An activatable labeling method for cell surface glycans

Abstract

The present invention relates to an activatable labeling method for cell surface polysaccharides. Atom transfer radical polymerization (ATRP) technology is used to connect chain initiators with disulfide bonds on the surface of galactose oxidase (GO), and in-situ controllable growth polymers (PN) are used to construct polysaccharide oxidation probes GO-PN. When the probe is co-incubated with cells, the PN on the probe acts as a physical barrier, making it difficult for cell surface polysaccharides to approach the enzyme active center, and GO-PN cannot oxidize galactose and N-acetylgalactosamine (Gal / GalNAc) at the end of the polysaccharide. When the reducing agent tris(2-carboxyethyl)phosphine (TCEP) is added to the system, the disulfide bond between PN and GO is disconnected to dissociate PN, the enzyme active site of the probe is exposed, and the Gal / GalNAc of the oxidized cell surface polysaccharides generates an aldehyde group. The aldehyde group can further react with hydrazide-modified molecules to efficiently connect, thereby realizing the activatable labeling of cell surface polysaccharides. The method realizes the time control of cell membrane polysaccharide labeling, and provides an important tool for in-situ detection and functional research of polysaccharides.

Description

1. Technical Field

[0001] The present invention relates to an activatable labeling method for cell surface glycans. 2. Background Technology

[0002] There are abundant sugar complexes on the cell surface. Sugar, together with nucleic acids, proteins and lipids, is one of the four basic components of cells, and is also the most abundant and diverse biological macromolecule in nature. More than 50% of human proteins are glycoproteins, and the glycosylation of proteins increases the heterogeneity at the molecular level and the diversity of functions. Glycans play an important role in a variety of biological processes. Their structure and expression level are closely related to the course of the disease, and they are very potential disease markers and therapeutic targets. Therefore, achieving controllable labeling of cellular glycans and then developing methods for detecting and editing glycans are of great significance for the diagnosis and intervention of diseases.

[0003] Common glycan labeling methods include lectin recognition, sugar metabolism, and sugar-modifying enzyme technology. Among them, lectin recognition has the disadvantages of poor specificity and low affinity; and the metabolic glycan labeling method relies on the metabolic process inside the cell and is difficult to regulate. Sugar-modifying enzyme technology has the advantages of high specificity and simple method by covalently labeling glycans outside the cell. In order to regulate the glycan remodeling activity of sugar-modifying enzymes, existing methods include the use of metal-organic framework materials or polyethylene glycol to encapsulate sugar-modifying enzymes. These methods have the disadvantages of low enzyme encapsulation rate, uncertain probe structure composition, and difficulty in controlling enzyme activity. Therefore, it is urgent to develop a glycan labeling method that is applicable to living cells and can be efficiently activated to provide new tools for in situ detection and editing of glycans.

[0004] In order to achieve efficient "off-on" conversion of enzyme activity, the present invention in situ grafts a polymer on the surface of galactose oxidase (GO) through a reductant-responsive disulfide bond, and uses the steric effect of the polymer to regulate the accessibility of the enzyme active center. After polymerization, the remodeling activity of GO on cell surface glycans is efficiently inhibited; in the presence of a reductant, the disulfide bond is disconnected to cause the polymer to leave, the enzyme active center is exposed again, and the cell surface glycans are oxidized to generate aldehyde groups. Further, the bioorthogonal chemical reaction of aldehyde groups and hydrazide-modified molecules is used to achieve activatable labeling of cellular glycans. This method provides a tool for the detection, intervention and functional research of cellular glycans, and has application prospects in the fields of bioanalysis, clinical diagnosis, glyco-tissue engineering, and glyco-immune checkpoint therapy. III. Summary of the invention

[0005] The purpose of the present invention is to grow a polymer in situ on the surface of a sugar-modified enzyme through a chemically responsive group, and the polymer has a steric hindrance effect. The enzyme activity is regulated by chemical stimulation to achieve the conversion of the enzyme active center from "closed" to "exposed", thereby activating the reconstruction of glycans on the cell surface, and further realizing the activatable labeling of cell surface glycans through the bioorthogonal chemical reaction between the reconstructed sugar and the labeling molecule.

[0006] The method first uses succinimidyl 3-(2-pyridyldisulfide)-propionate (SPDP) combined with thiol displacement reaction to connect the chain initiator to the amino group of GO through a disulfide bond. Then, atom transfer radical polymerization (ATRP) is used to in situ graft polyisopropylacrylamide (PN) on the surface of GO-chain initiator to prepare the polysaccharide oxidation probe GO-PN, such as Figure 1 shown.

