A method for enriching receptors on the cell surface that mediate tau protein diffusion

Abstract

The present invention provides a new method for discovering receptors mediating tau endocytosis by modifying biotin on microtubule-associated protein tau. The present invention discovers that tau protein can be taken up by mouse hippocampal neuronal cells HT22. After silver staining following StreptAvidin-Biotin enrichment, differential bands are observed. Further, differential protein candidates are identified by mass spectrometry. The research results of the present invention provide an effective method for discovering receptor proteins mediating tau diffusion.

Description

Technical Field

[0001] The present invention belongs to the fields of molecular biology and neurodegenerative diseases. More specifically, the present invention relates to the field of exploring the pathogenesis of neurodegenerative diseases. The present invention provides a method for discovering a receptor that mediates the diffusion of tau protein (i.e., an endocytic receptor for tau protein). Background Art

[0002] Alzheimer's disease (AD) is the most common neurodegenerative disease, accounting for an estimated 60-70% of all dementia cases worldwide, causing a serious impact on human health and reducing the quality of life of patients and their families. If there is still no effective treatment, it will reach epidemic levels by 2050 (H. Wesseling, W. Mair, M. Kumar, C. N. Schlaffner, S. Tang, P. Beerepoot, B. Fatou et al. Tau PTM Profiles Identify Patient Heterogeneity and Stages of Alzheimer's Disease, Cell 183(6)(2020)1699-1713e13).

[0003] Typical pathological features of AD are extracellular amyloid-β (Aβ) deposition, the formation of intracellular neurofibrillary tangles (NFTs), and neuroinflammation. In recent years, the popular amyloid cascade hypothesis holds that Aβ deposition in the brain is the initial event of AD. However, with the failure of clinical studies targeting Aβ, more and more evidence indicates that this hypothesis is insufficient to explain the pathogenesis of AD (F. Leng, P. Edison, Neuroinflammation and microglial activation in Alzheimer disease: where do we go from here?, Nature Reviews Neurology 17(3)(2020)157-172.). Some studies have shown that Aβ plaques also exist in the brains of normal elderly people, which also makes it a mystery whether Aβ is the pathogenic cause.

[0004] Microtubule-associated protein tau plays a role in stabilizing microtubules under physiological conditions. However, under pathological conditions, hyperphosphorylated tau protein detaches from microtubules and spontaneously forms aggregates, causing neurotoxicity. In recent years, the research and development of antibody drugs targeting tau protein have gradually become a hot topic. In the research, it has been found that related proteins such as tau and α-synuclein in common neurodegenerative diseases not only have their own toxicity but also have the property of spreading between cells similar to prion proteins. This leads to the spread of the disease from a very small area to the whole brain. For example, tau was first discovered in the entorhinal cortex, spread to the hippocampus, and finally reached the neocortex of the brain, resulting in atrophy of the whole brain. Although there are more and more studies on the above pathogenic causes, no effective therapeutic target has been found clinically, and the research on the pathogenesis of AD is still an unsolved problem. No receptor mediating the spread of tau protein in neurons has been reported by enrichment methods. The present invention uses techniques based on chemical synthesis, molecular biology, and mass spectrometry-based quantitative proteomics (such as flow cytometry, laser confocal microscopy, silver staining, on beads enzymatic digestion mass spectrometry identification) to find that: 1) Through flow cytometry analysis, tau-FITC can be taken up by neuronal cells; 2) Using laser confocal microscopy, tau-FITC co-localizes with neuronal cells; 3) In the MALDI characterization synthesis experiment, it is found that after coupling biotin, the peak pattern of tau protein shifts, indicating an increase in molecular weight. 4) Silver staining shows that compared with the control group without tau-biotin, there is an increase in differential bands in the tau-biotin incubation group. 5) Mass spectrometry identification of on beads enzymatic digestion samples finds a series of tau-interacting proteins, and some membrane proteins are enriched after their identification. The present invention discovers for the first time the role of tau-biotin as a bait to capture key membrane proteins; it is found that some of the captured interacting proteins have not been reported; therefore, tau-biotin can be used as a new method to discover receptors mediating the spread of tau protein. Summary of the Invention

[0005] One object of the present invention is to discover receptors mediating the spread of tau protein.

[0006] Another object of the present invention is to provide an effective tool for discovering receptors mediating the spread of tau protein.

