CN112851731A - O-GlcNAc glycosylation modification one-step enzyme labeling kit and application thereof - Google Patents
O-GlcNAc glycosylation modification one-step enzyme labeling kit and application thereof Download PDFInfo
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Abstract
The invention relates to an in-vitro detection kit for O-GlcNAc glycosylation modification, which mainly comprises bovine glycosyltransferase mutant solution, modified sugar donor aqueous solution, manganese chloride aqueous solution and buffer solution. The kit can realize the in-vitro efficient marking of O-GlcNAc glycosylation modification, and the number of proteins obtained by mass spectrometry (LC-MS/MS) detection and one-step enzymatic enrichment and purification is increased by about 1 time than the number of proteins identified by a two-step chemical enzymatic method; in addition, the kit can also be applied to the histological research of colon tumor samples.
Description
(I) technical field
The invention relates to an O-GlcNAc glycosylation modification one-step enzyme labeling kit and application thereof.
(II) background of the invention
Post-translational modification is an important factor in distinguishing mammals from lower organisms. Among them, glycosylation modification is a very complicated chemical biological process. The complexity of glycosylation modification is mainly related to two aspects: the diversity of monosaccharides and the diversity of individual monosaccharide linkages. Currently, monosaccharide molecules have at least 5 chiral centers, and in higher eukaryotes there are 9 basic monosaccharides, which can be linked at different positions by glycosidic linkages of two (α or β) configurations. The linear or branched sugar chain may also be subjected to secondary modifications, such as phosphorylation, sulfation or lipidation. The study of cellular glycosylation modifications is known as glycomics (glycome) and includes the spatiotemporal regulation of nucleotide sugar donors, glycoprotein or glycolipid acceptors, glycosyltransferases and glycosidases by cells.
Carbohydrate polymers linked to polypeptides through nitrogen and oxygen atoms are called N-glycans (N-glycans) and O-glycans (O-glycans), respectively, and in addition Glycosylphosphatidylinositol (GPI) -anchored proteins, glycosaminoglycans (GAGs), glycosphingolipids, and the like. Researchers have discovered as early as sixty years ago that changes in glycosylation modification are associated with cellular carcinogenesis. Glycosylation modifications in tumor cells are quite different from normal cell ratios, e.g., the amount of sialic acid and fucose-modified glycoproteins expressed in the blood of breast cancer patients correlates with cancer progression. Glycosylation modifications that occur specifically in tumor cells are called Tumor Associated Carbohydrate Antigens (TACAs).
An O-GlcNAc glycosylation modification is one in which an N-acetylglucosamine (GlcNAc) group is attached to a hydroxyl group on the serine/threonine side chain of a protein. Researches show that the O-GlcNAc glycosylation modification plays an important role in stem cell differentiation, immune cell maturation, cell metabolism regulation, nerve cell development and the like. At present, the detection means of O-GlcNAc glycosylation modification mainly comprises antibodies, agglutinin, a non-natural sugar metabolism labeling method and a chemical enzyme labeling method. Among them, the detection methods of antibodies and lectins have obvious disadvantages, sugar antibodies are difficult to prepare and have low affinity, and the recognition of most lectins lacks strict specificity, so that the detection of specific glycosylation modification is difficult to realize. Metabolic labeling is an effective means for detecting glycosylation modification of O-GlcNAc. With chemically-reactive groupsThe GlcNAc analogue is cultured, the modified GlcNAc analogue is integrated to cell glucoprotein via cell metabolism path, and the capture and mark with chemical reaction group glycosylation modification are realized by biological orthogonal reaction. However, metabolic labeling has two limitations: 1) GlcNAc analogs or metabolic precursors are involved in the biosynthetic pathway of various glycoconjugates, and markers that cannot achieve modification of O-GlcNAc glycosylation, 2) metabolic processes are involved with various glycosyltransferases, so metabolic markers cannot achieve the study of specific sugar chain structures. In addition, the patient is administered a metabolizable non-natural monosaccharide precursor (other than 2-deoxy-2-, (ii) to18F) fluoro-D-glucose) has potential safety hazard and the like. Therefore, metabolic markers cannot be used for the study of glycosylation modification of human tissue samples.
