Takanori Nakamura

Galectin-9 in physiological and pathological conditions.

We first cloned galectin-9 (Gal-9)/ecalectin as a T cell-derived eosinophil chemoattractant. Gal-9 plays a role in not only accumulation but also activation of eosinophils in experimental allergic models and human allergic patients, because Gal-9 induces eosinophil chemoattraction in vitro and in vivo and activates eosinophils in many aspects. Gal-9 requires divalent galactoside-binding activity but not the linker peptide of Gal-9 to exhibit its biological functions, and an unidentified matrix metalloproteinase is involved in the release of Gal-9. Our recent studies also showed that Gal-9 has other functions, such as cell differentiation, aggregation, adhesion, and death. Now, we and other groups are on the way of investigating the regulation and function of Gal-9 in a variety of physiological and pathological conditions. In this article, we will show the possible role of Gal-9 in physiological and pathological conditions by using our recent findings. Published in 2004.

Development of highly stable galectins: truncation of the linker peptide confers protease-resistance on tandem-repeat type galectins.

Galectin‐9 and galectin‐8, members of β‐galactoside‐binding animal lectin family, are promising agents for the treatment of immune‐related and neoplastic diseases. The proteins consist of two carbohydrate recognition domains joined by a linker peptide, which is highly susceptible to proteolysis. To increase protease resistance, we prepared mutant proteins by serial truncation of the linker peptide. As a result, mutant forms lacking the entire linker peptide were found to be highly stable against proteolysis and retained their biological activities. These mutant proteins might be useful tools for analyzing the biological functions and evaluating the therapeutic potential of galectin‐9 and galectin‐8.

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

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

Double-stranded RNA enhances the expression of galectin-9 in vascular endothelial cells

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.

Characterization of the Xenopus galectin family. Three structurally different types as in mammals and regulated expression during embryogenesis.

We have isolated six novel galectin cDNAs from a Xenopus laevis kidney cDNA library. The newly identified X. laevisgalectins (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.

Involvement of Galectin-9 in Guinea Pig Allergic Airway Inflammation

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.

Desulfated galactosaminoglycans are potential ligands for galectins: Evidence from frontal affinity chromatography

Galectins, a group of beta-galactoside-binding lectins, are involved in multiple functions through specific binding to their oligosaccharide ligands. No previous work has focused on their interaction with glycosaminoglycans (GAGs). In the present work, affinities of established members of human galectins toward a series of GAGs were investigated, using frontal affinity chromatography. Structurally-defined keratan sulfate (KS) oligosaccharides showed significant affinity to a wide range of galectins if Gal residue(s) remained unsulfated, while GlcNAc sulfation had relatively little effect. Consistently, galectins showed much higher affinity to corneal type I than cartilageous type II KS. Unexpectedly, galectin-3, -7, and -9 also exerted significant affinity to desulfated, GalNAc-containing GAGs, i.e., chondroitin and dermatan, but not at all to hyaluronan and N-acetylheparosan. These observations revealed that the integrity of 6-OH of betaGalNAc is important for galectin recognition of these galactosaminoglycans, which were shown, for the first time, to be implicated as potential ligands of galectins.

The Galβ-(syn)-gauche configuration is required for galectin-recognition disaccharides.

