Galectin-3 expression and subcellular localization in senescent human fibroblasts

Galectin-3 is a galactose/lactose-binding protein (M(r) approximately 30,000), identified as a required factor in the splicing of pre-mRNA. In the LG1 strain of human diploid fibroblasts, galectin-3 could be found in both the nucleus and the cytoplasm of young, proliferating cells. In contrast, the protein was predominantly cytoplasmic in senescent LG1 cells that have lost replicative competence through in vitro culture. Incubation of young cells with leptomycin B, a drug that disrupts the interaction between the leucine-rich nuclear export signal and its receptor, resulted in the accumulation of galectin-3 inside the nucleus. In senescent cells, galectin-3 staining remained cytoplasmic even in the presence of the drug, thus suggesting that the observed localization in the cytoplasm was due to a lack of nuclear import. In heterodikaryons derived from fusion of young and senescent LG1 cells, the predominant phenotype was galectin-3 in both nuclei. These results suggest that senescent LG1 cells might lack a factor(s) specifically required for galectin-3 nuclear import.     

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.     

Human galectin-2: nuclear presence in vitro and its modulation by quiescence/stress factors

Galectins have the particular capacity to interact with distinct proteins, in addition to the typical reactivity of lectins with glycans. Therefore, they can be functionally active when residing at places other than the membrane or extracellular matrix. In fact, nuclear presence of galectins-1 and -3 is solidly documented but it is an open question whether these two cases are exceptional within this lectin family. Thus, galectin-2, which shares 43% sequence identity on the protein level with galectin-1, warrants study in this respect. Based on initial immunohistochemical evidence we herein address the issue as to whether this galectin can join the category of nuclear lectins. To do so we studied different types of cell in vitro using an antibody preparation free of cross-reactivity against other tested galectins. The immunocytochemical experiments revealed that galectin-2 was present in nuclei of murine 3T3 fibroblasts and also genetically engineered human colon carcinoma cells with stable ectopic expression. Transport of galectin-2 to the nucleus could be enhanced by physical (UV light), chemical (mitomycin C, serum withdrawal) or cell biological (coculture with stromal cells) treatment modalities. As a means of further characterizing the staining profile cytochemically, a series of markers with well-defined site of residency within the nuclear compartment was tested in parallel. Importantly, no colocalization with galectins-1 and -3 and the splicing factor SC35 was detectable, the former cases also serving as inherent specificity control. In contrast, a similarity was uncovered in the case of the promyelocytic leukemia (PML) protein as marker of PML nuclear bodies. In aggregate, nuclear localization is documented for galectin-2. This attribute should thus not be considered as an exceptional finding confined to galectins-1 and -3. That even closely related family members, here galectins-1 and -2, exhibit distinct intranuclear localization patterns gives ensuing research a clear direction.     

Glycosylated nuclear lectin CBP70 also associated with endoplasmic reticulum and the Golgi apparatus: does the "classic pathway" of glycosylation also apply to nuclear glycoproteins?

The subcellular plurilocalization of some lectins (galectin-1, galectin-3, galectin-10, calreticulin, etc.) is an intriguing problem, implying different partners according to their localization, and involvement in a variety of cellular activities. For example, the well-known lectin, galectin-3, a lactose-binding protein, can act inside the nucleus in splicing events, and at the plasma membrane in adhesion, and it was demonstrated that galectin-3 interacts in the cytoplasm with Bcl-2, an antiapoptotic protein. Some years ago, our group isolated a nuclear lectin CBP70, capable of recognizing N-acetylglucosamine residues. This lectin, first isolated from the nucleus of HL60 cells, was also localized in the cytoplasm. It has been demonstrated that CBP70 is a glycosylated lectin, with different types of glycosylation, comparing cytoplasmic and nuclear forms. In this article, we have studied the localization of CBP70 in undifferentiated HL60 cells by electron microscopy, immunofluorescence analysis, and subcellular fractionation. The results obtained clearly demonstrated that CBP70 is a plurilocalized lectin that is found in the nucleus, at the endoplasmic reticulum, the Golgi apparatus, and mitochondria, but not at the plasma membrane. Because CBP70, a nuclear glycoprotein, was found to be associated also with the endoplasmic reticulum and the Golgi apparatus where the glycosylation take place, it raised the question: where does the glycosylation of nuclear proteins occur?     