[0007] The activatable labeling method of cell surface polysaccharides proposed by the present invention is as follows Figure 2 As shown. The polymer on the enzyme surface acts as a physical shield, making it difficult for cell surface polysaccharides to approach the enzyme active center of GO-PN, resulting in the apparent activity of the probe being in the "off" state. When cells are co-incubated with GO-PN probes and the reducing agent tri(2-carboxyethyl)phosphine (TCEP), TCEP cuts the disulfide bond between PN and GO, causing PN to leave. At this time, the galactose and N-acetylgalactosamine (Gal / GalNAc) at the end of the cell surface sugar chain are more accessible to the active site of the probe and are oxidized to form bioorthogonal aldehyde groups, which can further react with hydrazide-modified molecules to form stable hydrazone bonds, thereby achieving labeling of cell surface polysaccharides.

[0008] The present invention is achieved through the following technical solutions:

[0009] 1) If Figure 1 As shown, the chemical linker molecule succinimidyl 3-(2-pyridyldithio)-propionate (SPDP) was modified on GO, and the chain initiator bis[2-(2′-bromoisobutyryloxy)ethyl] disulfide (BiBOEDS) was labeled on the amino group of GO by tri(2-carboxyethyl)phosphine (TCEP) cleavage to prepare the GO-chain initiator complex. Then, with N-isopropylacrylamide (NIPAm) as a monomer, atom transfer radical polymerization was carried out on GO-chain initiator in the presence of N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA), CuCl2·2H2O and L-ascorbic acid (Vc) for 1 hour (in a mixed solution of methanol and water, oxygen-free) to prepare the glycan oxidation probe GO-PN.

[0010] 2) If Figure 2As shown, GO-PN and TCEP were co-incubated with cells for 60 minutes, the enzyme active site of the probe was exposed, and the Gal / GalNAc at the end of the sugar chain on the cell surface was oxidized to generate aldehyde groups. However, no glycan oxidation occurred in the cell sample with only GO-PN added. With the same concentration of GO as a reference, the oxidative activity of GO-PN on cell glycans was inhibited by 90%.

[0011] 3) If Figure 2 As shown, the aldehyde groups generated on the cell membrane sugar chains react with hydrazide-modified molecules (such as hydrazide-fluorescent molecules, hydrazide-biotin, etc.) under the catalysis of aniline for 1 hour to achieve activatable labeling of cell surface glycans. The labeling can be detected by laser confocal fluorescence microscopy, protein blotting, mass spectrometry and other technologies. The labeling efficiency of cell glycans can reach 74% of the highest labeling efficiency when GO is used as a probe.

[0012] Compared with the prior art, the present invention has the following characteristics:

[0013] The present invention proposes a new glycan reconstruction regulation platform, which utilizes the polymer grown in situ on GO to regulate the accessibility of the enzyme active center, and triggers the oxidation of cell surface glycans with chemical stimulation to achieve further labeling.

[0014] The probe synthesis conditions and polymer deblocking conditions do not damage the enzyme activity and glycosyl specificity of GO itself, and are cell-compatible and can perform activatable glycan labeling on living cells.

[0015] The polymers that are controllably grown in situ on GO can effectively block enzyme activity. Compared with directly grafting the polymers onto the GO surface, the probes prepared by this method have a higher enzyme activity inhibition rate and are easy to separate and purify.

[0016] This method is applicable to various hydrazide-labeled molecules (such as fluorescent molecules, biotin, etc.), and can be connected to a variety of detection methods according to the different functions of the molecules to achieve the analysis of cellular glycans, providing new technologies for clinical diagnosis and intervention. IV. Description of the drawings

[0017] Figure 1 .Schematic diagram of the preparation method of glycan oxidation probe GO-PN

[0018] Figure 2 . Schematic diagram of the method for labeling activatable cell surface glycans V. Specific implementation methods

[0019] Example 1: Combination Figure 1 , preparation of glycan oxidation probe GO-PN.

