Cell cycle regulation by galectin-12, a new member of the galectin superfamily

Galectins are a family of beta-galactoside-binding animal lectins with conserved carbohydrate recognition domains (CRDs). Here we report the identification and characterization of a new galectin, galectin-12, which contains two domains that are homologous to the galectin CRD. The N-terminal domain contains all of the sequence elements predicted to form the two beta-sheets found in other galectins, as well as conserved carbohydrate-interacting residues. The C-terminal domain shows considerable divergence from the consensus sequence, and many of these conserved residues are not present. Nevertheless, the protein has lactose binding activity, most likely due to the contribution of the N-terminal domain. The mRNA for galectin-12 contains features coding for proteins with growth-regulatory functions. These include start codons in a context that are suboptimal for translation initiation and AU-rich motifs in the 3'-untranslated region, which are known to confer instability to mRNA. Galectin-12 mRNA is sparingly expressed or undetectable in many tissues and cell lines tested, but it is up-regulated in cells synchronized at the G(1) phase or the G(1)/S boundary of the cell cycle. Ectopic expression of galectin-12 in cancer cells causes cell cycle arrest at the G(1) phase and cell growth suppression. We conclude that galectin-12 is a novel regulator of cellular homeostasis.     

Cell surface counter receptors are essential components of the unconventional export machinery of galectin-1

Galectin-1 is a component of the extracellular matrix as well as a ligand of cell surface counter receptors such as beta-galactoside-containing glycolipids, however, the molecular mechanism of galectin-1 secretion has remained elusive. Based on a nonbiased screen for galectin-1 export mutants we have identified 26 single amino acid changes that cause a defect of both export and binding to counter receptors. When wild-type galectin-1 was analyzed in CHO clone 13 cells, a mutant cell line incapable of expressing functional galectin-1 counter receptors, secretion was blocked. Intriguingly, we also find that a distant relative of galectin-1, the fungal lectin CGL-2, is a substrate for nonclassical export from Chinese hamster ovary (CHO) cells. Alike mammalian galectin-1, a CGL-2 mutant defective in beta-galactoside binding, does not get exported from CHO cells. We conclude that the beta-galactoside binding site represents the primary targeting motif of galectins defining a galectin export machinery that makes use of beta-galactoside-containing surface molecules as export receptors for intracellular galectin-1.     

Complex N-glycans are the major ligands for galectin-1, -3, and -8 on Chinese hamster ovary cells

Galectins are implicated in a large variety of biological functions, many of which depend on their carbohydrate-binding ability. Fifteen members of the family have been identified in vertebrates based on binding to galactose (Gal) that is mediated by one or two, evolutionarily conserved, carbohydrate-recognition domains (CRDs). Variations in glycan structures expressed on glycoconjugates at the cell surface may, therefore, affect galectin binding and functions. To identify roles for different glycans in the binding of the three types of mammalian galectins to cells, we performed fluorescence cytometry at 4 degrees C with recombinant rat galectin-1, human galectin-3, and three forms of human galectin-8, to Chinese hamster ovary (CHO) cells and 12 different CHO glycosylation mutants. All galectin species bound to parent CHO cells and binding was inhibited >90% by 0.2 M lactose. Galectin-8 isoforms with either a long or a short inter-CRD linker bound similarly to CHO cells. However, a truncated form of galectin-8 containing only the N-terminal CRD bound only weakly to CHO cells and the C-terminal galectin-8 CRD exhibited extremely low binding. Binding of the galectins to the different CHO glycosylation mutants revealed that complex N-glycans are the major ligands for each galectin except the N-terminal CRD of galectins-8, and also identified some fine differences in glycan recognition. Interestingly, increased binding of galectin-1 at 4 degrees C correlated with increased propidium iodide (PI) uptake, whereas galectin-3 or -8 binding did not induce permeability to PI. The CHO glycosylation mutants with various repertoires of cell surface glycans are a useful tool for investigating galectin-cell interactions as they present complex and simple glycans in a natural mixture of multivalent protein and lipid glycoconjugates anchored in a cell membrane.     

