Proteomic Analysis Of Lysine Acetylation In Human Sperm

As a dynamic and reversible post-translational modification, protein lysine acetylation is becoming one of the importmant post-modification. During the past half century, the researches on lysine acetylation mainly focused on histones and its role in transcription regulation. However, the research about none-histone lysine acetylation was paid attention in the last ten years. With the development of high-throughput proteomics technology, lysine acetylation has been studied a lot in eukaryotes, yeast, plant, Toxoplasma, Drosophila, rodent, human cells and so on. Therefore, a flood of transcription factors, cytoskeletal proteins, metabolic enzyme and other non-histone proteins were identified as (de)acetylation substrates and lots of acetylated non-histone proteins were found to function as regulators of many metabolic processes, such as glycolysis, tricarboxylic acid cycle, etc. Nevertheless, the composition and function of non-histone lysine acetylation in gametes remain unknown.With the advanced mass spectrometry and full-fledged proteomics research platform, the human sperm proteome has been studied in our labrotory before. And, a lot of deacetylases were identified in this proteome. Through immunoblot and immunofluorescence, we found many lysine acetylated proteins exist in capacitated human sperm. In physiological condition, only capacitated sperm can complete fertilization. In order to get the data of lysine acetylproteome in capacitated human sperm, we established the lysine acetylated peptides enrichment method. After immunopurification enrichment of acetylpeptides with anti-acetyllysine antibody and high-throughput liquid chromatography-tandem mass spectrometry identification, we characterized1206lysine acetylated sites, corresponding to576lysine acetylated proteins in human capacitated sperm. Bioinformatics analysis showed these proteins were widely located in mitochondrion (153genes), nucleoplasm (25genes), flagellum (21genes), cytosol (13genes) and plasma membrane (8genes). We also found that lysine acetylated proteins were enriched in acrosome vesicle and zona pellucida receptor complex. These indicated acetylation maybe associated with sperm functions, including motility, capacitation, acrosome reaction and sperm-egg interaction. Many acetylated proteins were found as components of several complexes such as espiratory chain complex I, ATP synthase complex, and proteasome complex. Inhibition of lysine deacetylases, the histone deacetylases, by trichostatin A and nicotinamide, could significantly suppress sperm motility. And in vitro fertilization inhibition assay by different concentrations of anti-acetyllysine antibody showed essential roles of lysine acetylation in fertilization. Therefore, lysine acetylation is expected to be an important regulatory mechanism for sperm functions.In summary, we constructed the lysine acetylproteome of capacitated human sperm and found a great number of lysine acetylated proteins associated with sperm motility and fertility. It could be a valuable resource for the further study of male fertility and provide the possible molecular targets for the diagnosis of male infertility.