Showing papers on "Galectin published in 2009"

Turning 'sweet' on immunity: galectin-glycan interactions in immune tolerance and inflammation.

Abstract: The function of deciphering the biological information encoded by the glycome, which is the entire repertoire of complex sugar structures expressed by cells and tissues, is assigned in part to endogenous glycan-binding proteins or lectins. Galectins, a family of animal lectins that bind N-acetyllactosamine-containing glycans, have many roles in diverse immune cell processes, including those relevant to pathogen recognition, shaping the course of adaptive immune responses and fine-tuning the inflammatory response. How do galectins translate glycan-encoded information into tolerogenic or inflammatory cell programmes? An improved understanding of the mechanisms underlying these functions will provide further opportunities for developing new therapies based on the immunoregulatory properties of this multifaceted protein family.

Roles of galectins in infection.

Abstract: Galectins, which were first characterized in the mid-1970s, were assigned a role in the recognition of endogenous ('self') carbohydrate ligands in embryogenesis, development and immune regulation. Recently, however, galectins have been shown to bind glycans on the surface of potentially pathogenic microorganisms, and function as recognition and effector factors in innate immunity. Some parasites subvert the recognition roles of the vector or host galectins to ensure successful attachment or invasion. This Review discusses the role of galectins in microbial infection, with particular emphasis on adaptations of pathogens to evasion or subversion of host galectin-mediated immune responses.

Galectins in innate immunity: dual functions of host soluble β‐galactoside‐binding lectins as damage‐associated molecular patterns (DAMPs) and as receptors for pathogen‐associated molecular patterns (PAMPs)

Abstract: The glycocalyx is a glycan layer found on the surfaces of host cells as well as microorganisms and enveloped virus. Its thickness may easily exceed 50 nm. The glycocalyx does not only serve as a physical protective barrier but also contains various structurally different glycans, which provide cell- or microorganism-specific 'glycoinformation'. This information is decoded by host glycan-binding proteins, lectins. The roles of lectins in innate immunity are well established, as exemplified by collectins, dectin-1, and dendritic cell (DC)-specific intracellular adhesion molecule-3-grabbing non-integrin (DC-SIGN). These mammalian lectins are synthesized in the secretory pathway and presented on the cell surface to bind to specific glycan 'epitopes'. As they recognize non-self glycans presented by microorganisms, they can be considered as receptors for pathogen-associated molecular patterns (PAMPs), i.e. pattern recognition receptors (PRRs). One notable exception is the galectin family. Galectins are synthesized and stored in the cytoplasm, but upon infection-initiated tissue damage and/or following prolonged infection, cytosolic galectins are either passively released by dying cells or actively secreted by inflammatory activated cells through a non-classical pathway, the 'leaderless' secretory pathway. Once exported, galectins act as PRR, as well as immunomodulators (or cytokine-like modulators) in the innate response to some infectious diseases. As galectins are dominantly found in the lesions where pathogen-initiated tissue damage signals appear, this lectin family is also considered as potential damage-associated molecular pattern (DAMP) candidates that orchestrate innate immune responses alongside the PAMP system.

Glycosylation in immune cell trafficking.

Abstract: Leukocyte recruitment encompasses cell adhesion and activation steps that enable circulating leukocytes to roll, arrest, and firmly adhere on the endothelial surface before they extravasate into distinct tissue locations. This complex sequence of events relies on adhesive interactions between surface structures on leukocytes and endothelial cells and also on signals generated during the cell-cell contacts. Cell surface glycans play a crucial role in leukocyte recruitment. Several glycosyltransferases such as alpha1,3 fucosyltransferases, alpha2,3 sialyltransferases, core 2 N-acetylglucosaminlytransferases, beta1,4 galactosyltransferases, and polypeptide N-acetylgalactosaminyltransferases have been implicated in the generation of functional selectin ligands that mediate leukocyte rolling via binding to selectins. Recent evidence also suggests a role of alpha2,3 sialylated carbohydrate determinants in triggering chemokine-mediated leukocyte arrest and influencing beta1 integrin function. The recent discovery of galectin- and siglec-dependent processes further emphasizes the significant role of glycans for the successful recruitment of leukocytes into tissues. Advancing the knowledge on glycan function into appropriate pathology models is likely to suggest interesting new therapeutic strategies in the treatment of immune- and inflammation-mediated diseases.

