Galectins and Immune Responses-Just How Do They Do Those Things They Do?

Galectins are a family of mammalian carbohydrate-binding proteins expressed by many cell types. Galectins can function intracellularly and can also be secreted to bind to cell surface glycoconjugate counterreceptors. Some galectins are made by immune cells, whereas other galectins are secreted by different cell types, such as endothelial or epithelial cells, and bind to immune cells to regulate immune responses. Galectin binding to a single glycan ligand is a low-affinity interaction, but the multivalency of galectins and the glycan ligands presented on cell surface glycoproteins results in high-avidity binding that can reversibly scaffold or cluster these glycoproteins. Galectin binding to a specific glycoprotein counterreceptor is regulated in part by the repertoire of glycosyltransferase enzymes (which make the glycan ligands) expressed by that cell, and the effect of galectin binding results from clustering or retention of specific glycoprotein counterreceptors bearing these specific ligands.

Secreted IgD Amplifies Humoral T Helper 2 Cell Responses by Binding Basophils via Galectin-9 and CD44.

B cells thwart antigenic aggressions by releasing immunoglobulin M (IgM), IgG, IgA, and IgE, which deploy well-understood effector functions. In contrast, the role of secreted IgD remains mysterious. We found that some B cells generated IgD-secreting plasma cells following early exposure to external soluble antigens such as food proteins. Secreted IgD targeted basophils by interacting with the CD44-binding protein galectin-9. When engaged by antigen, basophil-bound IgD increased basophil secretion of interleukin-4 (IL-4), IL-5, and IL-13, which facilitated the generation of T follicular helper type 2 cells expressing IL-4. These germinal center T cells enhanced IgG1 and IgE but not IgG2a and IgG2b responses to the antigen initially recognized by basophil-bound IgD. In addition, IgD ligation by antigen attenuated allergic basophil degranulation induced by IgE co-ligation. Thus, IgD may link B cells with basophils to optimize humoral T helper type 2-mediated immunity against common environmental soluble antigens.

Diverse immunological dysregulation, chronic inflammation, and impaired erythropoiesis in long COVID patients with chronic fatigue syndrome.

A substantial number of patients recovering from acute SARS-CoV-2 infection present serious lingering symptoms, often referred to as long COVID (LC). However, a subset of these patients exhibits the most debilitating symptoms characterized by ongoing myalgic encephalomyelitis or chronic fatigue syndrome (ME/CFS). We specifically identified and studied ME/CFS patients from two independent LC cohorts, at least 12 months post the onset of acute disease, and compared them to the recovered group (R). ME/CFS patients had relatively increased neutrophils and monocytes but reduced lymphocytes. Selective T cell exhaustion with reduced naïve but increased terminal effector T cells was observed in these patients. LC was associated with elevated levels of plasma pro-inflammatory cytokines, chemokines, Galectin-9 (Gal-9), and artemin (ARTN). A defined threshold of Gal-9 and ARTN concentrations had a strong association with LC. The expansion of immunosuppressive CD71+ erythroid cells (CECs) was noted. These cells may modulate the immune response and contribute to increased ARTN concentration, which correlated with pain and cognitive impairment. Serology revealed an elevation in a variety of autoantibodies in LC. Intriguingly, we found that the frequency of 2B4+CD160+ and TIM3+CD160+ CD8+ T cells completely separated LC patients from the R group. Our further analyses using a multiple regression model revealed that the elevated frequency/levels of CD4 terminal effector, ARTN, CEC, Gal-9, CD8 terminal effector, and MCP1 but lower frequency/levels of TGF-ß and MAIT cells can distinguish LC from the R group. Our findings provide a new paradigm in the pathogenesis of ME/CFS to identify strategies for its prevention and treatment.

Unveiling the molecular characteristics and antibacterial activity of tandem-repeat-type Galectin-8 in large yellow croaker (Larimichthys crocea).

