Galectins are a functionally diverse family of β-galactosyl-binding lectins that are ubiquitously present in animal species, with key roles in development and immune regulation. Recently, galectins have been found to recognize microbial glycosylated moieties, but the detailed mechanisms of their innate immune functions in mucosal epithelia have remained elusive. The zebrafish (Danio rerio) represents an ideal genetically tractable model to address these questions, as the skin, gills, and gut display mucosal surfaces exposed to the environment. In this study, we investigated the range of endogenous and microbial glycans that are recognized by zebrafish galectin Drgal1 present in epidermal mucus, which would be consistent with defense functions against a bacterial challenge. Results revealed that zebrafish galectin isoform Drgal1-L2 can recognize selected bacterial glycans, as well as zebrafish mucus glycans and cell-surface receptors for bacterial adhesins such as fibronectin (KD = 1.593 × 10-6 M) and CD147 (KD = 1.115 × 10-6 M). Furthermore, preliminary experiments revealed that Drgal1-L2 may hinder bacterial adhesion to epidermal mucus in about 50% at 2.5 μg/mL. Our results suggest that Drgal1-L2 present in epidermal mucus can prevent access of pathogenic bacteria to the epithelial cell surface by alternate or synergic binding to bacterial glycans and to zebrafish mucus components and epithelial receptors for bacterial adhesins. Thus, the present study provides key information for the testing of the abovementioned hypothesis by implementing gene-silencing approaches targeting both zebrafish Drgal1-L2 and its ligands.

Galectins are β-galactosyl-binding lectins with key roles in immune regulation and as pattern recognition receptors. To address their potential role(s) in viral infection of mucosal epithelia we currently investigate adhesion and entry mechanisms of the infectious hematopoietic necrosis virus (IHNV) using the zebrafish (Danio rerio) model system. We previously reported the recognition of IHNV envelope glycoprotein by the zebrafish galectin Drgal1-L2 and its inhibitory activity for viral adhesion to epithelial cells. Subsequently, we determined the structure of Drgal1-L2 and proposed a mechanism for Drgal1-mediated inhibition of IHNV spike fusion to the host epithelial cell. We now show that Drgal1 can also hinder viral adhesion and infection by binding to glycans on the host cell surface and epidermal mucus. We identified fibronectin, the reported IHNV receptor, as the cell surface glycoprotein recognized by Drgal1-L2. Surprisingly, IHNV also adhered in vitro to purified β1integrin, and pre-exposure of either IHNV or the immobilized β1integrin to Drgal1-L2 hindered IHNV adhesion. Binding of either anti-fibronectin or anti-β1integrin antibodies to the cell surface partially inhibited IHNV adherence. Drgal1-L2 also hindered IHNV adhesion by binding to mucus glycans. Taken together, our results suggest complementary mechanisms by which Drgal1-L2 may protect mucosal epithelial cells against IHNV infection and tentatively identify β1integrin as a novel receptor for IHNV.

Galectins are β-galactosyl-binding lectins with key roles in early development, immune regulation, and infectious disease. Influenza A virus (IAV) infects the airway epithelia, and in severe cases may lead to bacterial superinfections and hypercytokinemia, and eventually, to acute respiratory distress syndrome (ARDS) through the breakdown of airway barriers. The detailed mechanisms involved, however, remain poorly understood. Our prior in vivo studies in a murine model system revealed that upon experimental IAV and pneumococcal primary and secondary challenges, respectively, galectin-1 and galectin-3 (Gal-3) are released into the airway and bind to the epithelium that has been desialylated by the viral neuraminidase, contributing to secondary bacterial infection and hypercytokinemia leading to the clinical decline and death of the animals. Here we report the results of in vitro studies that reveal the role of the extracellular Gal-3 in additional detrimental effects on the host by disrupting the integrity of the airway epithelial barrier. IAV infection of the human airway epithelia cell line A549 increased release of Gal-3 and its binding to the A549 desialylated cell surface, notably to the transmembrane signaling receptors CD147 and integrin-β1. Addition of recombinant Gal-3 to A549 monolayers resulted in enhanced expression and release of matrix metalloproteinases, leading to disruption of cell-cell tight junctions, and a significant increase in paracellular permeability. This study reveals a critical mechanism involving Gal-3 that may significantly contribute to the severity of IAV infections by promoting disruption of tight junctions and enhanced permeability of the airway epithelia, potentially leading to lung edema and ARDS.

