (dd, J = 12.5, 1.6 Hz, 1H), 3.88 - 3.77 (m, 8H), 3.77(s, 4H), 3.74 - 3.60 (m, 5H), 3.57 - 3.49 (m, 4H), 3.41 - 3.33 (m, 1H), 2.10(s, 3H), 1.97 (p, J = 6.5 Hz, 2H). 13C NMR (151 MHz, D2O) δ 175.00, 102.95, 102.85, 102.13, 82.01, 78.40,75.66, 74.92, 74.79, 74.38, 73.58, 72.83, 70.03, 69.72, 68.36, 67.41, 61.00,60.51, 60.11, 55.69, 47.92, 28.27, 22.25. Example 3: Synthesis of type II tetrasaccharide compound LNnT-pro-N3 (Galβl-4GlcNAcβl-3Galβl-4GlcβPro-N3), the synthetic route is shown in the following formula: .

[0026] The receptor compound Lc3-pro-N3 (188 mg), galactose (107 mg), ATP (218 mg), UTP (217 mg), Tris-HCl buffer (100 mM, pH 7.5), and MgCl2 (50 mM, the amounts of Tris-HCl and MgCl2 added were calculated based on the final reaction volume) were dissolved in 50 mL centrifuge tubes. GalK (1.0–3.0 mg), USP (1.0–3.0 mg), and Nmβ4GalT (0.5–2.0 mg) were then added, and double-distilled water was added to a total volume of 50 mL. The reaction mixture was incubated in a shaker at 37 °C and 140 r / min for 4 h. The reaction was monitored by thin-layer chromatography (EtOAc:MeOH:H2O:EtOH = 4:2:1:0.5). After the reaction was complete, the reaction solution was boiled in boiling water for 10 min to terminate the reaction. The mixture was then centrifuged at 4 °C and 11000 r / min for 10 min, and the supernatant was collected and concentrated by rotary evaporation. The concentrate was purified by polyacrylamide gel column chromatography to obtain a white powder, namely the type II tetrasaccharide compound LNnT-pro-N3 (173 mg, yield 92%). 1 H-NMR and 13 The C-NMR spectra are shown below. Figure 5 and Figure 6 The specific parameters are as follows: 1 H NMR (600 MHz, Deuterium Oxide) δ 4.72 (d, J = 8.4 Hz, 1H), 4.50(dd, J= 7.9, 4.6 Hz, 2H), 4.45 (d, J = 7.9 Hz, 1H), 4.17 (d, J = 3.3 Hz,1H), 4.05 - 3.92 (m, 4H), 3.89 - 3.53 (m, 18H), 3.48 (t, J = 6.7 Hz, 2H),3.36 - 3.29 (m, 1H), 2.05 (s, 3H), 1.93 (p, J = 6.9, 6.5 Hz, 2H). 13 C NMR (151 MHz, D2O) δ 174.92, 102.94, 102.86, 102.74, 102.11,101.70, 82.08, 78.38, 78.16, 75.36, 74.89, 74.78, 74.55, 74.37, 73.08, 72.82,72.51, 72.19, 70.98, 69.98, 68.57, 68.33, 67.39, 66.26, 61.05, 60.97, 60.09,59.88, 57.45, 55.21, 52.31, 47.90, 43.52, 43.41, 42.96, 34.56, 33.36, 28.25, 22.23, 16.83, 14.29. Example 4: Synthesis of type II pentasaccharide compound LNnTA-pro-N3 (GlcAβl-4Galβl-4GlcNAcβl-3Galβl-4GlcβPro-N3), the synthetic route is shown in the following formula: .

