Antibody for specifically recognizing ATAD3A phosphorylation modification and application thereof

Abstract

The invention discloses an antibody for specifically recognizing ATAD3A phosphorylation modification and application of the antibody. According to the present invention, the research results show that the ATAD3A Ser321 site in senescent cells is the key phosphorylation site, and the antibody specifically recognizing ATAD3A Ser321 phosphorylation is obtained through immunization aiming at the site; the antibody has relatively strong specificity, and can be used for successfully detecting the expression of phosphorylated ATAD3A in senescent cells and the level of phosphorylated ATAD3A in tissues and organs of mice. According to the antibody, the market vacancy that a specific detection antibody for ATAD3A phosphorylation is lacked at present is filled, the antibody can be applied to Western blot and immunohistochemical detection of ATAD3A phosphorylation level in scientific research, and support is provided for mitochondrial related action mechanisms and anti-aging treatment research in the aging process.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and more specifically, relates to an antibody that specifically recognizes ATAD3A phosphorylation modification and its application. Background Technology

[0002] Mitochondria are essential for life because they play a crucial role in the most fundamental energy conversion processes within cells. With age, mitochondrial DNA (mtDNA) mutations, reduced mitochondrial production, instability of the electron transport chain (ETC) complex, and defects in mitochondrial dynamics lead to mitochondrial dysfunction, increasing levels of reactive oxygen species (ROS) and causing oxidative damage to nucleic acids, proteins, and lipids, ultimately accelerating aging. Therefore, exploring the mitochondrial-related mechanisms of action during aging is crucial for developing anti-aging therapies.

[0003] ATAD3A (the ATPase Family AAA Domain Containing 3A) is a mitochondrial protein that plays a crucial role in regulating mitochondrial dynamics, homeostasis, metabolism, and its interaction with the endoplasmic reticulum. ATAD3A is essential for maintaining mitochondrial homeostasis and plays a significant role in tumors and age-related neurodegenerative diseases. Researchers have found that acetylation of ATAD3A plays a key role in neurodegeneration in Huntington's disease (HD) patients, suggesting that ATAD3A may be associated with aging. However, the role of ATAD3A in cellular senescence remains unclear. The protein function of ATAD3A is regulated by its post-translational modifications, but research on these modifications is extremely limited. Therefore, further investigation into the mechanisms underlying ATAD3A post-translational modifications and aging, and the development of related diagnostic products, are of great significance. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide an antibody that specifically recognizes ATAD3A phosphorylation modification and its application.

[0005] A second object of the present invention is to provide applications of the antibody.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution:

[0007] This invention discovered that ATAD3A undergoes phosphorylation modification in senescent cells. Mass spectrometry detected that the Ser321 site (i.e., serine residue at position 321 of the ATAD3A protein) is a key phosphorylation site. The ATAD3A Ser321 site can serve as a biomarker for detecting senescent cells / senescence levels. Furthermore, this invention designed a phosphorylated peptide targeting this site as an immunogen to obtain an antibody that specifically recognizes ATAD3A Ser321 phosphorylation. This antibody can specifically recognize ATAD3A peptides phosphorylated at Ser321, but cannot recognize non-phosphorylated ATAD3A peptides. It can recognize changes in phosphorylation levels of wild-type ATAD3A, but cannot recognize ATAD3A Ser321 mutations that result in non-phosphorylated mutants, indicating that the antibody has strong specificity. The antibody can successfully detect the expression of phosphorylated ATAD3A in senescent cells and the level of phosphorylated ATAD3A in mouse tissues and organs.

[0008] Therefore, the present invention first provides the application of reagents for detecting the phosphorylation level of ATAD3A protein in the preparation of products for detecting senescent cells.

[0009] This invention also provides the application of reagents for detecting ATAD3A protein phosphorylation levels in the preparation of products for detecting aging-related diseases.

[0010] Specifically, the key phosphorylation site of the ATAD3A protein is the ATAD3A Ser321 site (i.e., serine at the 321st site of the ATAD3A protein); that is, the reagent for detecting the phosphorylation level of the ATAD3A protein is a reagent for detecting the phosphorylation level of the ATAD3A Ser321 site (i.e., serine at the 321st site of the ATAD3A protein).

