Thermodynamic binding studies of bivalent oligosaccharides to galectin-1, galectin-3, and the carbohydrate recognition domain of galectin-3

Galectins are a growing family of animal lectins with common consensus sequences that bind beta-Gal and LacNAc residues. There are at present 14 members of the galectin family; however, certain galectins possess different structures as well as biological properties. Galectin-1 is a dimer of two homologous carbohydrate recognition domains (CRDs) and possesses apoptotic and proinvasive activities. Galectin-3 consists of a C-terminal CRD and an N-terminal nonlectin domain implicated in the oligomerization of the protein and is often associated with antiapoptotic activity. Because many cellular oligosaccharide receptors are multivalent, it is important to characterize the interactions of multivalent carbohydrates with galectins-1 and -3. In the present study, binding of bovine heart galectin-1 and recombinant murine galectin-3 to a series of synthetic analogs containing two LacNAc residues separated by a varying number of methylene groups, as well as biantennary analogs possessing two LacNAc residues, were examined using isothermal titration microcalorimetry (ITC) and hemagglutination inhibition measurements. The thermodynamics of binding of the multivalent carbohydrates to the C-terminal CRD domain of galectin-3 was also investigated. ITC results showed that each bivalent analog bound by both LacNAc residues to the two galectins. However, galectin-1 shows a lack of enhanced affinity for the bivalent straight chain and branched chain analogs, whereas galectin-3 shows enhanced affinity for only lacto-N-hexaose, a naturally occurring branched chain carbohydrate. The CRD domain of galectin-3 was shown to possess similar thermodynamic binding properties as the intact molecule. The results of this study have important implications for the design of carbohydrate inhibitors of the two galectins.     

Quaternary solution structures of galectins-1, -3, and -7

Galectins are a growing family of animal lectins with functions in growth regulation and cell adhesion that bind beta-Gal residues in oligosaccharides. Evidence indicates that some of the biological properties of galectins are due to their cross-linking activities with multivalent glycoconjugate receptors. Therefore determination of the quaternary solution structures of these proteins is important in understanding their structure-function properties. The present study reports analytical sedimentation velocity and equilibrium data for galectins-1, -3, and -7 in the absence and presence of bound LacNAc, the natural ligand epitope. Galectin-1 from bovine heart and recombinant human galectin-7 were found to be stable dimers by both methods. In contrast, recombinant murine galectin-3, as well as its proteolytical derived C-terminal domain, are predominantly monomeric. The presence of LacNAc at concentrations sufficient to fully saturate the proteins had no significant effect on either the weight average molecular weight determined by sedimentation equilibrium or the hydrodynamic properties determined from sedimentation velocity experiments. These results show that binding of a monovalent ligand does not affect oligomerization of these galectins.     

Soluble beta-galactosyl-binding lectin (galectin) from toad ovary: crystallographic studies of two protein-sugar complexes

Galectin-1, S-type beta-galactosyl-binding lectins present in vertebrate and invertebrate species, are dimeric proteins that participate in cellular adhesion, activation, growth regulation, and apoptosis. Two high-resolution crystal structures of B. arenarum galectin-1 in complex with two related carbohydrates, LacNAc and TDG, show that the topologically equivalent hydroxyl groups in the two disaccharides exhibit identical patterns of interaction with the protein. Groups that are not equivalent between the two sugars present in the second moiety of the disaccharide, interact differently with the protein, but use the same number and quality of interactions. The structures show additional protein-carbohydrate interactions not present in previously reported lectin-lactose complexes. These contacts provide an explanation for the enhanced affinity of galectin-1 for TDG and LacNAc relative to lactose. Galectins are in dimer-monomer equilibrium at physiological protein concentrations, suggesting that this equilibrium may be involved in organ-specific regulation of activity. Comparison of B. arenarum with other galectin-1 structures shows that among different galectins there are significant changes in accessible surface area buried upon dimer formation, providing a rationale for the variations observed in the free-energies of dimerization. The structure of the B. arenarum galectin-1 has a large cleft with a strong negative potential that connects the two binding sites at the surface of the protein. Such a striking characteristic suggests that this cleft is probably involved in interactions of the galectin with other intra or extra-cellular proteins. Proteins 2000;40:378-388.     

Structural analysis of the recognition mechanism of poly-N-acetyllactosamine by the human galectin-9 N-terminal carbohydrate recognition domain

Galectins are a family of beta-galactoside-specific lectins bearing a conserved carbohydrate recognition domain. Interactions between galectins and poly-N-acetyllactosamine sequences are critical in a variety of biological processes. Galectin-9, a member of the galectin family, has two carbohydrate recognition domains at both the N- and C-terminal regions. Here we report the crystal structure of the human galectin-9 N-terminal carbohydrate recognition domain in complex with N-acetyllactosamine dimers and trimers. These complex structures revealed that the galectin-9 N-terminal carbohydrate recognition domain can recognize internal N-acetyllactosamine units within poly-N-acetyllactosamine chains. Based on these complex structures, we propose two putative recognition modes for poly-N-acetyllactosamine binding by galectins.     