Shuttling of galectin-3 between the nucleus and cytoplasm

In previous studies, we documented that galectin-3 (M(r) approximately 30,000) is a pre-mRNA splicing factor. Recently, galectin-3 was identified as a component of a nuclear and cytoplasmic complex, the survival of motor neuron complex, through its interaction with Gemin4. To test the possibility that galectin-3 may shuttle between the nucleus and the cytoplasm, human fibroblasts (LG-1) were fused with mouse fibroblasts (3T3). The monoclonal antibody NCL-GAL3, which recognizes human galectin-3 but not the mouse homolog, was used to monitor the localization of human galectin-3 in heterodikaryons. Human galectin-3 localized to both nuclei of a large percentage of heterodikaryons. Addition of the antibiotic leptomycin B, which inhibits nuclear export of galectin-3, decreased the percentage of heterodikaryons showing human galectin-3 in both nuclei. In a parallel experiment, mouse 3T3 fibroblasts, which express galectin-3, were fused with fibroblasts derived from a mouse in which the galectin-3 gene was inactivated. Mouse galectin-3 localized to both nuclei of a large percentage of heterodikaryons. Again, addition of leptomycin B restricted the presence of galectin-3 to one nucleus of a heterodikaryon. The results from both heterodikaryon assays suggest that galectin-3 can exit one nucleus, travel through the cytoplasm, and enter the second nucleus, matching the definition of shuttling.     

Transport of galectin-3 between the nucleus and cytoplasm. II. Identification of the signal for nuclear export

Galectin-3, a factor involved in the splicing of pre-mRNA, shuttles between the nucleus and the cytoplasm. Previous studies have shown that incubation of fibroblasts with leptomycin B resulted in the accumulation of galectin-3 in the nucleus, suggesting that the export of galectin-3 from the nucleus may be mediated by the CRM1 receptor. A candidate nuclear export signal fitting the consensus sequence recognized by CRM1 can be found between residues 240 and 255 of the murine galectin-3 sequence. This sequence was engineered into the pRev(1.4) reporter system, in which candidate sequences can be tested for nuclear export activity in terms of counteracting the nuclear localization signal present in the Rev(1.4) protein. Rev(1.4)-galectin-3(240-255) exhibited nuclear export activity that was sensitive to inhibition by leptomycin B. Site-directed mutagenesis of Leu247 and Ile249 in the galectin-3 nuclear export signal decreased nuclear export activity, consistent with the notion that these two positions correspond to the critical residues identified in the nuclear export signal of the cAMP-dependent protein kinase inhibitor. The nuclear export signal activity was also analyzed in the context of a full-length galectin-3 fusion protein; galectin-3(1-263; L247A) showed more nuclear localization than wild-type, implicating Leu247 as critical to the function of the nuclear export signal. These results indicate that residues 240-255 of the galectin-3 polypeptide contain a leucine-rich nuclear export signal that overlaps with the region (residues 252-258) identified as important for nuclear localization.     

Nuclear staining for the small heat shock protein alphaB-crystallin colocalizes with splicing factor SC35

AlphaB-Crystallin has for a long time been considered a specific eye lens protein. Later on it appeared that this protein belongs to the family of the small heat shock proteins and that it occurs also extra-lenticularly in many different cell types. AlphaB-Crystallin is mainly present in the cytoplasm, but there are some indications that it might have a function in the nucleus too. However, till now its presence in the nucleus is uncertain. We therefore compared the localization of alphaB-crystallin in nine cell lines cultured under normal conditions using four different antisera. All four antisera gave a diffuse staining for alphaB-crystallin in the cytoplasm, but one of the antibodies consistently showed nuclear staining in eight of the cell types, in the form of distinct speckles. These speckles are equally pronounced in the different cell types, whether or not cytoplasmic alphaB-crystallin is present. Preabsorption of the antiserum with alphaB-crystallin abolished the staining. Furthermore we demonstrate that if only minor amounts of alphaB-crystallin are present, the protein seems to be located exclusively in the nucleus. However, in case of higher amounts of protein, alphaB-crystallin is distributed between cytoplasm and nucleus. The nuclear alphaB-crystallin exists, like the cytoplasmic alphaB-crystallin, in non-phosphorylated and phosphorylated forms, is Triton-insoluble but can be extracted by 2 M NaCl. These data suggest that alphaB-crystallin might be bound to the nuclear matrix per se or to nuclear matrix proteins via other proteins. In agreement with other nuclear matrix proteins, nuclear alphaB-crystallin staining turns diffuse upon mitosis and leaves the chromosomes unstained. Double staining experiments revealed colocalization of alphaB-crystallin with the splicing factor SC35 in nuclear speckles, suggesting a role for alphaB-crystallin in splicing or protection of the splicing machinery.     