Adaptive Regulation at the Cell Surface by N-Glycosylation

Abstract: The association of receptors and solute transporters with components of the endocytic machinery regulates their surface levels, and thereby cellular sensitivity to cytokines, ligands and nutrients in the extracellular environment. Most transmembrane receptors and solute transporters are glycoproteins, and the Asn (N)-linked oligosaccharides (N-glycans) can bind animal lectins, forming multivalent lattices or microdomains that regulate glycoprotein mobility in the plane of membrane. The N-glycan number (sequence-encoded NXS/T) and context-dependent Golgi N-glycan branching cooperate to regulate glycoprotein affinities for the galectin family of lectins. Galectin-3 binding reduces EGF receptor trafficking into clathrin-coated pits and caveolae lipid rafts, decreases ligand-independent receptor activation and promotes alpha5beta1 integrin remodelling in focal adhesions. N-glycan branching in the medial Golgi increases glycan affinity for galectins, and the Golgi pathway is sensitive to uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) supply, in turn hexosamine pathway metabolites (fructose-6-P, glutamine and acetyl-CoA). Thus, lattice avidity and cellular responsiveness to extracellular cues are regulated in an adaptive manner by metabolism and Golgi modification to glycoproteins. Computational modelling of the hexosamine/Golgi/lattice has provided new insight on cell surface adaptation in cancer and autoimmune disease.

A primate subfamily of galectins expressed at the maternal–fetal interface that promote immune cell death

Abstract: Galectins are proteins that regulate immune responses through the recognition of cell-surface glycans. We present evidence that 16 human galectin genes are expressed at the maternal–fetal interface and demonstrate that a cluster of 5 galectin genes on human chromosome 19 emerged during primate evolution as a result of duplication and rearrangement of genes and pseudogenes via a birth and death process primarily mediated by transposable long interspersed nuclear elements (LINEs). Genes in the cluster are found only in anthropoids, a group of primate species that differ from their strepsirrhine counterparts by having relatively large brains and long gestations. Three of the human cluster genes (LGALS13, -14, and -16) were found to be placenta-specific. Homology modeling revealed conserved three-dimensional structures of galectins in the human cluster; however, analyses of 24 newly derived and 69 publicly available sequences in 10 anthropoid species indicate functional diversification by evidence of positive selection and amino acid replacements in carbohydrate-recognition domains. Moreover, we demonstrate altered sugar-binding capacities of 6 recombinant galectins in the cluster. We show that human placenta-specific galectins are predominantly expressed by the syncytiotrophoblast, a primary site of metabolic exchange where, early during pregnancy, the fetus comes in contact with immune cells circulating in maternal blood. Because ex vivo functional assays demonstrate that placenta-specific galectins induce the apoptosis of T lymphocytes, we propose that these galectins reduce the danger of maternal immune attacks on the fetal semiallograft, presumably conferring additional immune tolerance mechanisms and in turn sustaining hemochorial placentation during the long gestation of anthropoid primates.

The Role of Galectins in Protein Trafficking

Abstract: The galectins, a family of lectins, modulate distinct cellular processes, such as cancer progression, immune response and cellular development, through their specific binding to extracellular or intracellular ligands. In the past few years, research has unravelled interactions of different galectins with lipids and glycoproteins in the outer milieu or in the secretory pathway of cells. Interestingly, these lectins do not possess a signalling sequence to enter the endoplasmic reticulum as a starting point for the classical secretory pathway. Instead they use a so-called non-classical mechanism for translocation across the plasma membrane and/or into the lumen of transport vesicles. Here, they stabilize transport platforms for apical trafficking or sort apical glycoproteins into specific vesicle populations. Modes of ligand interaction as well as the modulation of binding activities and trafficking pathways are discussed in this review.