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.     

Homophilic Binding Properties of Galectin-3: Involvement of the Carbohydrate Recognition Domain

Abstract: Galectin-3, an animal lectin specific for β-galactosides, is composed of three different domains. The N-terminal half of the molecule (N domain) consists of a short N-terminal segment followed by glycine-, proline-, and tyrosine-rich tandem repeats. The C-terminal domain (C domain) harbors the carbohydrate recognition domain homologous to other members of the galectin family of lectins. Galectin-3 aggregates in solution, and participation of the N domain of the molecule in this process has already been demonstrated. Using a solid-phase radioligand binding assay, which allows the direct analysis of galectin-3 self-association, here we provide evidence that the carbohydrate recognition domain of the lectin is involved in carbohydrate-dependent homophilic interactions: (a) Radiolabeled galectin-3 binds to immobilized galectin-3, and the addition of unlabeled galectin-3 in solution increases the rate of binding of radiolabeled lectin; (b) binding of radiolabeled galectin-3 to immobilized galectin-3 is inhibited by the C domain; (c) binding of radiolabeled galectin-3 to immobilized galectin-3 or the C domain is inhibited by lactose but not by sucrose; and (d) the radiolabeled C domain does not bind to immobilized C domain. Taken together, these data suggest that in addition to the N domain, the homophilic interactions of galectin-3 are mediated by the C domain.     

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.     

It depends on the hinge: a structure-functional analysis of galectin-8, a tandem-repeat type lectin

Galectin-8, a member of the galectin family of mammalian lectins, is made of two carbohydrate-recognition domains (CRDs), joined by a "hinge" region. Ligation of integrins by galectin-8 induces a distinct cytoskeletal organization, associated with activation of the extracellular-regulated kinase (ERK) and phosphatidylinositol 3-kinase signaling cascades. We show that these properties of galectin-8 are mediated by the concerted action of its two CRDs and involve both protein-sugar and protein-protein interactions. Accordingly, the isolated N- or C-CRD domains of galectin-8 or galectin-8 mutated at selected residues implicated in sugar binding (E251Q; W85Y, W248Y, W[85,248]Y) exhibited reduced sugar binding, which was accompanied by severe impairment in the capacity of these mutants to promote the adhesive, spreading, and signaling functions of galectin-8. Other mutations that did not impair sugar binding (e.g. E88Q) still impeded the signaling and cell-adherence functions of galectin-8. Deletion of the "hinge" region similarly impaired the biological effects of galectin-8. These results provide evidence that cooperative interactions between the two CRDs and the "hinge" domain are required for the proper functioning of galectin-8.     

Molecular dynamics simulations of galectin-1-oligosaccharide complexes reveal the molecular basis for ligand diversity

Galectin-1 is a member of a protein family historically characterized by its ability to bind carbohydrates containing a terminal galactosyl residue. Galectin-1 is found in a variety of mammalian tissues as a homodimer of 14.5-kDa subunits. A number of developmental and regulatory processes have been attributed to the ability of galectin-1 to bind a variety of oligosaccharides containing the Gal-beta-(1,4)-GlcNAc (LacNAc(II)) sequence. To probe the origin of this permissive binding, solvated molecular dynamics (MD) simulations of several representative galectin-1-ligand complexes have been performed. Simulations of structurally defined complexes have validated the computational approach and expanded upon data obtained from X-ray crystallography and surface plasmon resonance measurements. The MD results indicate that a set of anchoring interactions between the galectin-1 carbohydrate recognition domain (CRD) and the LacNAc core are maintained for a diverse set of ligands and that substituents at the nonreducing terminus of the oligosaccharide extend into the remainder of a characteristic surface groove. The anionic nature of ligands exhibiting relatively high affinities for galectin-1 implicates electrostatic interactions in ligand selectivity, which is confirmed by a generalized Born analysis of the complexes. The results suggest that the search for a single endogenous ligand or function for this lectin may be inappropriate and instead support a more general role for galectin-1, in which the lectin is able to crosslink heterogeneous oligosaccharides displayed on a variety of cell surfaces. Such binding promiscuity provides an explanation for the variety of adhesion phenomena mediated by galectin-1.     

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.     