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.     

Transport of galectin-3 between the nucleus and cytoplasm. I. Conditions and signals for nuclear import

Galectin-3, a factor involved in the splicing of pre-mRNA, shuttles between the nucleus and the cytoplasm. We have engineered a vector that expresses the fusion protein containing the following: (a) green fluorescent protein as a reporter of localization, (b) bacterial maltose-binding protein to increase the size of the reporter polypeptide, and (c) galectin-3, whose sequence we wished to dissect in search of amino acid residues vital for nuclear localization. In mouse 3T3 fibroblasts transfected with this expression construct, the full-length galectin-3 (residues 1-263) fusion protein was localized predominantly in the nucleus. Mutants of this construct, containing truncations of the galectin-3 polypeptide from the amino terminus, retained nuclear localization through residue 128; thus, the amino-terminal half was dispensable for nuclear import. Mutants of the same construct, containing truncations from the carboxyl terminus, showed loss of nuclear localization. This effect was observed beginning with truncation at residue 259, and the full effect was seen with truncation at residue 253. Site-directed mutagenesis of the sequence ITLT (residues 253-256) suggested that nuclear import was dependent on the IXLT type of nuclear localization sequence, first discovered in the Drosophila protein Dsh (dishevelled). In the galectin-3 polypeptide, the activity of this nuclear localization sequence is modulated by a neighboring leucine-rich nuclear export signal.     

Nuclear localization of tetrahydrobiopterin biosynthetic enzymes

Biosynthesis of the tetrahydrobiopterin (BH(4)) cofactor, essential for catecholamines and serotonin production and nitric oxide synthase (NOS) activity, requires the enzymes GTP cyclohydrolase I (GTPCH), 6-pyruvoyl-tetrahydropterin synthase (PTPS), and sepiapterin reductase (SR). Upon studying the distribution of GTPCH and PTPS with polyclonal immune sera in cross sections of rat brain, prominent nuclear staining in many neurons was observed besides strong staining in peri-ventricular structures. Furthermore, localization studies in transgenic mice expressing a Pts-LacZ gene fusion containing the N-terminal 35 amino acids of PTPS revealed beta-galactosidase in the nucleus of neurons. In contrast, PTPS-beta-galactosidase was exclusively cytoplasmic in the convoluted kidney tubules but nuclear in other parts of the nephron, indicating again that nuclear targeting may occur only in specific cell categories. Furthermore, the N terminus of PTPS acts as a domain able to target the PTPS-beta-galactosidase fusion protein to the nucleus. In transiently transfected COS-1 cells, which do not express GTPCH and PTPS endogenously, we found cytoplasmic and nuclear staining for GTPCH and PTPS. To further investigate nuclear localization of all three BH(4)-biosynthetic enzymes, we expressed Flag-fusion proteins in transiently transfected COS-1 cells and analyzed the distribution by immunolocalization and sub-cellular fractionation using anti-Flag antibodies and enzymatic assays. Whereas 5-10% of total GTPCH and PTPS and approximately 1% of total SR were present in the nucleus, only GTPCH was confirmed to be an active enzyme in nuclear fractions. The in vitro studies together with the tissue staining corroborate specific nuclear localization of BH(4)-biosynthetic proteins with yet unknown biological function.     

Intracellular distribution of a nuclear localization signal binding protein.

The transport of proteins into the nucleus requires the recognition of a nuclear localization signal sequence. Several proteins that interact with these sequences have been identified, including one of about 66 kDa. We have prepared antibodies that recognize the 66-kDa nuclear localization signal binding protein (NLSBP) and inhibit nuclear localization in vitro. By immunofluorescence, it is seen that the NLSBP is predominantly cytoplasmic and is distributed peripherally around the nucleus and the microtubule organizing center. There is also a weak punctate staining of the surface of the nucleus. Methanol-fixed cells can also be stained directly with fluorescently labeled karyophilic proteins. These stains reveal the same cytoplasmic structures as anti-NLSBP. The expression of the NLSBP is growth dependent. When cells grown to confluence are examined, the cytoplasmic staining is greatly reduced, leaving the punctate nuclear staining as the predominant feature. In serum-starved cells, very little staining of either the cytoplasm or the nucleus can be seen. Upon simulation by the addition of serum, the original cytoplasmic and nuclear envelope staining is restored. Cells grown in the presence of colchicine or taxol have an altered NLSBP distribution but apparently normal cytoplasmic nuclear transport.     