Structural features of galectin-9 and galectin-1 that determine distinct T cell death pathways

The galectin family of lectins regulates multiple biologic functions, such as development, inflammation, immunity, and cancer. One common function of several galectins is the ability to trigger T cell death. However, differences among the death pathways triggered by various galectins with regard to glycoprotein receptors, intracellular death pathways, and target cell specificity are not well understood. Specifically, galectin-9 and galectin-1 both kill thymocytes, peripheral T cells, and T cell lines; however, we have found that galectin-9 and galectin-1 require different glycan ligands and glycoprotein receptors to trigger T cell death. The two galectins also utilize different intracellular death pathways, as galectin-9, but not galectin-1, T cell death was blocked by intracellular Bcl-2, whereas galectin-1, but not galectin-9, T cell death was blocked by intracellular galectin-3. Target cell susceptibility also differed between the two galectins, as galectin-9 and galectin-1 killed different subsets of murine thymocytes. To define structural features responsible for distinct activities of the tandem repeat galectin-9 and dimeric galectin-1, we created a series of bivalent constructs with galectin-9 and galectin-1 carbohydrate recognition domains connected by different peptide linkers. We found that the N-terminal carbohydrate recognition domain and linker peptide contributed to the potency of these constructs. However, we found that the C-terminal carbohydrate recognition domain was the primary determinant of receptor recognition, death pathway signaling, and target cell susceptibility. Thus, carbohydrate recognition domain specificity, presentation, and valency make distinct contributions to the specific effects of different galectins in initiating T cell death.     

Functional analyses of placental protein 13/galectin-13.

Placental protein 13 (PP13) was cloned from human term placenta. As sequence analyses, alignments and computational modelling showed its conserved structural and functional homology to members of the galectin family, the protein was designated galectin-13. Similar to human eosinophil Charcot-Leyden crystal protein/galectin-10 but not other galectins, its weak lysophospholipase activity was confirmed by 31P-NMR. In this study, recombinant PP13/galectin-13 was expressed and specific monoclonal antibody to PP13 was developed. Endogenous lysophospholipase activity of both the purified and also the recombinant protein was verified. Sugar binding assays revealed that N-acetyl-lactosamine, mannose and N-acetyl-glucosamine residues widely expressed in human placenta had the strongest binding affinity to both the purified and recombinant PP13/galectin-13, which also effectively agglutinated erythrocytes. The protein was found to be a homodimer of 16 kDa subunits linked together by disulphide bonds, a phenomenon differing from the noncovalent dimerization of previously known prototype galectins. Furthermore, reducing agents were shown to decrease its sugar binding activity and abolish its haemagglutination. Phosphorylation sites were computed on PP13/galectin-13, and phosphorylation of the purified protein was confirmed. Using affinity chromatography, PAGE, MALDI-TOF MS and post source decay, annexin II and beta/gamma actin were identified as proteins specifically bound to PP13/galectin-13 in placenta and fetal hepatic cells. Perinuclear staining of the syncytiotrophoblasts showed its expression in these cells, while strong labelling of the syncytiotrophoblasts' brush border membrane confirmed its galectin-like externalization to the cell surface. Knowing its colocalization and specific binding to annexin II, PP13/galectin-13 was assumed to be secreted to the outer cell surface by ectocytosis, in microvesicles containing actin and annexin II. With regard to our functional and immunomorphological results, PP13/galectin-13 may have special haemostatic and immunobiological functions at the lining of the common feto-maternal blood-spaces or developmental role in the placenta.     

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.     