[0020] GO was dissolved in a pH 8.0 phosphate buffer solution (PBS, containing 10 mM phosphate, 136.7 mM NaCl, 2.7 mM KCl) to prepare a 2 mg / mL GO stock solution. SPDP was dissolved in dimethyl sulfoxide (DMSO) to prepare a 100 mM stock solution. 20 μL of SPDP solution was added to 2 mL of GO solution, gently shaken at room temperature for 2 hours, and then washed 6 times with PBS (pH 8.0) solution through an ultrafiltration tube (30 kDa MWCO) to obtain GO-SPDP. The GO-SPDP solution was diluted to 1.8 mg / mL with PBS (pH 8.0), reacted with 60 mM TCEP for 30 minutes, and then ultrafiltered and washed 6 times with PBS (30 kDa MWCO) at pH 8.0 to obtain thiol-modified GO (GO-SH). 400 μL of chain initiator BiBOEDS (10 mM) was added drop by drop to GO-SH solution (1 mL, 2.7 mg / mL) and shaken on a homogenizer for 2 h. The product was dialyzed in PBS (pH 7.4) at 4 °C for 24 h using a 3500 Da MWCO dialysis bag to obtain GO-chain initiator. In a Shrek tube, NIPAm (200 mg), PMDETA (1.2 mg) and CuCl2·2H2O (1.2 mg) were dissolved in a mixture of H2O (2 mL) and methanol (1 mL) to obtain a blue monomer solution. The Shrek tube was sealed, frozen with liquid nitrogen, and thawed after nitrogen deoxygenation for 15 minutes. Under a nitrogen atmosphere, 200 μL of 6.2 mg / mL Vc solution was added to the monomer solution, and a "freeze-deoxygenation-thaw" operation was performed, and then 1 mL of deoxygenated GO-chain initiator solution (2 mg / mL) was added. After another freeze-deoxygenation-thaw operation, the Shrek tube was placed on a magnetic stirrer and reacted at room temperature for 1 hour, and the solution color turned light green. The reaction solution was exposed to air to terminate the polymerization reaction, and the solution color turned blue. The crude product was ultrafiltered (30 kDa MWCO) at 4°C for 6 times with PBS (pH 7.4) to obtain GO-PN.

[0021] Example 2: Combination Figure 2 , activatable oxidation and labeling of cell surface glycans.

[0022] Taking MCF-7 cells as an example, the attached cells (1×10 4After washing three times with PBS (pH 7.4), the cells were blocked with PBS containing 10% goat serum at 37°C for 30 minutes. The dishes were divided into two groups, group I (activated group) added a mixture of 100 μL GO-PN (equivalent enzyme concentration 0.05 mg / mL) and 5 mM TCEP, and group II (unactivated group) only added 100 μL GO-PN (equivalent enzyme concentration 0.05 mg / mL). After incubating the confocal dish at 4°C for 60 minutes, it was washed three times with PBS. 100 μL of PBS solution containing 10 mM aniline, 100 μM hydrazide-modified molecules and 5% fetal bovine serum was added to the confocal dish and incubated with the cells at 4°C for 1 hour. After washing the cells with PBS three times, the labeling was completed.

[0023] Example 3: According to Example 2, the aldehyde groups generated by oxidation of cell surface polysaccharides are fluorescently labeled.

[0024] 100 μL of PBS solution containing 10 mM aniline, 100 μM fluorescein-5-thiosemicarbazide and 5% fetal bovine serum was added to the oxidized cells and incubated for 1 hour at 4° C. After washing the cells three times with PBS, the cells were imaged using a Leica SP8 laser confocal scanning microscope, and the cell fluorescence intensity was recorded using Leica software to analyze the degree of labeling of cellular glycans.

Claims

1. An activatable labeling method for cell surface glycans, characterized in that a glycan oxidation probe is obtained by connecting polyisopropylacrylamide with a disulfide bond on the surface of galactose oxidase. The probe can only oxidize galactose and N-acetylgalactosamine at the end of the cell surface sugar chain to generate bioorthogonal groups for labeling cell glycans after being activated by a reducing agent that can break the disulfide bond.

2. The method according to claim 1, characterized in that The glycan oxidation probe was prepared by connecting the chain initiator bis[2-(2′-bromoisobutyryloxy)ethyl] disulfide to the amino group of galactose oxidase using succinimidyl 3-(2-pyridyldithio)-propionate combined with thiol displacement reaction, and further growing polyisopropylacrylamide in situ on the surface of galactose oxidase using atom transfer radical polymerization.

3. The method according to claim 1, characterized in that: The polymer on the glycan oxidation probe has a steric effect. Compared with galactose oxidase, the oxidation activity of the glycan oxidation probe on cellular glycans is inhibited by 90%. After the addition of the cell-compatible reducing agent tri(2-carboxyethyl)phosphine, the disulfide bond is broken and the polymer leaves. The glycan oxidation probe is converted into galactose oxidase, and the activity is restored, oxidizing cell surface galactose and N-acetylgalactosamine to generate bioorthogonal aldehyde groups.

4. The method according to claim 1, characterized in that: The aldehyde groups generated on the cell surface galactose and N-acetylgalactosamine can react with hydrazide-modified molecules to form hydrazone bonds, thereby achieving the labeling of cell surface glycans.

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