Chemical enzyme labeling (chemoenzymatic labeling) may ameliorate some of the disadvantages of metabolic labeling to some extent. The recombinant glycosyltransferase catalyzes the attachment of monosaccharide analogs to the cell surface or to specific receptors in cell lysates using nucleotide sugars as donors. The linked monosaccharide analog carries a radioactive label or a specific chemically reactive group-labeled by bio-orthogonal reaction with a specific tag molecule. The specificity of the enzyme for the acceptor substrate is critical for achieving labeling of a specific sugar chain structure. The bovine-derived galactosyltransferase (GalT1) is expressed as UDP-, [ 2 ]3H]Galactose is a donor to transfer radiolabeled galactose to GlcNAc residues in lymphocytes, and an O-linked GlcNAc glycosylation modification mainly present in cytoplasm and nucleus is found. The Hsieh-Wilson topic group reports the substitution of a chemically tagged nucleotide sugar analog for the labeling of an O-GlcNAc modified protein instead of a radioactive sugar donor. First, a bovine-derived glycosyltransferase mutant GalT1-Y289L linked a tag molecule with an azido group (GalNAz) to an O-GlcNAc-modified complex with UDP-GalNAz as the sugar donor. Then, a click chemistry reaction is used to attach a fluorophore with alkyne modification or an affinity (biotin) reporter to the O-GlcNAc glycosylation modification. However, the literature reports that the second click chemistry reaction is inefficient and less reproducible.
Disclosure of the invention
The invention aims to provide an O-GlcNAc glycosylation modification one-step enzyme labeling kit and application thereof.
The technical scheme adopted by the invention is as follows:
a modified sugar donor has a structure shown in formula (I):
the invention also relates to a method for preparing the modified sugar donor, which comprises the following steps: UDP-GalNAz is dissolved in water, triethylphosphine tetrahydrofuran solution is added to react fully, the reaction solution is freeze-dried to remove redundant triethylphosphine, water/methanol is added to dissolve and precipitate, N-hydroxysuccinimide Biotin is added to react fully under the protection of nitrogen, freeze-drying is carried out, silica gel column chromatography, desolventization and freeze-drying are carried out, and polyacrylamide gel column separation is carried out to obtain a target molecule UDP-GalNAc-Biotin, namely the modified sugar donor.
The invention also relates to application of the sugar donor with the modification in preparing an in-vitro detection kit for O-GlcNAc glycosylation modification.
The invention also relates to an O-GlcNAc glycosylation modified in-vitro detection kit, which mainly comprises bovine glycosyltransferase mutant solution, modified sugar donor aqueous solution, manganese chloride aqueous solution and buffer solution; the amino acid sequence of the bovine glycosyltransferase mutant is shown as SEQ ID No.1, and the structure of the modified sugar donor is shown as the formula (I):
the sequence of SEQ ID No.1 is as follows:
SLTACPEESPLLVGPMLIEFNIPVDLKLVEQQNPKVKLGGRYTPMDCISPHKVAIIIPFRNRQEHLKYWLYYLHPILQRQQLDYGIYVINQAGESMFNRAKLLNVGFKEALKDYDYNCFVFSDVDLIPMNDHNTYRCFSQPRHISVAMDA279FGFSLPYVQLFGGVSALSKQQFLSINGFPNNYWGWGGEDDDIYNRLAFRGMSVSRPNAVIGKTRMIRHSRDKKNEPNPQRFDRIAHTKETMLSDGLNSLTYMVLEVQRYPLYTKITVDIGTPS
through crystal structure analysis, a key site K279 is found, and experiments show that the GalT1-Y289L/K270A enzyme mutant can recognize a UDP-GalNAc-Biotin sugar donor modified in a large volume, so that the O-GlcNAc glycosylation modification of the one-step marker is realized.
The buffer solution comprises a buffer solution 1 and a buffer solution 2, wherein the buffer solution 1 is used for dissolving protein precipitates and comprises the following components: 20mM HEPES, 1% SDS, pH 7.9, solvent deionized water; the buffer solution 2 is used for ensuring the enzymatic activity of a reaction system and comprises the following components: 50mM HEPES, 125mM NaCl, 5% NP-40, pH 7.9, and deionized water as solvent.
