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Diabetes mellitus (DM) is now recognized as one of the risk factors for Alzheimer's disease (AD), and the disease‐modifying effects of anti‐diabetic drugs on AD have recently been attracting great attention. Sodium/glucose cotransporter 2 (SGLT2) inhibitors are a new class of anti‐diabetic drugs targeting the SGLT2/solute carrier family 5 member 2 (SLC5A2) protein, which is known to localize exclusively in the brush border membrane of early proximal tubules in the kidney. However, recent data suggest that it is also expressed in other tissues. In the present study, we investigated the expression of SGLT2/SLC5A2 in human and mouse brains. Immunohistochemical staining of paraffin sections from autopsied human brains and C3H/He mouse brains revealed granular cytoplasmic immunoreactivity in choroid plexus epithelial cells and ependymal cells. Immunoblot analysis of the membrane fraction of mouse choroid plexus showed distinct immunoreactive bands at 70 and 26 kDa. Band patterns around 70 kDa in the membrane fraction of the choroid plexus were different from those in the kidney. Reverse transcription‐polymerase chain reaction analysis confirmed the expression of Slc5a2 mRNA in the mouse choroid plexus. Our results provide in vivo evidence that SGLT2/SLC5A2 is expressed in cells facing the cerebrospinal fluid, in addition to early proximal tubular epithelial cells. These findings suggest that SGLT2 inhibitors may have another site of action in the brain. The effects of SGLT2 inhibitors on brain function and AD progression merit further investigation to develop better treatment options for DM patients.

The galectins are a family of β-galactoside-specific animal lectins, and have attracted much attention as novel regulators of the immune system. Galectin-10 is well-expressed in eosinophils, and spontaneously forms Charcot-Leyden crystals (CLCs), during prolonged eosinophilic inflammatory reactions, which are frequently observed in eosinophilic diseases. Although biochemical and structural characterizations of galectin-10 have been done, its biological role and molecular mechanism are still unclear, and few X-ray structures of galectin-10 in complex with monosaccharides/oligosaccharides have been reported. Here, X-ray structures of galectin-10 in complexes with seven monosaccharides are presented with biochemical analyses to detect interactions of galectin-10 with monosaccharides/oligosaccharides. Galectin-10 forms a homo-dimer in the face-to-face orientation, and the monosaccharides bind to the carbohydrate recognition site composed of amino acid residues from two galectin-10 molecules of dimers, suggesting that galectin-10 dimer likely captures the monosaccharides in solution and in vivo. d -Glucose, d -allose, d -arabinose, and D-N-acetylgalactosamine bind to the interfaces between galectin-10 dimers in crystals, and they affect the stability of molecular packing in crystals, leading to easy-dissolving of CLCs, and/or inhibiting the formation of CLCs. These monosaccharides may serve as effectors of G10 to form CLCs in vivo.

Modification of the domain architecture of galectins has been attempted to analyze their biological functions and to develop medical applications. Several types of galectin-1 repeat mutants were previously reported but, however, it was not clear whether the native structure of the wild type was retained. In this study, we determined the crystal structure of a galectin-1 tandem-repeat mutant with a short linker peptide, and compared the unfolding profiles of the wild type and mutant by chemical denaturation. The structure of the mutant was consistent with that of the dimer of the wild type, and both carbohydrate-binding sites were retained. The unfolding curve of the wild type with lactose suggested that the dimer dissociation and the tertiary structure unfolding was concomitant at micromolar protein concentrations. The midpoint denaturant concentration of the wild type was dependent on the protein concentration and lower than that of the mutant. Linking the two subunits significantly stabilized the tertiary structure. The mutant exhibited higher T-cell growth-inhibition activity and comparable hemagglutinating activity. Structural stabilization may prevent the oxidation of the internal cysteine residue.

The galectin family is a representative soluble lectin group, which is responsible for the modulation of various cell functions. Although the carbohydrate-binding specificity of galectins has been well-studied, the relationship between protein structure and specificity remains to be elucidated. We previously reported the characteristics of a Xenopus laevis skin galectin, xgalectin-Va, which had diverged from galectin-1. The carbohydrate selectivity of xgalectin-Va was different from that of human galectin-1 and xgalectin-Ib (a Xenopus laevis galectin-1 homolog). In this study, we clarified the key residues for this selectivity by site-directed mutagenesis. Substitution of two amino acids of xgalectin-Va, Val56Gly/Lys76Arg, greatly enhanced the binding ability to N-acetyllactosamine and conferred significant T-cell growth inhibition activity, although the wild type had no activity. These two residues, Gly54 and Arg74 in galectin-1, would cooperatively contribute to the N-acetyllactosamine recognition. The loop region between the S4 and S5 β-strands was involved in the binding to the TF-antigen disaccharide. The loop substitution successfully changed the carbohydrate selectivity of xgalectin-Va and xgalectin-Ib.

Glucose metabolism produces lactate and hydrogen ions in an anaerobic environment. Cerebral ischemia or hypoxia is believed to become progressively lactacidemic. Monocarboxylate transporters (MCTs) in endothelial cells are essential for the transport of lactate from the blood into the brain. In addition, it is considered that MCTs located in astrocytic and neuronal cells play a key role in the shuttling of energy metabolites between neurons and astrocytes. However, roles of lactate in the brain remain to be clarified. In this study, the localization of lactate transporters and a receptor for cellular uptake of lactate was immunohistochemically examined in autopsied human brains. Immunoreactivity for MCT1 was observed in the apical cytoplasmic membrane of some epithelial cells in the choroid plexus as well as astrocytes and the capillary wall, whereas that for MCT4 was found in the basolateral cytoplasmic membrane of small number of epithelial cells as well as astrocytes and the capillary wall. In addition, immunoreactivity for the hydroxy-carboxylic acid 1 receptor (HCA1 receptor), a receptor for cellular uptake of lactate, was also found on the basolateral cytoplasmic membrane of epithelial cells as well as astrocytic and neuronal cells. Immunoreactivity for lactate dehydrogenase (LDH)-B was observed in the cytoplasm of epithelial cells in the choroid plexus as well as astrocytes and the capillary wall. These immunohistochemical findings indicate the localization of MCT1, MCT4, the HCA1 receptor, and LDH-B in epithelial cells of the choroid plexus as well as astrocytes, and suggest the transport of intravascular lactate into the brain through epithelial cells of the choroid plexus as well as cerebral vessels and the possibility of lactate being utilized in epithelial cells.

Galectin-9 is the most potent inducer of cell death in lymphomas and other malignant cell types among the members of the galectin family. We investigated the mechanism of galectin-9-induced cell death in PC-3 prostate cancer cells in comparison with in Jurkat T cells. Galectin-9 induced apoptotic cell death in Jurkat cells, as typically revealed by DNA ladder formation. On the other hand, DNA ladder formation and other features of apoptosis were not apparent in PC-3 cells undergoing galectin-9-induced death. Exogenous galectin-9 was endocytosed and destined to the lysosomal compartment in PC-3 cells. The internalized galectin-9 was resistant to detergent solubilization but was solubilized with lactose. Agents inhibiting actin filament dynamics abolished the internalization and cytocidal effect of galectin-9 in PC-3 but not Jurkat cells. Galectin-9 induced accumulation of ubiquitinated proteins, possibly heterogeneously ubiquitinated and/or monoubiquitinated proteins, in PC-3 cells. PYR-41, an inhibitor of the ubiquitin-activating E1 enzyme, suppressed the cytocidal effect of galectin-9. Although ubiquitination was upregulated also in Jurkat cells by galectin-9, PYR-41 was ineffective against galectin-9-induced cell death. Colocalization of ubiquitinated proteins and LAMP-1 was detectable in PC-3 cells treated with galectin-9. The ubiquitinated proteins were recovered in the insoluble fraction upon cell fractionation. In contrast, ubiquitinated proteins that accumulated after treatment with proteasome inhibitors did not co-localize with LAMP-1 and were mainly recovered in soluble fraction. The results suggest that atypical ubiquitination and accumulation of ubiquitinated proteins in lysosomes play a pivotal role in galectin-9-induced non-apoptotic death in PC-3 cells.

