A glycan remodeling method for selective labeling of free glycoproteins in a cellular environment

Abstract

The present invention relates to a method for selectively labeling a glycan reconstruction of free glycoproteins in a cell environment. The method uses polymer-coated galactose oxidase (GO) as a glycan reconstruction probe (GO-PN). The shielding effect of the polymer on the enzyme active center on the probe varies with the size of the substrate. When the substrate is a cell, the sugar complex (including glycoproteins, glycolipids, etc.) anchored on the cell surface is difficult to approach the enzyme active center of the probe, so glycan reconstruction is difficult to occur; and when the substrate is a free glycoprotein, the sugar chain on the glycoprotein can approach the enzyme active center, and the galactose and N-acetylgalactosamine (Gal / GalNAc) at the end of the sugar chain are oxidized by GO to generate an aldehyde group. Based on this principle, the probe can selectively reconstruct the glycans of glycoproteins in a mixture of cells and free glycoproteins. The aldehyde group can further react with a hydrazide-modified molecule to achieve glycan labeling of free glycoproteins in a cell environment.

Description

1. Technical Field

[0001] The present invention relates to a polysaccharide reconstruction method for selectively marking free glycoproteins in a cell environment. 2. Background Technology

[0002] Glycosylation is the most abundant and diverse type of biomolecule modification. It mainly modifies dozens of monosaccharides with different compositions, sequences, and connection modes at multiple sites of proteins or lipids through the continuous synergistic action of the endoplasmic reticulum and the Golgi apparatus. Recent studies have shown that glycans can also directly modify RNA. These sugar chains not only expand the types of biomolecules, but also affect their intrinsic properties and mediate corresponding biological functions. Studies have shown that changes in cell surface glycans are closely related to the physiological and pathological processes of organisms. Therefore, the development of glycan analysis and editing methods is of great significance for the development of early diagnosis of diseases, prognosis monitoring technologies, and glycan-targeted therapeutic methods.

[0003] However, in animals, glycans exist in large quantities in various forms, from free monosaccharides, polysaccharides and secreted glycoconjugates (such as glycoproteins, glycolipids, and proteoglycans) in the extracellular matrix to glycocalyx (including membrane-bound glycoproteins and glycolipids) on the entire cell membrane. Existing glycan labeling and identification methods cannot distinguish the size of sugar-modified substrates. For example, when sugar-modifying enzymes are added to the cell system, both free glycoproteins and cell membranes carrying the same target glycans will be labeled, so there is an urgent need to develop a glycan labeling technology with "substrate size selectivity".

[0004] To address this problem, the present invention constructs a glycan reconstruction probe GO-PN by grafting a growth polymer (PN) on the surface of galactose oxidase (GO). The physical shielding effect of the polymer on the sugar-modified substrate depends on the substrate size. The probe has good stability and low cytotoxicity, and can only oxidize free glycoproteins in a mixed system of free glycoproteins and cells, successfully achieving substrate size-selective glycan reconstruction. The aldehyde group generated by oxidation can further react with a variety of hydrazide-modified molecules, providing a new tool for in situ labeling of glycoproteins in complex biological systems, and is expected to be used for the detection of secreted glycoproteins in living bodies. III. Summary of the invention

[0005] The purpose of the present invention is to prepare a polymer-encapsulated sugar-modifying enzyme, utilize the physical shielding effect of the polymer on the enzyme active center to achieve selective resolution of sugar substrates of different sizes, and only perform glycan reconstruction on free glycoproteins in a mixed system of free glycoproteins and cells for subsequent labeling operations.

[0006] This method first uses atom transfer radical polymerization (ATRP) to in situ graft polyisopropylacrylamide (PN) on the surface of GO to prepare the glycan reconstruction probe GO-PN. Figure 1As shown in Figure 2, natural GO can oxidize free or exposed galactose / N-acetylgalactosamine (Gal / GalNAc) to generate aldehyde groups, but cannot distinguish between free glycoproteins and sugar complexes on the cell surface; after PN is coated on the GO surface, the degree of PN's blocking of the substrate depends on the size of the substrate, thus giving GO substrate size selectivity.

[0007] The polysaccharide reconstruction method for selectively labeling free glycoproteins proposed by the present invention is as follows Figure 2 As shown, the GO-PN probe is first added to the mixed system of free glycoprotein and cells for co-incubation. The Gal / GalNAc on the free glycoprotein can be oxidized by GO-PN to generate aldehyde groups, while the Gal / GalNAc at the end of the sugar chain on the cell surface is difficult to pass through the PN layer to approach the enzyme active site, so it cannot be oxidized by the probe. The GO-PN treated system is further reacted with hydrazide modified molecules (such as fluorescent molecules, biotin, etc.). Only the aldehyde groups generated on the glycoprotein can be connected with hydrazide to form a stable hydrazone bond, thereby achieving the glycan labeling of the free glycoprotein in the mixed system.