The modified sugar donor is prepared by the following method: UDP-GalNAz is purchased in water, added with triethylphosphine tetrahydrofuran solution for full reaction, the reaction solution is freeze-dried, the redundant triethylphosphine is removed, water/methanol is used for dissolving and precipitating, N-hydroxysuccinimide Biotin is added, full reaction is carried out under the protection of nitrogen, freeze-drying, silica gel column chromatography, desolventizing and freeze-drying are carried out, and the target molecule UDP-GalNAc-Biotin is obtained by polyacrylamide gel column separation.
The invention also relates to application of the kit in-vitro labeling and detection of O-GlcNAc glycosylation modification.
The kit is improved on the existing method, mainly comprises mutation of an enzyme active site and change of a probe molecule, can realize one-step labeling of O-GlcNAc glycosylation, and is more sensitive and better in signal detection compared with the existing method.
The invention has the following beneficial effects: the kit can realize the in-vitro efficient marking of O-GlcNAc glycosylation modification, and the number of proteins obtained by mass spectrometry (LC-MS/MS) detection and one-step enzymatic enrichment and purification is increased by about 1 time than the number of proteins identified by a two-step chemical enzymatic method; in addition, the kit can also be applied to the histological research of colon tumor samples.
(IV) description of the drawings
FIG. 1 is a photograph of enzyme SDS-PAGE.
FIG. 2 shows the structure of a sugar donor.
FIG. 3 shows the O-GlcNAc glycosylation modification of the kit marker cell lysate.
FIG. 4 shows that the kit is used for identifying the O-GlcNAc glycosylation modification proteomics of cell lysate.
FIG. 5 is a marker colon cancer tissue O-GlcNAc glycosylation modification.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1:
preparation of bovine-derived glycosyltransferase:
construction of an expression strain: the bovine-derived glycosyltransferase GalT1 (NP-803478.1, 130-402 amino acids, pET23a including point mutation Y289L) gene vector was synthesized by the same company, and 100. mu.L of competent cells BL21 were transformed with 10-50ng of the plasmid (DE 3). The mixture was placed in an ice bath for 30 minutes. The water bath was heat shocked at 42 ℃ for 45 seconds and then the tubes were quickly transferred to ice and incubated for 2 minutes without shaking the centrifuge tubes. Add 500. mu.L of sterile LB medium (without antibiotics) to each tube, mix well, incubate at 37 ℃ for 2 hours at 200 rpm. Plating (ampicillin resistance) and culturing at 37 ℃ for 12-24h in an inverted way. Monoclonal colonies were picked. The protein vector GalT1-K279A is obtained by point mutation on the basis of a GalT1 vector. The primer sequence is as follows:
F—AATGGATGCGTTTGGATTTAGCCTACCTTA
R—ATCCAAACGCATCCATTGCTACAGAAATGT
the protein expression steps are as follows: using 1L of E.coli solution as an example, the protein extraction process was performed on ice and centrifuged at 4 ℃. Now, 2-nitro-5-sulfurous acid benzoic acid (NSTB) is prepared: 100mg of 5,5' -dithiobis (2-nitrobenzoic acid) was dissolved in 10mL of an aqueous sodium sulfite solution (1M) and the pH was adjusted to 7.5. Blowing air at 37 ℃ until the color of the solution changes from dark orange to light yellow, generally about 2-4 hours. The solution is prepared at present and is generally stored at 4 ℃ for 48 hours for use.
(1) The protein expression bacteria BL21 were picked, and added with 1mL LB medium (ampicillin resistance 100mg/L) for 2 hours at 37 ℃ until the bacteria became turbid. The volume of the culture was increased to 1L of LB medium (ampicillin resistance 100mg/L), and the culture was carried out at 37 ℃ to determine O.D.6000.7-0.9 (about 4h), IPTG (final concentration 1mM) was added to induce protein expression for 4h at 37 ℃. 5000rmp, 5 minutes harvest. The E.coli solid can be stored at-20 deg.C for several weeks.