We previously reported that galectin-9 (Gal-9), an immunomodulatory animal lectin, could bind to insoluble collagen preparations and exerted direct cytocidal effects on immune cells. In the present study, we found that mature insoluble elastin is capable of binding Gal-9 and other members of the human galectin family. Lectin blot analysis of a series of commercial water-soluble elastin preparations, PES-(A) ~ PES-(E), revealed that only PES-(E) contained substances recognized by Gal-9. Gal-9-interacting substances in PES-(E) were affinity-purified, digested with trypsin and then analyzed by reversed-phase HPLC. Peptide fragments derived from five members of the small leucine-rich repeat proteoglycan family, versican, lumican, osteoglycin/mimecan, prolargin, and fibromodulin, were identified by N-terminal amino acid sequence analysis. The results indicate that Gal-9 and possibly other galectins recognize glycans attached to small leucine-rich repeat proteoglycans associated with insoluble elastin and also indicate the possibility that mature insoluble elastin serves as an extracellular reservoir for galectins.

Study design An experimental study. Objective To evaluate the effectiveness of freeze-dried bone allograft (FDBA) with basic fibroblast growth factor (bFGF) fused with the polycystic kidney disease domain (PKD) and the collagen-binding domain (CBD) of Clostridium histolyticum collagenase, for the acceleration of lumbar posterolateral fusion in rats. Summary of background data Reports indicate bFGF is an effective growth factor with osteogenic potential for promoting bone regeneration, although its efficiency decreases rapidly following its diffusion in body fluid from the host site. We developed a bFGF fusion protein containing the PKD and the CBD of C histolyticum collagenase (bFGF-PKD-CBD), which markedly enhanced bone formation at a relatively low concentration when applied to the surface of rat femurs in a previous study. The potential of this novel protein to accelerate bone fusion in a rat model of lumbar posterolateral fusion has yet to be investigated. Methods Bilateral L4-L5 posterolateral fusions were performed, using 150 mg of FDBA powder per side. A total of 20 male Sprague-Dawley rats weighing 200 to 250 g/each were divided into two groups of 10 rats: FDBA was incubated with either phosphate-buffered saline (control group) or 0.58 nmol bFGF-PKD-CBD (bFGF-PKD-CBD group) before fusion surgery. The effect of bFGF-PKD-CBD was estimated using radiographs, microcomputed tomography, and histology (hematoxylin-eosin and von Kossa staining). Results Both grafted bone volume in the posterolateral lesion and the volume of new bone formation on the surface of laminae and spinal processes were significantly higher in the bFGF-PKD-CBD group than in the control group. Histologically, new bone formation and surrounding chondrocytes and fibroblasts were prominent in the bFGF-PKD-CBD group. Conclusion FDBA infused with bFGF-PKD-CBD may be a promising material for accelerating spinal fusion, and the FDBA-based delivery system for localizing bFGF-PKD-CBD may offer novel therapeutic approaches to augment spinal fusion. Level of evidence N/A.

Their aim was to examine whether microvascular leakage of endogenous albumin, a representative marker for blood-brain barrier (BBB) damage, was induced in the periventricular area of diabetic db/db mice because periventricular white matter hyperintensity formation in magnetic resonance images was accelerating in elderly patients with diabetes mellitus. Using light and electron microscopes, and semi-quantitative analysis techniques, immunoreactivity of endogenous albumin, indicating vascular permeability, was examined in the periventricular area and spinal cord of db/db mice and db/+m control mice. Greater immunoreactivity of albumin was observed in the vessel wall of the periventricular area of db/db mice than in controls. Additionally, weak immunoreactivity was observed in the spinal cord of both db/db mice and controls. The number of gold particles, indicating immunoreactivity of albumin, in the perivascular area of db/db mice was significantly higher than that of control mice, but there was no significant difference in the number of particles in the spinal cord between db/db mice and controls. These findings suggest that albumin microvascular leakage, or BBB breakdown, is induced in the periventricular area of diabetic mice. Microsc. Res. Tech. 79:833-837, 2016. © 2016 Wiley Periodicals, Inc.

Basic fibroblast growth factor 2 (bFGF) accelerates bone formation during fracture healing. Because the efficacy of bFGF decreases rapidly following its diffusion from fracture sites, however, repeated dosing is required to ensure a sustained therapeutic effect. We previously developed a fusion protein comprising bFGF, a polycystic kidney disease domain (PKD; s2b), and collagen-binding domain (CBD; s3) sourced from the Clostridium histolyticum class II collagenase, ColH, and reported that the combination of this fusion protein with a collagen-like peptide, poly(Pro-Hyp-Gly)10, induced mesenchymal cell proliferation and callus formation at fracture sites. In addition, C. histolyticum produces class I collagenase (ColG) with tandem CBDs (s3a and s3b) at the C-terminus. We therefore hypothesized that a bFGF fusion protein containing ColG-derived tandem CBDs (s3a and s3b) would show enhanced collagen-binding activity, leading to improved bone formation. Here, we examined the binding affinity of four collagen anchors derived from the two clostridial collagenases to H-Gly-Pro-Arg-Gly-(Pro-Hyp-Gly)12-NH2, a collagenous peptide, by surface plasmon resonance and found that tandem CBDs (s3a-s3b) have the highest affinity for the collagenous peptide. We also constructed four fusion proteins consisting of bFGF and s3 (bFGF-s3), s2b-s3b (bFGF-s2b-s3), s3b (bFGF-s3b), and s3a-s3b (bFGF-s3a-s3b) and compared their biological activities to those of a previous fusion construct (bFGF-s2b-s3) using a cell proliferation assay in vitro and a mouse femoral fracture model in vivo. Among these CB-bFGFs, bFGF-s3a-s3b showed the highest capacity to induce mesenchymal cell proliferation and callus formation in the mice fracture model. The poly(Pro-Hyp-Gly)10/bFGF-s3a-s3b construct may therefore have the potential to promote bone formation in clinical settings.

Xenopus laevis (African clawed frog) has two types of proto-type galectins that are similar to mammalian galectin-1 in amino acid sequence. One type, comprising xgalectin-Ia and -Ib, is regarded as being equivalent to galectin-1, and the other type, comprising xgalectin-Va and -Vb, is expected to be a unique galectin subgroup. The latter is considerably abundant in frog skin; however, its biological function remains unclear. We determined the crystal structures of two proto-type galectins, xgalectin-Ib and -Va. The structures showed that both galectins formed a mammalian galectin-1-like homodimer, and furthermore, xgalectin-Va formed a homotetramer. This tetramer structure has not been reported for other galectins. Gel filtration and other experiments indicated that xgalectin-Va was in a dimer-tetramer equilibrium in solution, and lactose binding enhanced the tetramer formation. The residues involved in the dimer-dimer association were conserved in xgalectin-Va and -Vb, and one of the Xenopus (Silurana) tropicalis proto-type galectins, but not in xgalectin-Ia and -Ib, and other galectin-1-equivalent proteins. Xgalectin-Va preferred Galβ1-3GalNAc and not Galβ1-4GlcNAc, while xgalectin-Ib preferred Galβ1-4GlcNAc as well as human galectin-1. Xgalectin-Va/Vb would have diverged from the galectin-1 group with accompanying acquisition of the higher oligomer formation and altered ligand selectivity.

