The in vivo roles of galectin-2 from Nile tilapia (Oreochromis niloticus) in immune response against bacterial infection.

Our previous study has recorded that the recombinant protein of Nile tilapia (Oreochromis niloticus) galectin-2 (rOnGal-2) can enhance immune response against Streptococcus agalactiae (S.agalactiae) infection in vitro. In this study, we further explored the effects of OnGal-2 in immune response against bacterial infection in vivo. The administration of rOnGal-2 could improve serum antibacterial activity (ALKP, ACP, and LZM) and antioxidant capacity (CAT, POD, and SOD). After S. agalactiae infection, rOnGal-2 injection could reduce bacterial burden and decrease tissue damage in head kidney, spleen, and liver of tilapia. Also, rOnGal-2 regulated the inflammatory-related genes expression including IL-6, IL-8 and IL-10 during bacterial infection. Furthermore, rOnGal-2 administration could increase the relative percentage survival of tilapia infected with S.agalactiae. Taken together, our results indicate that OnGal-2 can protect fish from bacterial infection through reducing bacterial burden, impairing tissue damage and modulating anti-bacterial immune response, which also can be applied as a potential vaccine adjuvant in O.niloticus culture.

Galectin-lattice sustains function of cationic amino acid transporter and insulin secretion of pancreatic ß cells.

Maintenance of cell surface residency and function of glycoproteins by lectins are essential for regulating cellular functions. Galectins are ß-galactoside-binding lectins and form a galectin-lattice, which regulates stability, clustering, membrane sub-domain localization and endocytosis of plasmalemmal glycoproteins. We have previously reported that galectin-2 (Gal-2) forms a complex with cationic amino acid transporter 3 (CAT3) in pancreatic ß cells, although the biological significance of the molecular interaction between Gal-2 and CAT3 has not been elucidated. In this study, we demonstrated that the structure of N-glycan of CAT3 was either tetra- or tri-antennary branch structure carrying ß-galactosides, which works as galectin-ligands. Indeed, CAT3 bound to Gal-2 using ß-galactoside epitope. Moreover, the disruption of the glycan-mediated bindings between galectins and CAT3 significantly reduced cell surface expression levels of CAT3. The reduced cell surface residency of CAT3 attenuated the cellular arginine uptake activities and subsequently reduced nitric oxide production, and thus impaired the arginine-stimulated insulin secretion of pancreatic ß cells. These results indicate that galectin-lattice stabilizes CAT3 by preventing endocytosis to sustain the arginine-stimulated insulin secretion of pancreatic ß cells. This provides a novel cell biological insight into the endocrinological mechanism of nutrition metabolism and homeostasis.

Brugia malayi galectin 2 is a tandem-repeat type galectin capable of binding mammalian polysaccharides.

Galectins are among the most abundant excretory/secretory (ES) products of filarial worms, but their role in filarial biology is poorly understood. Galectin-2 (Lec-2), a major component of Brugia malayi extracellular vesicles, is released by filarial worms, and was recently identified in the serum of persons with loiasis. We therefore sought to clone and characterize Lec-2, and to develop reagents to examine its potential as a biomarker and its role in parasite biology. We cloned and expressed recombinant B. malayi Lec-2 (rBmLec-2), generated a Lec-2-specific monoclonal antibody (4B4), and used it to confirm the presence of Lec-2 in B. malayi ES products and whole worm lysate. We show that Lec-2 is absent in B. malayi oocytes, and increases in concentration as embryos mature. Recombinant BmLec-2 hemagglutinates rabbit red blood cells at concentrations less than 1 µg/mL, and this is abrogated by single amino acid substitutions in the predicted carbohydrate recognition domains. rBmLec-2 binds multiple LacNAc oligosaccharides on a mammalian carbohydrate array. Sera from 17/23 (78 %) persons with microfilaremic loiasis and 4/10 (40 %) persons with bancroftian filariasis had detectable antibody to Lec-2 by western blot. Our studies confirm the functionality of BmLec-2 and indicate anti-Lec-2 antibody responses are common in persons with filariasis. These studies set the stage for further examination of the role of Lec-2 in filarial biology and in filarial-host interactions.