Selective striatal connections of midbrain dopaminergic nuclei in the chick (Gallus domesticus)

Histochemical monitoring of developmental processes is presently centered on protein-protein interactions. However, oligosaccharides have the potential to store and transmit biological information. Carbohydrate chains of cellular glycoconjugates present determinants for binding of endogenous lectins. This interaction can be relevant for developmental processes. In fact, beta-galactosides and their derivatives serve as ligands for members of the lectin family of galectins. Since it is unclear to what extent functions of different galectins differ or overlap, hereby introducing redundancy into this system, monitoring of galectin presence during tissue maturation should include more than one type of galectin (galectin fingerprinting). Here, we focus on the two most frequently described ones, namely the homodimeric prototype galectin-1 and the chimera-type galectin-3, the latter one so far not characterized from bovine tissue. In the first step, we have detected its presence biochemically in addition to the abundant galectin-1 in bovine respiratory and digestive tracts during development. Evidently, diversification of the primitive foregut will not lead to an alteration of this property. Immunohistochemistry revealed clear differences in the galectins' localization profiles. Galectin-1 expression is strong in mesenchymal cells, especially smooth muscle cells, while epithelial lining harbors galectin-3. A gradual increase in staining intensity with development is especially observed in the case of galectin-3. Notably, this change is accompanied by a shift from primarily nuclear localization to the cytoplasm, an alteration not seen for galectin-1. However, nuclear presence of galectin-1 is encountered. Thus, the delineation of differences in expression of galectin-1 and -3 with respect to cell types and in the developmental course of subcellular localization argues in favor of mediation of nonoverlapping functions by these two homologous, endogenous lectins.     

Purification of galectin-3 from ovine placenta: developmentally regulated expression and immunological relevance

Galectins, beta-galactoside-binding lectins, are extensively distributed in the animal kingdom and share some basic molecular properties. Galectin-3, a member of this family, is generally associated with differentiation, morphogenesis, and metastasis. In this study, galectin-3 was isolated from ovine placental cotyledons round the middle of the gestation period by lactose extraction followed by affinity chromatography on lactosyl-agarose, and separated from galectin-1 by size exclusion chromatography on a Superose 12 column. Under native conditions this lectin behaved as a monomer with an apparent molecular weight of approximately 29,000 and an isoelectric point of 9.0. The partial amino acid sequence of the peptides obtained by tryptic digestion of this protein followed by HPLC separation showed striking homology with other members of the galectin-3 subfamily. Furthermore, ovine placental galectin-3 exhibited specific mitogenic activity toward rat spleen mononuclear cells. Besides, this protein strongly reacted with a rabbit antiserum raised against a chicken galectin. Results obtained by Western blot analysis showed that its expression was greatly decreased in term placenta with respect to the middle of the gestation period, suggesting a regulated expression throughout development.      

Cellular and subcellular distribution of galectin-3 in the epiphyseal cartilage and bone of fetal and neonatal mice.

Galectin-3 is a 30 kDa beta-galactoside binding protein that belongs to the galectin family of animal lectins. By immunocytochemistry we show the presence of galectin-3 protein in the differentiated chondrocytes of the epiphyseal plate cartilage of long bones of both fetal and neonatal mice. The highest concentrations of galectin-3 are found in the cytoplasm of mature and early hypertrophic chondrocytes. Very little protein is detected in the late hypertrophic chondrocytes undergoing terminal maturation and cell death. Galectin-3 has also been found in osteoblasts and osteocytes of the woven bone of the metaphysis and the cortical bone of the diaphysis, as well as in osteoclasts and mononuclear cells within bone marrow cavities. Galectin-3 is never detected extracellularly, the protein seems restricted to the cytoplasm of chondrocytes and bone cells, although it is occasionally detected in the nuclei of dense non-hypertrophic chondrocytes in the zone of calcification and in young osteoblasts. The results indicate that galectin-3 is a marker of both chondrogenic and osteogenic cell lineages. They also suggest that galectin-3 could be involved in the process of endochondral bone formation, possibly as a regulator of chondrocyte survival.     

Comparative cross-linking activities of lactose-specific plant and animal lectins and a natural lactose-binding immunoglobulin G fraction from human serum with asialofetuin

Plant and animal lectins bind and cross-link certain multiantennaryoligosaccharides, glycopeptides, and glycoproteins. This canlead to the formation of homogeneous cross-linked complexes,which may differ in their stoichiometry depending on the natureof the sugar receptor involved. As a precisely defined ligand,we have employed bovine asialofetuin (ASF), a glycoprotein thatpossesses three asparagine-linked triantennary complex carbohydratechains with terminal LacNAc residues. In the present study,we have compared the carbohydrate cross-linking properties oftwo Lac-specific plant lectins, an animal lectin and a naturallyoccurring Lac-binding polyclonal iminunoglobulin G subfractionfrom human serum with the ligand. Quantitative precipitationstudies of the Lac-specific plant lectins, Viscum album agglutininand Ricinus communis agglutinin, and the Lac-specific 16 kDadimenc galectin from chicken liver demonstrate that these lectinsform specific, stoichiometric cross-linked complexes with ASF.At low concentrations of ASF, 1:9 ASF/lectin (monomer) complexesformed with both plant lectins and the chicken lectin. Withincreasing concentrations of ASF, 1:3 ASF/lectin (monomer) complexesformed with the lectins irrespective of their source or size.The naturally occurring polyclonal antibodies, however, revealeda different cross-linking behavior. They show the formationof 1:3 ASF/antibody (per Fab moiety) cross-linked complexesat all concentrations of ASF. These studies demonstrate thatLac-specific plant and animal lectins as well as the Lac-bindingimmunoglobulin subfraction form specific stoichiometric cross-linkedcomplexes with ASF. These results are discussed in terms ofthe structure-function properties of multivalent lectins andantibodies. asialofetuin Lac-specific lectins immunoglobulin subfraction     