Recombinant basic fibroblast growth factor (bFGF) is a potent mitogen for mesenchymal cells that accelerates bone union and repair when applied locally at defect sites. However, because bFGF diffuses rapidly from bone defect sites, repeated dosing is required for sustained therapeutic effect. We previously fused the collagen-binding domain (CBD) and polycystic kidney disease (PKD) domain of Clostridium histolyticum class II collagenase (ColH) to bFGF and demonstrated that the fusion protein markedly enhances bone formation when loaded onto collagen materials used for grafting. However, systemic injection of a fusion protein consisting of parathyroid hormone (PTH) and a CBD was shown to accelerate bone formation in an osteoporosis model more rapidly than treatment with a PTH-PKD-CBD fusion protein. Here, we compared the biological properties of two collagen-binding forms of bFGF, bFGF-CBD and bFGF-PKD-CBD. Both fusion proteins promoted the in vitro proliferation of periosteal mesenchymal cells, indicating that they had biological activity similar to that of native bFGF. In vivo periosteal bone formation assays in rat femurs showed that both bFGF-CBD and bFGF-PKD-CBD induced periosteal bone formation at higher rates than collagen sheet alone and bFGF. However, bFGF-PKD-CBD markedly enhanced bone formation and had higher collagen-binding ability than bFGF-CBD in in vitro protein release assays. Taken together, these results suggest that the PKD domain increases the retention of bFGF at graft sites by enhancing collagen-binding affinity. Therefore, bFGF-PKD-CBD-collagen composite appears to be a promising material for bone repair in the clinical setting. Copyright © 2015 John Wiley & Sons, Ltd.

Galectin-9 (G9) is a tandem-repeat type β-galactoside-specific animal lectin having N-terminal and C-terminal carbohydrate recognition domains (N-CRD and C-CRD, respectively) joined by a linker peptide that is involved in the immune system. G9 is divalent in glycan binding, and structural information about the spatial arrangement of the two CRDs is very important for elucidating its biological functions. As G9 is protease sensitive due to the long linker, the protease-resistant mutant form of G9 (G9Null) was developed by modification of the linker peptide, while retaining its biological functions. The X-ray structure of a mutant form of G9Null with the replacement of Arg221 by Ser (G9Null_R221S) having two CRDs was determined. The structure of G9Null_R221S was compact to associate the two CRDs in the back-to-back orientation with a large interface area, including hydrogen bonds and hydrophobic interactions. A metal ion was newly found in the galectin structure, possibly contributing to the stable structure of protein. The presented X-ray structure was thought to be one of the stable structures of G9, which likely occurs in solution. This was supported by structural comparisons with other tandem-repeated galectins and the analyses of protein thermostability by CD spectra measurements.

Agonistic anti–4-1BB suppresses autoimmune and allergic inflammation via binding to Galectin-9, which facilitates 4-1BB aggregation, signaling, and functional activity., Biologics to TNF family receptors are prime candidates for therapy of immune disease. Whereas recent studies have highlighted a requirement for Fcγ receptors in enabling the activity of CD40, TRAILR, and GITR when engaged by antibodies, other TNFR molecules may be controlled by additional mechanisms. Antibodies to 4-1BB (CD137) are currently in clinical trials and can both augment immunity in cancer and promote regulatory T cells that inhibit autoimmune disease. We found that the action of agonist anti–4-1BB in suppressing autoimmune and allergic inflammation was completely dependent on Galectin-9 (Gal-9). Gal-9 directly bound to 4-1BB, in a site distinct from the binding site of antibodies and the natural ligand of 4-1BB, and Gal-9 facilitated 4-1BB aggregation, signaling, and functional activity in T cells, dendritic cells, and natural killer cells. Conservation of the Gal-9 interaction in humans has important implications for effective clinical targeting of 4-1BB and possibly other TNFR superfamily molecules.

Growth factor delivered with implantable biomaterials has been used to both accelerate and ensure healing of open fractures in human patients. However, a major limitation of implantable biomaterials is the requirement for open surgical placement. Here, we developed an injectable collagen material-based bone formation system consisting of injectable collagen powder with fibril morphology and collagen triple helix conformation, and basic fibroblast growth factor (bFGF) fused to the collagen-binding domain (CBD) of Clostridium histolyticum collagenase. The affinity of the CBD towards collagen was confirmed by the results of collagen-binding assays. Moreover, the combination of the collagen binding-bFGF fusion protein (CB-bFGF) with injectable collagen powder induced bone formation at protein concentrations lower than those required for bFGF alone in mice fracture models. Taken together, these properties suggest that the CB-bFGF/collagen powder composite is a promising injectable material for bone repair in the clinical setting.

Basic fibroblast growth factor 2 (bFGF) is a potent mitogen for mesenchymal cells, and the local application of recombinant bFGF accelerates bone union and defect repair. However, repeated dosing is required for sustained therapeutic effect as the efficacy of bFGF decreases rapidly following its diffusion from bone defect sites. Here, we attempted to develop a collagen-based bone formation system using a fusion protein (collagen binding-bFGF, CB-bFGF) consisting of bFGF and the collagen-binding domain (CBD) of Clostridium histolyticum collagenase. The addition of the CBD to bFGF did not modify its native biological activity, as shown by the capacity of the fusion protein to promote the in vitro proliferation of periosteal mesenchymal cells. The affinity of the fusion protein towards collagen and demineralized bone matrix (DBM) was also confirmed by collagen-binding assays. Moreover, in vivo periosteal bone formation assays showed that the combination of CB-bFGF with a collagen sheet induced periosteal bone formation at protein concentrations lower than those required for bFGF alone. In addition, grafts of DBM loaded with CB-bFGF accelerated new bone formation in rat femurs compared to the same concentration of bFGF administered alone. Taken together, these properties suggest that the CB-bFGF/collagen composite is a promising material for bone repair in the clinical setting. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 1737–1743, 2014.

We previously developed a stable form of galectin-9, an immunomodulatory animal lectin with a truncated linker peptide (G9Null), to overcome the protease sensitivity of wild-type galectin-9. G9Null is highly resistant to proteolysis, while the modification marginally improved the low solubility of the wild-type protein. To increase its solubility, we further modified the remaining linker region of G9Null. A 10-amino acid deletion with a single amino acid substitution resulted in an ∼400% increase in solubility and yield without an adverse effect on its biological activity. This mutant protein might be useful for large-scale recombinant production needed for evaluation of the therapeutic potential of galectin-9.

Their aim was to examine whether microvascular leakage of endogenous albumin, a representative marker for blood-brain barrier (BBB) damage, was induced in the periventricular area of diabetic db/db mice because periventricular white matter hyperintensity formation in magnetic resonance images was accelerating in elderly patients with diabetes mellitus. Using light and electron microscopes, and semi-quantitative analysis techniques, immunoreactivity of endogenous albumin, indicating vascular permeability, was examined in the periventricular area and spinal cord of db/db mice and db/+m control mice. Greater immunoreactivity of albumin was observed in the vessel wall of the periventricular area of db/db mice than in controls. Additionally, weak immunoreactivity was observed in the spinal cord of both db/db mice and controls. The number of gold particles, indicating immunoreactivity of albumin, in the perivascular area of db/db mice was significantly higher than that of control mice, but there was no significant difference in the number of particles in the spinal cord between db/db mice and controls. These findings suggest that albumin microvascular leakage, or BBB breakdown, is induced in the periventricular area of diabetic mice. Microsc. Res. Tech. 79:833-837, 2016. © 2016 Wiley Periodicals, Inc.

