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.     

Expression of galectin-1 and -3 and of accessible binding sites during murine hair cycle

Although protein-carbohydrate interactions are supposed to play key roles in cell adhesion, signalling and growth control. Their exact role in skin physiology has only recently been investigated. The endogenous lectins galectin-1 and galectin-3 have been identified in skin including hair follicles. Here, we analyzed the expression and distribution of these galectins and their binding sites in C57BL/6 mice during hair cycle. The expression of galectin-1 and galectin-3 binding sites was found to be predominantly hair cycle-dependent showing some overlapping to the expression of galectin-1 and -3. The outer root sheath (ORS) expressed galectin-1 binding sites during anagen IV to VI and in early catagen, whereas galectin-1 was expressed from early anagen to late catagen. The ORS expressed galectin-3 binding sites during catagen transition corresponding to a galectin-3 expression during anagen V and catagen. The innermost layer of the ORS expressed galectin-3 binding sites during anagen VI until catagen VIII, but galectin-3 during anagen III to IV and catagen. The inner root sheath (IRS) expressed galectin-3 binding sites only in anagen IV but missed expression of any of the two galectins. The matrix cells expressed galectin-3 binding sites in catagen II-III as well as galectin-3 during anagen V to catagen IV. The present study provides the first evidence for a cycle-related expression of both galectin-1 and -3 and their binding sites during murine hair cycle.     

Expression of galectins on microvessel endothelial cells and their involvement in tumour cell adhesion

Lactoside-binding lectins (galectins) with molecular weights of about 14.5 kDa (galectin-1) and 29–35 kDa (galectin-3) bind preferentially to polylactosaminoglycan-containing glycoconjugates and have been found on the surface of tumour cells and implicated in cell-cell and cell-extracellular matrix adhesion and metastasis. We have demonstrated by immunoblotting that both galectin-1 and galectin-3 are present in extracts of endothelial cells cultured from bovine aorta, rat lung, mouse lung and mouse brain microvessels, whereas mouse hepatic sinusoidal endothelial cells expressed primarily galectin-1. These galectins were also localized by indirect immunofluorescent labelling on the surface of the different endothelial cells in culture and by immunohistochemical staining in human tissuesin vivo. Anti-galectin-1 antibodies inhibited the adhesion of liver-preferring murine RAW117-H10 large-cell lymphoma cells to hepatic sinusoidal endothelial cells or lung microvessel endothelial cellsin vitro. The data indicate that galectin-1 is expressed on the extracellular surface of endothelial cells and can mediate in part the adhesion of RAW117-H10 cells to liver microvessel endothelial cells.     

Galectin-1, -3 and -9 Expression and Clinical Significance in Squamous Cervical Cancer

Galectins are proteins that bind β-galactoside sugars and provide a new type of potential biomarkers and therapeutic targets in cancer. Galectin-1, -3 and -9 have become the focus of different research groups, but their expression and function in cervical cancer is still unclear. The aim of this study was to determine the phenotype of galectin-1, -3 and -9 expressing cells and the association with clinico-pathological parameters in cervical cancer. Galectin expression was scored in tumor cells, tumor epithelium infiltrating immune cells and stromal cells in squamous cervical cancer (n = 160). Correlations with clinico-pathological parameters and survival were studied according to the REMARK recommendations. We additionally investigated whether the galectins were expressed by tumor cells, fibroblasts, macrophages and T cells. Galectin-1 and -9 were both expressed by tumor cells in 11% of samples, while 84% expressed galectin-3. Strong galectin-1 expression by tumor cells was an independent predictor for poor survival (hazard ratio: 8.02, p = 0.001) and correlated with increased tumor invasion (p = 0.032) and receiving post-operative radiotherapy (p = 0.020). Weak and positive tumor cell galectin-3 expression were correlated with increased and decreased tumor invasion, respectively (p = 0.012). Tumor cell expression of galectin-9 showed a trend toward improved survival (p = 0.087). The predominant immune cell type expressing galectin-1, -3 and -9 were CD163⁺ macrophages. Galectin-1 and -3 were expressed by a minor population of T cells. Galectin-1 was mainly expressed by fibroblasts in the tumor stroma. To conclude, while tumor cell expression of galectin-9 seemed to represent a beneficial response, galectin-1 expression might be used as a marker for a more aggressive anti-cancer treatment.     