[0027] The receptor compound LNnT-pro-N3 (158 mg), glucuronic acid (78 mg), ATP (148 mg), UTP (147 mg), Tris-HCl buffer (100 mM, pH 7.5), and MgCl2 (50 mM, the amounts of Tris-HCl and MgCl2 added were calculated based on the final reaction volume) were dissolved in 50 mL centrifuge tubes. GlcAK (1.0–3.0 mg), USP (1.0–3.0 mg), and GlcAT-S (0.5–2.0 mg) were then added, and double-distilled water was added to a total volume of 50 mL. The reaction mixture was incubated in a shaker at 37 °C and 140 r / min for 12 h. The reaction was monitored by thin-layer chromatography (EtOAc:MeOH:H2O:EtOH = 4:2:1:0.5). After the reaction was complete, the reaction solution was boiled in boiling water for 10 min to terminate the reaction. The solution was then centrifuged at 4 °C and 11000 r / min for 10 min, and the supernatant was collected and concentrated by rotary evaporation. The concentrate was purified by polyacrylamide gel column chromatography to obtain a white powder, namely the type II pentasaccharide compound LNnTA-pro-N3 (142 mg, yield 90%). 1 H-NMR and 13 The C-NMR spectra are shown below. Figure 7 and Figure 8 The specific parameters are as follows: 1 H NMR (600 MHz, Deuterium Oxide) δ 4.72 (dd, J = 8.3, 1.8 Hz, 1H), 4.69 (dd, J = 7.8, 1.8 Hz, 1H), 4.55 (dd, J = 7.8, 1.9 Hz, 1H), 4.50 (dd, J =8.0, 1.8 Hz, 1H), 4.45 (dd, J = 7.9, 1.8 Hz, 1H), 3.76 - 3.74 (m, 2H), 3.68 -3.64 (m, 2H), 3.36 - 3.29 (m, 1H), 2.05 (d, J = 1.8 Hz, 3H), 1.93 (pd, J =6.8, 1.5 Hz, 2H). 13C NMR (151 MHz, D2O) δ 103.62, 102.94, 102.77, 102.45, 102.11,82.26, 82.05, 78.37, 78.05, 75.31, 75.08, 74.88, 74.77, 74.54, 74.36, 73.14,72.79, 72.16, 71.77, 70.12, 69.96, 68.32, 68.09, 67.37, 61.05, 60.95, 60.06,59.88, 55.19, 47.87, 28.23, 22.18. Example 5: Synthesis of type II sulfated pentasaccharide compound LNnTAS-pro-N3 (3S-GlcAβl-4Galβl-4GlcNAcβl-3Galβl-4GlcβPro-N3), the synthetic route is shown in the following formula: .

[0028] The receptor compound LNnTA-pro-N3 (87 mg), Na2SO4 (200 mM), ATP (50 mM), Tris-HCl buffer (100 mM, pH 7.5), and MgCl2 (50 mM, the amounts of Tris-HCl, Na2SO4, and MgCl2 added were calculated based on the final reaction volume) were dissolved in 50 mL centrifuge tubes. Then, ATPS (1.0–3.0 mg), APSK (1.0–3.0 mg), and HNK-1ST (0.5–2.0 mg) were added, and double-distilled water was added to a total volume of 50 mL. The reaction system was incubated in a shaker at 37 °C and 140 r / min for 56 h. The reaction progress was monitored by thin-layer chromatography (EtOAc:MeOH:H2O:EtOH = 4:2:1:0.5). After the reaction was complete, the reaction solution was boiled in boiling water for 10 min to terminate the reaction. The solution was then centrifuged at 4°C and 11000 r / min for 10 min, and the supernatant was collected and concentrated by rotary evaporation. The concentrate was purified by polyacrylamide gel column chromatography to obtain a white powder, namely the type II sulfated pentasaccharide compound LNnTAS-pro-N3 (69 mg, yield 80%). 1 H-NMR and 13 The C-NMR spectra are shown below. Figure 9 and Figure 10 The specific parameters are as follows: 1H NMR (600 MHz, Deuterium Oxide) δ 4.58 - 4.54 (m, 1H), 4.51 (dd, J = 8.0, 1.1 Hz, 1H), 4.49 - 4.44 (m, 1H), 4.36 (td, J = 9.1, 1.1 Hz, 1H), 4.19(dd, J = 16.0, 3.3 Hz, 2H), 4.06 - 3.95 (m, 3H), 3.91 - 3.69 (m, 12H), 3.72 -3.64 (m, 2H), 3.62 (s, 3H), 3.67 - 3.58 (m, 2H), 3.48 (t, J = 6.7 Hz, 2H),3.38 - 3.31 (m, 1H), 2.06 (d, J = 1.0 Hz, 3H), 1.94 (p, J = 6.5 Hz, 2H). 13 C NMR (151 MHz, D2O) δ 83.59, 82.32, 82.10, 78.38, 78.04, 75.08,74.90, 74.79, 74.55, 74.37, 72.82, 72.16, 71.97, 70.47, 70.08, 69.99, 68.33,68.15, 67.40, 61.07, 60.97, 60.07, 59.88, 55.21, 47.90, 28.24, 22.23. Example 6: Synthesis of type I tetrasaccharide compound LNT-pro-N3 (Galβl-3GlcNAcβl-3Galβl-4GlcβPro-N3), the synthetic route is shown in the following formula: .

