Evidence for a role for galectin-1 in pre-mRNA splicing.

Galectins are a family of beta-galactoside-binding proteins that contain characteristic amino acid sequences in the carbohydrate recognition domain (CRD) of the polypeptide. The polypeptide of galectin-1 contains a single domain, the CRD. The polypeptide of galectin-3 has two domains, a carboxyl-terminal CRD fused onto a proline- and glycine-rich amino-terminal domain. In previous studies, we showed that galectin-3 is a required factor in the splicing of nuclear pre-mRNA, assayed in a cell-free system. We now document that (i) nuclear extracts derived from HeLa cells contain both galectins-1 and -3; (ii) depletion of both galectins from the nuclear extract either by lactose affinity adsorption or by double-antibody adsorption results in a concomitant loss of splicing activity; (iii) depletion of either galectin-1 or galectin-3 by specific antibody adsorption fails to remove all of the splicing activity, and the residual splicing activity is still saccharide inhibitable; (iv) either galectin-1 or galectin-3 alone is sufficient to reconstitute, at least partially, the splicing activity of nuclear extracts depleted of both galectins; and (v) although the carbohydrate recognition domain of galectin-3 (or galectin-1) is sufficient to restore splicing activity to a galectin-depleted nuclear extract, the concentration required for reconstitution is greater than that of the full-length galectin-3 polypeptide. Consistent with these functional results, double-immunofluorescence analyses show that within the nucleus, galectin-3 colocalizes with the speckled structures observed with splicing factor SC35. Similar results are also obtained with galectin-1, although in this case, there are areas of galectin-1 devoid of SC35 and vice versa. Thus, nuclear galectins exhibit functional redundancy in their splicing activity and partition, at least partially, in the nucleoplasm with another known splicing factor.     

Dynamics of galectin-3 in the nucleus and cytoplasm

This review summarizes selected studies on galectin-3 (Gal3) as an example of the dynamic behavior of a carbohydrate-binding protein in the cytoplasm and nucleus of cells. Within the 15-member galectin family of proteins, Gal3 (M_r ∼ 30,000) is the sole representative of the chimera subclass in which a proline- and glycine-rich NH₂-terminal domain is fused onto a COOH-terminal carbohydrate recognition domain responsible for binding galactose-containing glycoconjugates. The protein shuttles between the cytoplasm and nucleus on the basis of targeting signals that are recognized by importin(s) for nuclear localization and exportin-1 (CRM1) for nuclear export. Depending on the cell type, specific experimental conditions in vitro, or tissue location, Gal3 has been reported to be exclusively cytoplasmic, predominantly nuclear, or distributed between the two compartments. The nuclear versus cytoplasmic distribution of the protein must reflect, then, some balance between nuclear import and export, as well as mechanisms of cytoplasmic anchorage or binding to a nuclear component. Indeed, a number of ligands have been reported for Gal3 in the cytoplasm and in the nucleus. Most of the ligands appear to bind Gal3, however, through protein–protein interactions rather than through protein–carbohydrate recognition. In the cytoplasm, for example, Gal3 interacts with the apoptosis repressor Bcl-2 and this interaction may be involved in Gal3's anti-apoptotic activity. In the nucleus, Gal3 is a required pre-mRNA splicing factor; the protein is incorporated into spliceosomes via its association with the U1 small nuclear ribonucleoprotein (snRNP) complex. Although the majority of these interactions occur via the carbohydrate recognition domain of Gal3 and saccharide ligands such as lactose can perturb some of these interactions, the significance of the protein's carbohydrate-binding activity, per se, remains a challenge for future investigations.     

