CARBOHYDRATE-BINDING PROTEIN 35

碳水化合物结合蛋白 35

• 批准号: 2684862
• 负责人: JOHN L WANG
• 金额: $ 20.96万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-04-01 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2684862
• 关键词: RNA splicing; binding proteins; chemical binding; chemical structure function; gene targeting; immunoprecipitation; intracellular transport; laboratory rabbit; lectin; posttranslational modifications; protein transport; ribonucleoproteins; site directed mutagenesis; tissue /cell culture; yeasts

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.

本项目建议继续我们对碳水化合物结合的研究。 蛋白35(CBP35)，最近被重新命名为Galectin-3。这个 半乳糖凝集素是由半乳糖/乳糖专一性糖组成的家族。 具有共同特征的氨基酸序列的结合蛋白 多肽的碳水化合物识别结构域。多肽 Galectin-3(Mr约30,000)是两个结构域的嵌合体：一个脯氨酸- 和NH2末端的富含甘氨酸的结构域和碳水化合物 在COOH-末端一半的识别结构域。在…的基础上 先前的研究表明，Galectin-3可以在 核糖核蛋白复合体形式的核，实验 用来检测Galectin-3在Pre-mRNA中的作用 正在处理。得出了几个重要结论：(A)核提取物 源自HeLa细胞，能够在无细胞的情况下进行剪接 含有Galectin-3的化验；(B)被 乳糖-琼脂糖亲和吸附在剪接中变得不足；(C) 乳糖-琼脂糖耗尽提取物的活性可能是 通过添加纯化的重组Galectin-3而重组的是一种 剪接因子/调节器。 根据这些和其他观察结果， 建议的研究包括：(1)通过现场定向生成 无糖结合的Galectin-3多肽的诱变 活性，并测试这些突变多肽的能力 在乳糖-琼脂糖耗尽提取物中重建剪接活性； (2)在酵母菌中寻找Galectin-3的同源物，并研究其在酵母中的作用。 整合破坏基因敲除的体内后果；(3) 为了继续我们对核糖核蛋白中Galectin-3的表征 复合体，特别是在p35多肽方面 与小核多肽的免疫共沉淀 核糖核蛋白复合体；(4)研究核转运 半乳糖凝集素-3的性质，就核的要求而言 本地化信号、翻译后修饰和调节 增殖的细胞与静止的细胞。