CN115960323A - Activatable labeling method of cell surface glycan - Google Patents
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- 239000000523 sample Substances 0.000 claims abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 18
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- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 claims abstract description 11
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- MBLBDJOUHNCFQT-UHFFFAOYSA-N N-acetyl-D-galactosamine Natural products CC(=O)NC(C=O)C(O)C(O)C(O)CO MBLBDJOUHNCFQT-UHFFFAOYSA-N 0.000 claims abstract description 10
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- UKODFQOELJFMII-UHFFFAOYSA-N pentamethyldiethylenetriamine Chemical compound CN(C)CCN(C)CCN(C)C UKODFQOELJFMII-UHFFFAOYSA-N 0.000 description 2
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
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Images
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to an activatable labeling method of cell surface glycan. A glycan oxidation probe GO-PN is constructed by utilizing an Atom Transfer Radical Polymerization (ATRP) technology, connecting a chain initiator on the surface of Galactose Oxidase (GO) by utilizing a disulfide bond, and controllably growing a Polymer (PN) in situ. When the probe and the cell are incubated together, PN on the probe is used as a physical barrier, so that the glycan on the cell surface is difficult to access the enzyme activity center, and GO-PN can not oxidize galactose and N-acetylgalactosamine (Gal/GalNAc) at the end of glycan. When a reducing agent tris (2-carboxyethyl) phosphine (TCEP) is added into the system, the disulfide bond between PN and GO is broken to dissociate PN, the enzyme active site of the probe is exposed, and Gal/GalNAc of glycan on the cell surface is oxidized to generate aldehyde group. The aldehyde group can further generate high-efficiency connection reaction with hydrazide modified molecules, thereby realizing the activatable marking of the cell surface glycan. The method realizes the time control of the cell membrane glycan marking, and provides an important tool for glycan in-situ detection and functional research.
Description
1. Field of the invention
The invention relates to an activatable labeling method for cell surface glycans.
2. Background of the invention
Glycocomplexes are abundant on the cell surface. Sugars, together with nucleic acids, proteins and lipids, are one of the four basic components that make up a cell, and are the most abundant and diverse biomacromolecules in nature. More than 50% of human proteins are glycoproteins, and glycosylation of proteins increases heterogeneity and functional diversity at the molecular level. The glycan plays an important role in various biological processes, and the structure and the expression level of the glycan are closely related to the disease process, so the glycan is a potential disease marker and a therapeutic target. Therefore, the controllable marking of the cell glycan is realized, and further, a glycan detection and editing method is developed, so that the method has great significance for diagnosis and intervention of diseases.
Common glycan labeling methods include lectin recognition, sugar metabolism, and sugar modifying enzyme techniques. Among them, lectin recognition has the disadvantages of poor specificity and low affinity; the metabolic glycan labeling method depends on the metabolic process in the cell and is difficult to regulate. The sugar modification enzyme technology has the advantages of high specificity, simple and convenient method and the like by carrying out covalent labeling on glycan outside cells. In order to regulate the glycan-remodeling activity of a sugar-modifying enzyme, existing methods include encapsulating the sugar-modifying enzyme with a metal-organic framework material or polyethylene glycol, and the like. The methods have the defects of low enzyme encapsulation rate, uncertain probe structure composition, difficult enzyme activity control and the like. Therefore, development of a glycan labeling method suitable for living cells and capable of being activated efficiently is urgently needed, and a new tool is provided for in-situ detection and editing of glycans.
In order to realize high-efficiency 'off-on' conversion of enzyme activity, the polymer is grown on the surface of Galactose Oxidase (GO) through in-situ grafting of a reducing agent response type disulfide bond, and the accessibility of an enzyme activity center is regulated by utilizing the steric effect of the polymer. The restructuring activity of the polymerized GO on cell surface glycan is efficiently inhibited; in the presence of a reducing agent, the disulfide bond is broken to remove the polymer, the active center of the enzyme is exposed again, and the cell surface glycan is oxidized to generate aldehyde group. Further utilizes the bioorthogonal chemical reaction of aldehyde group and hydrazide modified molecule to realize the activatable marking of the cell glycan. The method provides a tool for the detection, intervention and functional research of the cell glycan, and has application prospects in the fields of biological analysis, clinical diagnosis, glyco-tissue engineering and glyco-immune checkpoint treatment.
3. Summary of the invention
The purpose of the invention is: and (3) growing a polymer in situ on the surface of the sugar modified enzyme through a chemical response type group, wherein the polymer has a steric hindrance effect. The conversion of an enzymatic activity center from 'closed' to 'exposed' is realized by regulating the enzymatic activity by using chemical stimulation, thereby activating the glycan reconstruction on the cell surface, and further realizing the activatable marking of the glycan on the cell surface by the bioorthogonal chemical reaction of reconstructed sugar and marking molecules.