Galectin-3 regulates T-cell functions.

Abstract: Galectin-3 is absent in resting CD4+ and CD8+ T cells but is inducible by various stimuli. These include viral transactivating factors, T-cell receptor (TCR) ligation, and calcium ionophores. In addition, galectin-3 is constitutively expressed in human regulatory T cells and CD4+ memory T cells. Galectin-3 exerts extracellular functions because of its lectin activity and recognition of cell surface and extracellular matrix glycans. These include cell activation, adhesion, induction of apoptosis, and formation of lattices with cell surface glycoprotein receptors. Formation of lattices can result in restriction of receptor mobility and cause attenuation of receptor functions. Consistent with the presence of galectin-3 in intracellular locations, several functions have been described for this protein inside T cells. These include inhibition of apoptosis, promotion of cell growth, and regulation of TCR signal transduction. Studies of cell surface glycosylation have led to convergence of glycobiology and galectin biology and provided new clues on how galectin-3 may participate in the regulation of cell surface receptor activities. The rapid expansion of the field of galectin research has positioned galectin-3 as a key regulator in T-cell functions.

Galectin 3 induces a distinctive pattern of cytokine and chemokine production in rheumatoid synovial fibroblasts via selective signaling pathways

Abstract: Rheumatoid arthritis (RA) is a persistent systemic inflammatory disease characterized by inflammation involving multiple cell types, with the progressive destruction of involved joints (1). An essential component of the switch to persistence that underlies joint destruction is the production of chemokines, which recruit mononuclear cells, such as lymphocytes and monocytes, to the inflamed joint (2). Galectins, an evolutionarily conserved family of animal lectins, have diverse roles in cellular homeostasis and have been shown to modulate inflammatory responses, functioning as either proinflammatory or antiinflammatory regulators, in part through their ability to cluster and modulate signaling through glycan receptors associated with multiple ligands (3). This ability to influence immune responses has been demonstrated in animal models of a number of diseases, including arthritis (4). Galectin 3, a chimera-type member of the galectin family, has a C-terminal carbohydrate recognition domain responsible for carbohydrate binding but exhibits an N-terminal domain that is responsible for interactions between subunits facilitating its oligomerization (5,6). The biologic functions attributed to this lectin are likely to depend on both ligand crosslinking and oligomerization (6,7). Galectin 3 has been associated with a proinflammatory role in models of fibrotic disease affecting the lung and liver (8,9) and has been shown to promote monocyte chemotaxis and macrophage activation (10-12) in addition to neutrophil activation, degranulation, and superoxide production (13-15), suggesting a critical role in the development of innate immune responses. Furthermore, a key role of galectin 3 has also been shown in the survival and progression of tumor metastases by modulating different processes, including homotypic and heterotypic cell adhesion, migration, angiogenesis, and tumor-immune escape (16). In the context of synovitis, Ohshima and colleagues (17) have demonstrated increased levels of galectin 3 and its binding protein in RA synovial tissue compared with osteoarthritis (OA) synovial tissue. Furthermore, galectin 3 levels are increased in RA in both synovial fluid and peripheral blood compartments, where levels correlate with C-reactive protein (17). Interestingly, up-regulated expression of galectin 3 correlates with abnormal cell apoptosis in synovial tissue from patients with juvenile RA (18). In contrast, galectin 1, a prototype member of the galectin family composed of 1 conserved carbohydrate recognition domain that can dimerize, has a predominantly antiinflammatory role, suppressing experimental models of inflammatory diseases, such as hepatitis, experimental autoimmune encephalomyelitis, uveitis, colitis, and arthritis (19-22). Furthermore, this glycan-binding protein appears to play an important role in the mechanisms involved in Treg cell–mediated suppression of immune responses (23), inhibition of T cell receptor–mediated signal transduction (24), and differential regulation of T helper cell viability (25). Intriguingly, synovial fibroblasts engineered to overexpress galectin 1 ameliorated collagen-induced arthritis and induced a bias toward a Th2-mediated cytokine profile in vivo (22). Synovial fibroblasts have an established role as sentinel cells for immune cell activation in the joint (2), and in RA, these cells are responsible for secreting significant quantities of inflammatory cytokines (26). RA synovial fibroblasts actively contribute to destruction of cartilage and bone via secretion of matrix metalloproteinases (MMPs) and cathepsins, and via expression of RANKL, resulting in promotion of monocyte-to-osteoclast differentiation (27). The expanded population of synovial fibroblasts in RA is also a prolific source of chemokines responsible for the recruitment and retention of cells within the joint (2). It is clear that synovial fibroblasts are an important source of galectin 3 within the joint, as shown by messenger RNA (mRNA) and proteomic analyses (17,28). Following stimulation by the products of cartilage degradation, synovial fibroblasts also produce galectin 3 (29). However, there are likely to be many other sources of galectin 3 within the joint, including macrophages, which synthesize this glycan-binding protein in significant amounts (30). Although considerable information is available on the cellular sources of galectins in the synovium, the downstream effects of galectin 3 on different cell types in rheumatoid synovium remain largely unexplored. We therefore examined the effects of exogenous galectin 3 on RA synovial fibroblasts, comparing them with genetically matched control skin fibroblasts. We show that inflammatory cytokines, such as interleukin-6 (IL-6), and neutrophil-attracting chemokines, such as IL-8, are produced equally by galectin 3– and tumor necrosis factor α (TNFα)–stimulated synovial and skin fibroblasts. However, in response to galectin 3, synovial fibroblasts, but not skin fibroblasts, secrete mononuclear cell–attracting chemokines such as CCL2, CCL3, and CCL5. The molecular basis for this selectivity is due to the differential activation of MAPK and phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathways in response to galectin 3. The increased expression of galectin 3 found in fibroblast-rich areas of the synovium may therefore have significant functional consequences in terms of recruitment of monocyte and lymphocyte infiltrates.