[0011] Furthermore, the age-related disease is progeria.

[0012] Furthermore, the reagent used to detect the phosphorylation level of ATAD3A protein is an antibody that recognizes ATAD3A phosphorylation modification.

[0013] This invention provides an antibody that recognizes ATAD3A phosphorylation modification. The antibody is prepared from a naked peptide linker protein of EGVVLS(p)PSLEA, whose amino acid sequence is shown in SEQ ID No. 2, as an immunogen. The "(p)" in the amino acid sequence "EGVVLS(p)PSLEA" indicates that the "S" site of the amino acid has been phosphorylated.

[0014] Furthermore, the carrier protein is hemocyanin (KLH).

[0015] Furthermore, the antibody is prepared by immunizing animals with KLH-EGVVLS(p)PSLEA polypeptide as an immunogen, extracting serum and purifying the antibody to obtain the antibody.

[0016] Preferably, the animal is a New Zealand white rabbit.

[0017] Preferably, the immunization is performed in 5 stages. Specifically, the first immunization involves mixing the immunogen with Freund's complete adjuvant and injecting it subcutaneously at multiple points; the second to fifth booster immunizations involve mixing the immunogen with Freund's incomplete adjuvant and injecting it subcutaneously at multiple points.

[0018] Preferably, the mixture ratio of the immunogen to Freund's complete adjuvant or Freund's incomplete adjuvant is 1:1 (g / mL).

[0019] This invention provides the application of the antibody in the preparation of products for detecting senescent cells.

[0020] The present invention also provides the use of the antibody in the preparation of products for detecting aging-related diseases.

[0021] Furthermore, the age-related disease is progeria.

[0022] The present invention provides a kit for detecting aging and / or aging-related diseases, containing the antibodies described above.

[0023] Furthermore, the kit also contains reagents for immunoblotting detection.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This invention discovered that ATAD3A undergoes phosphorylation modification in senescent cells, with the ATAD3A Ser321 site being a key phosphorylation site. The ATAD3A Ser321 site can serve as a biomarker for detecting senescent cells / senescence levels. This invention designs a phosphorylated peptide targeting this site as an immunogen to obtain an antibody that specifically recognizes ATAD3A Ser321 phosphorylation through immunization. This antibody can specifically recognize ATAD3A peptides phosphorylated at Ser321, but cannot recognize non-phosphorylated ATAD3A peptides. It can recognize changes in phosphorylation levels of wild-type ATAD3A, but cannot recognize ATAD3A Ser321 mutations that result in non-phosphorylated mutants, indicating that the antibody has strong specificity. The antibody can successfully detect the expression of phosphorylated ATAD3A in senescent cells and the level of phosphorylated ATAD3A in mouse tissues and organs. This invention fills the market gap of lacking specific detection antibodies for ATAD3A phosphorylation. The antibody can be used in scientific research to detect ATAD3A phosphorylation levels using Western blot and immunohistochemistry, providing support for research on mitochondrial-related mechanisms of action during aging and anti-aging treatments, as well as for future research related to ATAD3A phosphorylation. Attached Figure Description

[0026] Figure 1 The results show that Ser321 is a key phosphorylation site for ATAD3A; where A is the β-gal staining result of senescent cells, B is the phosphorylation level of ATAD3A in senescent cells, C is the potential phosphorylation site of ATAD3A, and D is the phosphorylation level of ATAD3A after mutation of Ser289 and Ser321.

[0027] Figure 2 This study presents antibodies that specifically recognize ATAD3A phosphorylation modification and their application results. A shows a flowchart for obtaining ATAD3ASer321 phosphorylated antibodies; B shows a Dot blot experiment verifying the specificity of ATAD3ASer321 phosphorylated antibodies; C shows the results of ATAD3A Ser321 phosphorylated antibodies recognizing wild-type ATAD3A and non-phosphorylated mutants; D shows Western blot detection of ATAD3A phosphorylation levels in different aging cell models; E shows immunostaining results of different tissue sections from normal aging mice and HGPS progeria mice; and F shows Western blot detection of ATAD3A phosphorylation levels in normal aging mice and HGPS progeria mice. Detailed Implementation

[0028] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0029] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0030] Example 1: Ser321 is a key phosphorylation site of ATAD3A.