Human galectin-2: novel inducer of T cell apoptosis with distinct profile of caspase activation

Galectin-2 is structurally closely related to galectin-1, but has a distinct expression profile primarily confined to the gastrointestinal tract. Prominent differences in the proximal promoter regions between galectins-2 and -1 concern Sp1-, hepatocyte NF-3, and T cell-specific factor-1 binding sites. Of note, these sequence elements are positioned equally in the respective regions for human and rat galectins-2. Labeled galectin-2 binds to T cells in a beta-galactoside-specific manner. In contrast to galectin-1, the glycoproteins CD3 and CD7 are not ligands, while the shared affinity to beta1 integrin (or a closely associated glycoprotein) accounts for a substantial extent of cell surface binding. The carbohydrate-dependent binding of galectin-2 induces apoptosis in activated T cells. Fluorogenic substrate and inhibitor assays reveal involvement of caspases-3 and -9, in accordance with cleavage of the DNA fragmentation factor. Enhanced cytochrome c release, disruption of the mitochondrial membrane potential, and an increase of the Bax/Bcl-2 ratio by opposite regulation of expression of both proteins add to the evidence that the intrinsic apoptotic pathway is triggered. Cell cycle distribution and expression of regulatory proteins remained unaffected. Notably, galectins-1 and -7 reduce cyclin B1 expression, defining functional differences between the structurally closely related galectins. Cytokine secretion of activated T cells was significantly shifted to the Th2 profile. Our study thus classifies galectin-2 as proapoptotic effector for activated T cells, raising a therapeutic perspective. Of importance for understanding the complex galectin network, it teaches the lesson that selection of cell surface ligands, route of signaling, and effects on regulators of cell cycle progression are markedly different between structurally closely related galectins.     

Selective eosinophil adhesion to fibroblast via IFN-gamma-induced galectin-9

Among galectin family members, galectin-9 was first described as a potent eosinophil chemoattractant derived from Ag-stimulated T cells. In the present study a role of galectin-9 in the interaction between eosinophils and fibroblasts was investigated using a human lung fibroblast cell line, HFL-1. RT-PCR, real-time PCR, and Western blot analyses revealed that both galectin-9 mRNA and protein in HFL-1 cells were up-regulated by IFN-gamma stimulation. On the one hand, IL-4, known as a Th2 cytokine, did not affect the galectin-9 expression in HFL-1 cells. We further confirmed that IFN-gamma up-regulated the expression of galectin-9 in primary human dermal fibroblasts. Flow cytometric analysis revealed that IFN-gamma up-regulated surface galectin-9 expression on HFL-1 cells. Stimulation of HFL-1 cells with IFN-gamma up-regulated adhesion of eosinophils, but not neutrophils, to HFL-1 cells. This adherence of eosinophils to HFL-1 cells was inhibited by both lactose and anti-galectin-9 Ab. These findings demonstrate that IFN-gamma-induced galectin-9 expression in fibroblasts mediates eosinophil adhesion to the cells, suggesting a crucial role of galectin-9 in IFN-gamma-stimulated fibroblasts as a physiological modulator at the inflammatory sites.     

Thermodynamic binding studies of galectin-1, -3 and -7

The carbohydrate binding specificities of the galectin family of animal lectins has been the source of intense recent investigations. Isothermal titration microcalorimetry (ITC) provides direct determination of the thermodynamics of binding of carbohydrates to lectins, and has provided important insights into the fine carbohydrate binding specificities of a wide number of plant and animal lectins. Recent ITC studies have been performed with galectin-1, galectin-3 and galectin-7 and their interactions with sialylated and non-sialylated carbohydrates. The results show important differences in the specificities of these three galectins toward poly-N-acetyllactosamine epitopes found on the surface of cells.     