Like all aquatic vertebrates and invertebrates, teleost fish are subject to the constant pressure of bacterial, fungal, and parasitic organisms present in the environmental interface that can potentially cause disease. Numerous defense molecules, including galectins, have been isolated from the skin and gut tissues of several marine and freshwater fish species. To provide new insights into the potential role(s) of galectins in the teleost fish innate immune system, we carried out studies on the striped bass (Morone saxatilis), a keystone fish species in Chesapeake Bay. We purified from epidermal skin mucus, and skin and muscle tissue, a 15-kDa galectin that we designated Msgal1-L1 (M. saxatilis galectin1-like protein 1). Both the transcript sequence and gene organization of Msgal1-L1 suggested a close relationship to the zebrafish galectin Drgal1-L2 and other proto type galectins from vertebrates, including the mammalian galectin-1. Glycan microarray analysis of Msgal1-L1 revealed a binding preference for Galβ1,4GlcNAc, and a homology structural model identified the amino acids involved in ligand recognition, both observations consistent with proto type galectins. Immunohistological examination localized Msgal1-L1 to epithelial and macrophage-/fibroblast-like cells in mucosal tissues, including skin and gill. The preliminary localization of Msgal1-L1 in free macrophage-like cells in epidermal mucus was corroborated by immunofluorescence analysis of macrophages isolated from head kidney. Msgal1-L1 binds in a carbohydrate-specific manner to O-glycosylated components of epidermal mucus. Msgal1-L1 agglutinated environmental bacterial species and strains, some of which are recognized fish pathogens, such as Vibrio and Edwardsiella spp. A microbial microarray analysis revealed that it preferentially binds to bacterial exopolysaccharides (e.g., Streptococcus and Shigella spp.) as well as various lipopolysaccharide O-antigen serotypes of Proteus spp. A preliminary solid-phase assay showed that Msgal1-L1 strongly bound Streptococcus sp., but very weakly to Mycobacterium marinum, an endemic pathogen of striped bass in Chesapeake Bay. Taken together, this evidence suggests that Msgal1-L1 may function in defense recognition against environmental bacteria by agglutinating and/or cross-linking them to mucus oligosaccharides to immobilize them within the epidermal mucus film and prevent their access to the fish epithelial cell surface. M. marinum would evade this defense mechanism to reach and infect the fish skin epithelial layer.

The lung airway epithelial surface is heavily covered with sialic acids as the terminal carbohydrate on most cell surface glycoconjugates and can be removed by microbial neuraminidases or endogenous sialidases. By desia-lylating the lung epithelial surface, neuraminidase acts as an important virulence factor in many mucosal pathogens, such as influenza and S. pneumoniae. Desialylation exposes the subterminal galactosyl moieties - the binding glycotopes for galectins, a family of carbohydrate-recognition proteins playing important roles in various aspects of immune responses. Galectin-1 and galectin-3 have been extensively studied in their roles related to host immune responses, but some questions about their role(s) in leukocyte recruitment during lung bacterial infection remain unanswered. In this study, we found that both galectin-1 and galectin-3 bind to poly-morphonuclear leukocytes (PMNs) and enhance the interaction of endothelial intercellular adhesion molecule-1 (ICAM-1) with PMNs, which is further increased by PMN desialylation. In addition, we observed that in vitro galectin-1 mediates the binding of PMNs, particularly desialylated PMNs, onto the endothelial cells. Finally, in a murine model for LPS-mediated acute lung injury, we observed that galectin-1 modulates PMN infiltration to the lung without altering the expression of chemoattractant cytokines. We conclude that galectins, particularly galectin-1, may function as adhesion molecules that mediate PMN-endothelial cell interactions, and modulate PMN infiltration during acute lung injury.

To gain insight into sialic acid biology and sialidase/neuraminidase (NEU) expression in mature human neutrophil (PMN)s, we studied NEU activity and expression in PMNs and the HL60 promyelocytic leukemic cell line, and changes that might occur in PMNs undergoing apoptosis and HL60 cells during their differentiation into PMN-like cells. Mature human PMNs contained NEU activity and expressed NEU2, but not NEU1, the NEU1 chaperone, protective protein/cathepsin A(PPCA), NEU3, and NEU4 proteins. In proapoptotic PMNs, NEU2 protein expression increased > 30.0-fold. Granulocyte colony-stimulating factor protected against NEU2 protein upregulation, PMN surface desialylation and apoptosis. In response to 3 distinct differentiating agents, dimethylformamide, dimethylsulfoxide, and retinoic acid, total NEU activity in differentiated HL60 (dHL60) cells was dramatically reduced compared to that of nondifferentiated cells. With differentiation, NEU1 protein levels decreased > 85%, PPCA and NEU2 proteins increased > 12.0-fold, and 3.0-fold, respectively, NEU3 remained unchanged, and NEU4 increased 1.7-fold by day 3, and then returned to baseline. In dHL60 cells, lectin blotting revealed decreased α2,3-linked and increased α2,6-linked sialylation. dHL60 cells displayed increased adhesion to and migration across human bone marrow-derived endothelium and increased bacterial phagocytosis. Therefore, myeloid apoptosis and differentiation provoke changes in NEU catalytic activity and protein expression, surface sialylation, and functional responsiveness.