(2)10mL PBS solution heavy suspension of Escherichia coli, ice ultrasound (200V)10 minutes, 80mL PBS dilution, 10000 Xg, centrifugal 30 minutes. The supernatant was discarded. Washing the precipitate with 25% (w/v) sucrose in PBS for 6 times, mixing well each time, 10000 Xg, centrifuging for 20min, and discarding the supernatant. The pellet was stored in 5mL of 25% sucrose in PBS and stored overnight at 4 deg.C (where the first day experiment could be paused). The following day the supernatant was removed by centrifugation.
(3)20mL of an aqueous solution containing 0.3M sodium sulfite and 5M guanidine hydrochloride were resuspended in the pellet. Dissolve well, add 4mL of freshly prepared NSTB solution, stir well with magnetons at room temperature for 1-2 hours. The solution changed from dark orange to light yellow. The precipitate was diluted by adding 180mL of double distilled water (a large amount of precipitate appeared). 10000 Xg, centrifuge for 20 minutes, discard the supernatant. Resuspend the pellet in 10mL of aqueous solution, mix well, centrifuge and discard the supernatant, repeat twice. The residual NSTB was washed off (until the solution and precipitate became colorless).
(4)20mL of 5M guanidine hydrochloride solution dissolved the precipitate. 20mL of a folding buffer (500mM L-arginine, 50mM Tris, 5mM EDTA, 4mM cysteamine hydrochloride, and 2mM cystamine, pH 8.0) was added to the guanidine hydrochloride protein solution every 90 seconds at 4 ℃ for a total of 10 times. Equivalent to 10-fold dilution of the guanidinium hydrochloride protein solution with the folding solution over 15 minutes, the shaking was maintained during the addition, but vigorous stirring with magnetons was prohibited (breaking the protein folding). A small amount of protein may be separated out in the adding process, which belongs to a normal phenomenon. After dilution, the protein solution was left to stand at 4 ℃ and folded for 48 hours.
(5) The folded protein solution was filled into a pre-treated dialysis bag (cutoff <10kDa), at least 1/3 spaces were reserved, the dialysis bag was put into 4L of dialysate (50mM Tris, 5mM EDTA, 4mM cysteamine hydrochloride and 2mM cystamine, pH 8.0), dialyzed for 8 hours, and the dialysate was changed twice. A large amount of protein is separated out in the dialysis process, white precipitate is generated, and the protein is abnormally folded and belongs to a normal phenomenon.
(6) After dialysis, 10000 Xg was centrifuged for 20 minutes to remove the precipitate (which may be passed through a membrane). Protein solution ultrafiltration tubes (cutoff <10kDa), 5000 × g centrifuged, concentrated to 1-2 mL. About 3-4mg of protein can be extracted from 1L of bacteria.
(7) SDS-PAGE detects the purity and concentration of the protein. 10% SDS-PAGE (100V, 40 min), staining with Coomassie Brilliant blue, and washing with destaining solution for 2 h. Amino acids contained in the protein stock may interfere with BCA and Bradford protein assay methods. Therefore, the protein concentration was determined by SDS-PAGE staining using a BSA solution of a known concentration as a standard protein. The protein has a molecular weight of about 32.83kDa (FIG. 1).
Preparation of sugar donors:
the starting material UDP-GalNAz was purchased from Inc. (CAS Registry Number: 653600-61-4). UDP-GalNAz (0.046mmol, 30mg) was dissolved in 0.5mL of water, triethylphosphine (0.092mmol, 1M) in tetrahydrofuran (92. mu.L) was added, and the reaction was allowed to react at room temperature for 4 hours, with substantially complete mass spectrometric detection, EIS derived anion mode: starting material [ M-H]-Disappearance of 647 and reduction of the product [ M-H]-Appearing 621. The reaction solution was lyophilized to remove excess triethylphosphine. The precipitate was dissolved in water/methanol (0.5/0.5mL), and then N-hydroxysuccinimide biotin (CAS Registry Number:72040-63-2) was added to the solution, and the reaction was carried out at room temperature under nitrogen. Starting solution turbidity, clarifying basically after 2 hours, continuing reaction for 8 hours, detecting the reaction basically completely by mass spectrometry, freeze-drying, performing silica Gel column chromatography (isopropanol/ammonia water is 2/1) to obtain a target molecule crude product, performing desolventizing, freeze-drying, and separating by a polyacrylamide Gel column (Bio-Gel-P2, pure water) to obtain 20mg of a target molecule (UDP-GalNAc-Biotin), wherein the yield is 45%. The compound was a white solid.