Galectin-loaded cells as a platform for the profiling of lectin specificity by fluorescent neoglycoconjugates: a case study on galectins-1 and -3 and the impact of assay setting

The involvement of galectins as pleiotropic regulators of cell adhesion and growth in disease progression explains the interest to define their ligand-binding properties. Toward this end, it is desirable to approach in vivo conditions to attain medical relevance. In order to simulate physiological conditions with cell surface glycans as recognition sites and galectins as mediators of intercellular contacts we developed an assay using galectin-loaded Raji cells. The extent of surface binding of fluorescent neoglycoconjugates depended on the lectin presence and the type of lectin, the nature of the probes' carbohydrate headgroup and the density of unsubstituted beta-galactosides on the cell surface. Using the most frequently studied galectins-1 and -3, application of this assay led to rather equal binding levels for linear and branched oligomers of N-acetyllactosamine. A clear preference of galectin-3 for alpha1-3-linked galactosylated lactosamine was noted. In parallel, a panel of 24 neoglycoconjugates was tested as inhibitors of galectin binding from solution to N-glycans of surface-immobilized asialofetuin. These two assays differ in presentation of the galectin and ligand, facilitating identification of assay-dependent properties. Under the condition of the cell assay, selectivity among oligosaccharides for the lectins was higher, and extraordinary affinity of galectin-1 to 3'-O-sulfated probes in a solid-phase assay was lost in the cell assay. Having introduced and validated a cell assay, the comprehensive profiling of ligand binding to cell-surface-presented galectins is made possible.     

Recognition mechanism of galectin-4 for cholesterol 3-sulfate

Galectin-4 binds to glycosphingolipids carrying 3-O-sulfated Gal residues, and it co-localizes on the cell surface of human colonic adenocarcinoma cells with glycosphingolipids carrying SO(-)(3)-->3Galbeta1-->3(GalNAc) residues (Ideo, H., Seko, A., and Yamashita, K. (2005) J. Biol. Chem. 280, 4730-4737). In the present study, it was found that galectin-4 also binds to cholesterol 3-sulfate, which has no beta-galactoside moiety. This characteristic of galectin-4 is unique within the galectin family. The site-directed mutated galectin-4-R45A had diminished binding ability toward cholesterol 3-sulfate, suggesting that Arg(45) of galectin-4 is indispensable for cholesterol 3-sulfate recognition. Gel filtration and chemical cross-linking experiments revealed that some galectin-4 exists as dimers, and this multivalency seemed to enhance its avidity for cholesterol 3-sulfate binding. Cholesterol 3-sulfate and sulfatide co-existed with galectin-4 in detergent-insoluble fractions of porcine esophagus and intestine, respectively. These results suggested that not only sulfated glycosphingolipids but also cholesterol 3-sulfate are endogenous ligands for galectin-4 in vivo.     

Ganglioside GM1/Galectin-Dependent Growth Regulation in Human Neuroblastoma Cells: Special Properties of Bivalent Galectin-4 and Significance of Linker Length for Ligand Selection

Orchestrated upregulation of cell surface presentation of ganglioside GM1 and homodimeric galectin-1 is the molecular basis for growth regulation of human neuroblastoma (SK-N-MC) cells. Further study led to the discovery of competitive inhibition by galectin-3, prompting us to test tandem-repeat-type galectin-4 (two different lectin domains connected by a 42-amino-acid linker). This lectin bound to cells at comparably high affinity without involvement of the ganglioside, as disclosed by assays in the presence of cholera toxin B-subunit or galectin-1 and blocking glucosylceramide synthesis. Notably, when tested separately, binding of both lectin domains showed partial sensitivity to the bacterial agglutinin. Despite its ability for cross-linking surface association of galectin-4 did not affect proliferation, in contrast to homodimeric galectins. The truncation of linker length from 42 to 16 amino acids altered binding properties to let partial sensitivity to the bacterial lectin emerge. Cross-competition between parental and engineered proteins did not exceed 40%. No effect on cell growth was detected. This study reveals complete functional divergence between galectins differing in the spatial mode of lectin-site presentation and dependence of reactivity to distinct counter-receptor(s) on linker length. Due to the documented presence of galectin-4 in the nervous system and its affinity for sulfatide these in vitro results indicate the potential for a distinct functionality profile of this lectin in vivo, giving further research direction.     