[0007] To achieve the above objects, the present invention adopts the following technical solutions:

[0008] The present invention provides a synthesis method of tau-biotin.

[0009] Furthermore, the synthesized product is characterized by MALDI.

[0010] Furthermore, the ability of cells to take up tau-FITC is characterized.

[0011] The characterization methods described in the present invention are flow cytometry and laser confocal microscopy. The dyes involved are FITC carried by tau itself, nuclear dye DAPI, and cell membrane dye DIL.

[0012] Furthermore, the cells are incubated with tau-biotin. After crosslinking for half an hour in the group with crosslinking agent added, they are lysed and enriched. In the group without crosslinking agent added, they are directly lysed and enriched. The incubation time, tau-biotin concentration, composition of the lysis solution, and washing conditions are optimized.

[0013] Further, the enriched proteins are visualized by SDS-PAGE silver staining and compared with the control group.

[0014] Furthermore, the presence of tau protein in the enriched proteins is identified by immunoblotting.

[0015] The immunoblotting antibody used in the present invention is a monoclonal antibody, polyclonal antibody, or antibody fragment with immunological activity that has immunological activity against tau or its active fragment and can specifically recognize and bind to the amino acid sequence or spatial structure of tau, or one or more of them.

[0016] Further, the enriched proteins are identified by mass spectrometry.

[0017] For the mass spectrometry detection described in the present invention, the acquisition modes all include: one or more of multiple reaction monitoring (MRM), parallel reaction monitoring (PRM), multiple cumulative parallel reaction monitoring (MSX-PRM), data-dependent acquisition (DDA), data-independent acquisition (DIA), or parallel reaction monitoring with enlarged precursor ion fragmentation window.

[0018] The cells used in the present invention are mouse hippocampal neuron cells HT22.

[0019] The incubation times described in the present invention are 0 - 4 hours respectively, and the concentration is 50 - 200 nmol / L. -1 。

[0020] The present invention has the following advantages:

[0021] (1) Since the discovery of the receptor mediating tau protein diffusion currently still uses traditional biological methods, starting from related proteins, finding possible proteins and then verifying to prove reliability, this method is relatively indirect. The present invention provides a new method for directly discovering the receptor mediating tau diffusion.

[0022] (2) The present invention proves through a series of characterization experiments that tau-biotin can specifically capture related proteins.

[0023] (3) The present invention discovers for the first time a list of potential proteins that mediate tau diffusion receptors. Brief Description of the Drawings

[0024] Figure 1 . Synthetic characterization MALDI map of Tau-biotin;

[0025] Figure 2 . Flow cytometry shows the uptake of tau-FITC;

[0026] Figure 3 . Laser confocal microscopy shows the co-localization of tau-FITC with cells;

[0027] Figure 4 . Silver staining analysis of differential bands;

[0028] Figure 5 . Immunoblot shows the presence of tau protein in the enriched proteins. Detailed Description of the Invention

[0029] The present invention will be specifically described below in conjunction with specific embodiments. In the following embodiments, the experimental methods without specific conditions are usually carried out under conventional conditions, such as those described in Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) by Sambrook et al., or according to the conditions recommended by the manufacturer. The percentages in the solutions are volume ratios unless otherwise specified.

[0030] Example 1.

[0031] 1) Take a tube of 100 μg tau protein (purchased from rPeptide) and dissolve it in 100 μl of ultrapure water, and vortex it to dissolve it completely. Weigh biotin (EZ-Link™ Sulfo-NHS-LC-biotin, purchased from Thermo) into an EP tube, with a final concentration of 10 mmol / L -1 , and vortex it to dissolve it completely. Take 1 μl of EZ-Link TM Sulfo-NHS-LC-biotin and add it to the dissolved tau protein, vortex it to mix well, centrifuge at 1000 RPM to the bottom of the tube, and react at 4 °C for 2 hours. After 2 hours, add 5 μl of a 1 mol / L -1 ammonium bicarbonate solution to neutralize the unreacted EZ-Link TM Sulfo-NHS-LC-biotin to obtain a tau-biotin solution.