Export of Galectin-3 from Nuclei of Digitonin-Permeabilized Mouse 3T3 Fibroblasts

Galectin-3 is a galactose-/lactose-binding protein (M(r) approximately 30,000), identified as a required factor in the splicing of pre-mRNA. Immunofluorescence staining revealed that galectin-3 distributes differentially between the nucleus and the cytoplasm, depending on the proliferative state of the cells under analysis. Using digitonin-permeabilized mouse 3T3 fibroblasts, we provide evidence that galectin-3 is rapidly and selectively exported from the nucleus. Although both phosphorylated and nonphosphorylated isoforms of galectin-3 are found in the nuclear fraction, only phosphorylated galectin-3 is identified in the exported fraction, implying that phosphorylation is important for the nuclear export of the protein. The rate of galectin-3 export is temperature dependent and is decreased by the addition of wheat germ agglutinin. More strikingly, galectin-3 export can be inhibited by the addition of leptomycin B, a drug that disrupts the interaction between the leucine-rich nuclear export signal and its receptor, CRM1 (chromosome maintenance region 1). Indeed, a putative leucine-rich nuclear export signal can be found in residues 241-249 of the murine galectin-3 sequence. Finally, gel filtration of the exported material showed that galectin-3 can be found in at least two high molecular weight complexes (approximately 650 and approximately 60 kDa), both of which can be disrupted by lactose.     

Association of galectin-1 and galectin-3 with Gemin4 in complexes containing the SMN protein

In previous studies we showed that galectin-1 and galectin-3 are factors required for the splicing of pre-mRNA, as assayed in a cell-free system. Using a yeast two-hybrid screen with galectin-1 as bait, Gemin4 was identified as a putative interacting protein. Gemin4 is one component of a macromolecular complex containing approximately 15 polypeptides, including SMN (survival of motor neuron) protein. Rabbit anti-galectin-1 co-immunoprecipitated from HeLa cell nuclear extracts, along with galectin-1, polypeptides identified to be in this complex: SMN, Gemin2 and the Sm polypeptides of snRNPs. Direct interaction between Gemin4 and galectin-1 was demonstrated in glutathione S-transferase (GST) pull-down assays. We also found that galectin-3 interacted with Gemin4 and that it constituted one component of the complex co-immunoprecipitated with galectin-1. Indeed, fragments of either Gemin4 or galectin-3 exhibited a dominant negative effect when added to a cell-free splicing assay. For example, a dose-dependent inhibition of splicing was observed in the presence of exogenously added N-terminal domain of galectin-3 polypeptide. In contrast, parallel addition of either the intact galectin-3 polypeptide or the C-terminal domain failed to yield the same effect. Using native gel electrophoresis to detect complexes formed by the splicing extract, we found that with addition of the N-terminal domain the predominant portion of the radiolabeled pre-mRNA was arrested at a position corresponding to the H-complex. Inasmuch as SMN-containing complexes have been implicated in the delivery of snRNPs to the H-complex, these results provide strong evidence that galectin-1 and galectin-3, by interacting with Gemin4, play a role in spliceosome assembly in vivo.     

Nucleocytoplasmic shuttling of hexokinase II in a cancer cell

In yeast, the hexokinase type II enzyme (HXKII) translocates to the nucleus in the presence of excess glucose, and participates in glucose repression. However, no evidence has suggested a nuclear function for HXKII in mammalian cells. Herein, we present data showing nuclear localization of HXKII in HeLa cells, both by immunocytochemistry and subcellular fractionation. HXKII is extruded from the nucleus, at least in part, by the activity of the exportin 1/CrmA system, as demonstrated by increased nuclear expression and decreased cytoplasmic expression after incubation with leptomycin B, a bacterially-derived exportin inhibitor. Furthermore, cytoplasmic localization of HXKII is dependent on its enzymatic activity, as inhibiting HXKII activity using 2-deoxy-d-glucose (2DG) increased nuclear localization. This effect was more significant in cells incubated in the absence of glucose for 24 h prior to addition of 2DG. Regulated translocation of HXKII to the nucleus of mammalian cells could represent a previously unknown glucose-sensing mechanism.