Patricia G. Voss

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) approximately 30,000) is the sole representative of the chimera subclass in which a proline- and glycine-rich NH(2)-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 Gal3s 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 proteins carbohydrate-binding activity, per se, remains a challenge for future investigations.

Dissociation of the carbohydrate-binding and splicing activities of galectin-1

Galectin-1 (Gal1) and galectin-3 (Gal3) are two members of a family of carbohydrate-binding proteins that are found in the nucleus and that participate in pre-mRNA splicing assayed in a cell-free system. When nuclear extracts (NE) of HeLa cells were subjected to adsorption on a fusion protein containing glutathione S-transferase (GST) and Gal3, the general transcription factor II-I (TFII-I) was identified by mass spectrometry as one of the polypeptides specifically bound. Lactose and other saccharide ligands of the galectins inhibited GST-Gal3 pull-down of TFII-I while non-binding carbohydrates failed to yield the same effect. Similar results were also obtained using GST-Gal1. Site-directed mutants of Gal1, expressed and purified as GST fusion proteins, were compared with the wild-type (WT) in three assays: (a) binding to asialofetuin-Sepharose as a measure of the carbohydrate-binding activity; (b) pull-down of TFII-I from NE; and (c) reconstitution of splicing in NE depleted of galectins as a test of the in vitro splicing activity. The binding of GST-Gal1(N46D) to asialofetuin-Sepharose was less than 10% of that observed for GST-Gal1(WT), indicating that the mutant was deficient in carbohydrate-binding activity. In contrast, both GST-Gal1(WT) and GST-Gal1(N46D) were equally efficient in pull-down of TFII-I and in reconstitution of splicing activity in the galectin-depleted NE. Moreover, while the splicing activity of the wild-type protein can be inhibited by saccharide ligands, the carbohydrate-binding deficient mutant was insensitive to such inhibition. Together, all of the results suggest that the carbohydrate-binding and the splicing activities of Gal1 can be dissociated and therefore, saccharide-binding, per se, is not required for the splicing activity.

Carbohydrate-binding protein 35: molecular cloning and expression of a recombinant polypeptide with lectin activity in Escherichia coli

Affinity-purified antibodies directed against carbohydrate-binding protein 35 (CBP35), a galactose-specific lectin, were used to screen a lambda gt 11 expression library derived from mRNA of 3T3 fibroblasts. This screening yielded several putative clones containing cDNA for CBP35, one of which was characterized in terms of its expression of a fusion protein containing beta-galactosidase and CBP35 sequences. Limited proteolysis of lysates containing the fusion protein, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with anti-CBP35, yielded a peptide mapping pattern comparable to that obtained from parallel treatment of authentic CBP35. Such a limited proteolysis followed by affinity chromatography on a Sepharose column coupled with galactose also yielded a 30-kDa polypeptide that exhibited carbohydrate-binding activity. This polypeptide can be immunoblotted with anti-CBP35, but not with antibodies directed against beta-galactosidase. These results indicate that we have identified a cDNA clone for CBP35 that yields a recombinant polypeptide with lectin activity produced in Escherichia coli. Using this cDNA clone as a probe, Northern-blot analysis showed an increased expression of the CBP35 gene when quiescent 3T3 cells were activated by the addition of serum growth factors.

A mechanism for incorporation of galectin-3 into the spliceosome through its association with U1 snRNP

Previously, we showed that galectin-1 and galectin-3 are redundant pre-mRNA splicing factors associated with the spliceosome throughout the splicing pathway. Here we present evidence for the association of galectin-3 with snRNPs outside of the spliceosome (i.e., in the absence of pre-mRNA splicing substrate). Immunoprecipitation of HeLa nuclear extract with anti-galectin-3 resulted in the coprecipitation of the five spliceosomal snRNAs, core Sm polypeptides, and the U1-specific protein, U1 70K. When nuclear extract was fractionated on glycerol gradients, some galectin-3 molecules cosedimented with snRNP complexes. This cosedimentation represents bona fide galectin-3--snRNP complexes as (i) immunoprecipitation of gradient fractions with anti-galectin-3 yielded several complexes with varying ratios of snRNAs and associated proteins and (ii) the distribution of galectin-3--snRNP complexes was altered when the glycerol gradient was sedimented in the presence of lactose, a galectin ligand. A complex at approximately 10S showed an association of galectin-3 with U1 snRNP that was sensitive to treatment with ribonuclease A. We tested the ability of this U1 snRNP to recognize an exogenous pre-mRNA substrate. Under conditions that assemble early splicing complexes, we found this isolated galectin-3--U1 snRNP particle was sufficient to load galectin-3 onto a pre-mRNA substrate, but not onto a control RNA lacking splice sites. Pretreatment of the U1 snRNP with micrococcal nuclease abolished the assembly of galectin-3 onto this early complex. These data identify galectin-3 as a polypeptide associated with snRNPs in the absence of splicing substrate and describe a mechanism for the assembly of galectin-3 onto the forming spliceosome.

