STRUCTURE & FUNCTION OF THE EPIDIDYMIS AND VAS DEFERENS

结构

• 批准号: 2196866
• 负责人: DAVID W HAMILTON
• 金额: $ 17.48万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-09-29 至 1996-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2196866
• 关键词: SDS polyacrylamide gel electrophoresis; binding proteins; carbohydrate structure; cell membrane; chemical association; epididymis; gene expression; genetic regulation; glycoproteins; glycosylation; immunoprecipitation; laboratory mouse; laboratory rabbit; laboratory rat; membrane proteins; oligosaccharides; polymerase chain reaction; posttranslational modifications; protein purification; protein sequence; protein structure function; secretory protein; sperm; tissue /cell culture; western blottings

Sperm traverse the epididymis and acquire new proteins and glycoproteins on their plasma membranes even though they are incapable of nuclear DNA transcription and mRNA translation. We have been working for the past few years on two rat epididymis-specific secretory glycoproteins (proteins D & E) that are greater than 95% homologous at the amino acid level and are commonly considered to be identical, differing only in their glycosylation. Our preliminary data indicate that there are both carbohydrate and amino acid differences between the two proteins that could be very important to their behavior in the epididymis. We have raised a monoclonal antibody (4E9 MAb) that recognizes purified protein E, but not purified protein D. This MAb also localizes to the tail plasma membrane of caudal epididymal sperm, but not of caput epididymal sperm. All of our data, and published data, are consistent with the hypothesis that the 4E9 MAb antigen, is synthesized by epididymal epithelium, secreted into the epididymal lumen, and subsequently binds to sperm. The association of the antigen with sperm is very tight, since common biochemical assays for integral vs peripheral membrane proteins indicate that 4E9 antigen behaves as though it is an integral membrane molecule. Antibodies to protein D are available in other laboratories, and their immunolocalization studies indicate that protein D is primarily synthesized in regions of the epididymis different from those of protein E and the antibody localizes to the head of the sperm rather than to the tail. Therefore, the major question being asked in this grant is what signals mediate differential targeting to domain-specific regions on the sperm surface of two proteins with greater than 95% homology. In order to investigate this we propose five specific aims: The first is to study quantitatively and qualitatively binding of proteins D & E to the sperm surface and to determine structural motifs that mediate differential targeting. Secondly, it is important to complete studies on the primary amino acid sequences and oligosaccharide structures of proteins D and E and of their membrane counterparts. Thirdly, it is important to ascertain whether there are post-translational modifications of proteins D and E, both from fluid and sperm, other than glycosylation, since these modifications might act in some way to regulate signalling in the system. Fourth, there is evidence that protein E is processed prior to associating with the sperm surface and it is important, therefore, to study this process. Finally, I propose to study regulation of expression of proteins D&E.

精子穿越附睾并获得新的蛋白质和糖蛋白 即使它们无法使用核DNA，它们的质膜即使 转录和mRNA翻译。我们过去几个 在两个大鼠附加症特异性分泌糖蛋白上的年（蛋白D ＆e）在氨基酸水平上大于95％的同源物 通常认为是相同的 糖基化。我们的初步数据表明 两种蛋白质之间的碳水化合物和氨基酸差异 对于它们在附睾中的行为可能非常重要。 我们提出了一种识别纯化的单克隆抗体（4E9 mAb） 蛋白E，但不纯化的蛋白质D。此mAb也定位在尾巴上 尾骨精子的质膜，但没有cap子附子膜 精子。我们的所有数据和已发布的数据都与 假设4E9 mAb抗原是由附子合成的 上皮，分泌到附睾腔内，随后结合 精子。抗原与精子的关联非常紧，因为 积分与周围膜蛋白的常见生化测定 表明4E9抗原的行为好像是整体膜 分子。 蛋白质D的抗体在其他实验室中可用，它们的 免疫定位研究表明蛋白D主要是 在附睾区域合成的蛋白质不同 E和抗体定位于精子的头部，而不是 尾巴。因此，这笔赠款中提出的主要问题是什么 信号将差分定位到特定于域的区域上 两种蛋白质的精子表面，具有超过95％同源性。为了 调查这一点，我们提出了五个具体目标： 首先是定量研究蛋白质的质量结合 D＆E到精子表面并确定介导的结构基序 差分定位。其次，完成有关的研究很重要 主要的氨基酸序列和寡糖结构 蛋白质D和E及其膜的蛋白质。第三，是 确定是否存在翻译后修改很重要 除糖基化以外，来自液体和精子的蛋白质D和E的蛋白质 由于这些修改可能会以某种方式调节信号 系统。第四，有证据表明蛋白质在之前处理 与精子表面相关，因此很重要 研究此过程。最后，我建议研究表达的调节 蛋白质D＆E。