Distinct effects on splicing of two monoclonal antibodies directed against the amino-terminal domain of galectin-3

Previous experiments had established that galectin-3 (Gal3) is a factor involved in cell-free splicing of pre-mRNA. Addition of monoclonal antibody NCL-GAL3, whose epitope maps to the NH₂-terminal 14 amino acids of Gal3, to a splicing-competent nuclear extract inhibited the splicing reaction. In contrast, monoclonal antibody anti-Mac-2, whose epitope maps to residues 48-100 containing multiple repeats of a 9-residue motif PGAYPGXXX, had no effect on splicing. Consistent with the notion that this region bearing the PGAYPGXXX repeats is sequestered through interaction with the splicing machinery and is inaccessible to the anti-Mac-2 antibody, a synthetic peptide containing three perfect repeats of the sequence PGAYPGQAP (27-mer) inhibited the splicing reaction, mimicking a dominant-negative mutant. Addition of a peptide corresponding to a scrambled sequence of the same composition (27-mer-S) failed to yield the same effect. Finally, GST-hGal3(1-100), a fusion protein containing glutathione-S-transferase and a portion of the Gal3 polypeptide including the PGAYPGXXX repeats, also exhibited a dominant-negative effect on splicing.     

Caenorhabditis elegans proteins captured by immobilized Galβ1-4Fuc disaccharide units: assignment of 3 annexins

Galβ1-4Fuc is a key structural motif in Caenorhabditis elegans glycans and is responsible for interaction with C. elegans galectins. In animals of the clade Protostomia, this unit seems to have important roles in glycan–protein interactions and corresponds to the Galβ1-4GlcNAc unit in vertebrates. Therefore, we prepared an affinity adsorbent having immobilized Galβ1-4Fuc in order to capture carbohydrate-binding proteins of C. elegans, which interact with this disaccharide unit. Adsorbed C. elegans proteins were eluted with ethylenediaminetetraacetic acid (EDTA) and followed by lactose (Galβ1-4Glc), digested with trypsin, and were then subjected to proteomic analysis using LC–MS/MS. Three annexins, namely NEX-1, -2, and -3, were assigned in the EDTA-eluted fraction. Whereas, galectins, namely LEC-1, -2, -4, -6, -9, -10, and DC2.3a, were assigned in the lactose-eluted fraction. The affinity of annexins for Galβ1-4Fuc was further confirmed by adsorption of recombinant NEX-1, -2, and -3 proteins to the Galβ1-4Fuc column in the presence of Ca²⁺. Furthermore, frontal affinity chromatography analysis using an immobilized NEX-1 column showed that NEX-1 has an affinity for Galβ1-4Fuc, but no affinity toward Galβ1-3Fuc and Galβ1-4GlcNAc. We would hypothesize that the recognition of the Galβ1-4Fuc disaccharide unit is involved in some biological processes in C. elegans and other species of the Protostomia clade.     

Immunoprecipitation of spliceosomal RNAs by antisera to galectin-1 and galectin-3

We have shown that galectin-1 and galectin-3 are functionally redundant splicing factors. Now we provide evidence that both galectins are directly associated with spliceosomes by analyzing RNAs and proteins of complexes immunoprecipitated by galectin-specific antisera. Both galectin antisera co-precipitated splicing substrate, splicing intermediates and products in active spliceosomes. Protein factors co-precipitated by the galectin antisera included the Sm core polypeptides of snRNPs, hnRNP C1/C2 and Slu7. Early spliceosomal complexes were also immunoprecipitated by these antisera. When splicing reactions were sequentially immunoprecipitated with galectin antisera, we found that galectin-1 containing spliceosomes did not contain galectin-3 and vice versa, providing an explanation for the functional redundancy of nuclear galectins in splicing. The association of galectins with spliceosomes was (i) not due to a direct interaction of galectins with the splicing substrate and (ii) easily disrupted by ionic conditions that had only a minimal effect on snRNP association. Finally, addition of excess amino terminal domain of galectin-3 inhibited incorporation of galectin-1 into splicing complexes, explaining the dominant-negative effect of the amino domain on splicing activity. We conclude that galectins are directly associated with splicing complexes throughout the splicing pathway in a mutually exclusive manner and they bind a common splicing partner through weak protein–protein interactions.     

Purification and characterization of beta-galactoside-binding proteins from Caenorhabditis elegans.