Nuclear magnetic resonance and mass spectrometry identified the structure of the compound (fig. 2) with the following data:1H NMR(400MHz,D2O)δ7.96(d,J＝8.1Hz,1H),6.05-5.93(m,2H),5.57(dd,J＝7.3,3.4Hz,1H),4.62(dd,J＝7.9,4.8Hz,1H),4.43(dd,J＝7.9,4.5Hz,1H),4.40-4.35(m,2H),4.33-4.16(m,5H),4.09-3.91(m,4H),3.91-3.61(m,2H),3.45-3.30(m,1H),3.30-3.08(m,2H),3.01(dd,J＝13.1,5.0Hz,1H),2.79(d,J＝13.1Hz,1H),2.34(t,J＝7.4Hz,2H),2.26(t,J＝7.2Hz,2H),1.80-1.48(m,8H),1.46-1.28(m,4H).13C NMR(100MHz,D2O)δ177.4,176.6,172.0,166.1,165.3,151.7,141.7,102.6,94.6,94.5,88.5,83.1,75.8,73.8,72.1,69.6,68.4,67.5,62.1,61.1,60.2,55.4,42.3,39.7,39.1,35.5,35.4,28.0,27.8,27.6,25.6,25.2,24.8.MS(ESI)Calcd.for C33H53N7O20P2S[M-H]-960.25；found,960.31.
the enzyme and the glycosyl donor obtained by the method have certain stability. The enzyme can be stored for 6 months at 4 ℃ (the enzyme activity is not obviously reduced), and can be stored for one year at 20 ℃ to avoid repeated freeze thawing. The glycosyl donor powder is stored at-20 deg.C for 2 years, and the glycosyl donor solution is stored at 4 deg.C for 6 months and at-20 deg.C for one year.
An in vitro detection and labeling kit for O-GlcNAc glycosylation modification, comprising the following reagents:
(1) bovine glycosyltransferase mutant solution (abbreviated as enzyme, GalT1-K279A/Y289L, 0.02 mM);
(2) modified sugar donor aqueous solution (short for sugar donor, UDP-GalNAc-Biotin, 0.5 mM);
(3) manganese chloride (MnCl)2) Aqueous solution (. times.10,100 mM);
(4) buffer 1(20mM HEPES, 1% SDS, pH 7.9);
(5) buffer 2(50mM HEPES, 125mM NaCl, 5% NP-40, pH 7.9).
Example 2: O-GlcNAc glycosylation modification of marker cell lysate
(1) HEK293T cells (6cm dish) were harvested by centrifugation at 1000 Xg at 4 ℃ and 300. mu.L of cell lysate (RIPA lysate: 50mM Tris-HCl, pH 7.4, 150mM NaCl, 0.1% SDS, 1 XPIC protease inhibitor cocktail, 1mM PMSF) was added and lysed at 4 ℃ for 20min, 10000 Xg and centrifuged for 20 min. The supernatant was collected and the protein concentration was determined by BCA method.
(2) Adding about 1.2mg protein into 600 μ L methanol, 150 μ L dichloromethane and 400 μ L water in sequence, mixing (with white turbidity), centrifuging at 4 deg.C for 10min at 10000 × g for 10min, removing the upper solution, adding 450 μ L methanol, centrifuging, discarding supernatant, spin-drying again, and volatilizing methanol to obtain white protein precipitate.
(3) The PNGase kit (NEB, P0704S) excises protein N-glycosylation modifications. mu.L of 10 Xprotein inactivation buffer and 238. mu.L of water dissolved the precipitated protein, which was heated to 80 ℃ for 5min to dissolve the protein sufficiently. Then 80. mu.L of 10% NP-40 and 40. mu.L of 10 Xenzyme reaction buffer were added and mixed well, and finally 2. mu.L of PNGase enzyme solution was added and mixed well (400. mu.L system), and reacted at 4 ℃ for 20 hours. Precipitating the protein according to the method of step (2).
