Preparation of succinylated antibody to the K89 position of AIF and study of the interacting proteins with AIF
TANG Xilian1,3, LI Ning1, YU Huajun1,4, HE Liuyan1,3, HUANG Qianqian1,3, CHEN Rongrong1,3, CHEN Lili1,2, WU Jun2,3, ZHANG Haitao1,3,4
1. Medical University, Zhanjiang 524023; 2. Institute of Respiratory Diseases, Affiliated Hospital of Guangdong Medical University, Zhanjiang 524023; 3. Key Laboratory of Peptide and Protein Research and Application, Guangdong Medical University, Zhanjiang 524023; 4. Laboratory Animal Centre, Guangdong Medical University, Zhanjiang 524023
Abstract：Objective Our research team has previously found that AIF protein exhibits increased levels of lysine 89 succinylation modification in non-smoking female lung adenocarcinoma patients. Our team aims to prepare an antibody that can recognize the succinylation modification of lysine 89 residue on AIF, which will be used to detect AIF succinylation at this site. Methods Bioinformatics analysis was conducted using online databases to analyze the physicochemical properties, transmembrane structure, signal peptide, protein structure, and succinylation sites of AIF protein. Based on the amino acid sequence of AIF, three peptides were synthesized, including the lysine 89 residue (K89) site. Among them, two peptides containing AIF K89 were succinylated. The three synthesized peptides were coupled with KLH and then immunized in rabbits to prepare antibodies specifically recognizing AIF K89 succinylation modification. The antibody efficacy and specificity were evaluated using ELISA, Dot blot, Western blotting, and immunohistochemistry. Gel digestion and high-resolution mass spectrometry were used to analyze the proteins interacting with AIF. Results Bioinformatics analysis revealed that succinylation modification of AIF K89 led to a change in the α-helical structure formed by amino acid residues 79-99 of AIF: it transformed from a longer α-helix into two shorter α-helices with a gap. The two synthesized succinylated peptides and one non-succinylated peptide successfully generated corresponding antibodies in rabbits. The antibody titers in the immune sera detected by ELISA were 1: 486, 000, 1: 162, 000, and 1: 18, 000, respectively. The prepared antibody recognizing AIF K89 succinylation modification exhibited 27 times higher efficacy in recognizing succinylated peptides compared to non-succinylated peptides, as determined by antibody Dot blot. Western blotting results showed that the AIF succinylation antibody specifically recognized the AIF K89 succinylation modification site. Mass spectrometry identified proteins interacting with AIF, including XIAP/BIRC4, EIF3G, and PRELID1. Conclusion Bioinformatics analysis suggests that succinylation modification of AIF K89 affects the structure of AIF protein. The prepared AIF K89 succinylation antibody can specifically recognize AIF K89 succinylation modification, providing a foundation for further investigation into the mechanism of AIF succinylation.
汤喜连, 李宁, 余华军, 何柳燕, 黄倩倩, 陈融融, 陈丽丽, 伍俊, 张海涛. AIF的K89琥珀酰化抗体制备及与AIF的互作蛋白研究[J]. 湖南师范大学学报(医学版), 2023, 20(6): 20-27.
TANG Xilian, LI Ning, YU Huajun, HE Liuyan, HUANG Qianqian, CHEN Rongrong, CHEN Lili, WU Jun, ZHANG Haitao. Preparation of succinylated antibody to the K89 position of AIF and study of the interacting proteins with AIF. HuNan ShiFan DaXue XueBao(YiXueBan), 2023, 20(6): 20-27.
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