TO THE EDITOR In recent years, a regulatory role has emerged for the glycan-binding protein galectin-9 (Gal-9) in normal physiology and pathology (reviewed by Wiersma et al. (2011). In melanoma and other malignancies, the available data suggest that Gal-9 has a tumor-suppressor function, with loss of Gal-9 being closely associated with metastatic progression (Kageshita et al., 2002; Irie et al., 2005; Yamauchi et al., 2006; Liang et al., 2008). In particular, melanocytic nevi and primary melanoma lesions highly express Gal-9, whereas metastatic melanoma lesions have no or minimal expression of Gal-9 (Kageshita et al., 2002). Furthermore, ectopic expression of Gal-9 abrogates the formation of metastases by Gal-9-deficient B16F10 murine melanoma cells (Nobumoto et al., 2008). Similarly, treatment of Gal-9-deficient B16F10 cells with a recombinant form of Gal-9, designated Gal-9(0), strongly reduced metastasis formation (Nobumoto et al., 2008). This anti-metastatic activity of Gal-9(0) on B16F10 has been attributed mainly to inhibition of melanoma cell adhesion to endothelial cells and/or extracellular matrix components, such as collagen type I (collagen-I; Nobumoto et al., 2008). The data presented in the current letter suggest that within the 1-h time frame of adhesion-type assays, treatment with Gal-9(0) triggers early apoptotic cellular changes. In line with earlier findings, Gal-9(0) inhibits the adhesion of B16F10 and 7 human melanoma cell lines to collagen-I (Figure 1a). However, the morphology of Gal-9(0)-treated cells that had adhered to collagen-I-coated wells resembled that of dying cells (Figure 1b; illustrated for B16F10). Subsequent analysis of this melanoma cell line panel, as well as primary patient–derived malignant melanoma cells for the early apoptotic marker phosphatidyl serine (PS), revealed that treatment with Gal-9(0) induced ∼90% cell death within the time frame used in the adhesion assay (Figure 1c). Early apoptotic PS exposure was followed by apoptotic cell death within 24 h of treatment, as evidenced by loss of viability (Supplementary Figure S1a online), the presence of late apoptotic Annexin-V/PI double-positive cells (Supplementary Figure S1b and c online), and an increase in DNA fragmentation (Supplementary Figure S1d online). In primary human melanocytes, Gal-9(0) also triggered PS exposure, albeit to a lesser extent (Figure 1c; ∼55%). More importantly, the viability of these normal cells was not negatively affected after 24 h (Supplementary Figure S1a online). Thus, Gal-9(0) induces rapid apoptotic cell death in melanoma cells, but not in normal human melanocytes. PS exposure induced by Gal-9(0) was fully dependent on the glycan-binding specificity of Gal-9(0), as it was selectively blocked by the competitive Gal-9 inhibitor alpha-lactose but not by the irrelevant carbohydrate sucrose (Figure 1d). Sensitivity to Gal-9(0) did not or only weakly correlated with expression of endogenous Gal-9 (Supplementary Figure S2 online; r2=0.250). Time-course analysis in five of the human melanoma cell lines demonstrated that treatment with Gal-9(0) induced PS exposure in >60% of melanoma cells within 5 minutes of treatment (Figure 1e). Furthermore, the extent of PS exposure closely correlated with the inhibitory effect of Gal-9(0) on collagen I binding (Figure 1f, MM-RU; r2=0.693). Together, these data suggest that the biological effect of Gal-9(0) in adhesion assays is mediated, at least partly, through the induction of cell death. It is noteworthy that pan-caspase inhibition failed to block the anti-adhesive and apoptotic activity of Gal-9(0; Supplementary Figure S1e and f online). Thus, Gal-9(0)-mediated melanoma apoptosis does not require caspase activation, which is in line with e.g. reports on gal-1-mediated cell death of T cells (Hahn et al., 2004). Figure 1 Galectin-9 (Gal-9(0)) rapidly induces apoptosis in serum-free conditions. (a). Adhesion of B16F10 and a panel of human melanoma cell lines to collagen-I-coated wells is inhibited by recombinant Gal-9. In brief, 3 × 104 melanoma cells were added ... Adhesion assays are typically performed in serum-free conditions, whereas in normal physiological situations the presence of serum and/or plasma components may affect the biological activity of Gal-9. Indeed, it is well established that Gal-9 can interact with serum components (Cederfur et al., 2008). Therefore, the biological activity of Gal-9(0) was next evaluated in the presence of 10% fetal calf serum (FCS), the standard serum additive in cell death assays. The inclusion of 10% FCS in these apoptosis experiments completely abrogated the morphological changes in melanoma cells, with induction of PS exposure by Gal-9(0) being abrogated in six of the seven cell lines tested (Figure 2a). Similarly, PS exposure by Gal-9(0) was also strongly inhibited in the primary melanoma cells (Figure 2a). Indeed, FCS dose dependently inhibited PS exposure induced by Gal-9(0) (illustrated for cell line A2058 in Figure 2b) and blocked the binding of Gal-9(0) to A2058 cells (Figure 2c). When using dialysed FCS or heat-inactivated FCS, the activity of Gal-9(0) was still inhibited (Figure 2d). Thus, the inhibitory component present in FCS is not heat labile (i.e., complement factors) and is >10 kDa in size. Notably, FCS did not inhibit Gal-9(0) activity toward SK-MEL-28 cells (Figure 2a), which suggests that the inhibitory effect of FCS is not merely due to binding of a serum component to Gal-9(0). Possibly, a serum component may shield the receptor(s) of Gal-9 on most melanoma cells. On Sk-MEL-28 cells, Gal-9(0) may interact with an alternative receptor not subject to binding/inhibition by FCS. Figure 2 Cell death induction by galectin-9 (Gal-9(0)) is blocked by fetal calf serum (FCS) but not by human pooled plasma. (a) A panel of melanoma cells and primary patient–derived melanoma cells (n=1) were treated for 1 h with Gal-9(0) in standard ... Although FCS is the standard additive in in vitro cell death assays, a better approximation of physiological settings is the addition of human plasma. Importantly, 10% human pooled plasma did not abrogate Gal-9(0)-induced PS exposure in A2058 cells, with only a slight reduction in PS exposure compared with serum-free conditions (Figure 2d). Similar results were obtained in six melanoma cell lines and in primary patient–derived melanoma cells (Figure 2e). These experiments suggest that an important biological effect of Gal-9(0) on human melanoma cells is the induction of apoptosis. This biological effect of Gal-9(0) is masked by as yet unidentified components present in FCS, but is unmasked in serum-free conditions or when human plasma is used. Indeed, in standard FCS-containing culture conditions, apoptotic cell death of the melanoma cell line MM-RU was only detected after 72 h of treatment with Gal-9 (Kageshita et al., 2002). The use of FCS may similarly mask the biological activity of other gal family members. In this respect, gal-2, -3, -4, and -8 interact with various serum components (Cederfur et al., 2008). Notably, plasma levels of several of the gal family members are increased in malignancy (Barrow et al., 2011). The use of human pooled serum/plasma instead of FCS for in vitro biological assays with members of the Gal family therefore appears prudent. In conclusion, recombinant Gal-9 has a hitherto unrecognized cytotoxic effect toward human melanoma cells, which further highlights its potential therapeutic applicability for the treatment of human metastatic melanoma.