The method first utilizes succinimide 3- (2-pyridyldithio) -propionate (SPDP) in combination with a thiol displacement reaction to link a chain initiator via a disulfide bond to the amino group of GO. And then grafting poly (isopropyl acrylamide) (PN) on the surface of the GO-chain initiator in situ by adopting Atom Transfer Radical Polymerization (ATRP) to prepare a glycan oxidation probe GO-PN, as shown in figure 1.
The method for activatable labeling of cell surface glycans of the present invention is shown in FIG. 2. The polymer on the surface of the enzyme plays a role of physical shielding, so that glycan on the surface of a cell is difficult to approach the enzymatic activity center of GO-PN, and the apparent activity of the probe is in an off state. When the cell is incubated with a GO-PN probe and a reducing agent tris (2-carboxyethyl) phosphine (TCEP), the TCEP cuts off a disulfide bond between PN and GO to remove PN, galactose and N-acetylgalactosamine (Gal/GalNAc) at the tail end of a sugar chain on the cell surface are more accessible to the active site of the probe and oxidized to generate bioorthogonal aldehyde groups, and the aldehyde groups can further react with hydrazide modified molecules to form stable hydrazone bonds, so that the labeling of cell surface glycan is realized.
The invention is realized by the following technical scheme:
1) As shown in FIG. 1, chemical linker molecule succinimide 3- (2-pyridyldithio) -propionate (SPDP) was modified on GO, and the chain initiator bis [2- (2' -bromoisobutyryloxy) is cleaved by tris (2-carboxyethyl) phosphine (TCEP)Yl) ethyl]The disulfide (BiBOEDS) is labeled on the amino group of GO to prepare the GO-chain initiator compound. Then, using N-isopropyl acrylamide (NIPAm) as monomer, and adding N, N, N' -pentamethyl diethylenetriamine (PMDETA) and CuCl 2 ·2H 2 And (3) carrying out atom transfer radical polymerization on a GO-chain initiator for 1 hour (in a mixed solution of methanol and water, without oxygen) in the presence of O and L-ascorbic acid (Vc) to prepare a glycan oxidation probe GO-PN.
2) As shown in FIG. 2, GO-PN and TCEP were incubated with the cells for 60 min, the enzyme active site of the probe was exposed, and aldehyde groups were generated by oxidizing Gal/GalNAc at the sugar chain terminal on the cell surface. And glycan oxidation does not occur in a cell sample only added with GO-PN, and the oxidation activity of GO-PN on cell glycan is inhibited by 90% by taking GO with the same concentration as a reference.
3) As shown in FIG. 2, aldehyde groups generated on cell membrane sugar chains react with hydrazide modified molecules (such as hydrazide-fluorescent molecules, hydrazide-biotin, etc.) for 1 hour under the catalysis of aniline, so as to realize the activatable labeling of cell surface glycans. The mark can be detected by laser confocal fluorescence microscopy, western blotting, mass spectrometry and other technologies. The labeling efficiency of cellular glycans was able to reach 74% of the highest labeling efficiency when GO was the probe.
Compared with the prior art, the invention has the following characteristics:
the invention provides a new glycan reconstruction regulation platform, and further labeling is realized by utilizing accessibility of a polymer in situ growth on GO to regulate and control an enzymatic activity center and triggering oxidation of glycan on the cell surface by chemical stimulation.
The synthesis condition and the polymer deblocking condition of the probe do not damage the enzymatic activity and glycosyl specificity of GO, and the probe has cell compatibility and can be used for labeling activatable glycans on living cells.
Compared with the method that the polymer is directly grafted to the surface of GO, the probe prepared by the method has higher enzyme activity inhibition rate and is easy to separate and purify.
The method is suitable for various hydrazide labeled molecules (such as fluorescent molecules, biotin and the like), can be used for docking various detection means according to different molecular functions, realizes the analysis of the cell glycan, and provides a new technology for clinical diagnosis and intervention.
4. Description of the drawings
FIG. 1 is a schematic representation of a method for preparing a glycan oxidation probe GO-PN
FIG. 2 is a schematic diagram of an activatable cell surface glycan labeling method
5. Detailed description of the preferred embodiments
Example 1: and (3) preparing a glycan oxidation probe GO-PN by combining the figure 1.