Role of Tim-3/galectin-9 inhibitory interaction in viral-induced immunopathology: shifting the balance toward regulators.

Abstract: Controlling chronic immunoinflammatory diseases such as lesions in the eye caused by infection with HSV represents a therapeutic challenge Since CD4 + T cells are the primary orchestrators of lesions, targeting activated CD4 + T cell subsets and increasing the representation of cells that express regulatory function would be a logical therapeutic approach We show that this outcome can be achieved by therapy, systemic or local, with the lectin family member galectin-9 This molecule, which is a natural product of many cell types, acts as a ligand to the inhibitory molecule TIM-3 (T cell Ig and mucin-3) that is expressed by activated but not naive T cells We show that 50% or more of T cells in ocular lesions caused by HSV in mice express TIM-3 and that blocking signals from its natural ligand with a mAb results in more severe lesions More importantly, the provision of additional galectin-9, either systemically or more effectively by local subconjuctival administration, diminished the severity of stromal keratitis lesions as well as the extent of corneal neovascularization Multiple mechanisms were involved in inhibitory effects These included apoptosis of the orchestrating effector T cells with consequent reduction of proinflammatory cytokines and an increase in the representation of two separate subtypes of regulatory cells as well as inhibitory effects on the production of molecules involved in neovascularization, an essential component of stromal keratitis pathogenesis Our results indicate that galectin-9 therapy may represent a useful approach to control HSV-induced lesions, the most common cause of infectious blindness in the Western world