Galectin-8 modulates neutrophil function via interaction with integrin alphaM

The members of the galectin family are associated with diverse cellular events, including immune response. We investigated the effects of galectin-8 on neutrophil function. Human galectin-8 induced firm and reversible adhesion of peripheral blood neutrophils but not eosinophils to a plastic surface in a lactose-sensitive manner. Other human galectins, galectins-1, -3, and -9, showed low or negligible effects on neutrophil adhesion. Confocal microscopy revealed actin bundle formation in the presence of galectin-8. Cytochalasins inhibited both actin assembly and cell adhesion induced by galectin-8. Affinity purification of galectin-interacting proteins from solubilized neutrophil membrane revealed that N-terminal carbohydrate recognition domain (CRD) of galectin-8 bound promatrix metalloproteinase-9 (proMMP-9), and C-terminal CRD bound integrin alphaM/CD11b and proMMP-9. A mutant galectin-8 lacking the carbohydrate-binding activity of N-terminal CRD (galectin-8R69H) retained adhesion-inducing activity, but inactivation of C-terminal CRD (galectin-8R233H) abolished the activity. MMP-3-mediated processing of proMMP-9 was accelerated by galectin-8, and this effect was inhibited by lactose. Galectins-1 and -3 did not affect the processing. Superoxide production, an essential event in bactericidal function of neutrophils, was stimulated by galectin-8 to an extent comparable to that induced by fMLP. Galectin-8R69H but not galectin-8R233H could stimulate superoxide production. Taken together, these results suggest that galectin-8 is a novel factor that modulates the neutrophil function related to transendothelial migration and microbial killing.     

Modified citrus pectin reduces galectin-3 expression and disease severity in experimental acute kidney injury

Galectin-3 is a β-galactoside binding lectin with roles in diverse processes including proliferation, apoptosis, inflammation and fibrosis which are dependent on different domains of the molecule and subcellular distribution. Although galectin-3 is known to be upregulated in acute kidney injury, the relative importance of its different domains and functions are poorly understood in the underlying pathogenesis. Therefore we experimentally modulated galectin-3 in folic acid (FA)-induced acute kidney injury utilising modified citrus pectin (MCP), a derivative of pectin which can bind to the galectin-3 carbohydrate recognition domain thereby predominantly antagonising functions linked to this role. Mice were pre-treated with normal or 1% MCP-supplemented drinking water one week before FA injection. During the initial injury phase, all FA-treated mice lost weight whilst their kidneys enlarged secondary to the renal insult; these gross changes were significantly lessened in the MCP group but this was not associated with significant changes in galectin-3 expression. At a histological level, MCP clearly reduced renal cell proliferation but did not affect apoptosis. Later, during the recovery phase at two weeks, MCP-treated mice demonstrated reduced galectin-3 in association with decreased renal fibrosis, macrophages, pro-inflammatory cytokine expression and apoptosis. Other renal galectins, galectin-1 and -9, were unchanged. Our data indicates that MCP is protective in experimental nephropathy with modulation of early proliferation and later galectin-3 expression, apoptosis and fibrosis. This raises the possibility that MCP may be a novel strategy to reduce renal injury in the long term, perhaps via carbohydrate binding-related functions of galectin-3.     