Application of Recombinant Angiostrongylus cantonensis Galectin-2 Protein for Serodiagnosis of Human Angiostrongyliasis by Immunoblotting.

Angiostrongyliasis is a foodborne disease caused by a zoonotic nematode, Angiostrongylus cantonensis, which produces eosinophilic meningitis or meningoencephalitis (EOM) in humans. Definitive diagnosis is rarely possible because worms are almost never recovered from patients. Human disease can be diagnosed by clinical symptoms and serological tests. Presently, diagnosis is performed by serological detection of antibodies against specific somatic antigens (molecular mass 29-31 kDa) extracted from female worms. The life cycle of A. cantonensis must be maintained in the laboratory to provide a source of this diagnostic antigen. Here, we cloned and expressed recombinant A. cantonensis galectin-2 (rAcGal2) corresponding to a 31-kDa antigenic peptide. Recombinant protein was purified and used in immunoblot tests, which showed reactions with human serum panels consisting of six confirmed angiostrongyliasis and 24 clinically diagnosed cases of EOM-associated with angiostrongyliasis, 160 samples from patients with other parasitic infections, and 30 samples from normal healthy subjects. Accuracy, sensitivity, specificity, and positive and negative predictive values were 95.0%, 93.3%, 95.3%, 75.7%, and 98.9%, respectively. The test was nonreactive with sera of human gnathostomiasis and cysticercosis, two diseases that could present similar neurological symptoms. Recombinant AcGal2 has potential as a diagnostic antigen and could replace native parasite antigens in further development of an angiostrongyliasis serodiagnostic test kit.

Characterization of Galectin-2 from Nile tilapia (Oreochromis niloticus) involved in the immune response to bacterial infection.

galectin-2 plays important roles in innate and adaptive immunity. In this study, galectin-2 (OnGal-2) was identified from Nile tilapia (Oreochromis niloticus). Its tissue distribution and expression patterns following bacterial infection were also investigated. OnGal-2 is widely distributed in various tissues of healthy tilapia. After Streptococcus agalactiae challenge, OnGal-2 expressions were significantly up-regulated in all tested tissues. Meanwhile, the recombinant OnGal-2 (rOnGal-2) protein showed strong agglutinating activities against both Gram-negative bacteria and Gram-positive bacteria. Moreover, rOnGal-2 could promote phagocytosis of macrophages. Taken together, the present study indicated that OnGal-2 might play roles in the immune responses of Nile tilapia against bacterial pathogens.

Characterisation and functional comparison of single-CRD and multidomain containing galectins CgGal-2 and CgGal-3 from oyster Crassostrea gigas.

Galectins are ß-galactoside binding lectins that play crucial roles in innate immunity in vertebrates and invertebrates through their conserved carbohydrate-recognition domains (CRDs). In the present study, single- and four-CRD-containing galectins were identified in oyster Crassostrea gigas (designated CgGal-2 and CgGal-3). The open reading frames (ORFs) of CgGal-2 and CgGal-3 encode polypeptides of 200 and 555 amino acids, respectively. All CRDs of CgGal-3 include two consensus motifs essential for ligand-binding, and a novel motif is present in CgGal-2. Pathogen-associated molecular pattern (PAMP) profiles were determined for recombinant rCgGal-2 and rCgGal-3, and rCgGal-2 displayed low binding affinity for PAMPs, while rCgGal-3 bound various PAMPs including glucan, lipopolysaccharide (LPS), and peptidoglycan (PGN) with relatively high affinity. Furthermore, rCgGal-2 and rCgGal-3 exhibited different microbe binding profiles; rCgGal-2 bound to Gram-negative bacteria (Escherichia coli and Vibrio vulnificus) and fungi (Saccharomyces cerevisiae and Pichia pastoris), while rCgGal-3 bound to these microbes but also to Gram-positive bacteria (Micrococcus luteus). In addition, rCgGal-3 possessed microbial agglutinating activity and coagulation activity against fungi and erythrocytes, respectively, but rCgGal-2 lacked any agglutinating activity. Carbohydrate binding specificity analysis showed that rCgGal-3 specifically bound D-galactose. Furthermore, rCgGal-2 and rCgGal-3 functioned as opsonin participating in the clearance against invaders in C. gigas. Thus, CgGal-2 with one CRD and CgGal-3 with four CRDs are new members of the galectin family involved in immune responses against bacterial infection. Differences in the organisation and amino acid sequences of CRDs may affect their specificity and affinity for nonself substances.