Thermodynamic binding studies of galectin-1, -3 and -7

The carbohydrate binding specificities of the galectin family of animal lectins has been the source of intense recent investigations. Isothermal titration microcalorimetry (ITC) provides direct determination of the thermodynamics of binding of carbohydrates to lectins, and has provided important insights into the fine carbohydrate binding specificities of a wide number of plant and animal lectins. Recent ITC studies have been performed with galectin-1, galectin-3 and galectin-7 and their interactions with sialylated and non-sialylated carbohydrates. The results show important differences in the specificities of these three galectins toward poly-N-acetyllactosamine epitopes found on the surface of cells. Published in 2004.     

Ligand induced galectin-3 protein self-association

Many functions of galectin-3 entail binding of its carbohydrate recognition site to glycans of a glycoprotein, resulting in cross-linking thought to be mediated by its N-terminal noncarbohydrate-binding domain. Here we studied interaction of galectin-3 with the model glycoprotein asialofetuin (ASF), using a fluorescence anisotropy assay to measure the concentration of free galectin carbohydrate recognition sites in solution. Surprisingly, in the presence of ASF, this remained low even at high galectin-3 concentrations, showing that many more galectin-3 molecules were engaged than expected due to the about nine known glycan-based binding sites per ASF molecule. This suggests that after ASF-induced nucleation, galectin-3 associates with itself by the carbohydrate recognition site binding to another galectin-3 molecule, possibly forming oligomers. We named this type-C self-association to distinguish it from the previously proposed models (type-N) where galectin-3 molecules bind to each other through the N-terminal domain, and all carbohydrate recognition sites are available for binding glycans. Both types of self-association can result in precipitates, as measured here by turbidimetry and dynamic light scattering. Type-C self-association and precipitation occurred even with a galectin-3 mutant (R186S) that bound poorly to ASF but required much higher concentration (～50 μM) as compared with wild type (～1 μM). ASF also induced weaker type-C self-association of galectin-3 lacking its N-terminal domains, but as expected, no precipitation. Neither a monovalent nor a divalent N-acetyl-D-lactosamine-containing glycan induced type-C self-association, even if the latter gave precipitates with high concentrations of galectin-3 (>～50 μM) in agreement with published results and perhaps due to type-N self-association.     

Galectin-3 Protein Regulates Mobility of N-cadherin and GM1 Ganglioside at Cell-Cell Junctions of Mammary Carcinoma Cells

Galectin-3 binding to cell surface glycoproteins, including branched N-glycans generated by N-acetylglucosaminyltransferase V (Mgat5) activity, forms a multivalent, heterogeneous, and dynamic lattice. This lattice has been shown to regulate integrin and receptor tyrosine kinase signaling promoting tumor cell migration. N-cadherin is a homotypic cell-cell adhesion receptor commonly overexpressed in tumor cells that contributes to cell motility. Here we show that galectin-3 and N-cadherin interact and colocalize with the lipid raft marker GM1 ganglioside in cell-cell junctions of mammary epithelial cancer cells. Disruption of the lattice by deletion of Mgat5, siRNA depletion of galectin-3, or competitive inhibition with lactose stabilizes cell-cell junctions. It also reduces, in a p120-catenin-dependent manner, the dynamic pool of junctional N-cadherin. Proteomic analysis of detergent-resistant membranes (DRMs) revealed that the galectin lattice opposes entry of many proteins into DRM rafts. N-cadherin and catenins are present in DRMs; however, their DRM distribution is not significantly affected by lattice disruption. Galectin lattice integrity increases the mobile fraction of the raft marker, GM1 ganglioside binding cholera toxin B subunit Ctb, at cell-cell contacts in a p120-catenin-independent manner, but does not affect the mobility of either Ctb-labeled GM1 or GFP-coupled N-cadherin in nonjunctional regions. Our results suggest that the galectin lattice independently enhances lateral molecular diffusion by direct interaction with specific glycoconjugates within the adherens junction. By promoting exchange between raft and non-raft microdomains as well as molecular dynamics within junction-specific raft microdomains, the lattice may enhance turnover of N-cadherin and other glycoconjugates that determine junctional stability and rates of cell migration.     