[0032] 2) Take 2 μl of tau solution and tau-biotin solution respectively and spot them on the MALDI target plate. After air-drying in the fume hood, add 2 μl of SA matrix (sinapic acid, 3,5-dimethoxy-4-hydroxycinnamic acid) to each sample. After the SA is dried, place the target plate into the MALDI, select the molecular weight range of 10 - 80 KD, set the offset to 85%, and the range to 10%. Select the sample and strike 1000 times.

[0033] As Figure 1 shown, the results show that after coupling with biotin, the molecular weight of tau protein increases, proving the successful synthesis of tau-biotin.

[0034] Example 2. Characterization of cellular uptake of Tau-FITC by flow cytometry

[0035] Observe that HT22 cells (mouse hippocampal neuron cells, purchased from PROCELL, catalog number CL-0697) are in good growth condition. Discard the supernatant of the cells, digest the cells with trypsin (add 1 ml of trypsin to every 1x10 7 cells and digest in a 37 °C incubator for 2 minutes), after centrifugation, disperse the cells in DMEM medium with a volume concentration of 10% FBS (fetal bovine serum), and inoculate the cells into 2 six-well plates at a density of 2x10 5 cells / well. Place the cells in an air incubator at 37 °C with a volume concentration of 5% CO2. When the cell confluence reaches 90%, add tau-FITC (FITC-labeled form of tau-441, purchased from rPeptide, catalog number T-1113-2) with a final concentration of 100 nmol / L -1 to the medium in the first well and incubate for 8 hours. After 2 hours, add tau-FITC with a final concentration of 100 nmol / L -1 to the medium in the second well and incubate for 6 hours. After 4 hours, add tau-FITC with a final concentration of 100 nmol / L -1 to the medium in the third well and incubate for 4 hours. After 6 hours, add tau-FITC with a final concentration of 100 nmol / L -1 to the medium in the fourth well and incubate for 2 hours. After 7 hours, add tau-FITC with a final concentration of 100 nmol / L -1 to the medium in the fifth well and incubate for 1 hour. After 7.5 hours, add tau-FITC with a final concentration of 100 nmol / L -1tau-FITC was incubated in the culture medium for 0.5 hours. After all incubations were completed, the supernatant of the cell culture medium was discarded, and the cells were washed 3 times with PBS (pH 7.4) (purchased from Gibco, USA) to remove the adsorption on the cell surface. 300 μl of trypsin was added to each of the 7 wells to digest the cells. After 2 minutes, the digestion was terminated with 1 ml of 10% FBS DMEM culture medium by volume, and the cells were collected into flow cytometry tubes respectively. Centrifuge at 1000 rpm for 5 minutes respectively. After centrifugation, the supernatant was discarded, and 500 μl of PBS buffer was added to each tube to resuspend the cells. The samples were placed on ice in the dark and loaded onto the flow cytometer one by one.

[0036] As Figure 2 shown, the results showed that when incubated for 0.5 hours, the cells took up a small amount of tau-FITC. As the incubation time extended, the cells took up more and more tau-FITC. By 8 hours, 78.3% of the cells had taken up tau-FITC. This result indicated that tau-FITC could be taken up by HT22, proving the feasibility of carrying out subsequent experiments.

[0037] Example 3. Laser confocal characterization of the co-localization of tau-FITC and cells