Galectin-8 (Gal-8) is a versatile carbohydrate-binding protein with pivotal roles in immune regulation and cellular processes. This study introduces a novel galectin-8 protein, LcGal-8, from the large yellow croaker (Larimichthys crocea), showcasing typical characteristics of tandem-repeat-type galectins, including the absence of a signal peptide or transmembrane region and the presence of conserved sugar-binding motifs. Phylogenetic analysis reveals its conservation among fish species. Expression profiling indicates widespread distribution in immune tissues, particularly the spleen, implicating involvement in immune processes. The subcellular localization analysis reveals that LcGal-8 is present in both the cytoplasm and nucleus. Upon bacterial challenge, LcGal-8 is up-regulated in immune tissues, suggesting a role in host defense. Functional assays demonstrate that LcGal-8 can agglutinate gram-negative bacteria. The recombinant LcGal-8 protein agglutinates red blood cells from the large yellow croaker independently of Ca2⁺, however, this activity is inhibited by lipopolysaccharide (LPS) at 2.5 µg/mL. Fluorescence detection kits and scanning electron microscopy (SEM) confirm the agglutination and bactericidal effects of LcGal-8 against various gram-negative bacteria, including Vibrio harveyi, Aeromondaceae hydrophila, Aeromondaceae veronii, Pseudomonas plecoglossicida, Edwardsiella tarda. These findings contribute valuable insights into the genetic basis of disease resistance in the large yellow croaker and could support molecular breeding strategies to enhance disease resistance.

Sweet Immune Checkpoint Targets to Enhance T Cell Therapy.

Despite tremendous success against hematological malignancies, the performance of chimeric Ag receptor T cells against solid tumors remains poor. In such settings, the lack of success of this groundbreaking immunotherapy is in part mediated by ligand engagement of immune checkpoint molecules on the surface of T cells in the tumor microenvironment. Although CTLA-4 and programmed death-1 (PD-1) are well-established checkpoints that inhibit T cell activity, the engagement of glycans and glycan-binding proteins are a growing area of interest due to their immunomodulatory effects. This review discusses exemplary strategies to neutralize checkpoint molecules through an in-depth overview of genetic engineering approaches aimed at overcoming the inhibitory programmed death ligand-1 (PD-L1)/PD-1 axis in T cell therapies and summarizes current knowledge on glycoimmune interactions that mediate T cell immunosuppression.

T cells are Smad'ly in love with galectin-9.

In this issue of Immunity, Wu et al. (2014) report that galectin-9 is required for the formation and stability of iTreg cells. Galectin-9 interacts with CD44 in association with TGF-ß receptors to drive both Foxp3 and galectin-9 expression in a positive-feedforward loop involving Smad3 activation.

Galectin-9-CD44 interaction enhances stability and function of adaptive regulatory T cells.

The ß-galactoside-binding protein galectin-9 is critical in regulating the immune response, but the mechanism by which it functions remains unclear. We have demonstrated that galectin-9 is highly expressed by induced regulatory T cells (iTreg) and was crucial for the generation and function of iTreg cells, but not natural regulatory T (nTreg) cells. Galectin-9 expression within iTreg cells was driven by the transcription factor Smad3, forming a feed-forward loop, which further promoted Foxp3 expression. Galectin-9 increased iTreg cell stability and function by directly binding to its receptor CD44, which formed a complex with transforming growth factor-ß (TGF-ß) receptor I (TGF-ßRI), and activated Smad3. Galectin-9 signaling was further found to regulate iTreg cell induction by dominantly acting through the CNS1 region of the Foxp3 locus. Our data suggest that exogenous galectin-9, in addition to being an effector molecule for Treg cells, acts synergistically with TGF-ß to enforce iTreg cell differentiation and maintenance.

Galectin-9 Targets NLRP3 for Autophagic Degradation to Limit Inflammation.

NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome has been implicated in a variety of inflammatory disorders, and its activation should be tightly controlled to avoid detrimental effects. NLRP3 protein expression is considered as the rate-limiting step for NLRP3 inflammasome activation. In this study, we show that galectin-9 (encoded by lgals9) attenuated NLRP3 inflammasome activation by promoting the protein degradation of NLRP3 in primary peritoneal macrophages of C57BL/6J mice. Lgals9 deficiency enhances NLRP3 inflammasome activation and promotes NLRP3-dependent inflammation in C57BL/6J mice in vivo. Mechanistically, galectin-9 interacts with NLRP3, promotes the formation of NLRP3/p62 (an autophagic cargo receptor, also known as SQSTM1) complex, and thus facilitates p62-dependent autophagic degradation of NLRP3 in primary peritoneal macrophages of C57BL/6J mice and HEK293T cells. Therefore, we identify galectin-9 as an "eat-me" signal for selective autophagy of NLRP3 and uncover the potential roles of galectins in controlling host protein degradation. Furthermore, our work suggests galectin-9 as a priming therapeutic target for the diseases caused by improper NLRP3 inflammasome activation.

Perturbing dimer interactions and allosteric communication modulates the immunosuppressive activity of human galectin-7.

The design of allosteric modulators to control protein function is a key objective in drug discovery programs. Altering functionally essential allosteric residue networks provides unique protein family subtype specificity, minimizes unwanted off-target effects, and helps avert resistance acquisition typically plaguing drugs that target orthosteric sites. In this work, we used protein engineering and dimer interface mutations to positively and negatively modulate the immunosuppressive activity of the proapoptotic human galectin-7 (GAL-7). Using the PoPMuSiC and BeAtMuSiC algorithms, mutational sites and residue identity were computationally probed and predicted to either alter or stabilize the GAL-7 dimer interface. By designing a covalent disulfide bridge between protomers to control homodimer strength and stability, we demonstrate the importance of dimer interface perturbations on the allosteric network bridging the two opposite glycan-binding sites on GAL-7, resulting in control of induced apoptosis in Jurkat T cells. Molecular investigation of G16X GAL-7 variants using X-ray crystallography, biophysical, and computational characterization illuminates residues involved in dimer stability and allosteric communication, along with discrete long-range dynamic behaviors involving loops 1, 3, and 5. We show that perturbing the protein-protein interface between GAL-7 protomers can modulate its biological function, even when the overall structure and ligand-binding affinity remains unaltered. This study highlights new avenues for the design of galectin-specific modulators influencing both glycan-dependent and glycan-independent interactions.

Galectin-8, cytokines, and the storm.

Galectin-8 (Gal-8) belongs to a family of animal lectins that modulate cell adhesion, cell proliferation, apoptosis, and immune responses. Recent studies have shown that mammalian Gal-8 induces in an autocrine and paracrine manner, the expression and secretion of cytokines and chemokines such as RANKL, IL-6, IL-1ß, SDF-1, and MCP-1. This involves Gal-8 binding to receptor complexes that include MRC2/uPAR/LRP1, integrins, and CD44. Receptors ligation triggers FAK, ERK, Akt, and the JNK signaling pathways, leading to induction of NF-κB that promotes cytokine expression. Indeed, immune-competent Gal-8 knockout (KO) mice express systemic lower levels of cytokines and chemokines while the opposite is true for Gal-8 transgenic animals. Cytokine and chemokine secretion, induced by Gal-8, promotes the migration of cancer cells toward cells expressing this lectin. Accordingly, Gal-8 KO mice experience reduced tumor size and smaller and fewer metastatic lesions when injected with cancer cells. These observations suggest the existence of a 'vicious cycle' whereby Gal-8 expression and secretion promotes the secretion of cytokines and chemokines that further promote Gal-8 expression. This 'vicious cycle' could enhance the development of a 'cytokine storm' which is a key contributor to the poor prognosis of COVID-19 patients.

