Specific antibody for detecting or targeting ALDOA-K13 succinylation modification and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to a specific antibody for detecting or targeting ALDOA-K13 succinylation modification and application of the specific antibody. The specific antibody for detecting or targeting ALDOA-K13 succinylation modification can be specifically combined with a succinylation modification site of 13th lysine of human ALDOA protein, and is not combined with ALDOA of an unmodified type or other modified types. The invention provides a specific antibody for detecting or targeting ALDOA-K13 succinylation modification for the first time, and fills the blank of existing detection tools. The antibody not only has high specificity and sensitivity, but also can be used for developing a novel tumor diagnosis and prognosis detection method.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a specific antibody for detecting or targeting ALDOA-K13 succinylation modification and its application. Background Technology

[0002] Aldolase A (ALDOA) ​​is a key enzyme in the glycolysis pathway, primarily responsible for catalyzing the cleavage of fructose-1,6-bisphosphate into glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. In addition to its classic glycolytic function, recent studies have revealed that ALDOA also participates in various non-classical biological processes, including cytoskeleton remodeling, endocytosis, DNA repair, and tumorigenesis and development, indicating its multifunctional role in cellular physiology and pathology.

[0003] Previous studies have shown that ALDOA is abnormally highly expressed in various malignant tumors, including oral squamous cell carcinoma, liver cancer, lung cancer, pancreatic cancer, and osteosarcoma, and is closely related to poor patient prognosis. Inhibiting ALDOA can reduce glycolytic activity and suppress HIF-1α signaling, thereby slowing tumor cell growth. However, although existing small molecule ALDOA inhibitors (such as UM0112176 and TDZD-8) have shown certain inhibitory effects in vitro and in vivo, none have yet entered the effective clinical application stage due to insufficient target specificity, significant toxic side effects, and limited clinical translation.

[0004] Post-translational modifications (PTMs) are important molecular mechanisms regulating protein function. Among them, succinylation, a recently studied lysine modification, can significantly alter the charge state and spatial conformation of proteins, thereby affecting their activity, stability, subcellular localization, and molecular interaction networks. While multiple lysine post-translational modification sites have been reported for ALDOA, no studies have yet revealed the precise function of succinylation in tumorigenesis and development. Summary of the Invention

[0005] In order to overcome at least one of the technical problems existing in the prior art, the present invention provides a specific antibody for detecting or targeting ALDOA-K13 succinylation modification and its application.

[0006] The technical solution of the present invention is as follows: The present invention first provides a specific antibody for detecting or targeting ALDOA-K13 succinylation modification, which can specifically bind to the succinylation modification site of lysine 13 of human ALDOA protein, without binding to unmodified or other modified ALDOA. The antibody has the sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2.

[0007] The amino acid sequence of SEQ ID NO. 1 is as follows: ALTPEQKKELSDIC; the amino acid sequence of SEQ ID NO. 2 is as follows: YPALTPEQKKELSDC. Preferably, the antibody is selected from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, antibody fragments (Fab, scFv), nanobodies, or bispecific antibodies.

[0008] The present invention also provides the application of the above-mentioned specific antibody for detecting or targeting the ALDOA-K13 succinylation site in the preparation of a test kit for diagnosing tumors or predicting patient prognosis.

[0009] The present invention also provides the application of the above-mentioned specific antibody for detecting or targeting the ALDOA-K13 succinylation site in the preparation of a detection reagent for monitoring tumor recurrence, metastasis risk or drug responsiveness.

[0010] Beneficial Effects: This invention provides, for the first time, a specific antibody for detecting or targeting ALDOA-K13 succinylation modification, filling a gap in existing detection tools. This antibody not only possesses high specificity and sensitivity but can also be used to develop novel methods for tumor diagnosis and prognosis. Furthermore, the proposed strategy of combining the antibody with small molecule drugs has shown significant synergistic anti-tumor effects in animal experiments, demonstrating broad application prospects. Attached Figure Description

[0011] Figure 1 The image shows the specificity detection results of the ALDOA-K13 succinylated antibody.

[0012] Figure 2 This is a clinical efficacy diagram of ALDOA-K13 aberrant succinylation modification in esophageal cancer.

[0013] Figure 3 A diagram illustrating the regulation of esophageal cancer cell division and proliferation by ALDOA-K13 succinylation and acetylation.

[0014] Figure 4 A diagram illustrating how ALDOA-K13 succinylation and acetylation modifications regulate the migration and invasion of esophageal cancer cells.

[0015] Figure 5 ALDOA-K13 succinylation enhances its own protein stability by inhibiting ubiquitination.

[0016] Figure 6 The free property diagram of ALDOA-K13 was altered by succinylation and acetylation modifications. Detailed Implementation

[0017] The present invention will be further described in detail below with reference to specific embodiments, but the embodiments do not limit the present invention in any way.