[0031] To investigate the role of the mitochondrial protein ATAD3A in the aging process, we constructed different types of senescent cells and stained them with β-gal. Senescent cells showed a significant blue tint. Figure 1 A). ATAD3A phosphorylation levels were detected using a broad-spectrum phosphorylation antibody, and a significant increase in ATAD3A phosphorylation levels was found in senescent cells. Figure 1 B). Further mass spectrometry identification revealed two potential ATAD3A phosphorylation sites, Ser289 and Ser321. Figure 1 C). Mutations at these two sites inhibit phosphorylation at these sites. The Ser321 site mutation significantly suppressed ATAD3A phosphorylation levels, indicating that Ser321 is a key phosphorylation site for ATAD3A. Figure 1 D), The amino acid sequence of the ATAD3A Ser321 site is shown in SEQ ID No. 1.

[0032] Example 2: Preparation of antibodies that specifically recognize ATAD3A phosphorylation modification

[0033] Example 1 shows that the Ser321 site is a key phosphorylation site for ATAD3A. Based on this key site, we designed a phosphorylated peptide to immunize New Zealand white rabbits, and collected rabbit serum for affinity purification to obtain the ATAD3A Ser321 phosphorylated antibody. The specific method is as follows:

[0034] Synthesis of KLH-EGVVLS(p)PSLEA peptide: We conjugated the synthesized naked peptide EGVVLS(p)PSLEA (SEQ ID No. 2) with KLH to form the KLH-EGVVLS(p)PSLEA peptide. KLH solid was mixed with immunogen buffer (800mM sodium chloride + 100mM sodium dihydrogen phosphate + 100mM EDTA) to prepare a KLH solution with a concentration of 10 mg / mL. 10 mg / mL KLH solution was then mixed with 250 μL of 10 mg / mL SMCC to a concentration of 10 mg / mL, and incubated at 25°C for 2 hours. The solution was then dialyzed overnight in immunogen buffer. After dialyzing, the KLH solution was diluted with immunogen conjugation buffer to a concentration of 4 mg / mL. Dilute 100×TCEP to 1×TCEP with 125 μL of immunogen buffer to dissolve 1 mg of naked peptide (EGVVLS(p)PSLEA). Add 100 μL of 0.2 M disodium hydrogen phosphate to adjust the pH to 7.0 and 250 μL of KLH (4 mg / mL) for the reaction. After incubating at 37°C for 4 hours or at 4°C overnight, run a gel electrophoresis to check for successful coupling.

[0035] New Zealand White Rabbit Peptide Injection: Synthetic KLH-EGVVLS(p)PSLEA peptide was injected into New Zealand White rabbits. A total of 5 immunizations were administered, with an interval of 14 days between each immunization. Blood samples were collected 7 days after the 4th immunization. For the first immunization: 300 μg of immunogen was mixed evenly with the corresponding volume of Freund's complete adjuvant and injected subcutaneously at multiple sites. For the 2nd to 5th booster immunizations: 150 μg of immunogen was mixed evenly with the corresponding volume of Freund's incomplete adjuvant and injected subcutaneously at multiple sites.

[0036] Antibody purification: 1. Centrifuge the serum at 10,000 rpm, 4°C for 5 minutes (twice). Collect the supernatant and transfer it to a clean 50 mL centrifuge tube, discarding the precipitate. Remove the ProA column from the refrigerator and allow it to stand at room temperature for a few minutes. Then, pass 5 column volumes of dihydrochloric acid through the column, followed by equal volume of 20 mM sodium phosphate solution (pH 7.0) to equilibrate the column. Pass the centrifuged supernatant through the ProA column. After approximately 20 mL of serum has passed through the column, wash the column with 10 column volumes of 20 mM sodium phosphate solution (pH 7.0). In a 15 mL collection tube, pre-add 0.2 column volumes (0.4 mL) of 1M Tris-HCl (pH 9.0), then elute the column with 4 column volumes (8 mL) of 100 mM glycine (pH 2.5) and collect the eluent. Immediately after elution, invert the tube to mix the eluent with the neutralization solution to obtain the antibody. Figure 2 A is a flowchart for obtaining a phosphorylated antibody that specifically recognizes ATAD3ASer321.