T-cell growth, cell surface organization, and the galectin-glycoprotein lattice

Abstract: Basal, activation, and arrest signaling in T cells determines survival, coordinates responses to pathogens, and, when dysregulated, leads to loss of self-tolerance and autoimmunity. At the T-cell surface, transmembrane glycoproteins interact with galectins via their N-glycans, forming a molecular lattice that regulates membrane localization, clustering, and endocytosis of surface receptors. Galectin-T-cell receptor (TCR) binding prevents ligand-independent TCR signaling via Lck by blocking spontaneous clustering and CD4-Lck recruitment to TCR, and in turn F-actin transfer of TCR/CD4-Lck complexes to membrane microdomains. Peptide-major histocompatibility complexes overcome galectin-TCR binding to promote TCR clustering and signaling by Lck at the immune synapse. Galectin also localizes the tyrosine phosphatase CD45 to microdomains and the immune synapse, suppressing basal and activation signaling by Lck. Following activation, membrane turnover increases and galectin binding to cytotoxic T-lymphocyte antigen-4 (CTLA-4) enhances surface expression by inhibiting endocytosis, thereby promoting growth arrest. Galectins bind surface glycoproteins in proportion to the branching and number of N-glycans per protein, the latter an encoded feature of protein sequence. N-glycan branching is conditional to the activity of Golgi N-acetylglucosaminyl transferases I, II, IV and V (Mgat1, 2, 4, and 5) and metabolic supply of their donor substrate UDP-GlcNAc. Genetic and metabolic control of N-glycan branching co-regulate homeostatic set-points for basal, activation, and arrest signaling in T cells and, when disturbed, result in T-cell hyperactivity and autoimmunity.

Attenuation of Th1 response through galectin‐9 and T‐cell Ig mucin 3 interaction inhibits autoimmune diabetes in NOD mice

Abstract: Galectin-9 (gal-9), widely expressed in many tissues, regulates Th1 cells and induces their apoptosis through its receptor, T-cell Ig mucin 3, which is mainly expressed on terminally differentiated Th1 cells Type 1 diabetes is a Th1-dominant autoimmune disease that specifically destroys insulin-producing beta cells To suppress the Th1 immune response in the development of autoimmune diabetes, we overexpressed gal-9 in NOD mice by injection of a plasmid encoding gal-9 Mice treated with gal-9 plasmid were significantly protected from diabetes and showed less severe insulitis compared with controls Flow cytometric analyses in NOD-T1/2 double transgenic mice showed that Th1-cell population in spleen, pancreatic lymph node and pancreas was markedly decreased in gal-9 plasmid-treated mice, indicating a negative regulatory role of gal-9 in the development of pathogenic Th1 cells Splenocytes from gal-9 plasmid-treated mice were less responsive to mitogenic stimulation than splenocytes from the control group However, adoptive transfer of splenocytes from gal-9-treated or control mice caused diabetes in NOD/SCID recipients with similar kinetics, suggesting that gal-9 treatment does not induce active tolerance in NOD mice We conclude that gal-9 may downregulate Th1 immune response in NOD mice and could be used as a therapeutic target in autoimmune diabetes

The α-galactomannan Davanat binds galectin-1 at a site different from the conventional galectin carbohydrate binding domain