Galectin-3 interaction with Thomsen-Friedenreich disaccharide on cancer-associated MUC1 causes increased cancer cell endothelial adhesion

Patients with metastatic cancer commonly have increased serum galectin-3 concentrations, but it is not known whether this has any functional implications for cancer progression. We report that MUC1, a large transmembrane mucin protein that is overexpressed and aberrantly glycosylated in epithelial cancer, is a natural ligand for galectin-3. Recombinant galectin-3 at concentrations (0.2-1.0 microg/ml) similar to those found in the sera of patients with metastatic cancer increased adhesion of MUC1-expressing human breast (ZR-75-1) and colon (HT29-5F7) cancer cells to human umbilical vein endothelial cells (HUVEC) by 111% (111 +/- 21%, mean +/- S.D.) and 93% (93 +/- 17%), respectively. Recombinant galectin-3 also increased adhesion to HUVEC of MUC1 transfected HCA1.7+ human breast epithelial cells that express MUC1 bearing the oncofetal Thomsen-Friedenreich antigen (Galbeta1,3 GalNAc-alpha (TF)) but did not affect adhesion of MUC1-negative HCA1.7-cells. MUC1-transfected, Ras-transformed, canine kidney epithelial-like (MDE9.2+) cells, bearing MUC1 that predominantly carries sialyl-TF, only demonstrated an adhesive response to galectin-3 after sialidase pretreatment. Furthermore, galectin-3-mediated adhesion of HCA1.7+ to HUVEC was reduced by O-glycanase pretreatment of the cells to remove TF. Recombinant galectin-3 caused focal disappearance of cell surface MUC1 in HCA1.7+ cells, suggesting clustering of MUC1. Co-incubation with antibodies against E-Selectin or CD44H, but not integrin-beta1, ICAM-1 or VCAM-1, largely abolished the epithelial cell adhesion to HUVEC induced by galectin-3. Thus, galectin-3, by interacting with cancer-associated MUC1 via TF, promotes cancer cell adhesion to endothelium by revealing epithelial adhesion molecules that are otherwise concealed by MUC1. This suggests a critical role for circulating galectin-3 in cancer metastasis and highlights the functional importance of altered cell surface glycosylation in cancer progression.     

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.     

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.     

Expression of galectin-1 and galectin-3 in human fetal thyroid gland.

High levels of expression of galectin-1 and galectin-3, the beta-galactoside-binding proteins, have been recently described in malignant thyroid tumors but not in adenomas nor in normal thyroid tissue. However, there are no data about the expression of these galectins during fetal thyroid development. In this study we analyzed immunohistochemically the presence of galectin-1 and galectin-3 in human fetal thyroid glands (16-37 weeks of gestation). Weak to moderate cytoplasmic staining for galectin-1 was observed in follicular cells of all fetal thyroids. Galectin-3 could not be detected in thyroid follicular cells of any fetal thyroid investigated. Both galectins were detected in stromal tissue, but staining for galectin-1 was more intense. The absence of galectin-3 in thyroid cells during fetal development suggests that galectin-3 is expressed de novo during malignant transformation of thyroid epithelium, and that galectin-1 could be considered an oncofetal antigen. The results obtained indicated potential roles for galectin-1 and galectin-3 during the investigated period of human fetal thyroid gland development. Both galectins might participate in developmental processes regarding stromal fetal thyroid tissue organization, whereas galectin-1 might have a function in thyroid epithelium maturation.     

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.     

Galectin-4 in normal tissues and cancer

Galectin-4 belongs to a subfamily of galectins composed of two carbohydrate recognition domains within the same peptide chain. The two domains have all the conserved galectin signature amino acids, but their overall sequences are only approximately 40% identical. Both domains bind lactose with a similar affinity as other galectins, but their respective preferences for other disaccharides, and larger saccharides, are distinctly different. Thus galectin-4 has a property of a natural cross-linker, but in a modified sense since each domain prefers a different subset of ligands. Similarly to other galectins, galectin-4 is synthesized as a cytosolic protein, but can be externalized. During development and in adult normal tissues, galectin-4 is expressed only in the alimentary tract, from the tongue to the large intestine. It is often found in relatively insoluble complexes, as a component of either adherens junctions or lipid rafts in the microvillus membrane, and it has been proposed to stabilize these structures. Strong expression of galectin-4 can be induced, however, in cancers from other tissues including breast and liver. Within a collection of human epithelial cancer cell lines, galectin-4 is overexpressed and soluble in those forming highly differentiated polarized monolayers, but absent in less differentiated ones. In cultured cells, intracellular galectin-4 may promote resistance to nutrient starvation, whereas--as an extracellular protein--it can mediate cell adhesion. Because of its distinct induction in breast and other cancers, it may be a valuable diagnostic marker and target for the development of inhibitory carbohydrate-based drugs.     