(4) Dissolving the protein precipitate obtained in step (3) in 300. mu.L of the kit buffer 1 (which can be heated to 60 ℃), adding 600. mu.L of the kit buffer 2, and adding 60. mu.L of MnCl2(mother liquor concentration 100mM), mixed well and divided into 6 portions. mu.L of the glycosyl donor (final concentration 25. mu.M) and 3. mu.L of the corresponding enzyme (final concentration 0.3. mu.M) were added to each reaction, followed by 27. mu.L of water (200. mu.L system). The reaction was carried out at 4 ℃ for 20 hours. Precipitating the protein according to the method of step (2). The blank control group had no enzyme added.
The experimental group using UDP-GalNAz as the sugar donor needs to perform the next Click reaction, and the reaction system is as follows: mu.L of a solution (1% SDS, 50mM Tris-HCl, pH 8.0) was dissolved with protein, and then the following reagents were added in this order, mixed well, and reacted at room temperature for 2 hours to precipitate protein.
30 μ L of 2 × laoding buffer solubilized the protein pellet and was cooked at 100 ℃ for 10 minutes. Fully resolved, centrifuged, and analyzed by 8% SDS-PAGE. Coomassie blue staining was performed, and the amount of the sample was adjusted so that the total amount of the protein was the same for each group. 8% SDS-PAGE (100V, 60min) run gel, membrane transfer (120V, 100 min). The membrane was blocked with 3% BSA in PBST for 1h, and incubated with 3% BSA in PBST for 10 h at 4 ℃ with an antibody (horseradish peroxidase HRP-streptavidin, 1: 5000). PBST was washed three times at room temperature. Exposure apparatus detects fluorescence signal see FIG. 3.
As can be seen, the three blank controls (columns 1-3 on the left) have essentially no signal, and the labeling signal is significantly stronger in the one-step method (the kit recommends a combination of enzyme and sugar donor, column 4 on the right) than in the two-step method (column 4 from the left). The labeling signal of the original enzyme in combination with the new glycosyl donor (column 4 from the left) is also significantly weaker than in the one-step method. The above results indicate that the enzyme and sugar donor in the present kit are the optimal combination for the O-GlcNAc glycosylation modification of the tag.
Example 3: purification and omics identification of O-GlcNAc glycosylation modified protein of cell lysate
The cell lysates were labeled according to the procedure of example 2 (2 mg protein per group, reaction scale up), and then labeled proteins were enriched using streptavidin agarose resin, as follows:
(1) streptavidin agarose resin 50. mu.L, 2000 Xg centrifugation to remove the protective solution, then added 1mL binding buffer (100mM Na)2HPO4150mM NaCl, pH 7.2) and 1mL of neutral salt solution (6% NP-40, 100mM Na) was added2HPO4150mM NaCl): the column was equilibrated once with 1% aqueous SDS as 1:1 buffer.
(2) After the labeling of the protein had been completed, 200. mu.L of 1% SDS aqueous solution was added to dissolve the protein precipitate, and then 200. mu.L of neutral salt solution was added thereto, mixed well, 10000 Xg, and centrifuged for 10 min. The supernatant was added to the equilibrated resin and incubated at 4 ℃ for 10 hours.
(3) After the incubation, the supernatant was centrifuged and 1mL of a low salt buffer (100mM Na) was added2HPO4150mM NaCl, 1% triton-100, 0.5% sodium deoxycholate, 0.1% SDS, pH 7.5 and high salt buffer (100mM Na)2HPO4500mM NaCl, 0.2% triton-100, pH 7.5) 5 times, 5 resin washes in PBS solution, 50. mu.L of 2 × laminating buffer was added, mixed well and boiled at 100 ℃ for 10 minutes. Fully dissolving, centrifuging and taking supernatant. 1/10 solution was removed and analyzed by 8% SDS-PAGE for the amount of purified protein. Compared with the control group, the experimental group should have a obviously-stained band, which proves that the experimental group successfully marks and purifies the protein. And (3) sending the loading buffer containing the protein to be detected to a company for proteomics analysis under the low-temperature condition, wherein the result is shown in figure 4.