[0029] The receptor compound Lc3-pro-N3 (188 mg), galactose (107 mg), ATP (218 mg), UTP (217 mg), Tris-HCl buffer (100 mM, pH 7.5), and MgCl2 (50 mM, the amounts of Tris-HCl and MgCl2 added were calculated based on the final reaction volume) were dissolved in 50 mL centrifuge tubes. GalK (1.0–3.0 mg), USP (1.0–3.0 mg), and Cvβ3GalT (0.5–2.0 mg) were then added, and double-distilled water was added to a total volume of 50 mL. The reaction mixture was incubated in a shaker at 37 °C and 140 r / min for 4 h. The reaction progress was monitored by thin-layer chromatography (EtOAc:MeOH:H2O:EtOH = 4:2:1:0.5). After the reaction was complete, the reaction solution was boiled in boiling water for 10 min to terminate the reaction. The mixture was then centrifuged at 4 °C and 11000 r / min for 10 min, and the supernatant was collected and concentrated by rotary evaporation. The concentrate was purified by polyacrylamide gel column chromatography to obtain a white powder, namely the type I tetrasaccharide compound LNT-pro-N3 (167 mg, yield 89%). 1 H-NMR and 13 The C-NMR spectra are shown in Figure 11 and 12, respectively. Figure 12 The specific parameters are as follows: 1 H NMR (600 MHz, Deuterium Oxide) δ 4.78 (s, 0H), 4.53 (d, J = 8.0Hz, 1H), 4.50 (t, J = 7.8 Hz, 2H), 4.20 (d, J = 3.3 Hz, 1H), 4.07 - 4.00 (m,2H), 3.98 - 3.92 (m, 3H), 3.92 - 3.74 (m, 11H), 3.73 - 3.48 (m, 10H), 3.40 -3.32 (m, 1H), 2.08 (s, 3H), 1.96 (p, J = 6.6 Hz, 2H). 13C NMR (151 MHz, D2O) δ 175.03, 103.50, 102.94, 102.58, 102.13,82.10, 82.05, 78.42, 75.29, 75.20, 74.92, 74.79, 74.39, 72.84, 72.48, 70.72,70.04, 68.58, 68.48, 68.34, 67.43, 61.07, 61.00, 60.53, 60.11, 54.72, 47.93,28.27, 22.33. Example 7: Synthesis of type I pentasaccharide compound LNTA-pro-N3 (GlcAβl-4Galβl-3GlcNAcβl-3Galβl-4GlcβPro-N3), the synthetic route is shown in the following formula: .

[0030] The receptor compound LNT-pro-N3 (157 mg), glucuronic acid (78 mg), ATP (148 mg), UTP (147 mg), Tris-HCl buffer (100 mM, pH 7.5), and MgCl2 (50 mM, the amounts of Tris-HCl and MgCl2 added were calculated based on the final reaction volume) were dissolved in 50 mL centrifuge tubes. GlcAK (1.0–3.0 mg), USP (1.0–3.0 mg), and GlcAT-S (0.5–2.0 mg) were then added, and double-distilled water was added to a total volume of 50 mL. The reaction mixture was incubated in a shaker at 37 °C and 140 r / min for 12 h. The reaction was monitored by thin-layer chromatography (EtOAc:MeOH:H2O:EtOH = 4:2:1:0.5). After the reaction was complete, the reaction solution was boiled in boiling water for 10 min to terminate the reaction. The solution was then centrifuged at 4 °C and 11000 r / min for 10 min, and the supernatant was collected and concentrated by rotary evaporation. The concentrate was purified by polyacrylamide gel column chromatography to obtain a white powder, namely the type I pentasaccharide compound LNTA-pro-N3 (140 mg, yield 89%). 1 H-NMR and 13 The C-NMR spectra are shown in Figure 13 and 14, respectively. Figure 14 The specific parameters are as follows: 1 H NMR (600 MHz, Deuterium Oxide) δ 4.68 (s, 1H), 4.50 (dd, J= 8.0, 3.8 Hz, 2H), 4.45 (d, J = 7.8 Hz, 1H), 4.17 (dd, J = 12.0, 3.2 Hz, 2H), 4.04- 3.96 (m, 2H), 3.96 - 3.88 (m, 2H), 3.85 - 3.79 (m, 2H), 3.81 - 3.74 (m,7H), 3.76 - 3.73 (m, 1H), 3.73 (d, J = 3.3 Hz, 1H), 3.73 - 3.66 (m, 2H), 3.69- 3.40 (m, 12H), 3.36 - 3.29 (m, 1H), 2.04 (s, 2H), 1.92 (p, J = 6.6 Hz, 2H). 13 C NMR (151 MHz, D2O) δ 174.93, 103.61, 103.22, 102.92, 102.51,102.10, 82.27, 82.21, 81.95, 78.38, 75.30, 75.17, 74.99, 74.89, 74.76, 74.37,73.10, 72.79, 71.65, 70.00, 69.84, 68.50, 68.30, 68.07, 67.36, 61.02, 60.95,60.50, 60.07, 54.70, 54.62, 47.87, 28.22, 22.28, 22.23. Example 8. Synthesis of type I sulfated pentasaccharide compound LNTAS-pro-N3 (3S-GlcAβl-4Galβl-3GlcNAcβl-3Galβl-4GlcβPro-N3), the synthetic route is shown in the following formula: .

