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Galectin-3 (Mr 35,000) is a galactose/lactose-specific lectin found in association with ribonucleoprotein complexes in many animal cells. Cell-free-splicing assays have been carried out to study the requirement for galectin-3 in RNA processing by HeLa cell nuclear extracts by using 32P-labeled MINX as the pre-mRNA substrate. Addition of saccharides that bind galectin-3 with high affinity inhibited product formation in the splicing assay, while addition of carbohydrates that do not bind to the lectin did not inhibit product formation. Nuclear extracts depleted ofgalectin-3 by affinity adsorption on a lactoseagarose column were deficient in splicing activity. Extracts subjected to parallel adsorption on control celiobiose-agarose retained splicing activity. The activity of the galectin-3-depleted extract could be reconstituted by the addition of purified recombinant galectin-3, whereas the addition of other lectins, either with a similar saccharide binding specificity (soybean agglutinin) or with a different specificity (wheat germ agglutinin), did not restore splicing activity. The formation of splicing complexes was also sensitive to galectin-3 depletion and reconstitution. Together, these results define a requirement for galectin-3 in pre-mRNA splicing and identify it as a splicing factor.Galectin-3 (1) is the name for the galactose(Gal)/lactose (Lac)-specific lectin previously known under a number of different designations, including carbohydrate binding protein 35 (CBP35) (2), Mac-2 (3), IgE-binding protein (4), CBP30 (5), L-29 (6), and L-34 (7). In this communication, we will use the designation galectin-3 when we refer to the gene or protein in general, assuming that studies carried out on the gene or protein under any one of the above names is applicable to all of them. There are instances, however, in which the specific molecule used by one laboratory is slightly (but significantly) different from the corresponding molecule of another laboratory-e.g., the cDNAs reported for murine CBP35, Mac-2, and L-34 are of different lengths. In this case, we will use the old designation to highlight the specific source of the molecule.The galectin family of animal lectins is distinguished by the Gal/Lac specificity of its carbohydrate recognition domain (CRD), with highly conserved residues between members of the family (galectin-1, -2, -3, and -4) and between the homologs found in various species for any given single member (for reviews see refs. 8 and 9). The polypeptide of galectin-3 is delineated into two domains (2,8): an N-terminal half that is proline and glycine rich, with limited similarity to proteins of the heterogeneous nuclear ribonucleoprotein (RNP) complexes, and a C-terminal half that is similar to the CRD of other members of the galectin family. The majority of galectin-3 is found in the cytoplasm and nucleus of mouse 3T3 fibroblasts in the form of RNP complexes (10, 11). For example, treatment of permeabilized cells with ribonuclease A released the lectin from the nucleus with concomitant los...

Chemical derivatization of tetrameric concanavalin A (Con A) with succinic anhydride or acetic anhydride converts the protein to a dimeric molecule without altering its carbohydrate-binding specificity. At low concentrations, the dose-response curves for the mitogenic stimulation of mouse spleen cells by native Con A and succinyl-Con A are similar. Above lectin concentrations of 10 ug/ml, however, the response to Con A is diminished, while that for succinyl-Con A does not decrease until much higher doses are reached. We have attributed this difference mainly to the higher rate of cell death induced by the native Con A molecule. Con A also shows a greater capacity than succinyl-Con A to agglutinate sheep erythrocytes and to inhibit cap formation by immunoglobulin receptors on spleen cells. Moreover, at low concentrations, Con A induced its glycoprotein receptors to form caps, but succinyl-Con A did not induce cap formation. Addition of antibodies directed against Con A to succinyl-Con A bound on cells restored the properties of agglutination, inhibition of immunoglobulin receptor cap formation, and induction of cap formation by Con. A receptors. Similar results have been obtained for acetylCon A. These data suggest that the altered biological activities of succinyl-Con A and acetyl-Con A are attributable to their reduced valence.

The tentative amino-acid sequence and three-dimensional structure of the lectin concanavalin A have been determined. The amino-acid sequence, which was determined chemically, contains 238 residues. The sequences of three short stretches were assigned on the basis of x-ray crystallographic data. Interpretation of an electron density map at 2-A resolution indicates that the predominant structural element is extended polypeptide chain arranged in two anti-parallel pleated sheets or j8- Concanavalin A (Con A) is one of a group of plant proteins known as lectins (1), some of which have useful properties for studies of cell surfaces and cell division. Although the function of Con A in the jack bean (Canavalia eneiformis) is unknown, it can agglutinate various somatic (2, 3) and germ line cells (2), and it is mitogenic for lymphocytes (4). After suitable treatment, it can restore the growth pattern of virustransformed fibroblasts in tissue culture to that of normal cells (5). Various mammalian cells bind from 106 to 107 Con A -molecules per cell, presumably via surface glycoprotein receptors. This binding can be inhibited by a-D-gluco-and a-D-mannopyranosides. Very little is known, however, of the mechanism by which Con A leads to agglutination, mitogenesis, or cell-surface alteration.In order to understand the mode of action of Con A, we have undertaken structural and functional studies. We report here the tentative amino-acid sequence and an interpretation of an electron density map at 2-A resolution. The results suggest useful models for analyzing the biological activities of this protein. MATERIALS AND METHODSThe isolation of the intact subunit of Con A and the preparation of its CNBr fragments have been described (6, 7). The amino-acid sequence was determined by the dansyl-Edman procedure (8, 9), performed on peptides isolated after digestion of the CNBr fragments with trypsin, chymotrypsin, pepsin, thermolysin, and subtilisin.

An analysis of the inhibition by concanavalin A of the mobility of lymphocyte surface receptors is used to construct an hypothesis on membrane receptor-cytoplasmic interactions. It is proposed that binding of multivalent lectins alters the interaction of an assembly of colchicine-binding proteins with lectin receptors and other receptors, and reciprocally that the state of the colchicine-binding assembly alters the mobility and distribution of surface receptors on the cell membrane. Observations of the effect of colchicine and related drugs on the inhibition of receptor mobility by concanavalin A lend support to this hypothesis. The proposed model has several implications for studies of the initial events of mitogenesis in lymphocytes as well as for cellcell interactions in general.