Two carbohydrate-binding proteins (subunit molecular masses, 32 and 16 kDa, respectively) were isolated for the first time from a nematode, Caenorhabditis elegans. They were specifically extracted with lactose and adsorbed on asialofetuin-Sepharose in the absence of a metal ion. Although these two proteins were co-eluted from a gel filtration column at a position corresponding to an apparent molecular size of 30 kDa under non-denaturing conditions, they could be separated by reversed-phase chromatography. The 32 kDa protein, the main component, was further characterized. Together with its solubility, saccharide specificity and metal independence, some other structural properties, including its amino acid composition, UV spectrum, and partial amino acid sequence, strongly suggested that the 32 kDa protein is a member of a class of soluble beta-galactoside-binding lectins which had previously been only found in vertebrates.     

Binding of synthetic sulfated ligands by human splenic galectin 1, a β-galactoside-binding lectin

The carbohydrate-binding site of galectin 1, a vertebrate β-galactoside-binding lectin, has a pronounced specificity for the βGal(1→3)- and βGal(1→4)GlcNAc sequences. The binding inhibition study reported herein was carried out to determine whether sulfation of saccharides would influence their binding by galectin 1. The presence of 6′-OSO3- on LacNAc greatly reduces the inhibitory potency relative to LacNAc. 3′-OSO3-LacNAc, 3′-OSO3-Galβ(1→3)GlcNAcβ1-OBzl and 3-OSO3-Galβ1-OMe are more potent inhibitors than the non-sulfated parent compounds. Surprisingly, 2′-OSO3-LacNAc showed over 40 fold less inhibitory potency relative to LacNAc. Ovarian carcinoma A121 cells were shown to synthesize sulfated macromolecules that bind to galectin 1. Modulation in vivo of saccharide sulfation may lead to modulation of galectin 1 interaction with glycoconjugates; hence, sulfation could play a role in modulating lectin functions.     

Galectins in kidney development

Galectins are a family of proteins with overlapping but distinct carbohydrate-binding specificities. They differ in cell-type and tissue distribution, and have various functions. Extracellularly several galectins can modulate cellular adhesive interactions and signalling pathways, effects that may be important in the establishment and maintenance of tissue organization during normal development. This review will summarise recent progress in defining the roles of galectins that are expressed in the kidney in normal development, and discuss the evidence linking aberrant expression of galectins with kidney disease. Published in 2004.     

Human splenic galaptin: carbohydrate-binding specificity and characterization of the combining site.

A galactose-binding lectin (galaptin) from human spleen has been purified to homogeneity by affinity chromatography on asialofetuin-Sepharose. The carbohydrate-binding specificity of galaptin has been investigated by analyzing the binding of galaptin to asialofetuin in the presence of putative inhibitors. An enzyme-linked immunosorbent assay (ELISA) was developed that involved adsorption of asialofetuin to microtiter plates. Galaptin bound to asialofetuin was detected with polyclonal rabbit anti-galaptin serum followed by goat anti-rabbit IgG-peroxidase conjugate. The concentrations of inhibitors giving 50% inhibition of galaptin binding relative to controls were graphically determined and normalized relative to galactose or lactose. These analyses revealed that galaptin has a combining site at least as large as a disaccharide. The disaccharides having non-reducing-terminal beta-galactosyl residues linked (1,3), (1,4), and (1,6) to Glc or GlcNAc are better inhibitors than free Gal. GalNAc, either free or glycosidically linked, appears to have no affinity for the lectin. The nitrophenyl galactosides are better inhibitors than methyl galactosides, indicating the occurrence of hydrophobic interactions. The data indicate that OH groups at C-4 and C-6 of Gal and the OH at C-3 of GlcNAc in Gal beta(1,4)GlcNAc are important for lectin sugar interaction. Our data support the hypothesis that endogenous receptors for galaptin are most likely lactosaminoglycan moieties.     

Description of a monomeric prototype galectin from the lizard Podarcis hispanica

Galectins are a continuously expanding family of beta-galactoside-binding lectins present in a variety of evolutionarily divergent animal species. Here we report, for the first time, that expression of galectins extends to the reptilia lineage of lizards. Up to five lactose-binding proteins were isolated from the lizard Podarcis hispanica by affinity chromatography on asialofetuin-Sepharose. The main component, which is most abundantly expressed in skin, was purified from this tissue and further characterized. Under native conditions the protein behaved as a monomer with a molecular mass of 14,500 Da and an isoelectric point of 6.3. Based on sequence homology of the 58 N-terminal amino acid residues with galectins, and on its demonstrated galactoside-binding activity, this lectin we named LG-14 (from Lizard Galectin and 14 kDa) is classified as a new member of the galectin family. LG-14 falls into and strengthen the still thinly populated category of monomeric prototype galectins.     