FCER1G positively relates to macrophage infiltration in clear cell renal cell carcinoma and contributes to unfavorable prognosis by regulating tumor immunity.

BACKGROUND: Tumor-associated macrophages (TAMs) are closely related to unfavorable prognosis of patients with clear cell renal cell carcinoma (ccRCC). However, the important molecules in the interaction between ccRCC and TAMs are unclear. METHODS: TCGA-KIRC gene expression data of tumor tissues and normal tissues adjacent to tumor were compared to identify differentially expressed genes in ccRCC. TAMs related genes were discovered by analyzing the correlation between these differentially expressed genes and common macrophage biomarkers. Gene set enrichment analysis was performed to predict functions of TAMs related gene. The findings were further validated using RNA sequencing data obtained from the CheckMate 025 study and immunohistochemical analysis of samples from 350 patients with ccRCC. Kaplan-Meier survival curve, Cox regression analysis and Harrell's concordance index analysis were used to determine the prognostic significance. RESULTS: In this study, we applied bioinformatic analysis to explore TAMs related differentially expressed genes in ccRCC and identified 5 genes strongly correlated with all selected macrophage biomarkers: STAC3, LGALS9, TREM2, FCER1G, and PILRA. Among them, FCER1G was abundantly expressed in tumor tissues and showed prognostic importance in patients with ccRCC who received treatment with Nivolumab; however, it did not exhibit prognostic value in those treated with Everolimus. We also discovered that high expression levels of FCER1G are related to T cell suppression. Moreover, combination of FCER1G and macrophage biomarker CD68 can improve the prognostic stratification of patients with ccRCC from TCGA-KIRC. Based on the immunohistochemical analysis of samples from patients with ccRCC, we further validated that FCER1G and CD68 are both highly expressed in tumor tissue and correlate with each other. Higher expression of CD68 or FCER1G in ccRCC tissue indicates shorter overall survival and progression-free survival; patients with high expression of both CD68 and FCER1G have the worst outcome. Combining CD68 and FCER1G facilitates the screening of patients with a worse prognosis from the same TNM stage group. CONCLUSIONS: High expression of FCER1G in ccRCC is closely related to TAMs infiltration and suppression of T cell activation and proliferation. Combining the expression levels of FCER1G and macrophage biomarker CD68 may be a promising postoperative prognostic index for patients with ccRCC.

Molecular characterization and expression profiling of tandem-repeat galectin-8 from red-spotted grouper (Epinephelus akaara): Potential antibacterial, antiviral, and wound healing activities.

Galectin-8 is a typical ß-galactoside binding lectin, which primarily functions as a pattern recognition receptor and/or danger receptor that is engaged in pathogen recognition by the host innate immune system. Although several fish galectins have been identified, the role of galectin-8 in teleost immunity is still not fully understood. In this study, molecular, transcriptional, and immune-related functions of galectin-8 (EaGal8) from red-spotted grouper (Epinephelus akaara) were analyzed. The open reading frame of EaGal8 comprised 960 bp encoding 319 amino acids of a ∼35 kDa protein, composed of the N- and C-terminal carbohydrate recognition domains joined by a short hinge peptide. Phylogenetic analysis revealed that EaGal8 was closely related to the Epinephelus lanceolatus galectin-8-like protein. Although EaGal8 showed ubiquitous tissue expression, the highest expression level was observed in the blood. Immunostimulants, including lipopolysaccharide, poly(I:C), and nervous necrosis virus, significantly upregulated the EaGal8 transcription level in a time-dependent manner (p < 0.05). Furthermore, recombinant EaGal8 (rEaGal8) showed a binding affinity toward seven different carbohydrates in a concentration-dependent manner. In addition, rEaGal8 caused strong agglutination of fish red blood cells and several gram-positive and gram-negative bacteria, including Streptococcus iniae, Streptococcus parauberis, Lactococcus garvieae, Escherichia coli, Edwardsiella tarda, Vibrio alginolyticus, Vibrio parahaemolyticus, and Pseudomonas aeruginosa. For the first time in teleosts, we report the wound healing ability of galectin-8 in this study. At low concentrations, rEaGal8 showed potential wound healing responses in FHM cells, in vitro. Thus, this study reinforces the role of EaGal8 in innate immune responses against bacterial and viral infections and wound healing in red-spotted grouper.