[0037] To verify antibody specificity, we used a Dot blot experiment to add the synthesized phosphorylated peptide BSA-ATAD3A(p) and non-phosphorylated peptide BSA-ATAD3A to a PVDF membrane. The Dot blot assay was then used to detect the specificity of the phosphorylated ATAD3A (p-ATAD3A) antibody. The results showed that the antibody specifically recognized the ATAD3A peptide phosphorylated at Ser321, but not the non-phosphorylated ATAD3A peptide, indicating that the antibody has good specificity. Figure 2 B). To further verify the antibody's specificity, we mutated the ATAD3ASer321 site to a non-phosphorylated mutant. The results showed that our prepared antibody could recognize changes in the phosphorylation level of wild-type ATAD3A, but could not recognize the non-phosphorylated mutant, further demonstrating the high specificity of this antibody. Figure 2 C).

[0038] Example 3

[0039] To test the application of this antibody in scientific research, this invention uses an ATAD3A Ser321 phosphorylation antibody to detect the expression of phosphorylated ATAD3A in senescent cells and the level of phosphorylated ATAD3A in mouse tissues and organs.

[0040] ATAD3A phosphorylation levels in different senescent cell models were detected by Western blot. Normal cells: WI-38P4 / MEF P3 / MSC-WT / A549DMSO; senescent cells: WI-38P45 / MEF P8 / MSC-HGPS / A549Dox. Results showed that ATAD3A phosphorylation levels were elevated in senescent cells. Figure 2 D).

[0041] We further performed immunostaining on different tissue sections from young mice (Y), normally aging mice (N), and HGPS progeria mice (H). The results showed that the phosphorylation level of ATAD3A was significantly increased in the tissues of normally aging mice and HGPS progeria mice. Figure 2 E). Western blot analysis also confirmed this result. Figure 2 F).

[0042] The above results demonstrate that the ATAD3A Ser321-specific phosphorylation antibody provided by this invention can successfully detect the expression of phosphorylated ATAD3A in senescent cells and the level of phosphorylated ATAD3A in mouse tissues and organs. It also indicates that the ATAD3A Ser321 site can serve as a biomarker for detecting senescent cells / senescence levels. This antibody, which specifically recognizes ATAD3A phosphorylation modification, fills a market gap and can be applied to Western blot and immunohistochemical detection of ATAD3A phosphorylation levels in scientific research.

Claims

1. Use of a reagent for detecting the phosphorylation level of ATAD3A protein in the preparation of a product for detecting senescent cells.

2. Use of a reagent for detecting the phosphorylation level of ATAD3A protein in the preparation of a product for detecting senescence-related diseases.

3. Use according to claim 2, characterized in that, The senescence-related disease is progeria.

4. Use according to claim 1 or 2, characterized in that, The reagent for detecting the phosphorylation level of ATAD3A protein is an antibody recognizing the phosphorylation modification of ATAD3A.

5. An antibody that recognizes a phosphorylation modification of ATAD3A, characterized in that, The naked peptide linker carrier protein with the amino acid sequence "EGVVLS(p)PSLEA" is used as an immunogen.

6. The antibody of claim 5, wherein The carrier protein is hemocyanin.

7. Use of the antibody of claim 5 or 6 in the preparation of a product for detecting senescent cells.

8. Use of the antibody of claim 5 or 6 in the preparation of a product for detecting senescence-related diseases.

9. A kit for detecting aging and / or an aging-related disease, characterized by, The antibody of claim 5 or 6 is contained.

10. The kit of claim 9, wherein A reagent for immunoblotting detection is also contained.

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