MOLECULAR STRUCTURE OF ENDOSOMES IN DEVELOPING INTESTINE

在发育过程中，肠道的吸收细胞经历了 细胞高度专一的吞噬细胞的阶段 肠腔中的大分子。在这段时间(这段时间， 根据物种的不同，可能发生在子宫内或出生后) 细胞含有一个精致的内体复合体。这个建筑群已经被 提示在选择性跨上皮细胞转运中起重要作用 对儿童的正常发展至关重要的生长因素 肠道和其他器官系统。然而，人们对此知之甚少 控制选择性跨上皮转运的细胞机制 同时防止病原体的机会性 通过相同的途径感染。找出那些 作为选择性跨上皮运输的基础，有必要 描述内部化产品分类所涉及的隔间的特征 大分子。目前的方案的目标是获得cDNA 发现一种完整的膜糖蛋白(Entubin)的序列 在几个物种发育中的肠道的内体中， 包括人类，但不在成熟的肠道或任何其他地方 单元类型。将通过以下方法筛选新生大鼠肠道cDNA文库 利用获得后产生的寡核苷酸探针进行杂交 来自亲和纯化的Enubin的内部氨基酸序列。在老鼠身上， Enubin以两种在电泳学上可区分的形式存在， 表明mRNA、多个基因的选择性剪接，或者 差异糖基化。因此，这一基因将被用于 描述编码转录单位的数量和大小 并确定编码内毒素的基因数量。在……里面 此外，该cDNA还将用于筛选其他上皮细胞和非上皮细胞。 Enubin相关蛋白的上皮细胞类型。如果与Enubin相关 在其他类型的细胞中也检测到蛋白质，内毒素可能 (或内毒素样蛋白)是正常功能所必需的。 内噬菌体。此外，这个的碳水化合物组成 糖蛋白将通过特定的糖苷酶消化来测定 亲和纯化的Enubin及其推导的氨基酸 用于预测膜中蛋白质的拓扑结构的序列。 Enubin的这种特征是理解 内体膜的分子结构。此外，尽管该函数 是未知的，它提供了一个独特而有价值的标志物 内胚体隔室。通过将该基因导入细胞系，以获得 Enubin将有可能进行详细的形态和 内体蛋白靶向性的生物化学研究及解决 内体隔室的生物发生问题 动力学和内体成分的循环利用。从长远来看，这些 研究将提供对分类和定向的机制的洞察 发展重要的配体，如生长因子。

During development, the absorptive cells of the intestine go through a stage in which they are highly specialized for the endocytosis of macromolecules from the intestinal lumen. During this time (which, depending upon the species, may occur in utero or after birth) these cells contain an elaborate endosomal complex. This complex has been suggested to be important in the selective transepithelial transport of growth factors that are crucial for the normal development of the intestine and other organ systems. However, little is known about the cellular mechanisms that control the selective transepithelial transport of macromolecules while preventing pathogens from opportunistically infecting through the same route. To identify the mechanisms that underlie selective transepithelial transport, it is necessary to characterize the compartment involved in sorting of internalized macromolecules. The goals of the current proposal are to obtain the cDNA sequence for an integral membrane glycoprotein (entubin), which is found in the endosomes of the developing intestine of several species, including human, but is absent from the mature intestine or any other cell type. A rat neonatal intestine cDNA library will be screened by hybridization using oligonucleotide probes generated after obtaining internal amino acid sequence from affinity purified entubin. In the rat, entubin is present in two electrophoretically distinguishable forms, indicating either alternative splicing of mRNAs, multiple genes, or differential glycosylation. Therefore, this cDNA will be used to characterize the number and size of transcriptional units encoding entubin and to determine the number of genes encoding entubin. In addition, the cDNA will be used to screen other epithelial cell and non- epithelial cell types for entubin-related proteins. If entubin related proteins are detected in other cell types, it is possible that entubin (or entubin-like proteins) are required for the normal function of endosomes. In addition, the carbohydrate composition of this glycoprotein will be determined by specific glycosidase digestion of affinity purified entubin and together with the deduced amino acid sequence used to predict the topology of the protein in the membrane. This characterization of entubin is a first step in understanding the molecular structure of endosomal membranes. Also, although the function of entubin is unknown, it provides a unique and valuable marker of the endosomal compartment. By transfecting cell lines with the cDNA for entubin it will be possible to carry out detailed morphological and biochemical studies of the targeting of endosomal proteins and to address the questions of biogenesis of endosomal compartments, endosomal dynamics, and recycling of endosomal components. In the long term, these studies will provide insight into the mechanism of sorting and targeting of developmentally important ligands such as growth factors.