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.     

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.     

Molecular dynamics simulations elucidate oligosaccharide recognition pathways by galectin-3 at atomic resolution

The recognition of carbohydrates by lectins plays key roles in diverse cellular processes such as cellular adhesion, proliferation, and apoptosis, which makes it a therapeutic target of significance against cancers. One of the most functionally active lectins, galectin-3 is distinctively known for its specific binding affinity toward β-galactoside. However, despite the prevalence of high-resolution crystallographic structures, the mechanistic basis and more significantly, the dynamic process underlying carbohydrate recognition by galectin-3 are currently elusive. To this end, we employed extensive Molecular Dynamics simulations to unravel the complete binding event of human galectin-3 with its native natural ligand N-acetyllactosamine (LacNAc) at atomic precision. The simulation trajectory demonstrates that the oligosaccharide diffuses around the protein and eventually identifies and binds to the biologically designated binding site of galectin-3 in real time. The simulated bound pose correlates with the crystallographic pose with atomic-level accuracy and recapitulates the signature stabilizing galectin-3/oligosaccharide interactions. The recognition pathway also reveals a set of transient non-native ligand poses in its course to the receptor. Interestingly, kinetic analysis in combination with a residue-level picture revealed that the key to the efficacy of a more active structural variant of the LacNAc lay in the ligand’s resilience against disassociation from galectin-3. By catching the ligand in the act of finding its target, our investigations elucidate the detailed recognition mechanism of the carbohydrate-binding domain of galectin-3 and underscore the importance of ligand–target binary complex residence time in understanding the structure–activity relationship of cognate ligands.     

Nucleocytoplasmic lectins

This review summarizes studies on lectins that have been documented to be in the cytoplasm and nucleus of cells. Of these intracellular lectins, the most extensively studied are members of the galectin family. Galectin-1 and galectin-3 have been identified as pre-mRNA splicing factors in the nucleus, in conjunction with their interacting ligand, Gemin4. Galectin-3, -7, and -12 regulate growth, cell cycle progression, and apoptosis. Bcl-2 and synexin have been identified as interacting ligands of galectin-3, involved in its anti-apoptotic activity in the cytoplasm. Although the annexins have been studied mostly as calcium-dependent phospholipid-binding proteins mediating membrane-membrane and membrane-cytoskeleton interactions, annexins A4, A5 and A6 also bind to carbohydrate structures. Like the galectins, certain members of the annexin family can be found both inside and outside cells. In particular, annexins A1, A2, A4, A5, and A11 can be found in the nucleus. This localization is consistent with the findings that annexin A1 possesses unwinding and annealing activities of a helicase and that annexin A2 is associated with a primer recognition complex that enhances the activity of DNA polymerase alpha. Despite these efforts and accomplishments, however, there is little evidence or information on an endogenous carbohydrate ligand for these lectins that show nuclear and/or cytoplasmic localization. Thus, the significance of the carbohydrate-binding activity of any particular intracellular lectin remains as a challenge for future investigations.     

Different roles of galectin-9 isoforms in modulating E-selectin expression and adhesion function in LoVo colon carcinoma cells

Galectin-9, a member of galectin family, plays multiple roles in a variety of cellular functions, including cell adhesion, aggregation, and apoptosis. Galectin-9 also has three isoforms (named galectin-9L, galectin-9M, and galectin-9S), but whether these isoforms differ in their functions remains poorly understood. In this study, we showed that transient expression of galectin-9L decreased E-selectin levels, while transient expression of galectin-9M or galectin-9S increased E-selectin levels in LoVo cells, which do not express endogenous galectin-9. We also found that over-expression of three galectin-9 isoforms led to increased attachment of LoVo cells to extracellular matrix proteins respectively, while over-expression of galectin-9M or galectin-9S increased the adhesion of LoVo cells to human umbilical vein endothelial cells in vitro. In summary, these findings indicate that different isoforms of galectin-9 exhibit distinct biological functions.