A galectin contributes to the innate immune recognition and elimination of pathogens in the freshwater mussel Hyriopsis cumingii.

Galectins are members of the lectin superfamily. They function as pattern recognition receptors in the innate immune system of vertebrates and invertebrates. A galectin homolog from the triangle sail mussel Hyriopsis cumingii (HcGal2) was cloned and characterized. HcGal2 mRNA was expressed in all tissues examined, displaying particular enrichment in mantle tissue. Interestingly, rHcGAL2 protein was only detected in the mantle, hemocytes, and gills, suggesting that post-transcriptional regulation may occur. HcGal2 expression was induced in the mantle, liver, and hemocytes after exposure to lipopolysaccharides, Gram-negative bacteria (Aeromonas hydrophila), and Gram-positive bacteria (Staphylococcus aureus). The transcript significant upregulated was also detected after implantation in the mantle, pearl sac, liver, and hemocytes. Recombinant HcGAL2 protein (rHcGAL2) agglutinated Gram-positive and Gram-negative bacteria. In addition, rHcGAL2 promoted phagocytosis by hemocytes in vivo. Our data suggest that HcGal2 functioned as a pattern recognition receptor in against the pathogenic microbes and contributed to the "non-self" recognition and elimination in H. cumingii.

Autoantibodies against galectin-2 peptides as biomarkers for the antiphospholipid syndrome.

Autoantibodies against opsonins of dying and dead cells mediate Fcγ receptor-dependent phagocytosis of autologous apoptotic and necrotic cells and hereby tend to elicit inflammation instead of silent clearance. We analysed sera of patients with chronic autoimmune diseases for the occurrence of IgG autoantibodies recognizing galectins. These pluripotent effectors can also bind to apoptotic or necrotic cells. Patients with antiphospholipid syndrome (APS; n = 104) and systemic lupus erythematosus (SLE; n = 62) were examined, healthy donors (n = 31) served as controls. Selected peptides of galectin (Gal)-2 were employed for peptide-based ELISAs. Levels of anti-Gal-2(PEP)-IgG were significantly increased in SLE and APS when compared with controls. In addition, patients with APS showed significantly higher levels of anti-Gal-2(PEP)-IgG compared with patients with SLE. Anti-Gal-2(PEP)-IgG may, therefore, be considered novel biomarkers for APS.

Galectins distinctively regulate central monocyte and macrophage function.

Monocytes and macrophages link the innate and adaptive immune systems and protect the host from the outside world. In inflammatory disorders their activation leads to tissue damage. Galectins have emerged as central regulators of the immune system. However, if they regulate monocyte/macrophage physiology is still unknown. Binding of Gal-1, Gal-2, Gal-3 and Gal-4 to monocytes/macrophages, activation, cytokine secretion and apoptosis were determined by FACS, migration by Transwell system and phagocytosis by phagotest. Supernatants from macrophages co-cultured with galectins revealed their influence on T-cell function. In our study Gal-1, Gal-2, Gal-4, and partly Gal-3 bound to monocytes/macrophages. Galectins prevented Salmonella-induced MHCII upregulation. Cytokine release was distinctly induced by different galectins. T-cell activation was significantly restricted by supernatants of macrophages co-cultured in the presence of Gal-2 or Gal-4. Furthermore, all galectins tested significantly inhibited monocyte migration. Finally, we showed for the first time that galectins induce potently monocyte, but not macrophage apoptosis. Our study provides evidence that galectins distinctively modulate central monocyte/macrophage function. By inhibiting T-cell function via macrophage priming, we show that galectins link the innate and adaptive immune systems and provide new insights into the action of sugar-binding proteins.

Caenorhabditis elegans N-glycan core beta-galactoside confers sensitivity towards nematotoxic fungal galectin CGL2.