Human galectin 3 (Gal-3) has been implicated to play important roles in different biological recognition processes such as tumor growth and cancer metastasis. High-affinity Gal-3 ligands are desirable for functional studies and as inhibitors for potential therapeutic development. We report here a facile synthesis of β-cyclodextrin (CD)-based Tn and TF antigen-containing multivalent ligands via a click reaction. Binding studies indicated that the synthetic multivalent glycan ligands demonstrated a clear clustering effect in binding to human Gal-3, with up to 153-fold enhanced relative affinity in comparison with the monomeric glycan ligand. The GalNAc (Tn antigen) containing heptavalent ligand showed the highest affinity for human Gal-3 among the synthetic ligands tested, with an EC50 of 1.4 μM in binding to human Gal-3. A cell-based assay revealed that the synthetic CD-based multivalent ligands could efficiently inhibit Gal-3 binding to human airway epithelial cells, with an inhibitory capacity consistent with their binding affinity measured by SPR. The synthetic cyclodextrin-based ligands described in this study should be valuable for functional studies of human Gal-3 and potentially for therapeutic applications.

Pregnancy-specific beta 1 glycoprotein (PSG1) is secreted from trophoblast cells of the human placenta in increasing concentrations as pregnancy progresses, becoming one of the most abundant proteins in maternal serum in the third trimester. PSG1 has seven potential N-linked glycosylation sites across its four domains. We carried out glycomic and glycoproteomic studies to characterize the glycan composition of PSG1 purified from serum of pregnant women and identified the presence of complex N-glycans containing poly LacNAc epitopes with α2,3 sialyation at four sites. Using different techniques, we explored whether PSG1 can bind to galectin-1 (Gal-1) as these two proteins were previously shown to participate in processes required for a successful pregnancy. We confirmed that PSG1 binds to Gal-1 in a carbohydrate-dependent manner with an affinity of the interaction of 0.13 μM. In addition, we determined that out of the three N-glycosylation-carrying domains, only the N and A2 domains of recombinant PSG1 interact with Gal-1. Lastly, we observed that the interaction between PSG1 and Gal-1 protects this lectin from oxidative inactivation and that PSG1 competes the ability of Gal-1 to bind to some but not all of its glycoprotein ligands.

Two cosegregating single-nucleotide polymorphisms (SNPs) in human TLR4, an A896G transition at SNP rs4986790 (D299G) and a C1196T transition at SNP rs4986791 (T399I), have been associated with LPS hyporesponsiveness and differential susceptibility to many infectious or inflammatory diseases. However, many studies failed to confirm these associations, and transfection experiments resulted in conflicting conclusions about the impact of these SNPs on TLR4 signaling. Using advanced protein modeling from crystallographic data of human and murine TLR4, we identified homologous substitutions of these SNPs in murine Tlr4, engineered a knock-in strain expressing the D298G and N397I TLR4 SNPs homozygously, and characterized in vivo and in vitro responses to TLR4 ligands and infections in which TLR4 is implicated. Our data provide new insights into cellular and molecular mechanisms by which these SNPs decrease the TLR4 signaling efficiency and offer an experimental approach to confirm or refute human data possibly confounded by variables unrelated to the direct effects of the SNPs on TLR4 functionality.

The purification of fucose-binding lectins from the liver of striped bass (Morone saxatilis), a teleost fish, and the identification of a novel lectin sequence motif led to the identification of a new family of lectins, the F-type lectins (FTLs) (see overview of the FTL family in Chapter 23 ). Isolation and purification of these proteins from liver extracts of striped bass was accomplished by affinity chromatography and size exclusion, and their identification as FTLs, by direct Edman sequencing, and protein, transcript, and gene sequence analysis. The development of specific antibodies against the M. saxatilis FTL provided an additional tool for the identification of FTLs. These methods have been successfully used for the purification of the FTL family members from tissues and body fluids of various animal species. Production and characterization of FTLs has been facilitated by the expression of the recombinant proteins. In this chapter, the biochemical characterization of FTLs is focused on the analysis of their carbohydrate specificity.