[0038] HT22 cells were observed until they were in good growth condition. The supernatant of the cells was discarded, and the cells were digested with trypsin (1 ml of trypsin was added to every 1x10 7 cells and digested in a 37 °C incubator for 2 minutes). After centrifugation, the cells were dispersed in DMEM culture medium with 10% FBS (fetal bovine serum) by volume, and the cells were seeded into the confocal at a density of 2x10 4 cells / well, and the cells were placed in an air incubator at 37 °C with 5% CO2 by volume for culture. When the cell confluence reached 90%, 100 nmol / L -1 of tau-FITC was added to the culture medium and incubated for another 4 hours. After 2 hours, 100 nmol / L -1 of tau-FITC was added to the culture medium and incubated for another 2 hours. After 3 hours, 100 nmol / L -1 of tau-FITC was added to the culture medium and incubated for another 1 hour. After 3.5 hours, 100 nmol / L -1Add tau-FITC to the culture medium and continue incubation for 0.5 hours. When all incubations are completed, discard the cell supernatant and wash the cells three times with PBS (pH 7.4) (purchased from Gibco, USA) to remove cell surface adsorption. Add 1 ml of 4% paraformaldehyde to fix the cells for 10 minutes. After 10 minutes, discard the formaldehyde and wash the cells three times with PBS, 3 minutes each time, and shake at room temperature. Add 1 μl (1 mg / ml) of nuclear dye DAPI (4',6-diamidino-2-phenylindole) to 1 ml PBS, add the dye to the cells, stain at room temperature for 10 minutes, discard DAPI after 10 minutes, wash the cells three times with PBS, 3 minutes each time, and shake at room temperature. Add 1 μl (10 mg / ml) of cell membrane dye DIL (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) to 1 ml of PBS, add the dye to the cells after DAPI staining, stain at room temperature for 10 minutes, discard DIL after 10 minutes, wash the cells 3 times with PBS, 3 minutes each time, shake at room temperature. After the samples are prepared, take pictures one by one with a laser confocal microscope. The excitation wavelength of fluorescein FITC is 495nm, and the emission wavelength is 519nm. The excitation wavelength of nuclear dye DAPI is 358nm, and the emission wavelength is 461nm. The excitation wavelength of membrane dye DIL is 549nm, and the emission wavelength is 565nm.

[0039] like Figure 3 As shown, the results showed that tau-FITC co-localized with the cell membrane at 2 hours and localized in the cytoplasm at 4 hours.

[0040] Example 4. Silver staining analysis of differential bands

[0041] Observe HT22 cells until they grow well, discard the supernatant, and digest the cells with trypsin (1x10 7 Add 1 ml of trypsin to each cell and digest in a 37°C incubator for 2 minutes. After centrifugation, the cells are dispersed in three volumes of a 10% FBS DMEM (fetal bovine serum) culture medium, inoculated into a 10 cm dish, and cultured in a 37°C incubator with a 5% CO2 air volume concentration.

[0042] When the cell confluence reached 90%, 100 nmol / L -1 The cells were incubated with the tau-biotin obtained in Example 1 for 2 hours, and no tau-biotin was added as a control group. After the incubation, the supernatant was discarded and the cells were washed 3 times with PBS. 1 ml of cell lysis buffer was added to each dish, and the lysis buffer composition was 50 mmol / L -1 Tris (pH 7.4), 150 mmol / L-1 NaCl, 1% Triton X-100, 1% protease inhibitor (purchased from Sigma, catalog number P8340). Cells were scraped into EP tubes respectively and sonicated for 2 minutes. After sonication, the proteins were centrifuged at 15,000 g for 40 minutes at 4 °C. After centrifugation, the supernatant was transferred to a new tube, and 20 μl of Streptavidin Agarose (purchased from Thermo, catalog number 20353) was added to each tube for enrichment at room temperature for 4 hours. After enrichment, the supernatant was discarded by centrifugation. The Streptavidin Agarose was washed 3 times with lysis buffer, 10 minutes each time. After washing, 50 μl of 1x loading buffer (purchased from Beijing TransGen Biotech Co., Ltd., catalog number DL101-02) was added to the Streptavidin Agarose, and the proteins were denatured and detached at 95 °C for 5 minutes. The proteins were collected.

[0043] The control group and the tau-biotin group were loaded respectively. After electrophoresis, according to the operation guide steps of the ProteoSilver Silver Stain Kit (purchased from Sigma Corporation, catalog number PROTSIL1): fixation - ethanol washing - sensitization - water washing - silver staining - water washing - color development - termination. The silver-stained gel was photographed and saved with a chemiluminescence imager.

[0044] As Figure 4 shown, the experimental results showed that compared with the control group without tau-biotin, there were obvious differential bands in the tau-biotin group.

[0045] Example 5. Immunoblotting indicated that there was tau protein in the enriched proteins

[0046] The operation of the sample preparation process was the same as that in Example 4.

[0047] A 12.5% SDS-PAGE gel was prepared. The control group and the tau-biotin group were loaded respectively. After gel running, according to the immunoblotting operation process: membrane transfer - blocking - primary antibody incubation - membrane washing - secondary antibody incubation - membrane washing - development. The Bio rad electrophoresis apparatus and the Bio rad membrane transfer apparatus were purchased from Bio rad Company in the United States, and the Tau 4R antibody was purchased from CST Company.