Abstract: Galectins are a sub-family of lectins, defined by their highly conserved beta-sandwich structures and ability to bind to beta-galactosides, like Gal beta1-4 Glc (lactose). Here, we used (15)N-(1)H HSQC and pulse field gradient (PFG) NMR spectroscopy to demonstrate that galectin-1 (gal-1) binds to the relatively large galactomannan Davanat, whose backbone is composed of beta1-4-linked d-mannopyranosyl units to which single d-galactopyranosyl residues are periodically attached via alpha1-6 linkage (weight-average MW of 59 kDa). The Davanat binding domain covers a relatively large area on the surface of gal-1 that runs across the dimer interface primarily on that side of the protein opposite to the lactose binding site. Our data show that gal-1 binds Davanat with an apparent equilibrium dissociation constant (K(d)) of 10 x 10(-6) M, compared to 260 x 10(-6) M for lactose, and a stiochiometry of about 3 to 6 gal-1 molecules per Davanat molecule. Mannan also interacts at the same galactomannan binding domain on gal-1, but with at least 10-fold lower avidity, supporting the role of galactose units in Davanat for relatively strong binding to gal-1. We also found that the beta-galactoside binding domain remains accessible in the gal-1/Davanat complex, as lactose can still bind with no apparent loss in affinity. In addition, gal-1 binding to Davanat also modifies the supermolecular structure of the galactomannan and appears to reduce its hydrodynamic radius and disrupt inter-glycan interactions thereby reducing glycan-mediated solution viscosity. Overall, our findings contribute to understanding gal-1-carbohydrate interactions and provide insight into gal-1 function with potentially significant biological consequences.

Endogenous galectins and the control of the host inflammatory response

Abstract: A new era of research is being devoted to deciphering endogenous mediators and mechanisms that are in place to resolve the inflammatory response. Accruing evidence indicates that galectins fall into this category of immunoregulatory mediators signifying their use as prospective novel anti-inflammatory agents. The focus of this review is to depict the immunoregulatory bioactivities of three members of the galectin superfamily, Galectin (Gal)-1, Gal-3 and Gal-9. Emphasis is given to the studies investigating the properties of these endogenous lectins. Gal-1, Gal-3 and Gal-9 are emerging as pertinent players in the modulation of acute and chronic inflammatory diseases, autoimmunity and cancer, and thus being increasingly recognised as molecular targets for innovative drug discovery.

Chemo-enzymatic synthesis of poly-N-acetyllactosamine (poly-LacNAc) structures and their characterization for CGL2-galectin-mediated binding of ECM glycoproteins to biomaterial surfaces

Abstract: Poly-N-acetyllactosamine (poly-LacNAc) struc- tures have been identified as important ligands for galectin- mediated cell adhesion to extra-cellular matrix (ECM) proteins. We here present the biofunctionalization of surfaces withpoly-LacNAc structuresand subsequentbinding ofECM glycoproteins. First, we synthesized β-GlcNAc glycosides carrying a linker for controlled coupling onto chemically functionalized surfaces. Then we produced poly-LacNAc structures with defined lengths using human β1,4-galactosyl- transferase-1 and β1,3-N-acetylglucosaminyltransferase from Helicobacter pylori. These compounds were also used for kinetic characterization of glycosyltransferases and lectin binding assays. A mixture of poly-LacNAc-structures cova- lently coupled to functionalized microtiter plates were identi- fied for best binding to our model galectin His6CGL2. We further demonstrate for the first time that these poly-LacNAc surfaces are suitable for further galectin-mediated binding of the ECM glycoproteins laminin and fibronectin. This new technology should facilitate cell adhesion to biofunctionalized surfaces by imitating the natural ECM microenvironment.

Galectin-8 provides costimulatory and proliferative signals to T lymphocytes

Abstract: Galectin (Gal) constitute a family of carbohydrate-recognizing molecules ubiquitously expressed in mammals. In the immune system, they regulate many processes such as inflammation, adhesion, and apoptosis. Here, we report the expression in the spleen of the two same Gal-8 splice variants described previously in the thymus. Gal-8 was found to induce two separate biological activities on T lymphocytes: a robust naive CD4(+) T cell proliferation in the absence of antigen and notably, a costimulatory signal that synergized the cognate OVA peptide in DO11.10 mice transgenic for TCR(OVA). The antigen-independent proliferation induced by Gal-8 displayed increased expression of pro- and anti-inflammatory cytokines, thus suggesting the polyclonal expansion of Th1 and Th2 clones. The costimulatory effect on antigen-specific T cell activation was evidenced when the Gal and the peptide were assayed at doses suboptimal to induce T cell proliferation. By mass spectra analysis, several integrins and leukocyte surface markers, including CD45 isoforms, as well as other molecules specific to macrophages, neutrophils, and platelets, were identified as putative Gal-8 counter-receptors. Gal-8 triggered pZAP70 and pERK1/2. Moreover, pretreatment with specific inhibitors of CD45 phosphatase or ERK1/2 prevented its antigen-dependent and -independent T cell-proliferative activities. This seems to be associated with the agonistic binding to CD45, which lowers the activation threshold of the TCR signaling pathway. Taken together, our findings support a distinctive role for locally produced Gal-8 as an enhancer of otherwise borderline immune responses and also suggest that Gal-8 might fuel the reactivity at inflammatory foci.

