An evolutionary and proteomic search for human gamete recognition proteins that mediate zona pellucida dissolution

项目摘要 确定相互作用的雄性-雌性生殖蛋白是分子理解的必要条件 受精的过程。这项R21提案的目标是利用DNA识别技术识别一组人类配子识别蛋白。 基因组学和蛋白质组学方法，然后进行进化引导的计算生物信息学 寻找鲍鱼精子溶素的同源物。从三维蛋白质晶体学 哺乳动物卵透明带（ZP）和鲍鱼卵黄膜（VE）主要蛋白的研究 它们含有同源的ZP-N结构域。考虑到ZP-N结构域的分子同源性和总体 由于卵子外壳的生化相似性，一个明显的问题是精子是否使用相似的 机制（或同源蛋白质），以产生一个路径，通过鸡蛋外套。鲍鱼的溶解 精子蛋白溶素对卵子VE的作用已被广泛地表征。然而，仍然不知道如何 哺乳动物的精子能穿透卵的外壳，目前在哺乳动物中还没有发现溶素同源物。 基因组我们的建议通过以下方式解决这个问题：（1）使用长读序测序来产生对以下基因的深度覆盖： 用于精子蛋白质组学的全长非嵌合cDNA，（2）使用这些资源作为数据库， 进行详细的生物信息学分析，旨在生成假定的人类配子识别数据库 鲍鱼精子溶素的人同源物的鉴定和（3）预测的 蛋白质破坏小鼠ZP的能力。如果我们鉴定出一种人类精子溶素， 研究哺乳动物配子识别和人类精子如何通过ZP的途径。如果我们 如果没有成功鉴定出人类溶素，我们将产生一个有价值的基因组和蛋白质组资源， 人类精子生物学未来研究的数据库。

Project Summary The identification of interacting male-female reproductive proteins is imperative for a molecular understanding of fertilization. The goal of this R21 proposal is to identify a set of human gamete recognition proteins using genomic and proteomic approaches, and then perform evolutionary guided computational bioinformatic searches for a homolog of abalone sperm lysin. It is known from three-dimensional protein crystallographic studies of the major proteins from the mammalian Zona Pellucida (ZP) and abalone Vitelline Envelope (VE) that they contain homologous ZP-N domains. Given the molecular homology of ZP-N domains and overall biochemical similarity of the egg coats, an obvious question to address is whether sperm use similar mechanisms (or homologous proteins) to generate a path through the egg coat. The dissolution of the abalone egg VE by the sperm protein lysin has been extensively characterized. However, it remains unknown how mammalian sperm penetrate egg coats, and no lysin homolog is currently identifiable in any mammalian genome. Our proposal address this question by (1) using long-read sequencing to generate deep coverage of full-length non-chimeric cDNAs for use in sperm proteomics, (2) using these resources as a database to perform detailed bioinformatic analyses aimed at generating a database of putative human gamete recognition proteins and identification of a human homolog of abalone sperm lysin and (3) functional tests of predicted protein's ability to disrupt the mouse ZP. If we identify a human sperm lysin, it will immediately open up new avenues of research into mammalian gamete recognition and how the human sperm passes the ZP. If we are unsuccessful at identifying a human lysin, we will have generated a valuable genomic and proteomic resource database for future studies of human sperm biology.