[0018] First, different doses of ALDOA-K13 succinylated modified peptides and unmodified peptides were immobilized on a solid-phase membrane, incubated with antibody, followed by the addition of enzyme-labeled secondary antibody and chemiluminescent substrate. The binding of the peptides and antibodies was then detected. Dot hybridization results showed that the ALDOA-K13 succinylated specific antibody showed virtually no binding to the negative unmodified peptide but exhibited strong binding to the positive peptide, even at a concentration of 1 ng of positive peptide, indicating good specificity and sensitivity of the customized antibody. Figure 1 (A) To further detect the specificity of the ALDOA-K13 succinylated modified antibody, this invention used a single ALDOA-K13 succinylated antibody and 20-fold concentration of succinylated modified peptide 1, an antibody and 20-fold concentration of succinylated modified peptide 2, and an antibody and 20-fold concentration of unmodified peptide for Western blotting detection on purified GST-Aldolase protein from human embryonic kidney cells HEK293T, human esophageal cancer cells (KYSE30, KYSE150, KYSE510, and TE1), and prokaryotic cells. The results showed that ALDOA-K13 succinylation modification is widely present in human embryonic kidney cells and various types of esophageal cancer cell lines. The peptide detection results also further verified the specificity of the ALDOA-K13 succ antibody. Figure 1 (B in the middle).

[0019] To investigate ALDOA and its clinical efficacy in esophageal cancer, this invention first analyzed the expression of ALDOA in paired samples of cancerous and adjacent normal tissues from 30 esophageal cancer patients. Immunohistochemical staining results showed that, compared with normal adjacent tissues, the ALDOA-K13 succinylation level in esophageal cancer patients was significantly increased in cancerous tissues. Figure 2 (A in the original text). Then, this invention further evaluated the clinical significance of aberrant ALDOA-K13 succinylation in esophageal cancer prognostic prediction. KM curve survival analysis showed that ALDOA-K13 succinylation levels were closely related to poor prognosis in esophageal cancer patients; high succinylation levels in esophageal cancer patients significantly shortened overall survival and disease-free survival (DFS). Figure 2 (BD in the middle).

[0020] To elucidate the biological functions of ALDOA-K13 aberrant succinylation and acetylation modifications in esophageal cancer and whether there is a competitive relationship between them, this invention used site-directed mutagenesis to construct K13E (simulating succinylation), K13Q (simulating acetylation), and K13R (simulating the unmodified state) at the K13 site. These mutants were then overexpressed in endogenous ALDOA knockdown esophageal cancer cells. A series of cellular function experiments showed that, compared with wild-type ALDOA, the succinylation-mimicking K13E exhibited stronger proliferative and invasive abilities. Figure 3 ), while the oncogenic ability of K13Q modified with acetylation was significantly lower than that of wild-type ( Figure 4 ).

[0021] To investigate whether ALDOA-K13 succinylation affects protein stability, this invention used cycloheximide (CHX) for different time periods in esophageal cancer cells expressing ALDOA-K13 succinylation-modified mutant K13E, unmodified mutant K13R, and ALDOA wild-type WT. Western blotting was then performed to detect the protein. The results showed that ALDOA-K13 succinylation prolonged the half-life of the ALDOA protein. Figure 5 Further ubiquitination experiments showed that ALDOA-K13succ prolongs the half-life of ALDOA protein by inhibiting ubiquitination degradation (A in the text). Figure 5 (B in the middle).

[0022] This invention utilizes protein immunoprecipitation and competitive peptide elution to obtain purified ALDOA-K13-mimicked proteins, which are then detected using an aldolase activity assay kit. The results show that neither the succinylated ALDOA-K13E nor the acetylated ALDOA-K13Q alters the aldolase activity. Figure 6 (A in the original text). Based on the biological characteristics of ALDOA, this invention hypothesizes that post-translational modification of ALDOA-K13 may affect its dissociation and activation from the cytoskeleton. To verify this hypothesis, this invention first overexpressed the aforementioned mimic mutant in esophageal cancer cells, then used digitalis saponins to permeate cells to obtain free proteins in the cytoplasm, and simultaneously used a soluble / cytoskeleton component separation method to obtain the corresponding cellular components, followed by Western blotting detection. Experimental results show that, compared with wild-type ALDOA, succinylation of ALDOA-K13 regulates its dissociation from F-actin; succinylation modification K13E significantly enhances its freeness and activates it, while acetylation modification K13Q significantly inhibits this process. Figure 6 (B in 6 and C in 6).

Claims

1. A specific antibody for detecting or targeting ALDOA-K13 succinylation modification, characterized in that, This antibody can specifically bind to the succinylation modification site of lysine at position 13 of human ALDOA protein, without binding to unmodified or other modified ALDOA. The antibody has the sequence shown in SEQ ID NO. 1 or SEQ ID NO.

2.

2. The specific antibody for detecting or targeting ALDOA-K13 succinylation modification according to claim 1, characterized in that, The antibody is selected from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, antibody fragments, nanobodies, or bispecific antibodies.

3. The use of the specific antibody according to any one of claims 1 to 2 for detecting or targeting the ALDOA-K13 succinylation site in the preparation of a diagnostic kit for diagnosing tumors or predicting patient prognosis.

4. The use of the specific antibody according to any one of claims 1 to 2 for detecting or targeting the ALDOA-K13 succinylation site in the preparation of a diagnostic reagent for monitoring tumor recurrence, metastasis risk, or drug responsiveness.

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