Immunohistochemical Localization of Six Galectin Subtypes in the Mouse Digestive Tract

Abstract: Galectin, an animal lectin that recognizes beta-galactoside of glycoconjugates, is abundant in the gut. This IHC study showed the subtype-specific localization of galectin in the mouse digestive tract. Mucosal epithelium showed region/cell-specific localization of each galectin subtype. Gastric mucous cells exhibited intense immunoreactions for galectin-2 and galectin-4/6 with a limited localization of galectin-3 at the surface of the gastric mucosa. Electron microscopically, galectin-3 immunoreactivity coated indigenous bacteria on the gastric surface mucous cells. Epithelial cells in the small intestine showed characteristic localizations of galectin-2 and galectin-4/6 in the cytoplasm of goblet cells and the baso-lateral membrane of enterocytes in association with maturation, respectively. Galectin-3 expressed only at the villus tips was concentrated at the myosin-rich terminal web of fully matured enterocytes. Epithelial cells of the large intestine contained intense immunoreactions for galectin-3 and galectin-4/6 but not for galectin-2. The stratified squamous epithelium of the forestomach was immunoreactive for galectin-3 and galectin-7, but the basal layer lacked galectin-3 immunoreactivity. Outside the epithelium, only galectin-1 was localized in the connective tissue, smooth muscles, and neuronal cell bodies. The subtype-specific localization of galectin suggests its important roles in host-pathogen interaction and epithelial homeostasis such as membrane polarization and trafficking in the gut.

Intracellular galectin-9 activates inflammatory cytokines in monocytes

Abstract: Whether galectin-9 plays a role in inflammatory responses remains elusive. The present study was designed to determine the role of intracellular galectin-9 in activation of inflammatory cytokine genes in human monocytes. Galectin-9 expression vector pBKCMV3-G9 was transiently co-transfected into THP-1 monocytic cells along with luciferase reporters carrying gene promoters of IL-1alpha (IL1A), IL-1beta (IL1B) and IFNgamma. Transient transfection studies showed that galectin-9 over-expression activated all three gene promoters, suggesting that intracellular galectin-9 induces inflammatory cytokine genes in monocytes. Galectin-9 over-expression also activated NF-IL6 (C/EBP beta) and AP-1, but not NF-kappaB. In contrast, extracellular galectin-9 is not involved in regulation of inflammatory cytokines. Immunoprecipitation/Western blotting, using anti-galectin-9 Ab and anti-NF-IL6 Ab, showed physical association of intracellular galectin-9 with NF-IL6. RT-PCR confirmed that galectin-9 over-expression increased IL-1alpha and IL-1beta mRNA levels in THP-1 cells. The interaction of galectin-9 with NF-IL6 was enhanced following LPS treatment in THP-1 cells. Intracellular galectin-9 synergized with LPS to activate NF-IL6. Nuclear translocation of galectin-9 was also observed in THP-1 cells treated with LPS. Our results indicate that galectin-9 is a LPS-responsive factor, and further demonstrate that intracellular galectin-9 transactivates inflammatory cytokine genes in monocytes through direct physical interaction with NF-IL6.