Both vertebrates and invertebrates display active innate immune mechanisms for defense against microbial infection, including diversified repertoires of soluble and cell-associated lectins that can effect recognition and binding to potential pathogens, and trigger downstream effector pathways that clear them from the host internal milieu. Galectins are widely distributed and highly conserved lectins that have key regulatory effects on both innate and adaptive immune responses. In addition, galectins can bind to exogenous (“non-self”) carbohydrates on the surface of bacteria, enveloped viruses, parasites, and fungi, and function as recognition receptors and effector factors in innate immunity. Like most invertebrates, eastern oysters (Crassostrea virginica) and softshell clams (Mya arenaria) can effectively respond to most immune challenges through soluble and hemocyte-associated lectins. The protozoan parasite Perkinsus marinus, however, can infect eastern oysters and cause “Dermo” disease, which is highly detrimental to both natural and farmed oyster populations. The sympatric Perkinsus chesapeaki, initially isolated from infected M. arenaria clams, can also be present in oysters, and there is little evidence of pathogenicity in either clams or oysters. In this review, we discuss selected observations from our studies on the mechanisms of Perkinsus recognition that are mediated by galectin-carbohydrate interactions. We identified in the oyster two galectins that we designated CvGal1 and CvGal2, which strongly recognize P. marinus trophozoites. In the clam we also identified galectin sequences, and focused on one (that we named MaGal1) that also recognizes Perkinsus species. Here we describe the biochemical characterization of CvGal1, CvGal2, and MaGal1 with focus on the detailed study of the carbohydrate specificity, and the glycosylated moieties on the surfaces of the oyster hemocytes and the two Perkinsus species (P. marinus and P. chesapeaki). Our goal is to gain further understanding of the biochemical basis for the interactions that lead to recognition and opsonization of the Perkinsus trophozoites by the bivalve hemocytes. These basic studies on the biology of host-parasite interactions may contribute to the development of novel intervention strategies for parasitic diseases of biomedical interest.

F-type lectins (FTLs) are characterized by a fucose recognition domain (F-type lectin domain; FTLD) that displays a novel jellyroll fold ("F-type" fold) and unique carbohydrate- and calcium-binding sequence motifs. This novel lectin family comprises widely distributed proteins exhibiting single, double, or greater multiples of the FTLD, either tandemly arrayed or combined with other structurally and functionally distinct domains. Further, differences in carbohydrate specificity among tandemly arrayed FTLDs present in any FTL polypeptide subunit, together with the expression of multiple FTL isoforms in a single individual supports a striking diversity in ligand recognition. Functions of FTLs in self/nonself recognition include innate immunity, fertilization, microbial adhesion, and pathogenesis, among others, revealing an extensive structural/functional diversification. The taxonomic distribution of FTLDs is surprisingly discontinuous, suggesting that this lectin family has been subject to secondary loss, lateral transfer, and functional co-option along evolutionary lineages.

Soft-tissue bacterial infection can progress to severe sepsis and septic shock as a result of a disproportionate inflammatory response, characterized by an excessive release of cytokines and influx of immune cells. Reltecimod (previously known as AB103 or p2TA), a peptide derived from the T-cell receptor CD28, modulates the host immune response by targeting the co-stimulatory pathway, which is essential for the induction of multiple pro-inflammatory cytokines. Consequently, reltecimod has demonstrated beneficial effects against different bacterial infections, their exotoxins and endotoxins, and ionizing radiation. The dosing regimen of reltecimod was evaluated in three mouse models of infection. The effect of the number of reltecimod doses with respect to survival, cytokine/chemokine levels, and blood leukocyte profiles was assessed. Overall, mice treated with a single intravenous dose of reltecimod (5 mg/kg) at 1–2 h after infection showed significantly greater survival as compared with saline-treated controls. Mice treated with a second doses demonstrated improved survival compared with saline-treated controls. However, in all models of infection, administration of a single therapeutic dose of reltecimod was superior to two or multiple doses. Further examination showed that the single therapeutic dose of reltecimod was associated with an early (within 24 h) decrease in cytokine/chemokine levels and most circulating leukocyte subpopulations. A second dose of reltecimod did not improve these early positive effects and appeared to attenuate further changes. These results provided insight into the mechanism of action of reltecimod and established a basis for the dosing regimen utilized in clinical trials, where reltecimod is administered as a single dose.