BACKGROUNDnGalectins form a large family of animal lectins, individual members having variously divergent carbohydrate-recognition domains (CRDs) responsible for extensive physiological phenomena. Sugar-binding affinities of galectins were previously investigated by us using frontal affinity chromatography (FAC) with a relatively small set (i.e., 41) of oligosaccharides. However, total understanding of a consensus rule for galectin-recognition saccharides is still hampered by the lack of fundamental knowledge about their sugar-binding specificity toward a much larger panel of oligosaccharides in terms of dissociation constant (K(d)).nnnMETHODSnIn the present study, we extended a FAC analysis from a more systematic viewpoint by using 142 fluorescent-labeled oligosaccharides, initially with focus on functional human galectins-1-9. Binding characteristics were further validated with 11 non-human galectins and 13 non-galectin Gal/GalNAc-binding lectins belonging to different families.nnnRESULTSnAn empirical [Galβ-equatorial] rule for galectin-recognition disaccharides was first derived by our present research and previous works by others. However, this rule was not valid for a recently reported nematode disaccharide, Galβ1-4-L-Fuc [Butschi et al. PLoS Pathog, 2010; 6(1):e1000717], because this glycosidic linkage was directed to axial 4-OH of L-Fuc. After careful reconsideration of the structural data, we reached an ultimate rule of galectin-recognition disaccharides, which all of the galectins so far identified fulfilled, i.e., under the re-defined configuration Galβ-(syn)-gauche. The rule also worked perfectly for differentiation of galectins from other types of lectins.nnnGENERAL SIGNIFICANCEnThe present attempt should provide a basis to solve the riddle of the glyco-code as well as to develop therapeutic inhibitors mimicking galectin ligands.

Aldosterone: a mediator of retinal ganglion cell death and the potential role in the pathogenesis in normal-tension glaucoma.

Glaucoma is conventionally defined as a chronic optic neuropathy characterized by progressive loss of retinal ganglion cells (RGCs) and optic nerve fibers. Although glaucoma is often associated with elevated intraocular pressure (IOP), significant IOP reduction does not prevent progression of the disease in some glaucoma patients. Thus, exploring IOP-independent mechanisms of RGC loss is important. We describe chronic systemic administration of aldosterone and evaluate its effect on RGCs in rat. Aldosterone was administered via an osmotic minipump that was implanted subcutaneously into the mid-scapular region. Although systemic administration of aldosterone caused RGC loss associated with thinning of the retinal nerve fiber layer without elevated IOP, the other cell layers appeared to be unaffected. After chronic administration of aldosterone, RGC loss was observed at 2 weeks in the peripheral retina and at 4 weeks in the central retina. However, administration of mineralocorticoid receptor blocker prevented RGC loss. These results demonstrate aldosterone is a critical mediator of RGC loss that is independent of IOP. We believe this rat normal-tension glaucoma (NTG) animal model not only offers a powerful system for investigating the mechanism of neurodegeneration in NTG, but can also be used to develop therapies directed at IOP-independent mechanisms of RGC loss.

Direct cytocidal effect of galectin-9 localized on collagen matrices on human immune cell lines

BACKGROUNDnThere is a continuous demand for new immunosuppressive agents for organ transplantation. Galectin-9, a member of the galactoside-binding animal lectin family, has been shown to suppress pathogenic T-cell responses in autoimmune disease models and experimental allograft transplantation. In this study, an attempt has been made to develop new collagen matrices, which can cause local, contact-dependent immune suppression, using galectin-9 and collagen-binding galectin-9 fusion proteins as active ingredients.nnnMETHODSnGalectin-9 and galectin-9 fusion proteins having collagen-binding domains (CBDs) derived from bacterial collagenases and a collagen-binding peptide (CBP) were tested for their ability to bind to collagen matrices, and to induce Jurkat cell death in solution and in the collagen-bound state.nnnRESULTSnGalectin-9-CBD fusion proteins exhibited collagen-binding activity comparable to or lower than that of the respective CBDs, while their cytocidal activity toward Jurkat cells in solution was 80~10% that of galectin-9. Galectin-9 itself exhibited oligosaccharide-dependent collagen-binding activity. The growth of Jurkat cells cultured on collagen membranes treated with galectin-9 was inhibited by~90%. The effect was dependent on direct cell-to-membrane contact. Galectin-9-CBD/CBP fusion proteins bound to collagen membranes via CBD/CBP moieties showed a low or negligible effect on Jurkat cell growth.nnnCONCLUSIONSnAmong the proteins tested, galectin-9 exhibited the highest cytocidal effect on Jurkat cells in the collagen-bound state. The effect was not due to galectin-9 released into the culture medium but was dependent on direct cell-to-membrane contact.nnnGENERAL SIGNIFICANCEnThe study demonstrates the possible use of galectin-9-modified collagen matrices for local, contact-dependent immune suppression in transplantation.