Intracellular Galectin-9 Enhances Proximal TCR Signaling and Potentiates Autoimmune Diseases.

Galectin-9 is a risk gene in inflammatory bowel disease. By transcriptomic analyses of ileal biopsies and PBMCs from inflammatory bowel disease patients, we identified a positive correlation between galectin-9 expression and colitis severity. We observed that galectin-9-deficient T cells were less able to induce T cell-mediated colitis. However, several mouse-based studies reported that galectin-9 treatment induces T cell apoptosis and ameliorates autoimmune diseases in an exogenously modulated manner, indicating a complicated regulation of galectin-9 in T cells. We found that galectin-9 is expressed mainly inside T cells, and its secreted form is barely detected under physiological conditions. Endogenous galectin-9 was recruited to immune synapses upon T cell activation. Moreover, proximal TCR signaling was impaired in galectin-9-deficient T cells, and proliferation of these cells was decreased through an intracellularly modulated manner. Th17 cell differentiation was downregulated in galectin-9-deficient T cells, and this impairment can be rescued by strong TCR signaling. Taken together, these findings suggest that intracellular galectin-9 is a positive regulator of T cell activation and modulates the pathogenesis of autoimmune diseases.

BST2, a Novel Inhibitory Receptor, Is Involved in NK Cell Cytotoxicity through Its Cytoplasmic Tail Domain.

Bone Marrow Stromal Cell Antigen 2 (BST2) is a type II transmembrane protein expressed on various cell types that tethers the release of viruses. Natural killer (NK) cells express low levels of BST2 under normal conditions but exhibit increased expression of BST2 upon activation. In this study, we show for the first time that murine BST2 can control the cytotoxicity of NK cells. The cytoplasmic tail of murine BST2 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM). The absence of BST2 on NK cells can enhance their cytotoxicity against tumor cells compared to wild type NK cells. NK cells isolated from NZW mice, which express ITIM-deficient BST2, also showed higher cytotoxicity than wild type NK cells. In addition, we found that galectin-8 and galectin-9 were ligands of BST2, since blocking galectin-8 or -9 with monoclonal antibodies enhanced the cytotoxicity of NK cells. These results suggested that BST2 might be a novel NK cell inhibitory receptor as it was involved in regulating NK cell cytotoxicity through its interaction with galectins.

Galectin-9 regulates follicular helper T cells to inhibit humoral autoimmunity-induced pulmonary fibrosis.

Interstitial pneumonia with autoimmune features (IPAF) is an unexplained disease state characterized by autoimmunity and pulmonary fibrosis. Exploring the pathogenesis of IPAF is helpful for the treatment of interstitial pneumonia and idiopathic pulmonary fibrosis. In this study, we observed that the lung Galectin-9 (Gal-9) of IPAF patients was significantly reduced, which was significantly related to lung dysfunction and abnormal humoral immunity. Moreover, an overreactive germinal center (GC) reaction in the lung lymph nodes (LNs) of Gal-9-deficient mice was found to be related to abnormally active follicular helper T cells (Tfh) cells. The lack of Gal-9 ligand in Tfh cells can lead to excessive transcriptional programming and differentiation and help GC B cells. Gal-9 deficiency caused an abnormal humoral immune response in mice, leading to excessive deposition of nonspecific autoantibodies in mice and chronic lung fibrosis. Our research reveals the important regulatory role of gal-9 in Tfh cells and a possible target for the treatment of IPAF.