GO was dissolved in phosphate buffered saline (PBS, containing 10mM phosphate, 136.7mM NaCl,2.7mM KCl) at pH8.0 to make a 2mg/mL stock solution of GO. SPDP was dissolved in dimethyl sulfoxide (DMSO) to make 100mM stock. Add 20. Mu.L of SPDP solution to 2mL of GO solution, shake gently at room temperature for 2 hours, then wash 6 times with PBS (pH8.0) solution through a ultrafiltration tube (30 kDa MWCO) to obtain GO-SPDP. GO-SPDP solution was diluted to 1.8mg/mL with PBS (pH 8.0), reacted with 60mM TCEP for 30 minutes and then washed 6 times with PBS ultrafiltration (30 kDa MWCO) at pH8.0 to make thiol-modified GO (GO-SH). mu.L of the chain initiator BiBOEDS (10 mM) was added drop by drop to a GO-SH solution (1 mL,2.7 mg/mL) and shaken on a homogenizer for 2 hours. The product was dialyzed using 3500Da MWCO dialysis bags in PBS (pH 7.4) at 4 ℃ for 24 hours to obtain GO-chain initiators. In Schlenk tube, NIPAm (200 mg), PMDETA (1.2 mg) and CuCl 2 ·2H 2 O (1.2 mg) dissolved in H 2 A blue monomer solution was obtained in a mixed solution of O (2 mL) and methanol (1 mL). The schleck tube was sealed, frozen with liquid nitrogen, deoxygenated with nitrogen for 15 minutes and thawed. Under a nitrogen atmosphere, 200 μ L of a 6.2mg/mL Vc solution was added to the monomer solution, a "freeze-deoxygenation-thaw" operation was performed, followed by the addition of 1mL of the GO-chain initiator solution (2 mg/mL) that had been deoxygenated. After another "freeze-deoxidation-thawing" operation, the Schlenk tube was placed on a magnetic stirrer and reacted at room temperature for 1 hour, the solution turned pale green. The reaction solution was exposed to air to terminate the polymerization reaction, at which time the solution color changed to blue. The crude product was ultrafiltered (30 kDa MWCO) 6 times with PBS (pH 7.4) at 4 ℃ to yield GO-PN.
Example 2: in conjunction with fig. 2, activatable oxidation and labeling of cell surface glycans.
Using MCF-7 cells as an example, adherent cells (1X 10 cells) were placed in a confocal dish 4 One) was washed 3 times with PBS (pH 7.4) and then the cells were blocked with PBS containing 10% sheep serum for 30 minutes at 37 ℃. The dishes were divided into 2 groups, group I (activated) added 100. Mu.L GO-PN (equivalent enzyme concentration 0.05 mg/mL) mixed with 5mM TCEP, and group II (unactivated) added only 100. Mu.L GO-PN (equivalent enzyme concentration 0.05 mg/mL). After incubating the confocal dish at 4 ℃ for 60 minutes, it was washed 3 times with PBS. mu.L of PBS solution containing 10mM aniline, 100. Mu.M hydrazide-modified molecule and 5% fetal bovine serum was added to the confocal dish and incubated with cells for 1 hour at 4 ℃. After the cells were washed 3 times with PBS, labeling was completed.
Example 3: aldehyde groups generated by oxidation of cell surface glycans were fluorescently labeled according to example 2.
mu.L of PBS containing 10mM aniline, 100. Mu.M fluorescein-5-thiosemicarbazide and 5% fetal bovine serum was added to the oxidized cells and incubated at 4 ℃ for 1 hour. Washing the cells with PBS for 3 times, imaging the cells with a come card SP8 laser confocal scanning microscope, recording the fluorescence intensity of the cells with come card software, and analyzing the labeling degree of cell glycan.
Claims (4)
1. An activated labeling method of cell surface glycan is characterized in that a glycan oxidizing probe is obtained by connecting a polymer on the surface of Galactose Oxidase (GO) by using a disulfide bond, and the probe can oxidize galactose and N-acetylgalactosamine (Gal/GalNAc) at the tail end of a sugar chain on the cell surface to generate a bio-orthogonal group for labeling the cell glycan only after being activated by a reducing agent.
2. The method as claimed in claim 1, characterized in that the glycan oxidation probe GO-PN is prepared by using succinimide 3- (2-pyridyldithio) -propionate in combination with a mercapto-displacement reaction to link the chain initiator bis [2- (2' -bromoisobutyryloxy) ethyl ] disulfide and further using atom transfer radical polymerization to grow Polyisopropylacrylamide (PN) in situ on the GO surface.
3. The method of claim 1, wherein the polymer on GO-PN is sterically hindered, the oxidation activity of GO-PN to cellular glycans is inhibited by 90% compared to GO, upon addition of the cytocompatible reducing agent tris (2-carboxyethyl) phosphine, disulfide bond cleavage leaves the polymer, GO-PN is converted to GO, activity is restored, and oxidation of cell surface Gal/GalNAc generates bioorthogonal aldehyde groups.
4. The method of claim 1, wherein aldehyde groups generated on the cell surface Gal/GalNAc can be linked with hydrazide modified molecules to form hydrazone bonds, thereby labeling glycans on the cell surface.
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