M. Ueno, T. Nakagawa, Y. Nagai, N. Nishi, T. Kusaka, K. Kanenishi, M. Onodera, N. Hosomi, C. Huang, H. Yokomise, H. Tomimoto and H. Sakamoto (2011) Neuropathology and Applied Neurobiology37, 727–737 The expression of CD36 in vessels with blood–brain barrier impairment in a stroke-prone hypertensive model Aims: The class B scavenger receptor CD36, the receptor for oxidized low-density lipoprotein, mediates free radical production and brain injury in cerebral ischaemia. Free radical production is known to be involved in the remodelling of the cerebral vasculature of stroke-prone spontaneously hypertensive rats (SHRSP). Accordingly, we examined whether the expression of CD36 is increased in the vasculature with blood–brain barrier (BBB) impairment and collagen deposition of SHRSP. Methods: The gene and protein expression of CD36 was examined in the vessels of the hippocampus of SHRSP with BBB impairment and those of Wistar Kyoto rats without the impairment, by real-time RT-PCR, Western blotting and immunohistochemical techniques. Results: The gene and protein expression of CD36 was increased in the hippocampus of SHRSP compared with that of Wistar Kyoto rats. Confocal microscopic examination revealed CD36 immunoreactivity in perivascular microglial cells immunopositive for ED1. Immunoelectron microscopic examination revealed that the immunosignals for CD36 were located mainly in the cytoplasm of perivascular cells in vessels showing increased vascular permeability and a few in the cytoplasmic membranes of endothelial cells. Conclusions: These findings indicate that the expression of CD36 was increased in vessels with BBB impairment in the hippocampus of SHRSP and was mainly seen in the cytoplasm of perivascular microglial cells, suggesting a role of CD36 in cerebrovascular injury.

IFN-γ plays a central role in antitumor immunity. T cell Ig and mucin domain (Tim-3) is expressed on IFN-γ–producing Th1 cells; on interaction with its ligand, galectin-9, Th1 immunity is terminated. In this study, we show that transgenic overexpression of Tim-3 on T cells results in an increase in CD11b+Ly-6G+ cells and inhibition of immune responses. Molecular characterization of CD11b+Ly-6G+ cells reveals a phenotype consistent with granulocytic myeloid-derived suppressor cells. Accordingly, we find that modulation of the Tim-3/galectin-9 (Gal-9) pathway impacts on tumor growth. Similarly, overexpression of Tim-3 ligand, Gal-9, results in an increase in CD11b+Ly-6G+ cells and inhibition of immune responses. Loss of Tim-3 restores normal levels of CD11b+Ly-6G+ cells and normal immune responses in Gal-9 transgenic mice. Our data uncover a novel mechanism by which the Tim-3/Gal-9 pathway regulates immune responses and identifies this pathway as a therapeutic target in diseases where myeloid-derived suppressor cells are disadvantageous.

Galectin-9 (Gal-9) inhibits the metastasis of tumor cells by blocking their adhesion to endothelium and the extracellular matrix. In this study, we addressed the involvement of Gal-9 in anti-tumor activity. Gal-9 significantly prolonged the survival of B16F10 melanoma-bearing mice. Gal-9 increased the numbers of NK cells, CD8 T cells and macrophages in tumor-bearing mice. Gal-9-mediated anti-tumor activity was not induced in NK cell-, macrophage- and CD8 T cell-depleted mice. NK cells from Gal-9-treated mice, compared to PBS-treated mice, exhibited significantly higher cytolytic activity. Co-culture of naïve NK cells with macrophages from Gal-9-treated mice resulted in enhanced NK activity, although Gal-9 itself did not enhance the NK activity. We also found that Ly-6C(+)CD11b(+)F4/80(+) macrophages with plasmacytoid cell (pDC)-like phenotypes (PDCA-1 and B220) were responsible for the enhanced NK activity. These results provide evidence that Gal-9 promotes NK cell-mediated anti-tumor activity by expanding unique macrophages with a pDC-like phenotype.

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.

The rat prostate is dependent on androgen for growth and differentiation. In an effort to find novel genes involved in androgen-induced growth of the rat prostate, we carried out PCR-based subtractive hybridization and identified several factors that were transiently up-regulated after androgen stimulation in castrated rat prostate. Among them, a novel member of the Spot 14 family has been identified. This protein (Spot 14-like androgen-inducible protein, SLAP) exhibited the highest homology (about 50%) with zebrafish gastrulation-specific G12 protein and about 30% homology with rat Spot 14. The SLAP mRNA level decreased following castration and transiently increased after testosterone replacement, attaining a peak 3 days after the treatment. The change in the protein level was similar to that in mRNA except that it was low in both untreated and castrated rat prostate tissue. In normal adult rats, SLAP was expressed at relatively high levels in the lung, stomach and liver. Unlike the prostate, SLAP expression in the lung was not affected by the androgen status. Like other members of the Spot 14 family, SLAP has a leucine-zipper motif in its C-terminal region, making it possible to form a stable homodimer. Though the physiological role of SLAP remains to be clarified, the current results suggest that SLAP expression is associated with some growth-related processes in regrowing rat prostate.

We previously described an inverse correlation between galectin-9 (Gal-9) expression and metastasis in patients with malignant melanoma and breast cancer. This study verified the ability of Gal-9 to inhibit lung metastasis in experimental mouse models using highly metastatic B16F10 melanoma and Colon26 colon cancer cells. B16F10 cells transfected with a secreted form of Gal-9 lost their metastatic potential. Intravenous Gal-9 administration reduced the number of metastases of both B16F10 and Colon26 cells in the lung, indicating that secreted Gal-9 suppresses metastasis. Analysis of adhesive molecule expression revealed that B16F10 cells highly express CD44, integrin alpha1, alpha 4, alpha V, and beta1, and that Colon26 cells express CD44, integrin alpha2, alpha 5, alpha V, and beta1, suggesting that Gal-9 may inhibit the adhesion of tumor cells to vascular endothelium and the extracellular matrix (ECM) by binding to such adhesive molecules. Indeed, Gal-9 suppressed the binding of hyaluronic acid to CD44 on both B16F10 and Colon26 cells, and also suppressed the binding of vascular cell adhesion molecule-1 to very late antigen-4 on B16F10 cells. Furthermore, Gal-9 inhibited the binding of tumor cells to ECM components, resulting in the suppression of tumor cell migration. The present results suggest that Gal-9 suppresses both attachment and invasion of tumor cells by inhibiting the binding of adhesive molecules on tumor cells to ligands on vascular endothelium and ECM.

The effects of galectin-9 on a mouse collagen-induced arthritis (CIA) model were assessed to clarify whether galectin-9 suppresses CIA by regulating T cell immune responses. Galectin-9 suppressed CIA in a dose-dependent manner, and such suppression was observed even when treatment was started on 7 days after the booster, indicating its preventive and therapeutic effects. Galectin-9 induced the decreased levels of pro-inflammatory cytokines, IL-17, IL-12, and IFNgamma in the joint. Galectin-9 induced the decreased number of CD4(+) TIM-3(+) T cells in peripheral blood. Galectin-9-deficient mice became susceptible to CIA may be by increased number of CD4(+) TIM-3(+) T cells and decreased number of Treg cells. We further found that galectin-9 induces differentiation of naive T cells to Treg cells, and it suppresses differentiation to Th17 cells in vitro. The present results suggested that galectin-9 ameliorates CIA by suppressing the generation of Th17, promoting the induction of regulatory T cells.