[0048] As Figure 5 shown, the results showed that there was no band of tau in the control group, while there was a band that could incubate tau in the tau-biotin group. It indicated that tau-biotin could be enriched by Streptavidin Agarose.

[0049] Example 6. Mass spectrometry identification of differential proteins

[0050] The sample enrichment process was the same as that in Example 4.

[0051] After the enrichment was completed, the supernatant was discarded by centrifugation. The Streptavidin Agarose was washed with lysis buffer three times, 10 minutes each time. Then it was washed with 20 mmol / L -1 ammonium bicarbonate three times, 10 minutes each time. 10 μl of trypsin was added to the Streptavidin Agarose and enzymolysis was carried out overnight at 37 °C. The next day, it was centrifuged, and the enzymolyzed peptide segments were aspirated. After centrifugation at 16,000 g for 40 minutes, the supernatant was taken and transferred to a sample bottle for mass spectrometry detection.

[0052] The results in the following table show that more differential proteins can be identified after tau-biotin enrichment, and some of these differential proteins are membrane proteins, laying a foundation for the discovery of key receptor proteins mediating tau diffusion.

[0053] P68040 Q9DBG3 P26040 P35922 P62071 Q6PHZ2 Q8C0T5 Q9CQW9

[0054] In summary, the present invention synthesized a targeted tau-biotin enrichment tool and proved the effectiveness of this method.

[0055] Example 7

[0056] 1) The process and conditions were the same as those in Example 1;

[0057] 2) The process was the same as that in Example 4, except that after the cells were collected, they were incubated with a PEG5 cross-linking agent at a final concentration of 1 mmol / L -1 for 30 minutes. The operations of lysing the cells and enriching the proteins were the same as those in Example 4.

[0058] Silver staining proved that more differential proteins could be enriched by using the cross-linking agent.

[0059] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for enriching receptors that mediate tau protein diffusion on the cell surface, characterized in that: 1) Modify biotin on tau protein to obtain tau-biotin; 2) Co-incubate tau-biotin with cells, collect the cells for cell lysis, and then add 10 - 100 μl of Streptavidin Agarose to the lysed cell solution to enrich the target protein; The specific process of the method is as follows: Step 1) Dissolve 50 - 200 μg of tau protein in 100 μl of water; Prepare a final concentration of 5 - 20 mmol / L -1 EZ-Link™ Sulfo-NHS-LC-biotin aqueous solution; Take 1 - 5 μl of the aqueous solution of EZ-Link™ Sulfo-NHS-LC-biotin and add it to the dissolved tau protein. Mix well and react at 4 - 25 °C for 1 - 4 hours. Then add 1 - 10 μl of ammonium bicarbonate with a concentration of 1 mol / L to neutralize the unreacted EZ-Link™ Sulfo-NHS-LC-biotin to obtain a tau-biotin solution; -1 ​ Step 2) Add tau-biotin with a final concentration of 50 - 200 nmol / L to the cell-containing culture medium, incubate the cells for 1 - 4 hours. After the incubation, discard the supernatant and wash the cells with PBS; -1 ​ Add 1 ml of cell lysate to 1 - 5 x 10 7 cells; after lysis, centrifuge at 12,000 - 15,000 g for 20 - 40 minutes at 4°C; collect the supernatant by centrifugation, add 10 - 100 μl of Streptavidin Agarose purification resin to the supernatant and enrich at room temperature for 2 - 6 hours; after enrichment, centrifuge and discard the supernatant; add cell lysate to wash the Streptavidin Agarose; after washing, add 20 - 60 μl of 1x loading buffer to the Streptavidin Agarose, heat at 95°C for 5 - 10 minutes to denature and release the protein; collect the protein by centrifugation.

2. The method according to claim 1, wherein: The cells are nerve cells.

3. The method according to claim 2, characterized in that: The cells include one or two of mouse hippocampal neuron cells and microglial cells.

4. The method according to claim 1, characterized in that: After tau-biotin is co-incubated with cells in step 2), the cells are collected and a crosslinking agent is added, and incubated for 10 - 40 minutes. The crosslinking agent is one or two of BSP and PEG5, and the concentration of the crosslinking agent in the cells is 1 - 5 mg of the crosslinking agent per 1x10 7 cells; then the cells are collected again and cell lysis is performed to obtain the lysed cell lysate.

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