Newcastle disease virus neuraminidase primes neutrophils for stimulation by galectin-3 and formyl-Met-Leu-Phe

Human neutrophils are activated by the β-galactoside-binding lectin galectin-3, provided that the cells are primed by in vivo extravasation or by in vitro preactivation with, for example, LPS. Removal of terminal sialic acid can change neutrophil functionality and responsiveness due to exposure of underlying glycoconjugate receptors or change in surface charge. Here, we investigated whether such alteration of the cell surface carbohydrate composition can alter the responsiveness of the cells to galectin-3. Neutrophils were treated with neuraminidases (NA) of different origins: Clostridium perfringens (CP), Salmonella typhimurium, Vibrio cholerae, and Newcastle disease virus (NDV). In the presence of NDV-NA, but no other NA, the otherwise non-responding neutrophils responded readily to galectin-3 by activation of the NADPH-oxidase. The galectin-3 priming effect was inhibited by the sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetyl-neuraminic acid. Earlier studies have shown that priming of the neutrophil response to galectin-3 with, for example, LPS is paralleled by degranulation of intracellular vesicles and granules and upregulation of potential galectin-3 receptors. Also, NDV-NA (but not CP-NA) treatment induced degranulation, shown as an upregulation of complement receptor 3. Since not only the galectin response but also the response to the chemoattractant fMLF was primed, NDV-NA appears to induce a general priming phenomenon, possibly due to receptor upregulation by degranulation.     

Visualization of galectin-3 oligomerization on the surface of neutrophils and endothelial cells using fluorescence resonance energy transfer

Galectin-3, a member of the galectin family of carbohydrate binding proteins, is widely expressed, particularly in cells involved in the immune response. Galectin-3 has also been indicated to play a role in various biological activities ranging from cell repression to cell activation and adhesion and has, thus, been recognized as an immunomodulator. Whereas those activities are likely to be associated with ligand cross-linking by this lectin, galectin-3, unlike other members of the galectin family, exists as a monomer. It has consequently been proposed that oligomerization of the N-terminal domains of galectin-3 molecules, after ligand binding by the C-terminal domain, is responsible for this cross-linking. The oligomerization status of galectin-3 could, thus, control the majority of its extracellular activities. However, little is known about the actual mode of action through which galectin-3 exerts its function. In this report we present data suggesting that oligomerization of galectin-3 molecules occurs on cell surfaces with physiological concentrations of the lectin. Using galectin-3 labeled at the C terminus with Alexa 488 or Alexa 555, the oligomerization between galectin-3 molecules on cell surfaces was detected using fluorescence resonance energy transfer. We observed this fluorescence resonance energy transfer signal in different biological settings representing the different modes of action of galectin-3 that we previously proposed; that is, ligand crosslinking leading to cell activation, cell-cell interaction/adhesion, and lattice formation. Furthermore, our data suggest that galectin-3 lattices are robust and could, thus, be involved, as previously proposed, in the restriction of receptor clustering.     

Galectin-3 but not galectin-1 induces mast cell death by oxidative stress and mitochondrial permeability transition

Galectin-1 and galectin-3 are the most ubiquitously expressed members of the galectin family and more importantly, these two molecules are shown to have opposite effects on pro-inflammatory responses and/or apoptosis depending on the cell type. Herein, we demonstrate for the first time that galectin-3 induces mast cell apoptosis. Mast cells expressed substantial levels of galectin-3 and galectin-1 and to a lesser extent the receptor for advanced glycation end products (RAGE) on their surfaces. Treatment of cells with galectin-3 at concentrations of > or =100 nM for 18-44 h resulted in cell death by apoptosis. Galectin-3-induced apoptosis was completely prevented by lactose, neutralizing antibody to RAGE, and the caspase-3 inhibitor z-DEVD-fmk. Galectin-3-induced apoptosis was also completely abolished by dithiothreitol and superoxide dismutase, but not inhibited by catalase. Moreover, galectin-3 but not galectin-1 induced the release of superoxide, which was blocked by lactose, anti-RAGE, and dithiothreitol. Finally, galectin-3-induced apoptosis was blocked by bongkrekic acid, an antagonist of the mitochondrial permeability transition pore (PTP), while atractyloside, an agonist of the PTP, greatly facilitated galectin-1-induced apoptosis. These data suggest that galectin-3 induces oxidative stress, PTP opening, and the caspase-dependent death pathway by binding to putative surface receptors including RAGE via the carbohydrate recognition domain.     