[0031] The receptor compound LNTA-pro-N3 (85 mg), Na2SO4 (200 mM), ATP (50 mM), Tris-HCl buffer (100 mM, pH 7.5), and MgCl2 (50 mM, the amounts of Tris-HCl, Na2SO4, and MgCl2 added were calculated based on the final reaction volume) were dissolved in 50 mL centrifuge tubes. Then, ATPS (1.0–3.0 mg), APSK (1.0–3.0 mg), and HNK-1ST (0.5–2.0 mg) were added, and double-distilled water was added to a total volume of 50 mL. The reaction system was incubated in a shaker at 37 °C and 140 r / min for 56 h. The reaction progress was monitored by thin-layer chromatography (EtOAc:MeOH:H2O:EtOH = 4:2:1:0.5). After the reaction was complete, the reaction solution was boiled in boiling water for 10 min to terminate the reaction. The solution was then centrifuged at 4°C and 11,000 r / min for 10 min, and the supernatant was collected and concentrated by rotary evaporation. The concentrate was purified by polyacrylamide gel column chromatography to obtain a white powder, namely the type I sulfated pentasaccharide compound LNTAS-pro-N3 (64 mg, yield 75%). 1 H-NMR and 13 The C-NMR spectra are shown in Figure 15 and 16, respectively. Figure 16 The specific parameters are as follows: 1 H NMR (600 MHz, Deuterium Oxide) δ 4.75 (d, J = 8.8 Hz, 1H), 4.51(ddd, J = 8.0, 5.0, 1.6 Hz, 2H), 4.46 (dd, J = 7.9, 1.6 Hz, 1H), 4.34 (td, J = 9.1, 1.6 Hz, 1H), 4.21 - 4.15 (m, 2H), 4.05 - 3.97 (m, 2H), 3.95 - 3.89 (m,2H), 3.87 - 3.73 (m, 7H), 3.75 - 3.72 (m, 1H), 3.74 - 3.64 (m, 3H), 3.67 -3.55 (m, 4H), 3.53 - 3.45 (m, 2H), 3.36 - 3.30 (m, 1H), 2.09 (s, 0H), 2.04(d, J= 1.6 Hz, 3H), 1.98 - 1.89 (m, 2H). 13 C NMR (151 MHz, D2O) δ 175.45, 174.94, 103.24, 103.21, 102.93,102.51, 102.10, 83.63, 82.41, 82.28, 81.94, 78.38, 75.18, 75.01, 74.89,74.76, 74.36, 72.78, 71.96, 70.44, 69.99, 69.75, 68.52, 68.30, 68.07, 67.36,61.04, 60.95, 60.51, 60.05, 54.59, 47.87, 28.22, 22.22. To detect the affinity of two types of antigens for MAG antibodies, the antigens were conjugated with biotin for SPR detection. Example 9: Synthesis of type II biotinylated pentasaccharide compound LNnTAS-Biotin (3S-GlcAβl-3Galβl-4GlcNAcβl-3Galβl-4Glcβ-Biotin). The synthetic route is shown in the following formula: .

