CARBOHYDRATE-BINDING PROTEIN 35

本项目拟继续开展碳水化合物结合的研究

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.