The mass spectrometry results show that the original two-step method yields 204 proteins (red) and the new one-step method yields 432 proteins (green). The total number of proteins in the one-step process is more than 2 times that in the two-step process, wherein only 14.7% of the proteins in the two-step process are unique (30/204), and 59.7% of the proteins in the two-step process are not identified in the one-step process. This shows that the one-step method can identify more potential O-GlcNAc glycosylated proteins, which is beneficial to the discovery of more potential protein targets.
Example 4: histological analysis of colon tumor samples
The kit for carrying out tissue marking comprises sample treatment and marking, and comprises the following specific steps:
(1) and (4) dewaxing. And (3) drying the mounting sample in an oven at 65 ℃ for half an hour, and then sequentially soaking the mounting sample in the following solutions for 5min for dewaxing treatment: xylene, ethanol, 95% ethanol, 85% ethanol and ddH 2O. The above dissolving dewaxing treatment step was repeated once. The deparaffinized samples were then soaked in PBS solution for 10 min.
(2) Antigen recovery. Preparing antigen retrieval solution A (0.1M sodium citrate): b (0.1M citric acid) ═ 10: 1, adjusting the pH to 6.0. The dewaxed sample is placed in an antigen retrieval solution and boiled at 98 ℃ for 2h for antigen retrieval. The sample was then washed 2 times with pure water and PBS in sequence.
(3) The kit marks glycosylated protein. Each sample slide was placed in 2mL of an enzymatic reaction solution (15. mu.M GalT1-K279A, 250. mu.M UDP-GalANc-Biotin, 5mM MnCl)2in PBS, pH 7.9), at 4 ℃ for 12 h. Then three times TBST solution washes.
(4) And (4) carrying out fluorescent labeling. The sample slides were incubated with Alexa Fluor 488-streptavidin (1: 1000) in PBS for 12h at 4 ℃ and washed three times with TBST solution. Finally, the samples were incubated with DAPI in PBS (1: 10) protected from light for 3h at room temperature and washed three times with TBST solution.
(5) And (4) detecting fluorescence. Samples were examined by Nikon Eclipse Ti fluorescence microscopy (blue light: DAPI; green light: O-GlcNAc marker) and the images were analyzed by ImageJ software with the results shown in FIG. 5.
As can be seen, the experimental group (added with enzyme group) has obvious green fluorescence signal (right, blue is cell nucleus signal), the statistical result shows that the green fluorescence signal of the experimental group is obviously stronger than that of the control group (not added with enzyme group), and the results of the 7 groups of data have significant difference. This demonstrates that the kit can be used for immunohistochemical studies to assay tissues for O-GlcNAc glycosylation modifications.
Sequence listing
<110> Zhejiang university
<120> O-GlcNAc glycosylation modification one-step enzyme labeling kit and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
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<211> 273
<212> PRT
<213> Unknown (Unknown)
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Ser Leu Thr Ala Cys Pro Glu Glu Ser Pro Leu Leu Val Gly Pro Met
1 5 10 15
Leu Ile Glu Phe Asn Ile Pro Val Asp Leu Lys Leu Val Glu Gln Gln
20 25 30
Asn Pro Lys Val Lys Leu Gly Gly Arg Tyr Thr Pro Met Asp Cys Ile
35 40 45
Ser Pro His Lys Val Ala Ile Ile Ile Pro Phe Arg Asn Arg Gln Glu
50 55 60
His Leu Lys Tyr Trp Leu Tyr Tyr Leu His Pro Ile Leu Gln Arg Gln
65 70 75 80
Gln Leu Asp Tyr Gly Ile Tyr Val Ile Asn Gln Ala Gly Glu Ser Met
85 90 95
Phe Asn Arg Ala Lys Leu Leu Asn Val Gly Phe Lys Glu Ala Leu Lys
100 105 110