Phosphorylation of the beta-galactoside-binding protein galectin-3 modulates binding to its ligands

The beta-galactoside-binding protein galectin-3 has pleiotropic biological functions and has been implicated in cell growth, differentiation, adhesion, RNA processing, apoptosis, and malignant transformation. Galectin-3 may be phosphorylated at N-terminal Ser(6), but the role of phosphorylation in determining interactions of this endogenous lectin with its ligands remains to be elucidated. We therefore studied the effect of phosphorylation on binding of galectin-3 to two of its reported ligands, laminin and purified colon cancer mucin. Human recombinant galectin-3 was phosphorylated in vitro by casein kinase I, and separated from the native species by isoelectric focusing for use in solid phase binding assays. Non-phosphorylated galectin-3 bound to laminin and asialomucin in a dose-dependent manner with half-maximal binding at 1.5 microg/ml. Phosphorylation reduced saturation binding to each ligand by >85%. Ligand binding could be fully restored by dephosphorylation with protein phosphatase type 1. Mutation of galectin-3 at Ser(6) (Ser to Glu) did not alter galectin ligand binding. Metabolic labeling or separation by isoelectric focusing confirmed the presence of phosphorylated galectin-3 species in vivo in the cytosol of human colon cancer cells from which ligand mucin was purified. Phosphorylation significantly reduces the interaction of galectin-3 with its ligands. The process by which phosphorylation modulates protein-carbohydrate interactions has important implications for understanding the biological functions of this protein, and may serve as an "on/off" switch for its sugar binding capabilities.     

Tyrosine-phosphorylated galectin-3 protein is resistant to prostate-specific antigen (PSA) cleavage

Galectin-3 is a chimeric carbohydrate-binding protein, which interacts with cell surface carbohydrate-containing molecules and extracellular matrix glycoproteins and has been implicated in various biological processes such as cell growth, angiogenesis, motility, and metastasis. It is expressed in a wide range of tumor cells and is associated with tumor progression. The functions of galectin-3 are dependent on its localization and post-translational modifications such as cleavage and phosphorylation. Recently, we showed that galectin-3 Tyr-107 is phosphorylated by c-Abl; concomitantly, it was also shown that galectin-3 can be cleaved at this site by prostate-specific antigen (PSA), a chymotrypsin-like serine protease, after Tyr-107, resulting in loss of galectin-3 multivalency while preserving its carbohydrate binding activity. Galectin-3 is largely a monomer in solution but may form a homodimer by self-association through its carbohydrate recognition domain, whereas, in the presence of a ligand, galectin-3 polymerizes up to pentamers utilizing its N-terminal domain. Oligomerization is a unique feature of secreted galectin-3, which allows its function by forming ordered galectin-glycan structures, i.e. lattices, on the cell surface or through direct engagement of specific cell surface glycoconjugates by traditional ligand-receptor binding. We questioned whether Tyr-107 phosphorylation by c-Abl affects galectin-3 cleavage by PSA. The data suggest a role for galectin-3 in prostate cells associated with increased activity of c-Abl kinase and loss of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) activity. In addition, the ratio of phosphorylated/dephosphorylated galectin-3 might be used as a complementary value to that of PSA for prognosis of prostate cancer and a novel therapeutic target for the treatment of prostate cancer.