STRUCTURE & FUNCTION OF THE EPIDIDYMIS AND VAS DEFERENS

精子穿过附睾并获得新的蛋白质和糖蛋白 即使它们不能将核DNA 转录和mRNA翻译。过去几年我们一直在努力 两种大鼠附睾特异性分泌糖蛋白（蛋白D & E），其在氨基酸水平上大于95%同源，并且 通常被认为是相同的，不同的只是他们的 糖基化我们的初步数据显示， 两种蛋白质之间的碳水化合物和氨基酸差异， 可能对它们在附睾中的行为非常重要。 我们已经提出了一种单克隆抗体（4E9单克隆抗体）， 蛋白E，但不是纯化的蛋白D。这种MAb也定位于尾部 附睾尾部精子质膜，附睾头部无 精子我们所有的数据和公布的数据都与 假设4E9 MAb抗原由附睾合成， 上皮细胞，分泌到附睾腔，随后结合到 精子抗原与精子的结合非常紧密，因为 积分与外周膜蛋白的常见生化测定 表明4E9抗原表现得好像它是一个完整膜 分子。 蛋白D的抗体可在其他实验室获得， 免疫定位研究表明，蛋白D主要是 在附睾中合成的区域不同于蛋白质的区域 抗体定位于精子的头部，而不是精子的头部。 尾巴因此，在这项赠款中提出的主要问题是什么 信号介导差异靶向至细胞上的结构域特异性区域， 精子表面的两种蛋白具有大于95%的同源性。为了 为此，我们提出了五个具体目标： 首先是定量和定性研究蛋白质的结合 D & E到精子表面，并确定介导的结构基序 差异靶向其次，重要的是要完成研究， 主要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。三是 重要的是确定是否存在翻译后修饰 蛋白质D和E，无论是从流体和精子，除了糖基化， 因为这些修饰可能以某种方式调节信号传导， the system.第四，有证据表明，蛋白质E是加工之前， 与精子表面结合，因此， 研究这个过程。最后，我建议研究表达调控 蛋白质D&E

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.