[0032] The receptor compound LNnTAS-pro-N3 (1 mg, 1 eq) and DBCO-PEG8-Biotin (1.4 eq) were dissolved in PBS-P in 15 mL centrifuge tubes, and the pH was adjusted to 8. The reaction system was incubated in a shaker at 37 °C and 140 r / min for 2 h. The reaction progress was monitored by thin-layer chromatography (EtOAc:MeOH:H2O:EtOH = 4:2:1:0.5). After the reaction was completed, the reaction solution was directly purified by polyacrylamide gel column chromatography to obtain a white powder, namely the type II biotinylated pentasaccharide compound LNnTAS-Biotin (1.50 mg, yield 78%).

[0033] Example 10: Synthesis of type I biotinylated pentasaccharide compound LNTAS-Biotin (3S-GlcAβl-3Galβl-3GlcNAcβl-3Galβl-4Glcβ-Biotin). The synthetic route is shown in the following formula: .

[0034] The receptor compound LNTAS-pro-N3 (1 mg, 1 eq) and DBCO-PEG8-Biotin (1.4 eq) were dissolved in PBS-P in 15 mL centrifuge tubes, and the pH was adjusted to 8. The reaction system was incubated in a shaker at 37 °C and 140 r / min for 2 h. The reaction progress was monitored by thin-layer chromatography (EtOAc:MeOH:H2O:EtOH = 4:2:1:0.5). After the reaction was completed, the reaction solution was directly purified by polyacrylamide gel column chromatography to obtain a white powder, namely the type I biotinylated pentasaccharide compound LNTAS-Biotin (1.58 mg, yield 83%).

[0035] Example 11: Affinity test of two types of HNK-1 antigens prepared in this invention against HNK-1 IgM antibodies. Surface plasmon resonance (SPR) experiments were performed using a Biacore T200 instrument. The mobile phase was PBS-P (20 mM phosphate buffer, 2.7 mM KCl, 137 mM NaCl, and 0.05% Surfactant P2O). First, streptavidin (prepared with pH 5.0 NaOAc buffer, concentration 50 µg / µL, volume 35 µL) was immobilized on the surface of a CM5 chip using an amino-coupling method. Then, 2 µL of biotin-labeled oligosaccharide solutions (0.6–60 ng / µL) were dissolved in PBST and immobilized on the streptavidin-modified CM5 chip surface at a flow rate of 10 µL / min. Each biotin-labeled oligosaccharide occupied one horizontal channel, with one horizontal channel left untreated as a reference channel. Antibody binding assays were performed by sequentially passing antibody solutions of different concentrations through the chip surface after immobilizing oligosaccharide structures. The experimental conditions were: 35 µL of antibody solution (dissolved in PBST) was injected at a flow rate of 30 µL / min, with a binding time of 70 s and a dissociation time of 300 s. The equilibrium dissociation constant (KD) was calculated using 5-6 antibody solutions of different concentrations. After each antibody flow through the chip surface, the surface was regenerated with 0.1 mM NaOH and re-equilibrated with the mobile phase. All experimental data were analyzed and processed using Biacore T200 Evaluation software.

[0036] like Figure 17As shown, the KD(1) value of the type II HNK-1 glycan (9.21 nM) is about 30 times higher than that of the type I HNK-1 glycan (0.28 nM), indicating that the latter has a higher affinity for HNK-1 IgM antibody. The KD(2) values ​​of the two glycans are not significantly different (0.251 nM for type II and 0.388 nM for type I), indicating that the conformational changes of the two glycans are similar upon binding.

Claims

1. A high-affinity antigen for autoimmune diseases, characterized in that: This antigen is a type I HNK-1 antigen, and has the following structural formula: ； Where R represents glycolipids or glycoproteins.