Functional characterization of an eosinophil-specific galectin,ovine galectin-14

Across mammalian species, human galectin-10 and ovine galectin-14 are unique in their expression in eosinophils and their release into lung and gastrointestinal tissues following allergen or parasite challenge. Recombinant galectin-14 is active in carbohydrate binding assays and has been used in this study to unravel the function of this major eosinophil constituent. In vitro cultures revealed that galectin-14 is spontaneously released by eosinophils isolated from allergen-stimulated mammary gland lavage, but not by resting peripheral blood eosinophils. Galectin-14 secretion from peripheral blood eosinophils can be induced by the same stimuli that induce eosinophil degranulation. Flow cytometric analysis showed that recombinant galectin-14 can bind in vitro to eosinophils, neutrophils and activated lymphocytes. Glycan array screening indicated that galectin-14 recognizes terminal N-acetyllactosamine residues which can be modified with α1-2-fucosylation and, uniquely for a galectin, prefers α2- over α2-sialylation. Galectin-14 showed the greatest affinity for lacto-N-neotetraose, an immunomodulatory oligosaccharide expressed by helminths. Galectin-14 binds specifically to laminin in vitro, and to mucus and mucus producing cells on lung and intestinal tissue sections. In vivo, galectin-14 is abundantly present in mucus scrapings collected from either lungs or gastrointestinal tract following allergen or parasite challenge, respectively. These results suggest that in vivo secretion of eosinophil galectins may be specifically induced at epithelial surfaces after recruitment of eosinophils by allergic stimuli, and that eosinophil galectins may be involved in promoting adhesion and changing mucus properties during parasite infection and allergies.     

Unique sequence and expression profiles of rat galectins-5 and -9 as a result of species-specific gene divergence

Presence of species-specific gene divergence in a protein family prompts to thoroughly study structural aspects and expression profiles of the products. We herein focus on two members of an adhesion/growth-regulatory group of endogenous lectins, i.e. galectins-5 and -9. After first ascertaining species specificity of occurrence of galectin-5, constituted by a short section of rat galectin-9's N-terminal part and its C-terminal carbohydrate recognition domain, by database mining, we next detected and defined sequence differences in the proximal promoter region between the two genes. The ensuing hypothesis for distinct expression profiles was tested first by RT-PCR and then by immunohistochemistry. For the latter purpose, we employed antibodies rigorously controlled for absence of cross-reactivity including assays with various other galectins and, if necessary, refined by chromatographic removal of bi- or oligospecific activities. Indeed, the galectins have non-identical expression profiles, qualitative differences, e.g. seen for galectin-5-positive bone marrow and erythrocytes or for hitherto unknown expression in cells of the theca folliculi and galectin-9-positive skin epidermis and esophageal epithelium. Lack of hepatocyte or renal cortex staining separates these two expression profiles in rat from localization of galectin-9 in mouse. Interspecies extrapolation in a case of a galectin involved in unique gene divergence may thus not be valid. The presented results on galectin-5 relative to galectin-9 intimate distinct functions especially in erythropoiesis and imply currently unknown mechanisms to compensate its absence from the galectin network in other mammals.     