Kinetic studies of galectin-10 release from eosinophils exposed to proliferating T cells.

Galectin-10 is involved in the T cell suppressive activity of regulatory T cells and eosinophils alike. We have identified a subpopulation of T cell suppressive eosinophils that express CD16 on the surface and contain more galectin-10 compared with conventional CD16-negative eosinophils. Our main goal was to determine how the intracellular protein galectin-10 is released from eosinophils when exposed to proliferating T cells and if such release could be inhibited. Confocal microscopy and imaging flow cytometry were used to study the release of galectin-10 from eosinophils incubated with polyclonally activated T cells. T cell proliferation was monitored by measurement of the incorporation of [3 H]-thymidine. Initially, galectin-10-containing synapses formed between eosinophils and T cells. Subsequently, the plasma membrane of eosinophils began to disintegrate and cap-like accumulations of galectin-10 budded on the eosinophil cell surface. Lastly, eosinophil extracellular traps composed of nuclear DNA and galectin-10 were freed. It was solely the CD16-expressing suppressive eosinophils that formed synapses and eosinophil extracellular traps containing galectin-10. Dissolution of the extracellular traps by DNase I partly abrogated the T cell suppression exerted by eosinophils. Extracellular trap formation has mainly been associated with anti-bacterial defense, but we show a new putative function of these cellular formations, as mediators of T cell suppression by enabling the release of galectin-10 from eosinophils.

Galectins from Onchocerca ochengi and O. volvulus and their immune recognition by Wistar rats, Gudali zebu cattle and human hosts.

BACKGROUND: During the last two decades research on animal filarial parasites, especially Onchocerca ochengi, infecting cattle in savanna areas of Africa revealed that O. ochengi as an animal model has biological features that are similar to those of O. volvulus, the aetiological agent of human onchocerciasis. There is, however, a paucity of biochemical, immunological and pathological data for O. ochengi. Galectins can be generated by parasites and their hosts. They are multifunctional molecules affecting the interaction between filarial parasites and their mammalian hosts including immune responses. This study characterized O. ochengi galectin, verified its immunologenicity and established its immune reactivity and that of Onchocerca volvulus galectin. RESULTS: The phylogenetic analysis showed the high degree of identity between the identified O. ochengi and the O. volvulus galectin-1 (ß-galactoside-binding protein-1) consisting only in one exchange of alanine for serine. O. ochengi galectin induced IgG antibodies during 28 days after immunization of Wistar rats. IgG from O. ochengi-infected cattle and O. volvulus-infected humans cross-reacted with the corresponding galectins. Under the applied experimental conditions in a cell proliferation test, O. ochengi galectin failed to significantly stimulate peripheral blood mononuclear cells (PBMCs) from O. ochengi-infected cattle, regardless of their parasite load. CONCLUSION: An O. ochengi galectin gene was identified and the recombinantly expressed protein was immunogenic. IgG from Onchocerca-infected humans and cattle showed similar cross-reaction with both respective galectins. The present findings reflect the phylogenetic relationship between the two parasites and endorse the appropriateness of the cattle O. ochengi model for O. volvulus infection research.

Molecular characterization, expression analysis and immune effect of Galectin-8 from Japanese flounder (Paralichthys olivaceus).