2. The enzymatic synthesis method for high-affinity antigens of autoimmune disease antibodies according to claim 1, characterized in that: This method utilizes a "one-pot multi-enzyme" system, using compound 1 as the initiator acceptor and UDP-GlcNAc as the glycosyl donor, to link N-acetylglucosamine to the non-reducing end of compound 1 via a β1,3-glycosidic bond, generating a trisaccharide compound; using the same system, UDP-Gal is used as the glycosyl donor to link galactose to the non-reducing end of the trisaccharide compound via a β1,3-glycosidic bond, generating a tetrasaccharide compound; and using UDP-GlcA as the glycosyl donor, glucuronic acid is linked to the non-reducing end of the tetrasaccharide compound via a β1,3-glycosidic bond, generating a pentasaccharide compound. Using a "one-pot, multiple-enzyme" system, sulfate groups were obtained from PAPS and transferred to the glucuronic acid terminus of a pentasaccharide compound to form the HNK-1 epitope, thus synthesizing a type I sulfated pentasaccharide compound; the structural formula of compound 1 is as follows: ; The "one-pot multi-enzyme" system used in the reaction to synthesize trisaccharide compounds includes β1,3-N-acetylglucosamine transferase, N-acetylglucosamine kinase and UDP-glucose pyrophosphorylase. The "one-pot multi-enzyme" system used in the reaction to synthesize tetrasaccharide compounds includes β-1,3-galactosyltransferase, galactokinase, and UDP-glucose pyrophosphorylase. In the reaction that synthesizes the pentasaccharide compound, the "one-pot multi-enzyme" system used includes human glucuronyltransferase, glucuronyl kinase and UDP-glucose pyrophosphorylase; the amino acid sequence of the human glucuronyltransferase is shown in SEQ ID NO.1 of the sequence listing; The "one-pot multi-enzyme" system used in the synthesis of type I sulfated pentasaccharide compounds includes HNK-1 sulfotransferase, adenosine triphosphate thioylase, and adenosine sulfate kinase.

3. The application of the high-affinity antigen of the autoimmune disease antibody according to claim 1, characterized in that: This antigen is used to prepare drugs for treating MAG neuropathy.

4. A human glucuronyl transferase, characterized in that, The amino acid sequence of this enzyme is shown in SEQ ID NO.1 of the sequence listing. This enzyme is used for the addition of GlcA in the enzymatic synthesis of high-affinity antigens of autoimmune disease antibodies.

5. A DNA molecule encoding the human glucuronyl transferase of claim 4, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.2 or is a complementary sequence to the sequence shown in SEQ ID NO.

2.

6. The application of the human glucuronyl transferase of claim 4 or the DNA molecule of claim 5 in the enzymatic synthesis of high-affinity antigens for autoimmune diseases; wherein the high-affinity antigen for autoimmune diseases is type I HNK-1 antigen, type II HNK-1 antigen, or a carbohydrate compound containing type I HNK-1 antigen or type II HNK-1 antigen.

7. An enzymatic method for synthesizing high-affinity antigens for autoimmune diseases, characterized in that: This method utilizes a "one-pot multi-enzyme" system, using compound 1 as the initiator acceptor and UDP-GlcNAc as the glycosyl donor, to link N-acetylglucosamine to the non-reducing end of compound 1 via a β1,3-glycosidic bond, generating a trisaccharide compound; using the same system, UDP-Gal is used as the glycosyl donor to link galactose to the non-reducing end of the trisaccharide compound via a β1,4-glycosidic bond, generating a tetrasaccharide compound; and using UDP-GlcA as the glycosyl donor, glucuronic acid is linked to the non-reducing end of the tetrasaccharide compound via a β1,3-glycosidic bond, generating a pentasaccharide compound. Using a "one-pot, multiple-enzyme" system, sulfate groups were obtained from PAPS and transferred to the glucuronic acid at the end of a pentasaccharide compound to form the HNK-1 epitope, thus synthesizing a type II sulfated pentasaccharide compound; the structural formula of compound 1 is as follows: ; Where R represents glycolipids or glycoproteins; The "one-pot multi-enzyme" system used in the reaction to synthesize trisaccharide compounds includes β1,3-N-acetylglucosamine transferase, N-acetylglucosamine kinase and UDP-glucose pyrophosphorylase. The "one-pot multi-enzyme" system used in the reaction to synthesize tetrasaccharide compounds includes β-1,4-galactosyltransferase, galactokinase, and UDP-glucose pyrophosphorylase. In the reaction that synthesizes the pentasaccharide compound, the "one-pot multi-enzyme" system used includes human glucuronyltransferase, glucuronyl kinase and UDP-glucose pyrophosphorylase; the amino acid sequence of the human glucuronyltransferase is shown in SEQ ID NO.1 of the sequence listing; The "one-pot multi-enzyme" system used in the synthesis of type I sulfated pentasaccharide compounds includes HNK-1 sulfotransferase, adenosine triphosphate thioylase, and adenosine sulfate kinase.

8. A drug for treating MAG neuropathy, characterized in that, It contains the type I HNK-1 antigen as described in claim 1.

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