Institute of Anthropology, University Mainz , Anselm-Franz-von-Bentzel-Weg 7, Mainz 55128, Germany.
J Proteome Res. 2013 Dec 6;12(12):5370-82. doi: 10.1021/pr400228c. Epub 2013 Sep 27.
We investigated possible associations between sequence evolution of mammalian sperm proteins and their phosphorylation status in humans. As a reference, spermatozoa from three normozoospermic men were analyzed combining two-dimensional gel electrophoresis, immunoblotting, and mass spectrometry. We identified 99 sperm proteins (thereof 42 newly described) and determined the phosphorylation status for most of them. Sequence evolution was studied across six mammalian species using nonsynonymous/synonymous rate ratios (dN/dS) and amino acid distances. Site-specific purifying selection was assessed employing average ratios of evolutionary rates at phosphorylated versus nonphosphorylated amino acids (α). According to our data, mammalian sperm proteins do not show statistically significant sequence conservation difference, no matter if the human ortholog is a phosphoprotein with or without tyrosine (Y) phosphorylation. In contrast, overall phosphorylation of human sperm proteins, i.e., phosphorylation at serine (S), threonine (T), and/or Y residues, associates with above-average conservation of sequences. Complementary investigations suggest that numerous protein-protein interactants constrain sequence evolution of sperm phosphoproteins. Although our findings reject a special relevance of Y phosphorylation for sperm functioning, they still indicate that overall phosphorylation substantially contributes to proper functioning of sperm proteins. Hence, phosphorylated sperm proteins might be considered as prime candidates for diagnosis and treatment of reduced male fertility.
我们研究了哺乳动物精子蛋白的序列进化与其在人类中的磷酸化状态之间可能存在的联系。作为参考，我们结合二维凝胶电泳、免疫印迹和质谱分析了来自三名正常精子的精子。我们鉴定了 99 种精子蛋白（其中 42 种是新描述的），并确定了它们中的大多数的磷酸化状态。我们使用非同义/同义比率（dN/dS）和氨基酸距离在六个哺乳动物物种中研究了序列进化。使用磷酸化与非磷酸化氨基酸的进化速率平均比值（α）评估了特定部位的纯化选择。根据我们的数据，哺乳动物精子蛋白没有表现出统计学上显著的序列保守性差异，无论人类直系同源物是否为具有或不具有酪氨酸（Y）磷酸化的磷酸蛋白。相比之下，人类精子蛋白的整体磷酸化，即丝氨酸（S）、苏氨酸（T）和/或 Y 残基的磷酸化，与序列的平均以上保守性相关。补充研究表明，许多蛋白质-蛋白质相互作用物限制了精子磷酸蛋白的序列进化。尽管我们的发现否定了 Y 磷酸化对精子功能的特殊重要性，但它们仍然表明整体磷酸化对精子蛋白的正常功能有很大贡献。因此，磷酸化的精子蛋白可能被视为诊断和治疗男性生育力降低的主要候选物。
