CARBOHYDRATE-BINDING PROTEIN 35

本计画将继续进行碳水化合物结合的研究 蛋白质35（CBP 35），最近被重新命名为半乳糖凝集素-3。 的 半乳糖凝集素包括半乳糖/乳糖特异性糖家族， 结合蛋白，其在所述结合蛋白中共享特征性氨基酸序列， 多肽的碳水化合物识别结构域。 所述多肽 半乳糖凝集素-3（Mr约30，000）是一个嵌合体的两个结构域：脯氨酸- 和富含甘氨酸的结构域在NH 2-末端的一半和碳水化合物 在COOH-末端的一半识别域。 的基础上 先前的研究表明，半乳糖凝集素-3可以在 核，在核糖核蛋白复合物的形式，实验 以测试半乳糖凝集素-3在前体mRNA中的作用 处理. 取得了几项关键发现：（a）核提取物 来源于HeLa细胞，能够在无细胞的 测定法，含有半乳糖凝集素-3;（B）通过 在乳糖-琼脂糖上的亲和吸附变得缺乏剪接;（c） 乳糖-琼脂糖耗尽的提取物的活性可以 通过添加纯化的重组半乳糖凝集素-3重建的是 剪接因子/调节子。 根据这些意见和其他意见， 建议的研究包括：（1）生成，通过定点 突变，缺乏碳水化合物结合的半乳糖凝集素-3多肽 活性，并测试这些突变多肽的能力， 在乳糖-琼脂糖耗尽的提取物中重建剪接活性; (2)在酵母中寻找半乳糖凝集素-3的同源物，并研究半乳糖凝集素-3在酵母中的作用。 通过整合破坏的基因敲除的体内后果;（3） 为了继续我们对核糖核蛋白中半乳糖凝集素-3的表征， 复合物，特别是就p35多肽而言 与小核蛋白多肽共免疫沉淀 核糖核蛋白复合物;（4）研究核运输 半乳糖凝集素-3的性质，在核要求方面， 定位信号，翻译后修饰和调控， 增殖细胞与静止细胞。

This project proposes to continue our studies on Carbohydrate Binding Protein 35 (CBP35), which has recently been renamed galectin-3. The galectins comprise a family of galactose/lactose-specific-saccharide- binding proteins that share characteristic amino acid sequences in the carbohydrate recognition domain of the polypeptides. The polyeptide of galectin-3 (Mr about 30,000) is a chimera of two domains: a proline- and glycine-rich domain at the NH2-terminal half and a carbohydrate recognition domain at the COOH-terminal half. On the basis of previous studies demonstrating that galectin-3 can be found in the nucleus, in the form of a ribonucleoprotein complex, experiments were performed to test for a role of galectin-3 in pre-mRNA processing. Several key findings were made: (a) nuclear extracts derived from HeLa cells, capable of carrying out splicing in a cell-free assay, contain galectin-3; (b) nuclear extracts depleted of galectin-3 by affinity adsorption on lactose-agarose become deficient in splicing; (c) the activity of the lactose-agarose depleted extract could be reconstituted by the addition of purified recombinant galectin-3 is a splicing factor/regulator. On the basis of these and other observations, the specific objectives of the proposed research include: (1) to generate, by site-directed mutagenesis, galectin-3 polypeptides devoid of carbohydrate-binding activity and to test the ability of these mutant polypeptides to reconstitute the splicing activity in a lactose-agarose depleted extract; (2) to search for a homolog of galectin-3 in yeasts and to study the in vivo consequences of a gene knockout via integrative disruption; (3) to continue our characterization of galectin-3 in the ribonucleoprotein complex, particularly in terms of the p35 polypeptide coimmunoprecipitated with the polypeptides of the small nuclear ribonucleoprotein complexes; and (4) to study the nuclear transport properties of galectin-3, in terms of requirements for a nuclear localization signal, post-translational modifications, and regulation in proliferative versus quiescent cells.