Galectin-3: differential accumulation of distinct mRNAs in serum-stimulated mouse 3T3 fibroblasts

The murineGalectin-3 gene spans ∼12 kb of DNA and contains six exons, with the translation initiation codon located in exon II. On the basis of restriction mapping and sequence analysis of the DNA upstream of exon II, primer extension assays, rapid amplification of cDNA ends, and ribonuclease protection assays were designed and carried out to determine the initiation site of transcription and the sequence of exon I. The results revealed at least two transcription initiation sites (α and δ), each of which appears to be specifically associated with the use of alternative donor splice sites, resulting in distinct mRNA species. Type I message initiates at transcription start site δ, uses splice donor site No. 2, retaining a 27 bp sequence, whereas type II message initiates at transcription start site α, uses splice donor site No. 1, resulting in the loss of the 27 bp sequence. Primer extension assays carried out with mRNA isolated from 3T3 fibroblasts at various times after serum stimulation indicate that while the type II message varies in level only a little over the first 20 h, there is dramatic accumulation of the type I message, which peaks at 16 h post mitogen addition.

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.

Examination of the role of galectins in pre-mRNA splicing.

Several lines of evidence have been accumulated to indicate that galectin-1 and galectin-3 are two of the many proteins involved in nuclear splicing of pre-mRNA. First, nuclear extracts, capable of carrying out splicing of pre-mRNA in a cell-free assay, contain both of the galectins. Second, depletion of the galectins from nuclear extracts, using either lactose affinity chromatography or immunoadsorption with antibodies, results in concomitant loss of splicing activity. Third, addition of either galectin-1 or galectin-3 to the galectin-depleted extract reconstitutes the splicing activity. Fourth, the addition of saccharides that bind to galectin-1 and galectin-3 with high affinity (e.g., lactose or thiodigalactoside) to nuclear extract results in inhibition of splicing whereas parallel addition of saccharides that do not bind to the galectins (e.g., cellobiose) fail to yield the same effect. Finally, when a splicing reaction is subjected to immunoprecipitation by antibodies directed against galectin-1, radiolabeled RNA species corresponding to the starting pre-mRNA substrate, the mature mRNA product, and intermediates of the splicing reaction are coprecipitated with the galectin. Similar results were also obtained with antibodies against galectin-3. This chapter describes two key assays used in our studies: one reports on the splicing activity by looking at product formation on a denaturing gel; the other reports on the intermediates of spliceosome assembly using non-denaturing or native gels.

Inhibition of Cell-Free Splicing by Saccharides That Bind Galectins and SR Proteins

The carbohydrate-binding proteins galectin-1 and galectin-3 are found in the nucleus of cells. Using a cell-free assay, depletion and reconstitution experiments have documented that these two proteins are required factors in pre-mRNA splicing. Thiodigalactoside, which binds to both galectins, inhibited the splicing reaction, whereas cellobiose, which binds neither protein, failed to yield the same effect. Although L-rhamnose does not bind to either galectin-1 or galectin-3, it does inhibit the cell-free splicing assay. The effect of rhamnose is best explained by its strong binding to Sfrs1, one member of another family of nuclear splicing factors (the SR proteins) that exhibit carbohydrate-binding activity.

A 10S galectin-3-U1 snRNP complex assembles into active spliceosomes

In previous studies, we reported that fractionation of HeLa cell nuclear extracts on glycerol gradients revealed an endogenous ∼10S particle that contained galectin-3 and U1 snRNP and this particle was sufficient to load the galectin polypeptide onto a pre-mRNA substrate. We now document that this interaction between the galectin-3–U1 snRNP particle and the pre-mRNA results in a productive spliceosomal complex, leading to intermediates and products of the splicing reaction. Nuclear extracts were depleted of U1 snRNP with a concomitant loss of splicing activity. Splicing activity in the U1-depleted extract can be reconstituted by the galectin-3–U1 snRNP particle, isolated by immunoprecipitation of the 10S region (fractions 3–5) of the glycerol gradient with anti-galectin-3 antibodies. In contrast, parallel anti-galectin-3 immunoprecipitation of free galectin-3 molecules not in a complex with U1 snRNP (fraction 1 of the same gradient), failed to restore splicing activity. These results indicate that the galectin-3–U1 snRNP-pre-mRNA ternary complex is a functional E complex and that U1 snRNP is required to assemble galectin-3 onto an active spliceosome.