Galectins are a family of beta-galactoside-binding lectins that contain a conserved carbohydrate recognition domain (CRD). They exhibit high affinities for small beta-galactosides as well as variable binding specificities for complex glycoconjugates. Structural and biochemical analyses of the mechanism governing specific carbohydrate recognition provide a useful template to elucidate the function of these proteins. Here we report the crystal structures of the human galectin-9 N-terminal CRD (NCRD) in the presence of lactose and Forssman pentasaccharide. Mouse galectin-9 NCRD, the structure of which was previously solved by our group, forms a non-canonical dimer in both the crystal state and in solution. Human galectin-9 NCRD, however, exists as a monomer in crystals, despite a high sequence identity to the mouse homologue. Comparative frontal affinity chromatography analysis of the mouse and human galectin-9 NCRDs revealed different carbohydrate binding specificities, with disparate affinities for complex glycoconjugates. Human galectin-9 NCRD exhibited a high affinity for Forssman pentasaccharide; the association constant for mouse galectin-9 NCRD was 100-fold less than that observed for the human protein. The combination of structural data with mutational studies demonstrated that non-conserved amino acid residues on the concave surface were important for determination of target specificities. The human galectin-9 NCRD exhibited greater inhibition of cell proliferation than the mouse NCRD. We discuss the biochemical and structural differences between highly homologous proteins from different species.

We previously showed that tandem-repeat type galectin-8, which has two covalently linked carbohydrate recognition domains (CRDs), induces neutrophil-adhesion through binding to integrin alphaM. Here, we analysed the function of galectin-8 in Jurkat T-cells. Galectin-8, as well as tandem-repeat galectin-9, and several multivalent plant lectins, induced Jurkat T-cell adhesion to a culture plate, whereas single-CRD galectins-1 and -3 did not. Galectin-8 also induced the adhesion of peripheral blood leucocytes to human umbilical vein endothelial cells. These results suggest that the di- or multi-valent structure of galectin-8 is essential for the induction of cell adhesion and that this ability exhibits broad specificity for leucocytes. The galectin-8-induced cell adhesion was accompanied by stress fibre formation, which suggests that intracellular signalling is required. We have identified integrin alpha4 as one of the candidate target molecules associated with the induction of cell adhesion. Indeed, inhibition of the function of integrin alpha4 by treating cells with a blocking-antibody reduced the sensitivity to galectin-8. Also, the phosphorylation of Pyk and ERK1/2, indicators of integrin-mediated signalling, was up-regulated on treatment with galectin-8. Thus, a primary target of galectin-8 must be the sugar chains on members of the integrin family, which are abundantly expressed on the surface of leucocytic cells.

Objective. To compare the expression of galectin 9 (Gal-9) in synovial tissue (ST) from rheumatoid arthritis (RA) patients and osteoarthritis (OA) patients and to evaluate the effects of Gal-9 on fibroblast-like synoviocytes (FLS) in these patients. Methods. The expression of Gal-9 in ST and FLS was compared using immunohistochemical techniques. Apoptotic cells in RA and OA ST samples were detected by TUNEL assay. Apoptosis of FLS was analyzed by the sub-G1 method in vitro. The in vivo suppressive effects of Gal-9 on collagen-induced arthritis (CIA) in a mouse model were also elucidated. Results. The percentage of Gal-9–positive cells in ST samples and the amount of Gal-9 in synovial fluid samples were significantly higher in patients with RA than in patients with OA, suggesting the involvement of Gal-9 in the development of RA. Compared with the 2 wild-type Gal-9 forms, stable Gal-9, a mutant protein resistant to proteolysis, significantly induced apoptosis of FLS from RA patients. In contrast, other galectins, such as Gal-1, Gal-3, and Gal-8, did not induce apoptosis or suppress the proliferation of human RA FLS. Stable Gal-9 preferentially induced apoptosis and suppressed the proliferation of RA FLS in vitro. It also induced apoptosis of cells in RA ST implanted into SCID mice in vivo. In a mouse model of CIA, apoptotic cells were detected in the joints of stable Gal-9–treated mice, but not phosphate buffered saline–treated mice, and suppressed CIA characterized by pannus formation with inflammatory cell infiltration and bone/cartilage destruction. Conclusion. Gal-9–induced apoptosis of hyperproliferative RA FLS may play a critical role in the suppression of RA.

Galectin-9 (Gal-9) belongs to the galectin family, which exhibits affinity for beta-galactosides. Gal-9 has a variety of biological activities; however, its role in allergic inflammation is unknown.We evaluated the effect of a stable form of the human protein on allergic airway inflammation in a mite allergen-induced asthma model.Human stable Gal-9 was given by intravenous injection to mice during antigen challenge. The effect of Gal-9 on airway inflammation and airway hyperresponsiveness (AHR) was then evaluated.Gal-9 reduced AHR as well as Th2-associated airway inflammation. Furthermore, administration of Gal-9 as well as anti-CD44 monoclonal antibody inhibited the infiltration of peripheral blood Th2 cells into the airway. Interestingly, Gal-9 directly bound the CD44 adhesion molecule and inhibited interactions with hyaluronan (HA). Consistent with the concept that CD44-HA interactions mediate the migration of T cells into the lung, Gal-9 blocked CD44-dependent adhesion of BW5147 mouse T cells to HA.We conclude that Gal-9 inhibits allergic inflammation of the airway and AHR by modulating CD44-dependent leukocyte recognition of the extracellular matrix.

Background: There is little information about the involvement of galectin-9 (Gal-9) in allergic inflammation. Thus, we investigated the role of Gal-9 in asthma model guinea pigs. Methods: Airway resistance (Raw) was measured using a double-flow plethysmograph system. Gal-9 expression in the lung was assessed by Western blot and immunohistochemistry. Eosinophil chemotactic activity was evaluated in a chamber containing a polyvinylpyrolidone-free membrane. Cell apoptosis was analyzed on a flowcytometry with propidium iodide. Results: In cloning guinea pig Gal-9 we identified three isoforms that differ only in the length of their linker peptides, just as with human Gal-9. Guinea pig Gal-9 was found to be a chemoattractant for eosinophils and to promote induction of apoptosis in sensitized but not non-sensitized T lymphocytes. In allergic airway hypersensitivity model, a low level of Gal-9 expression was observed in the nonsensitized/nonchallenged group, but upregulation was detected at 7 h after challenge and sustained up to 24 h. Such upregulation correlated with elevation of eosinophil peroxidase activity but not with increased Raw. Conclusions: The present results provide evidence that Gal-9 is not involved in airway hypersensitivity, but is partly involved in prolonged eosinophil accumulation in the lung.

Oncogenic mutation of KRAS (Kirsten rat sarcoma viral oncogene homolog) in colorectal cancer (CRC) confers resistance to both chemotherapy and EGFR (epidermal growth factor receptor)-targeted therapy. We uncovered that KRAS mutant (KRASmut) CRC is uniquely sensitive to treatment with recombinant LGALS9/Galectin-9 (rLGALS9), a recently established regulator of epithelial polarity. Upon treatment of CRC cells, rLGALS9 rapidly internalizes via early- and late-endosomes and accumulates in the lysosomal compartment. Treatment with rLGALS9 is accompanied by induction of frustrated autophagy in KRASmut CRC, but not in CRC with BRAF (B-Raf proto-oncogene, serine/threonine kinase) mutations (BRAFmut). In KRASmut CRC, rLGALS9 acts as a lysosomal inhibitor that inhibits autophagosome-lysosome fusion, leading to autophagosome accumulation, excessive lysosomal swelling and cell death. This antitumor activity of rLGALS9 directly correlates with elevated basal autophagic flux in KRASmut cancer cells. Thus, rLGALS9 has potent antitumor activity towards refractory KRASmut CRC cells that may be exploitable for therapeutic use.

