Yu Heguo, Diao Hua, Wang Chunmei, Lin Yan, Yu Fudong, Lu Hui, Xu Wei, Li Zheng, Shi Huijuan, Zhao Shimin, Zhou Yuchuan, Zhang Yonglian
From the NPFPC Key Laboratory of Contraceptives and Devices, Shanghai Institute of Planned Parenthood Research, Institutes of Reproduction and Development.
Shanghai Key Laboratory for Molecular Andrology, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Mol Cell Proteomics. 2015 Apr;14(4):1009-23. doi: 10.1074/mcp.M114.041384. Epub 2015 Feb 13.
Male infertility is a medical condition that has been on the rise globally. Lysine acetylation of human sperm, an essential posttranslational modification involved in the etiology of sperm abnormality, is not fully understood. Therefore, we first generated a qualified pan-anti-acetyllysine monoclonal antibody to characterize the global lysine acetylation of uncapacitated normal human sperm with a proteomics approach. With high enrichment ratios that were up to 31%, 973 lysine-acetylated sites that matched to 456 human sperm proteins, including 671 novel lysine acetylation sites and 205 novel lysine-acetylated proteins, were identified. These proteins exhibited conserved motifs XXXKYXXX, XXXKFXXX, and XXXKHXXX, were annotated to function in multiple metabolic processes, and were localized predominantly in the mitochondrion and cytoplasmic fractions. Between the uncapacitated and capacitated sperm, different acetylation profiles in regard to functional proteins involved in sperm capacitation, sperm-egg recognition, sperm-egg plasma fusion, and fertilization were observed, indicating that acetylation of functional proteins may be required during sperm capacitation. Bioinformatics analysis revealed association of acetylated proteins with diseases and drugs. Novel acetylation of voltage-dependent anion channel proteins was also found. With clinical sperm samples, we observed differed lysine acetyltransferases and lysine deacetylases expression between normal sperm and abnormal sperm of asthenospermia or necrospermia. Furthermore, with sperm samples impaired by epigallocatechin gallate to mimic asthenospermia, we observed that inhibition of sperm motility was partly through the blockade of voltage-dependent anion channel 2 Lys-74 acetylation combined with reduced ATP levels and mitochondrial membrane potential. Taken together, we obtained a qualified pan-anti-acetyllysine monoclonal antibody, analyzed the acetylproteome of uncapacitated human sperm, and revealed associations between functional protein acetylation and sperm functions.
男性不育是一种在全球范围内呈上升趋势的医学病症。人类精子的赖氨酸乙酰化是一种参与精子异常病因的重要翻译后修饰，但目前尚未完全了解。因此，我们首先制备了一种合格的泛抗乙酰赖氨酸单克隆抗体，采用蛋白质组学方法来表征未获能的正常人类精子的整体赖氨酸乙酰化情况。通过高达31%的高富集率，鉴定出了973个与456种人类精子蛋白匹配的赖氨酸乙酰化位点，其中包括671个新的赖氨酸乙酰化位点和205种新的赖氨酸乙酰化蛋白。这些蛋白呈现出保守基序XXXKYXXX、XXXKFXXX和XXXKHXXX，被注释为在多个代谢过程中发挥作用，并且主要定位于线粒体和细胞质部分。在未获能精子和获能精子之间，观察到了与精子获能、精卵识别、精卵质膜融合及受精相关的功能蛋白的不同乙酰化谱，这表明在精子获能过程中可能需要功能蛋白的乙酰化。生物信息学分析揭示了乙酰化蛋白与疾病和药物的关联。还发现了电压依赖性阴离子通道蛋白的新乙酰化情况。利用临床精子样本，我们观察到在正常精子与弱精子症或死精子症的异常精子之间，赖氨酸乙酰转移酶和赖氨酸脱乙酰酶的表达存在差异。此外，用表没食子儿茶素没食子酸酯损伤精子样本以模拟弱精子症，我们观察到精子活力的抑制部分是通过阻断电压依赖性阴离子通道2的赖氨酸-74乙酰化，同时伴有ATP水平降低和线粒体膜电位下降。综上所述，我们获得了一种合格的泛抗乙酰赖氨酸单克隆抗体，分析了未获能人类精子的乙酰蛋白质组，并揭示了功能蛋白乙酰化与精子功能之间的关联。