Developmental aspects of galectin-3 expression in the lens

In order to investigate the temporal and spatial expression pattern of the lectin galectin-3 during lens development we performed immunohistochemical studies using monoclonal and polyclonal antibodies against galectin-3 on paraffin sections of human, mouse and rat eyes. Galectin-3 has been shown to be involved in various biological functions related to cell adhesion, proliferation, apoptosis and differentiation in other tissues. In the human lens, galectin-3 shows a selective expression pattern during lens development. It is present in all cells of the early lens vesicle and at later stages it is strongly expressed during the elongation phase in differentiating primary lens fibres. From about 7 weeks onwards the anterior lens epithelium fails to express galectin-3. Adult lenses, however, exhibit immunoreactivity in the anterior epithelial cells and in the early differentiating secondary fibres of the lens' outer cortex prior to the onset of degradation of the nuclei. In contrast to the observed expression pattern in prenatal human lenses, mouse and rat lenses exhibited immunoreactivity for galectin-3 during postnatal and adult stages only. At these stages, the expression pattern closely resembles that seen in the corresponding human lenses. The spatiotemporal pattern of galectin-3 distribution during lens development favours a role of this lectin in adhesion processes and in the regulation of programmed organelle elimination during lens cell differentiation.     

Intestinal colonization with bifidobacteria affects the expression of galectins in extraintestinal organs

This study aimed at determining the contribution of intestinal bifidobacteria to the immune system activation using widely distributed galectins as markers of immune cell homoeostasis. In human flora-associated mice, bacteria were enumerated in the gut, blood, spleen, liver and lungs, while the expression of galectin-1 (Gal-1) and galectin-3 (Gal-3) was estimated by PCR in the intestine and real-time quantitative PCR in the other organs. Gal-1 and -3 were rarely expressed in the intestine. In blood, only Gal-1 was expressed while both galectins were expressed in all other organs. A high prevalence of colonic bifidobacteria was associated with a lower expression of both pulmonary galectins, whose levels negatively correlated with bifidobacterial counts. Caecal bifidobacterial counts also negatively correlated with pulmonary Gal-3 mRNA levels. The spleen was the only organ showing an upregulation of Gal-1 expression related to its bacterial contamination. However, this upregulation was only observed when bifidobacteria were not detected in the colon. A putative mechanism explaining the reduced expression of galectins when bifidobacteria highly colonize the mouse intestine could be that, by reducing the bacterial translocation, bifidobacteria also lead to a decreased blood concentration of substances produced by intestinal bacteria.     

Analogs of tetrahydroisoquinoline natural products that inhibit cell migration and target galectin-3 outside of its carbohydrate-binding site

Cell migration is central to a number of normal and disease processes. Small organic molecules that inhibit cell migration have potential as both research probes and therapeutic agents. We have identified two tetrahydroisoquinoline natural product analogs with antimigratory activities on Madin-Darby canine kidney epithelial cells: a semisynthetic derivative of quinocarmycin (also known as quinocarcin), DX-52-1, and a more complex synthetic molecule, HUK-921, related to the naphthyridinomycin family. It has been assumed that the cellular effects of reactive tetrahydroisoquinolines result from the alkylation of DNA. We have reported previously that the primary target of DX-52-1 relevant to cell migration appears to be the membrane-cytoskeleton linker protein radixin. Here we extend the analysis of the protein targets of DX-52-1, reporting that the multifunctional carbohydrate-binding protein galectin-3 is a secondary target of DX-52-1 that may also be relevant to the antimigratory effects of both DX-52-1 and HUK-921. All known inhibitors of galectin-3 target its beta-galactoside-binding site in the carbohydrate recognition domain. However, we found that DX-52-1 and HUK-921 bind galectin-3 outside of its beta-galactoside-binding site. Intriguingly HUK-921, although a less potent inhibitor of cell migration than DX-52-1, had far greater selectivity for galectin-3 over radixin, exhibiting little binding to radixin, both in vitro and in cells. Overexpression of galectin-3 in cells led to a dramatic increase in cell adhesion on different extracellular matrix substrata as well as changes in cell-cell adhesion and cell motility. Galectin-3-overexpressing cells had greatly reduced sensitivity to DX-52-1 and HUK-921, and these compounds caused a change in localization of the overexpressed galectin-3 and reversion of the cells to a more normal morphology. The converse manipulation, RNA interference-based silencing of galectin-3 expression, resulted in reduced cell-matrix adhesion and cell migration. In aggregate, the data suggest that DX-52-1 and HUK-921 inhibit a carbohydrate binding-independent function of galectin-3 that is involved in cell migration.