Galectin-9, a mammalian lectin with affinity for beta-galactosides, is known as an apoptosis inducer of activated T lymphocytes. In the present study, we examined the properties of galectin-9-mediated cell death of Jurkat T cells. Galectin-9NC (wild-type), consisting of two CRDs (N-terminal and C-terminal carbohydrate recognition domains), and derivatives of it, galectins-9-NN and -9-CC, induced Jurkat T-cell apoptosis. However, a single CRD (galectin-9NT or -CT) had no effect, suggesting the stable dimeric structure of two CRDs is required for the activity. The apoptosis was inhibited by pretreatment with an N-glycan synthesis inhibitor, indicating that the expression of N-glycans in the cells is essential for galectin-9-induced apoptosis. We previously showed that the apoptosis of MOLT-4 cell is mediated by galectin-9 via a Ca(2+)-calpain-caspase-1-dependent pathway. In Jurkat cells, the cell death by galectin-9, was insufficiently suppressed by caspase inhibitors, Ca(2+)-chelator or calpain inhibitor. Furthermore, we observed the loss of mitochondrial membrane potential and significant AIF release in galectin-9-treated cells. These findings suggest that caspase-dependent and-independent death pathways exist in Jurkat cells, and the main pathway might vary with the T-cell type.

The galectins are a family of beta-galactoside-binding animal lectins with a conserved carbohydrate recognition domain (CRD). They have a high affinity for small beta-galactosides, but binding specificity for complex glycoconjugates varies considerably within the family. The ligand recognition is essential for their proper function, and the structures of several galectins have suggested their mechanism of carbohydrate binding. Galectin-9 has two tandem CRDs with a short linker, and we report the crystal structures of mouse galectin-9 N-terminal CRD (NCRD) in the absence and the presence of four ligand complexes. All structures form the same dimer, which is quite different from the canonical 2-fold symmetric dimer seen for galectin-1 and -2. The beta-galactoside recognition mechanism in the galectin-9 NCRD is highly conserved among other galectins. In the apo form structure, water molecules mimic the ligand hydrogen-bond network. The galectin-9 NCRD can bind both N-acetyllactosamine (Galbeta1-4GlcNAc) and T-antigen (Galbeta1-3GalNAc) with the proper location of Arg-64. Moreover, the structure of the N-acetyllactosamine dimer (Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc) complex shows a unique binding mode of galectin-9. Finally, surface plasmon resonance assay showed that the galectin-9 NCRD forms a homophilic dimer not only in the crystal but also in solution.

Galectin-9, a member of the beta-galactoside-binding animal lectin family, is involved in various cellular biological events, including aggregation and apoptosis, adhesion of cancer cells, and dendritic cell maturation. We recently reported the relationship between galectin-9 expression in tumor tissue and distant metastasis in breast cancer. Tumors in 42 of the 84 patients were galectin-9-positive, and tumors in 19 of the 21 patients with distant metastasis were galectin-9-negative, assessed by immunohistochemistry. The cumulative distant metastasis-free survival ratio for galectin-9-positive patients was better than for the galectin-9-negative group (p < 0.0001). Multivariate analysis revealed that galectin-9 status influenced distant metastasis independent of and much more than lymph node metastasis. MCF-7 subclones with a high level of galectin-9 expression formed tight clusters during proliferation in vitro, whereas a subclone (K10) with the lowest level of galectin-9 expression did not. However, K10 cells stably transfected with a galectin-9 expression vector aggregated in nude mice as well as in culture. Ectopic expression of galectin-9 also reduced MCF-7 cell adhesion to extracellular matrix proteins.

We have identified members of the Xenopus cortical granule lectin (xCGL) family as candidate target glycoproteins of Xenopus galectin-VIIa (xgalectin-VIIa) in Xenopus embryos. In addition to the original xCGL, we also identified a novel member of the xCGL family, xCGL2. Expression of the mRNAs of xCGL and xCGL2, as well as that of xgalectin-VIIa, was observed throughout early embryogenesis. Two and three potential N-glycosylation sites were deduced from the amino acid sequences of xCGL and xCGL2, respectively, and xgalectin-VIIa recognizes N-glycans linked to a common site in xCGL and xCGL2 and also recognizes N-glycans linked to a site specific to xCGL2. However, interaction between xgalectin-Ia and xCGLs was not detectable. We also obtained consistent results on surface plasmon resonance analysis involving xCGLs as ligands and xgalectins as analytes. The Kd value of the interaction between xgalectin-VIIa and xCGLs was calculated to be 35.9 nM. The structures of the N-glycans of xCGLs, which were recognized by xgalectin-VIIa, were analyzed by the two-dimensional sugar map method, and three kinds of N-acetyllactosamine type, biantennary N-glycans were identified as the major neutral N-glycans. The binding specificity of oligosaccharides for xgalectin-VIIa was analyzed by frontal affinity chromatography (FAC). The oligosaccharide specificity pattern of xgalectin-VIIa was similar to that of the human homolog galectin-3, and it was also shown that the N-acetyllactosamine type, biantennary N-glycans exhibit high affinity for xgalectin-VIIa (Kd = 11 microM). These results suggest that xgalectin-VIIa interacts with xCGLs through binding to N-acetyllactosamine type N-glycans and that this interaction might make it possible to organize a lectin network involving members of different lectin families.

Treatment of cells with double-stranded RNA (dsRNA) in vitro mimics viral infection and regulates expression of various genes. We addressed the mechanisms of leucocyte traffic across the vascular endothelium induced by dsRNA. The present study focused on the expression of galectin-9, which is one of key molecules in the regulation of the interaction between vascular wall and white blood cells. Human umbilical vein endothelial cells (HUVEC) in culture were treated with polyinosinic-polycytidylic acid (poly IC), and expression of mRNA and protein of galectin-9 was analysed by reverse transcription polymerase-chain reaction (RT-PCR) and Western blotting. Poly IC enhanced the expression of galectin-9 mRNA and protein in concentration- and time-dependent manners. This effect of poly IC was almost completely suppressed by the pretreatment with 2-aminopurine, an inhibitor of dsRNA-dependent kinase. Poly IC treatment of HUVEC also enhanced the adherence of EoL-1 cells to the cells, which was inhibited by co-treatment with lactose. We conclude that poly IC upregulates galectin-9 expression in the vascular endothelium and this may explain part of the mechanism for leucocyte traffic through the vascular wall.