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

[0009] 1) If Figure 1 As shown, GO was reacted with succinimidyl 3-(2-pyridyldithio)-propionate (SPDP) and tri-(2-carboxyethyl)phosphine to generate thiol-modified GO (GO-SH), and the chain initiator bis[2-(2′-bromoisobutyryloxy)ethyl] disulfide (BiBOEDS) was further labeled on the amino group of GO by thiol displacement reaction to generate GO-chain initiator. N-isopropylacrylamide (NIPAm), N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA), CuCl2·2H20, methanol and water were pre-mixed and deoxygenated, and then L-ascorbic acid (Vc) and GO-chain initiator were added under anaerobic conditions, and atom transfer radical polymerization was performed at room temperature under closed conditions for 1 hour to obtain the probe GO-PN.

[0010] 2) If Figure 2 As shown in the figure, GO-PN was added to the mixed system of cells and glycoproteins and incubated for 1 hour. The probe oxidized the Gal / GalNAc at the end of the sugar chain on the glycoprotein to generate aldehyde groups. The enzyme activity was 54% with the same concentration of GO as the control. However, the Gal / GalNAc on the cell surface was not oxidized, and the enzyme activity was less than 10% with the same concentration of GO as the control.

[0011] 3) If Figure 2 As shown, the glycoprotein reconstructed with GO-PN glycans was reacted with hydrazide-modified molecules under aniline catalysis for 1 hour to achieve the labeling of glycans on the glycoprotein.

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

[0013] The present invention proposes a glycan reconstruction method for selectively labeling free glycoproteins in a complex cell environment. The polymer grafted on the surface of GO has a "molecular sieve" function, which gives the sugar-modified enzyme substrate size selectivity, so that the probe can selectively reconstruct glycans on small-sized substrates. At the same time, the probe has good stability and cell compatibility.

[0014] Compared with the prior art, the present invention realizes the in situ labeling of glycans of free glycoproteins in a mixed system of cells and free glycoproteins for the first time. The method is simple in steps and easy to operate, and only requires the co-incubation of the probe with the target system. Subsequently, the aldehyde group generated on the glycoprotein can be connected to the hydrazide-modified functional molecule as needed to achieve glycan labeling of the glycoprotein. The method provides a new idea for the detection and separation of glycoproteins in a mixed system, and has the potential to be applied to the selective labeling of glycoproteins at the in vivo level. IV. Description of the drawings

[0015] Figure 1 . Schematic diagram of the preparation method of glycan remodeling probe GO-PN

[0016] Figure 2 Schematic diagram of selective reconstitution and labeling of free glycoproteins in a cellular environment V. Specific implementation methods

[0017] Example 1: Combination Figure 1 , preparation of GO-PN glycan reconstructed probe.

[0018] 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.

[0019] Example 2: Combination Figure 2 , using GO-PN to selectively reconstitute and label free glycoproteins in a cellular environment.

[0020] Taking the mixed system of MCF-7 cells and mucin 2 (MUC2) as an example, MCF-7 cells were seeded in confocal dishes (1×10 4After washing the cells three times with PBS (pH 7.4, containing 136.7mM NaCl, 2.7mM KCl, 8.72mM Na2HPO4, 1.41mM KH2PO4), the cells were blocked with PBS containing 10% goat serum at 37°C for 30 minutes. After washing three times with PBS, 50μL of 1.28mg / mL mucin 2 (MUC2) in PBS solution was added to the cells to prepare a mixed model system of cells and free glycoproteins (cell-MUC2). 50μL GO-PN (equivalent enzyme concentration of 0.1mg / mL) was added to the cell-MUC2 system and incubated at 4°C for 1 hour to complete the glycan reconstruction of free glycoproteins in the cell environment. 100 μL of PBS solution containing 20 mM aniline, 200 μM hydrazide-modified molecules and 200 mM K4Fe(CN)6 was added to the confocal dish and reacted at 4°C for 1 hour to achieve labeling of glycoprotein glycans in the cell environment.

Claims

1. A glycan reconstruction method for selectively labeling free glycoproteins in a cell environment, characterized in that galactose oxidase is encapsulated with poly-N-isopropylacrylamide as a glycan reconstruction probe, which can selectively oxidize the glycans of free glycoproteins in a mixed system of free glycoproteins and cells to generate aldehyde groups, and the aldehyde groups further react with hydrazide-modified molecules to achieve glycan labeling of free glycoproteins in a cell environment.

2. The reconstruction method according to claim 1 is characterized in that the blocking effect of the poly (N-isopropylacrylamide) coated on the galactose oxidase on the sugar-modified substrate depends on the size of the substrate, and the free glycoprotein can approach the enzyme active center, while the cell glycocalyx cannot approach the enzyme active center.

3. The reconstruction method according to claim 1 is characterized in that in a cellular environment, the glycan reconstruction probe can oxidize galactose and N-acetylgalactosamine of free glycoproteins to generate aldehyde groups, and its oxidation activity is 54% of galactose oxidase at the same concentration, but cannot oxidize galactose and N-acetylgalactosamine on the cell membrane sugar chain, and its oxidation activity is less than 10%.

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