The physiological role of fungal galectins has remained elusive. Here, we show that feeding of a mushroom galectin, Coprinopsis cinerea CGL2, to Caenorhabditis elegans inhibited development and reproduction and ultimately resulted in killing of this nematode. The lack of toxicity of a carbohydrate-binding defective CGL2 variant and the resistance of a C. elegans mutant defective in GDP-fucose biosynthesis suggested that CGL2-mediated nematotoxicity depends on the interaction between the galectin and a fucose-containing glycoconjugate. A screen for CGL2-resistant worm mutants identified this glycoconjugate as a Galbeta1,4Fucalpha1,6 modification of C. elegans N-glycan cores. Analysis of N-glycan structures in wild type and CGL2-resistant nematodes confirmed this finding and allowed the identification of a novel putative glycosyltransferase required for the biosynthesis of this glycoepitope. The X-ray crystal structure of a complex between CGL2 and the Galbeta1,4Fucalpha1,6GlcNAc trisaccharide at 1.5 A resolution revealed the biophysical basis for this interaction. Our results suggest that fungal galectins play a role in the defense of fungi against predators by binding to specific glycoconjugates of these organisms.

Galectin-2 induces apoptosis of lamina propria T lymphocytes and ameliorates acute and chronic experimental colitis in mice.

Galectins have recently emerged as central regulators of the immune system. We have previously demonstrated that carbohydrate-dependent binding of galectin-2 induces apoptosis in activated T cells. Here, we investigate the potential therapeutic effect of galectin-2 in experimental colitis. Galectin-2 expression and its binding profile were determined by immunohistochemistry. Acute and chronic colitis was induced by dextran sodium sulfate administration and in a T-cell transfer colitis model. Apoptosis was assessed by TdT-mediated dUTP-biotin nick-end labeling, and cytokine secretion was determined by cytometric bead array. We show that galectin-2 was constitutively expressed mainly in the epithelial compartment of the mouse intestine and bind to lamina propria mononuclear cells. During colitis, galectin-2 expression was reduced, but could be restored to normal levels by immunosuppressive treatment. Galectin-2 treatment induced apoptosis of mucosal T cells and thus ameliorated acute and chronic dextran-sodium-sulfate-induced colitis and in a T-helper-cell 1-driven model of antigen-specific transfer colitis. Furthermore, the pro-inflammatory cytokine release was inhibited by galectin-2 treatment. In preliminary toxicity studies, galectin-2 was well tolerated. Our study provides evidence that galectin-2 induces apoptosis in vivo and ameliorates acute and chronic murine colitis. Furthermore, galectin-2 has no significant toxicity over a broad dose range, suggesting that it may serve as a new therapeutic agent in the treatment of inflammatory bowel disease.

Co-expression and functional interaction of silicatein with galectin: matrix-guided formation of siliceous spicules in the marine demosponge Suberites domuncula.

Sponges (phylum Porifera) of the class of Demospongiae are stabilized by a siliceous skeleton. It is composed of silica needles (spicules), which provide the morphogenetic scaffold of these metazoans. In the center of the spicules there is an axial filament that consists predominantly of silicatein, an enzyme that catalyzes the synthesis of biosilica. By differential display of transcripts we identified additional proteins involved in silica formation. Two genes were isolated from the marine demosponge Suberites domuncula; one codes for a galectin and the other for a fibrillar collagen. The galectin forms aggregates to which silicatein molecules bind. The extent of the silicatein-mediated silica formation strongly increased if associated with the galectin. By applying a new and mild extraction procedure that avoids hydrogen fluoride treatment, native axial filaments were extracted from spicules of S. domuncula. These filaments contained, in addition to silicatein, the galectin and a few other proteins. Immunogold electron microscopic studies underscored the role of these additional proteins, in particular that of galectin, in spiculogenesis. Galectin, in addition to silicatein, presumably forms in the axial canal as well as on the surface of the spicules an organized net-like matrix. In the extraspicular space most of these complexes are arranged concentrically around the spicules. Taken together, these additional proteins, working together with silicatein, may also be relevant for potential (nano)-biotechnological applications of silicatein in the formation of surface coatings. Finally, we propose a scheme that outlines the matrix (galectin/silicatein)-guided appositional growth of spicules through centripetal and centrifugal synthesis and deposition of biosilica.