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.

精子横穿附睾会获得新的蛋白质和糖蛋白 在它们的质膜上，即使它们不能产生核DNA 转录和信使核糖核酸翻译。我们在过去的几年里一直在工作 两种附睾特异性分泌型糖蛋白(蛋白D)的研究 &E)在氨基酸水平上同源性大于95%，并且 通常被认为是相同的，不同的只是 糖基化。我们的初步数据表明，两者都有 两种蛋白质之间的碳水化合物和氨基酸差异 可能对他们在附睾处的行为非常重要。 我们已经提出了一种可识别纯化的单抗(4E9单抗) 蛋白E，但不是纯化的蛋白D。这种单抗也定位于尾巴 附睾尾精子的质膜，附睾头精子的质膜 精子。我们所有的数据和发布的数据都与 假设4E9单抗是由附睾体合成的 上皮，分泌到附睾腔，随后结合到 精子。这种抗原与精子的联系非常紧密，因为 整体膜蛋白与外周膜蛋白的常用生化分析方法 表明4E9抗原表现为一个完整的膜 分子。 其他实验室也有针对蛋白质D的抗体，他们的 免疫定位研究表明，蛋白D主要是 附睾区合成的蛋白质与附睾区不同 E和抗体定位于精子头部，而不是精子 尾巴。因此，这笔赠款中提出的主要问题是 信号介导域特定区域的差异靶向 精子表面的两种蛋白质具有95%以上的同源性。为了 为此，我们提出了五个具体目标： 第一是定量和定性研究蛋白质的结合。 D&E到精子表面，并确定中介结构基序 差异化目标定位。其次，完成对……的研究很重要 黄曲霉的初级氨基酸序列和寡糖结构 蛋白质D和E以及它们的膜对应物。第三，它是 重要的是确定是否存在翻译后修改 蛋白质D和E，除了糖基化外，还来自液体和精子， 由于这些修改可能在某种程度上调节 这个系统。第四，有证据表明，蛋白质E是在 与精子表面相联系，因此，重要的是 研究这一过程。最后，我建议对表达的调控进行研究 蛋白质D&E。