MOLECULAR STRUCTURE OF ENDOSOMES IN DEVELOPING INTESTINE

在发育过程中，肠道的吸收细胞通过一个 在这个阶段，它们高度专门化为 从肠腔排出的大分子。 在此期间（其中， 取决于物种，可能发生在子宫内或出生后）这些 细胞含有复杂的内体复合物。 这座建筑群 表明在选择性跨上皮转运中很重要， 这些生长因子对正常发育至关重要， 肠和其他器官系统。 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 同时防止病原体伺机而动 通过相同的途径感染。 为了确定 作为选择性跨上皮转运的基础， 表征参与内化的分选的隔室 大分子 目前的目标是获得cDNA 一个完整的膜糖蛋白（entubin）的序列，发现 在几个物种的发育肠的内体中， 包括人，但不存在于成熟肠或任何其它肠中。 细胞类型。 通过筛选大鼠新生肠cDNA文库， 使用获得后产生的寡核苷酸探针进行杂交 来自亲和纯化Entubin的内部氨基酸序列。 在老鼠体内， Entubin以两种在药理学上可区分的形式存在， 表明mRNA的选择性剪接，多个基因，或 差异糖基化 因此，该cDNA将用于 表征编码的转录单位的数量和大小 Entubin的表达，并确定编码Entubin的基因的数目。 在 此外，该cDNA还将用于筛选其他上皮细胞和非上皮细胞。 上皮细胞类型的微管相关蛋白。 如果与Entubin相关 在其他细胞类型中检测到蛋白质，可能是Entubin (or微管蛋白样蛋白）是正常功能所必需的。 核内体 此外，这种碳水化合物的组成 糖蛋白将通过特异性糖苷酶消化 亲和纯化的Entubin与推导的氨基酸一起 用于预测膜中蛋白质拓扑结构的序列。 Entubin的这种表征是理解Entubin的 内体膜的分子结构。 此外，虽然函数 Entubin是未知的，它提供了一个独特的和有价值的标志物， 内体区室 通过用cDNA转染细胞系， 因此，将有可能进行详细的形态学和 靶向内体蛋白的生物化学研究， 内体区室的生物起源问题， 动力学和内体组分的再循环。 从长远来看，这些 研究将提供深入了解的机制， 发育重要的配体，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.