Cloning of a protein arginine methyltransferase PRMT1 homologue from Schistosoma mansoni: evidence for roles in nuclear receptor signaling and RNA metabolism

The most studied arginine methyltransferase is the type I enzyme, which catalyzes the transfer of an S-adenosyl-L-methionine to a broad spectrum of substrates, including histones, RNA-transporting proteins, and nuclear hormone receptor coactivators. We cloned a cDNA encoding a protein arginine methyltransferase in Schistosoma mansoni (SmPRMT1). SmPRMT1 is highly homologous to the vertebrate PRMT1 enzyme. In vitro methylation assays showed that SmPRMT1 recombinant protein was able to specifically methylate histone H4. Two schistosome proteins likely to be involved in RNA metabolism, SMYB1 and SmSmD3, that display a number of RGG motifs, were strongly methylated by SmPRMT1. In vitro GST pull-down assays showed that SMYB1 and SmSmD3 physically interacted with SmPRMT1. Additional GST pull-down assay suggested the occurrence of a ternary complex including SmPRMT1, SmRXR1 nuclear receptor, and the p160 (SRC-1) nuclear receptor coactivator. Together, these data suggest a mechanism by which SmPRMT1 plays a role in nuclear receptor-mediated chromatin remodeling and RNA transactions.     

Discoidin I from Dictyostelium discoideum and Interactions with Oligosaccharides: Specificity, Affinity, Crystal Structures, and Comparison with Discoidin II

Discoidin I (DiscI) and discoidin II (DiscII) are N-acetylgalactosamine (GalNAc)-binding proteins from Dictyostelium discoideum. They consist of two domains: an N-terminal discoidin domain and a C-terminal H-type lectin domain. They were cloned and expressed in high yield in recombinant form in Escherichia coli. Although both lectins bind galactose (Gal) and GalNAc, glycan array experiments performed on the recombinant proteins displayed strong differences in their specificity for oligosaccharides. DiscI and DiscII bind preferentially to Gal/GalNAcβ1-3Gal/GalNAc-containing and Gal/GalNAcβ1-4GlcNAcβ1-6Gal/GalNAc-containing glycans, respectively. The affinity of the interaction of DiscI with monosaccharides and disaccharides was evaluated using isothermal titration calorimetry experiments. The three-dimensional structures of native DiscI and its complexes with GalNAc, GalNAcβ1-3Gal, and Galβ1-3GalNAc were solved by X-ray crystallography. DiscI forms trimers with involvement of calcium at the monomer interface. The N-terminal discoidin domain presents a structural similarity to F-type lectins such as the eel agglutinin, where an amphiphilic binding pocket suggests possible carbohydrate-binding activity. In the C-terminal H-type lectin domain, the GalNAc residue establishes specific hydrogen bonds that explain the observed affinity (K_d = 3 × 10^− 4 M). The different specificities of DiscI and DiscII for oligosaccharides were rationalized from the different structures obtained by either X-ray crystallography or molecular modeling.     

SOX2 modulates alternative splicing in transitional cell carcinoma

Aberrant alternative splicing of key cellular regulators may play a pivotal role in cancer development. To investigate the potential influence of altered alternative splicing on the development of transitional cell carcinoma (TCC), splicing activity in the TCC cell lines TSGH8301 and BFTC905 was examined using the SV40-immortalized uroepithelial cell line SV-HUC-1 as a reference. Our results indicate a significant alteration in splice site selection in the TCC cell lines. By gene expression profiling and subsequent validation, we discovered that sex-determining region Y-box protein 2 (SOX2) is specifically upregulated in BFTC905. Furthermore, ectopic expression of SOX2 modulates alternative splicing of the splicing reporter in vivo. More significantly, using an in vitro pull-down assay, it was found that SOX2 exhibits RNA-binding capability. Our observations suggest that SOX2 modulates alternative splicing by functioning as a splicing factor.