Carbohydrate structures on the cell surface encode complex information that through specific recognition by carbohydrate-binding proteins (lectins) modulates interactions between cells, cells and the extracellular matrix, or mediates recognition of potential microbial pathogens. Galectins are a family of ß-galactoside-binding lectins, which are evolutionary conserved and have been identified in most organisms, from fungi to invertebrates and vertebrates, including mammals. Since their discovery in the 1970s, their biological roles, initially understood as limited to recognition of endogenous carbohydrate ligands in embryogenesis and development, have expanded in recent years by the discovery of their roles in tissue repair and regulation of immune homeostasis. More recently, evidence has accumulated to support the notion that galectins can also bind glycans on the surface of potentially pathogenic microbes, and function as recognition and effector factors in innate immunity, thus establishing a new paradigm. Furthermore, some parasites 'subvert' the recognition roles of the vector/host galectins for successful attachment or invasion. These recent findings have revealed a striking functional diversification in this structurally conserved lectin family.

LvMKK4, a homologue of the mammalian mitogen-activated protein kinase kinase 4 (MKK4), was isolated and identified from Litopenaeus vannamei in the present study. The full-length cDNA of LvMKK4 is 1947 bp long, with an open reading frame (ORF) of 1185 bp encoding a putative protein of 388 amino acids. LvMKK4 contains several characteristic domains such as D domain, SIAKT motif and kinase domain, all of which are conserved in MAP kinase kinase family. Like mammalian MKK4 but not Drosophila MKK4, LvMKK4 could bind to, phosphorylate and activate p38 MAPK, which provided some insights into the signal transduction mechanism of MKK4-p38 cascade in invertebrates. Our real-time PCR data indicated that LvMKK4 was ubiquitously expressed in all tested tissues and extraordinarily abundant in muscle. Dual luciferase reporter assays in Drosophila S2 cells revealed that LvMKK4 activated the transcription of antimicrobial peptide genes (AMPs), including Drosophila Attacin A, Drosomycin, and shrimp Penaeidins. Additionally, LvMKK4 was up-regulated in both intestine and hepatopancreas by a variety of inflammatory stimuli including LPS, Vibrio parahaemolyticus, Staphhylococcu saureus, Poly (I: C) and white spot syndrome virus. Furthermore, RNAi-mediated knockdown of LvMKK4 enhanced the sensitivity of L. vannamei to V. parahaemolyticus infection. These findings suggested that LvMKK4 played an important role in anti-bacterial response and could be a potential target for inflammation treatment.

Sialic acids (Sias) play multifarious roles in immunity, and likely contribute to inflammation seen in xenograft failure. The ‘net’ cellular sialyation state depends on many things including transient loss of Sia moieties with exposure of subterminal galactosyl structures (mediated by sialyltransferases and sialidases [neuraminidases]), which can function as receptors for extracellular lectins, such as galectins. Here, we report the role of Sias in modulating adhesion of human neutrophils (NΦ’s) to pig endothelial cells. Human (hAECs) and pig (pAECs) aortic endothelial cells were pretreated with Clostridium perfringens neuraminidase (NA, to remove Sias), human tumor necrosis factor (hTNF, 25 ug/ml), or both. CalceinAM-labelled human NΦ’s were treated with rhIL-8, NA, or N-acetyllactosamine (LacNAc, a galectin inhibitor). NΦ adhesion was measured and expressed as percent adhesion. NA treatment of AECs increased NΦ adhesion to pig (36.3% vs 22.4%) more than to human (24.2% vs 20.7%) AECs. NΦ adhesion was similarly increased by TNF activation of AECs in static and flow conditions. hIL-8 dramatically increased NΦ adhesion to pAECs (76.5%, p<.001) and hAECs (45.3%, p<.001), with the highest adhesion observed with NA-treated pAECs and hIL-8-activated NΦ’s. Pre-exposure of the NΦ’s to LacNAc prevented their galectin-mediated adhesion to galactose moieties exposed on the NA-treated pAECs (13.7%, p<.0001). In summary, AEC de-sialylation promotes xenogenic NΦ adhesion, an effect completely inhibited by pre-incubation of Nf’s with LacNAc. These results suggest the inhibition of sialidase activity or galectin binding as promising therapeutic approaches to limit inflammation in xenogeneic organ injury.