Galectin-8 gene belongs to the agglutinin family, which can specifically recognize ß-galactoside bonds and play essential roles in many biological processes. In this study, we researched the sequence characteristics and immune-related function of Galectin-8 gene in Japanese flounder Paralichthys olivaceus, named PoGalectin-8. The results showed that the open reading frame of PoGalectin-8 was 891 bp, which encoding a protein with 296 amino acid residues and containing typical HXNPR and WGXEE motifs in the N-terminal and C-terminal CRD domains. Sequence alignment showed that PoGalectin-8 was conserved in different aquatic animals and exhibited the highest similarity (95.27%) with Seriola dumerili. PoGalectin-8 expressed in all detected tissues and exhibited the highest expression level in spleen, followed by skin and kidney. After infected by Edwardsiella tarda, the expression of PoGalectin-8 was down-regulated in the spleen and skin tissues of P. olivaceus. Further to study its immune-related functions, the recombinant PoGalectin-8 (rPoGalectin-8) was expressed and purified. The rPoGalectin-8 can specifically bind to lipopolysaccharide and peptidoglycan, the main components of cell walls from Gram-negative and Gram-positive bacteria. Bacteria binding and the microbial agglutinating experiments showed that the rPoGalectin-8 could bind and agglutinate all examined Gram-positive and Gram-negative bacteria. This study implied that PoGalectin-8, as a pattern recognition receptor, may play important roles during immune responses against bacterial infection, which laid a foundation for further functional identification of Galectin-8 in aquatic animal immunity.

Molecular characterization and functional study of a tandem-repeat Galectin-9 from Japanese flounder (Paralichthys olivaceus).

Galectin-9 is a ß-galactoside-binding lectin which could modulate a variety of biological functions including recognition, aggregation and clearance of pathogen. In this study, one Galectin-9 (named PoGalectin-9) was identified from Japanese flounder Paralichthys olivaceus. PoGalectin-9 belongs to the tandem-repeat type, containing one 127-amino acids CRD domain within N terminal and one 122-amino acids CRD domain within C-terminal. The open reading frame of PoGalectin-9 cDNA was 921 bp encoding 306 amino acids. Sequence similarity comparison confirmed that PoGalectin-9 shared high homology with other Galectin-9. The tissue distribution and expression profiles after bacterial infection were also investigated. PoGalectin-9 was widely distributed in all of the examined tissues of Japanese flounder but was predominantly expressed in the spleen, kidney and intestine. After Edwardsiella tarda challenge, the expression of PoGalectin-9 was up-regulated in spleen and down regulated in kidney. ELISA experiment showed that recombinant PoGalectin-9 (rPoGalectin-9) exhibit binding capacity to lipopolysaccharide (LPS) and peptidoglycan (PGN), which is significantly correlated with the concentration of rPoGalectin-9. Meanwhile, the rPoGalectin-9 protein showed strong agglutinating activities against both Gram-negative bacteria and Gram-positive bacteria. Bacterial binding experiments showed that rPoGalectin-9 could bind all examined bacteria. In conclusion, the present study indicate that PoGalectin-9 might play important roles during the immune responses of Japanese flounder against bacterial pathogens.

The N- and C-terminal carbohydrate recognition domains of galectin-9 from Carassius auratus contribute differently to its immunity functions to Aeromonas hydrophila and Staphylococcus aureus.

Galectin-9, an important pathogen recognition receptor (PRR), could recognize and bind pathogen-associated molecular patterns (PAMPs) on the surface of invading microorganisms, initiating the innate immune responses. A galectin-9 was identified from Qihe crucian carp Carassius auratus and designated as CaGal-9. The predicted CaGal-9 protein contained two non-identical carbohydrate recognition domains (CRDs), namely, N-CRD and C-CRD. The recombinant proteins (rCaGal-9, rN-CRD and rC-CRD) were purified from Escherichia coli BL21 (DE3) and exhibited strong agglutinating activity with erythrocytes of rabbit. The haemagglutination was inhibited by D-galactose, α-lactose and N-acetyl-D-galactose. Results of microbial agglutination assay showed that three recombinant proteins agglutinated Gram-negative bacterium Aeromonas hydrophila and Gram-positive bacterium Staphylococcus aureus. With regard to the binding activity, each recombinant protein could bind to LPS, PGN and the examined microorganisms (A. hydrophila and S. aureus) with different binding affinities. The integrated analyses suggested that CaGal-9 with two CRD domains could play an important role in immune defence against pathogenic microorganisms for C. auratus.