Leishmania parasites are the causative agents of leishmaniasis, manifesting itself in a species-specific manner. The glycan epitopes on the parasite are suggested to be involved in the Leishmania pathogenesis. One of such established species-unique glycan structures is the poly-β-galactosyl epitope (Galβ1–3)n found on L. major, which can develop cutaneous infections with strong inflammatory responses. Interestingly, the polygalactosyl epitope is also suggested to be involved in the development of the parasites in its host vector, sand fly. Thus, the recognition of the galactosyl epitope by lectins expressed in host or sand fly should be implicated in the species-specific manifestations of leishmaniasis and in the parasite life cycle, respectively. We recently reported that one host β-galactoside-binding protein, galectin-3, can distinguish L. major from the other species through its binding to the poly-β-galactosyl epitope, proposing a role for galectin-3 as an immunomodulator that could influence the L. major-specific immune responses in leishmaniasis. Here we report that galectin-9 can also recognize L. major by binding to the L. major-specific polygalactosyl epitope. Frontal affinity analysis with different lengths of poly-β-galactosyllactose revealed that the galectin-9 affinity for polygalactose was enhanced in proportion to the number of Galβ1–3 units present. Even though both galectins have comparable affinities toward the polygalactosyl epitopes, only galectin-9 can promote the interaction between L. major and macrophages, suggesting distinctive roles for the galectins in the L. major-specific development of leishmaniasis in the host.

Galectin-9 (Gal-9) induced the apoptosis of not only T cell lines but also of other types of cell lines in a dose- and time-dependent manner. The apoptosis was suppressed by lactose, but not by sucrose, indicating that β-galactoside binding is essential for Gal-9-induced apoptosis. Moreover, Gal-9 required at least 60 min of Gal-9 binding and possibly de novo protein synthesis to mediate the apoptosis. We also assessed the apoptosis of peripheral blood T cells by Gal-9. Apoptosis was induced in both activated CD4+ and CD8+ T cells, but the former were more susceptible than the latter. A pan-caspase inhibitor (Z-VAD-FMK) inhibited Gal-9-induced apoptosis. Furthermore, a caspase-1 inhibitor (Z-YVAD-FMK), but not others such as Z-IETD-FMK (caspase-8 inhibitor), Z-LEHD-FMK (caspase-9 inhibitor), and Z-AEVD-FMK (caspase-10 inhibitor), inhibited Gal-9-induced apoptosis. We also found that a calpain inhibitor (Z-LLY-FMK) suppresses Gal-9-induced apoptosis, that Gal-9 induces calcium (Ca2+) influx, and that either the intracellular Ca2+ chelator BAPTA-AM or an inositol trisphosphate inhibitor 2-aminoethoxydiphenyl borate inhibits Gal-9-induced apoptosis. These results suggest that Gal-9 induces apoptosis via the Ca2+-calpain-caspase-1 pathway, and that Gal-9 plays a role in immunomodulation of T cell-mediated immune responses.

We have isolated six novel galectin cDNAs from a Xenopus laevis kidney cDNA library. The newly identified X. laevis galectins (xgalectins) comprise one proto type (xgalectin-Vb), one chimera type (xgalectin-VIIa), and four tandem repeat types (xgalectin-IIb, -IIIb, -VIa, and -VIIIa). Thus, together with those mentioned in our previous work (Shoji, H., Nishi, N., Hirashima, M., and Nakamura, T. (2002) Glycobiology 12, 163-172), the 12 xgalectins are classified into three types based on their domain structures, as in mammals. The xgalectins whose counterparts in other species have not been identified (xgalectin-IVa, -Vb, and -VIa) were confirmed to possess lactose-binding activity by expression of their recombinant forms. This shows that they truly function as animal lectins. The protein purification study revealed that the major xgalectins in kidney are xgalectin-Ib, -IIa, -IIb, -IIIa, and -VIIa. The mRNAs of xgalectin-IIb, -IIIb, -Vb, and -VIa were localized to specific adult tissues, whereas those of xgalectin-VIIa and -VIIIa were broadly distributed. The temporal expression patterns of the mRNAs of the 12 xgalectins during embryogenesis were analyzed and categorized into three groups: 1) mRNA observed to exist throughout embryogenesis, i.e. maternal mRNA also exists (xgalectin-Ia, -IIa, -IIIa, -IIIb, -Va, -VIIa, and -VIIIa); 2) mRNA observed from the gastrula stage (xgalectin-VIa); and 3) mRNA observed from the tail bud or the tadpole stage (xgalectin-Ib, -IIb, -IVa, and -Vb). The mRNA of the most abundant xgalectin in embryos, xgalectin-VIIa, was localized to the surface layer of embryos, the epidermis, the cement gland, and various placodes. Xgalectin-VIIa protein was also observed to exist throughout embryogenesis by Western blot analysis with specific antiserum. These results show that the expression of each member is spatiotemporally regulated from eggs to adulthood, suggesting that galectins play multiple roles not only in adults, but also in development.

Background: Galectin-9 is involved in chemotaxis and adhesion of eosinophils, and is induced in vascular endothelial cells by interferon-γ (IFN-γ). 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is a ligand for peroxisome proliferator-activated receptor-γ (PPAR-γ), and known to modulate the expression of various genes. Methods: We have studied the effect of 15d-PGJ2 on the IFN-γ-induced galectin-9 expression in human umbilical vein endothelial cells (HUVEC) in culture. Results: 15d-PGJ2 inhibited the IFN-γ-induced galectin-9 expression in a PPAR-γ-independent manner, and also inhibited the adhesion of EoL-1 cells to an HUVEC monolayer treated with IFN-γ. 15d-PGJ2 partially inhibited IFN-γ-induced phosphorylation of STAT-1 in HUVEC. Conclusions: 15d-PGJ2 may regulate inflammatory reactions through the inhibition of galectin-9 expression.

Galectin-9 is a member of the galectin family and has been identified as an eosinophil chemoattractant produced by activated T lymphocytes. Vascular endothelial cells play an important role in the initial step of eosinophil recruitment and activation in immune and inflammatory responses. We have addressed the stimulation of galectin-9 expression in endothelial cells. Galectin-9 was detected in membrane and cytosolic fractions of human umbilical vein endothelial cells stimulated with interferon-γ (IFN-γ). IFN-γ also enhanced the adhesion of human eosinophilic leukemia-1 cells to endothelial monolayers, and it was inhibited by the presence of lactose. Interleukin-4, which induces eotaxin expression, did not affect the expression of galectin-9. The in situ endothelium from patients with inflammatory diseases was found to express galectin-9. IFN-γ-induced production of galectin-9 by endothelial cells may play an important role in immune responses by regulating interactions between the vascular wall and eosinophils.

We have characterized galectin family proteins in adult tissues of Xenopus laevis and purified 14-kDa and 36-kDa proteins from the liver. The liver galectins showed comparable hemagglutination activities to those of mammalian galectins. Furthermore, we isolated five galectin cDNAs from a Xenopus liver library. These cDNAs revealed that X. laevis galectins (xgalectins) form a family consisting of at least proto and tandem repeat types based on their domain structures, like the mammalian galectin family. Two proto-type xgalectins, -Ia and -Ib, exhibited a high sequence identity (91 %) with each other at the amino acid level and were most similar (49-50% identity) to human galectin-1. From their sequence similarity and ubiquitous tissue distributions, xgalectins-Ia and -Ib both seemed to be Xenopus homologues of mammalian galectin-1. Three tandem repeat-type xgalectins were newly identified. Two of them, xgalectins-IIa and -IIIa, seemed to be homologous to human galectins-4 and -9, respectively, judging from their high sequence similarities (42-50 % identity). However, xgalectin-IVa seemed to be a novel type. Distributions of mRNAs of xgalectins were analyzed by northern hybridization. In addition to adult tissues, either of three tandem repeat-type xgalectins were expressed in whole embryos. Moreover, amino acid sequence analysis of liver proteins indicated that xgalectins-Ia, -IIa, and -IIIa are produced as abundant galectins in the adult liver.