Asp Tyr Asp Tyr Asn Cys Phe Val Phe Ser Asp Val Asp Leu Ile Pro
115 120 125
Met Asn Asp His Asn Thr Tyr Arg Cys Phe Ser Gln Pro Arg His Ile
130 135 140
Ser Val Ala Met Asp Ala Phe Gly Phe Ser Leu Pro Tyr Val Gln Leu
145 150 155 160
Phe Gly Gly Val Ser Ala Leu Ser Lys Gln Gln Phe Leu Ser Ile Asn
165 170 175
Gly Phe Pro Asn Asn Tyr Trp Gly Trp Gly Gly Glu Asp Asp Asp Ile
180 185 190
Tyr Asn Arg Leu Ala Phe Arg Gly Met Ser Val Ser Arg Pro Asn Ala
195 200 205
Val Ile Gly Lys Thr Arg Met Ile Arg His Ser Arg Asp Lys Lys Asn
210 215 220
Glu Pro Asn Pro Gln Arg Phe Asp Arg Ile Ala His Thr Lys Glu Thr
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Met Leu Ser Asp Gly Leu Asn Ser Leu Thr Tyr Met Val Leu Glu Val
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Gln Arg Tyr Pro Leu Tyr Thr Lys Ile Thr Val Asp Ile Gly Thr Pro
260 265 270
Ser
Claims (7)
1. A modified sugar donor has a structure shown in formula (I):
2. a method of making the modified sugar donor of claim 1, the method comprising: UDP-GalNAz is dissolved in water, triethylphosphine tetrahydrofuran solution is added to react fully, the reaction solution is freeze-dried to remove redundant triethylphosphine, water/methanol is added to dissolve and precipitate, N-hydroxysuccinimide Biotin is added to react fully under the protection of nitrogen, freeze-drying is carried out, silica gel column chromatography, desolventization and freeze-drying are carried out, and polyacrylamide gel column separation is carried out to obtain a target molecule UDP-GalNAc-Biotin, namely the modified sugar donor.
3. Use of the modified sugar donor of claim 1 in the preparation of an in vitro test kit for the modification of O-GlcNAc glycosylation.
4. An in-vitro detection kit for O-GlcNAc glycosylation modification mainly comprises bovine glycosyltransferase mutant solution, modified sugar donor aqueous solution, manganese chloride aqueous solution and buffer solution; the amino acid sequence of the bovine glycosyltransferase mutant is shown as SEQ ID No.1, and the structure of the modified sugar donor is shown as the formula (I):
5. the kit of claim 4, wherein the buffer comprises a first buffer and a second buffer, wherein the buffers comprise: 20mM HEPES, 1% SDS, pH 7.9, solvent deionized water; the buffer solution comprises the following components: 50mM HEPES, 125mM NaCl, 5% NP-40, pH 7.9, and deionized water as solvent.
6. Use of the kit of claim 4 for the in vitro detection of a modification of O-GlcNAc glycosylation.
7. Use of the kit of claim 4 for in vitro labelling of a modification of O-GlcNAc glycosylation.
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Non-Patent Citations (5)
| Title |
|---|
| BONANDI, ELISA ET AL.: "The 1,2,3-triazole ring as a bioisostere in medicinal chemistry", 《DRUG DISCOVERY TODAY》, vol. 22, no. 10, 31 December 2017 (2017-12-31), pages 1572 - 1581, XP085215112, DOI: 10.1016/j.drudis.2017.05.014 * |
| LIUQING WEN ET AL.: "Chemoenzymatic Synthesis of Unnatural Nucleotide Sugars for Enzymatic Bioorthogonal Labeling", 《ACS CATAL.》, vol. 8, 12 July 2018 (2018-07-12), pages 7659 - 7666, XP055689275, DOI: 10.1021/acscatal.8b02081 * |
| THOMAS BÜLTER ET AL.: "Chemoenzymatic Synthesis of Biotinylated Nucleotide Sugars as Substrates for Glycosyltransferases", 《CHEMBIOCHEM》, vol. 2, 31 December 2001 (2001-12-31), pages 884 - 894, XP072142552, DOI: 10.1002/1439-7633(20011203)2:12<884::AID-CBIC884>3.0.CO;2-2 * |
| 郁美娟 等: "新型罗丹明类荧光标记探针的研究（II）", 《染料与染色》, vol. 41, no. 5, 31 October 2004 (2004-10-31), pages 249 - 251 * |
| 闫新豪: "5′位氨基修饰的核苷化合物的合成", 《广州化工》, vol. 38, no. 5, 31 December 2010 (2010-12-31), pages 172 * |
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