The carbohydrate-binding domain on galectin-1 is more extensive for a complex glycan than for simple saccharides: implications for galectin–glycan interactions at the cell surface

Abstract: gal-1 (galectin-1) mediates cell–cell and cell–extracellular matrix adhesion, essentially by interacting with β-galactoside-containing glycans of cell-surface glycoconjugates. Although most structural studies with gal-1 have investigated its binding to simple carbohydrates, in particular lactose and N-acetyl-lactosamine, this view is limited, because gal-1 functions at the cell surface by interacting with more complex glycans that are heterogeneous in size and composition. In the present study we used NMR spectroscopy to investigate the interaction of human gal-1 with a large (120 kDa) complex glycan, GRG (galactorhamnogalacturonate glycan), that contains non-randomly distributed mostly terminal β(1→4)-linked galactose side chains. We used 15N–1H-HSQC (heteronuclear single quantum coherence) NMR experiments with 15N-enriched gal-1 to identify the GRG-binding region on gal-1 and found that this region covers a large surface area on gal-1 that includes the quintessential lactose-binding site and runs from that site through a broad valley or cleft towards the dimer interface. HSQC and pulsed-field-gradient NMR diffusion experiments also show that gal-1 binds GRG with a gal-1:GRG stoichiometry of about 5:1 (or 6:1) and with average macroscopic and microscopic equilibrium dissociation constants (Kd) of 8×10−6 M and 40×10−6 M (or 48×10−6 M) respectively, indicating stronger binding than to lactose (Kd=520×10−6 M). Although gal-1 may bind GRG in various ways, the glycan can be competed for by lactose, suggesting that there is one major mode of interaction. Furthermore, even though terminal motifs on GRG are Gal-β(1→4)-Gal rather than the traditional Gal-β(1→4)-Glc/GlcNAc (where GlcNAc is N-acetylglucosamine), we show that the disaccharide Gal-β(1→4)-Gal can bind gal-1 at the lactose-binding domain. In addition, gal-1 binding to GRG disrupts inter-glycan interactions and decreases glycan-mediated solution viscosity, a glycan decongestion effect that may help explain why gal-1 promotes membrane fluidity and lateral diffusion of glycoconjugates within cell membranes. Overall, our results provide an insight into the function of galectin in situ and have potential significant biological consequences.

Glycosylation pattern of brush border-associated glycoproteins in enterocyte-like cells: involvement of complex-type N-glycans in apical trafficking.

Abstract: We have previously reported that galectin-4, a tandem repeat-type galectin, regulates the raft-dependent delivery of glycoproteins to the apical brush border membrane of enterocyte-like HT-29 cells. N-Acetyllactos-amine-containing glycans, known as galectin ligands, were found enriched in detergent-resistant membranes. Here, we analyzed the potential contribution of N- and/or O-glycans in this mechanism. Structural studies were carried out on the brush border membrane-enriched fraction using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and nano-ESI-QTOF-MS/MS. The pattern of N-glycans was very heterogeneous, with the presence of high mannose- and hybrid-type glycans as well as a multitude of complex-type glycans. In contrast, the pattern of O-glycans was very simple with the presence of two major core type 1 O-glycans, sialylated and bisialylated T-antigen structures [Neu5Acalpha2-3Galbeta1-3GalNAc-ol and Neu5Acalpha2- 3Galbeta1-3(Neu5Acalpha2-6)GalNAc-ol]. Thus, N-glycans rather than O-glycans contain the N-acetyllactosamine recognition signals for the lipid raft-based galectin-4-dependent apical delivery. In the presence of 1-deoxymannojirimycin, a drug which inhibits the generation of hybrid-type or complex type N-glycans, the extensively O-glycosylated mucin-like MUC1 glycoprotein was not delivered to the apical brush border but accumulated inside the cells. Altogether, our data demonstrate the crucial role of complex N-glycans in the galectin-4-dependent delivery of glycoproteins to the apical brush border membrane of enterocytic HT-29 cells.