A tandem-repeat galectin-4 from Nile tilapia (Oreochromis niloticus) is involved in immune response to bacterial infection via mediating pathogen recognition and opsonization.

Galectins are the family of carbohydrate-binding proteins that participate in host-pathogen interaction. In this study, a galectin-4 homolog (OnGal-4) from Nile tilapia (Oreochromis niloticus) was characterized. The open reading frame of OnGal-4 was 1194 bp, encoding a peptide of 397 amino including two CRD regions and two carbohydrate recognition sites. OnGal-4 mRNA was expressed in all examined tissues with the highest level in spleen. After Streptococcus agalactiae (S.agalactiae) challenge, the OnGal-4 expression was up-regulated in the spleen, head kidney, brain, and monocytes/macrophages (Mo/MΦ). The in vitro experiments showed that recombinant OnGal-4 (rOnGal-4) protein could bind and agglutinate S.agalactiae and A.hydrophila. Also, rOnGal-4 could induce cytokines expressions and increased bactericidal activity of Mo/MΦ. Further in vivo analysis indicated that OnGal-4 overexpression could protect O.niloticus from S.agalactiae infection through modulating inflammation response. Our study suggested that OnGal-4 could improve immune response against bacterial infection by mediating pathogen recognition and opsonization.

How presence of a signal peptide affects human galectins-1 and -4: Clues to explain common absence of a leader sequence among adhesion/growth-regulatory galectins.

BACKGROUND: Galectins are multifunctional effectors, which all share absence of a signal sequence. It is not clear why galectins belong to the small set of proteins, which avoid the classical export route. METHODS: Products of recombinant galectin expression in P. pastoris were analyzed by haemagglutination, gel filtration and electrophoresis and lectin blotting as well as mass spectrometry on the level of tryptic peptides and purified glycopeptides(s). Density gradient centrifugation and confocal laser scanning microscopy facilitated localization in transfected human and rat cells, proliferation assays determined activity as growth mediator. RESULTS: Directing galectin-1 to the classical secretory pathway in yeast produces N-glycosylated protein that is active. It cofractionates and -localizes with calnexin in human cells, only Gal-4 is secreted. Presence of N-glycan(s) reduces affinity of cell binding and growth regulation by Gal-1. CONCLUSIONS: Folding and activity of a galectin are maintained in signal-peptide-directed routing, N-glycosylation occurs. This pathway would deplete cytoplasm and nucleus of galectin, presence of N-glycans appears to interfere with lattice formation. GENERAL SIGNIFICANCE: Availability of glycosylated galectins facilitates functional assays to contribute to explain why galectins invariably avoid classical routing for export.

Expression profiling and microbial ligand binding analysis of galectin-4 in turbot (Scophthalmus maximus L.).

Galectins are a family of galactoside-binding proteins with an affinity for ß-galactosides, involved in mediating fundamental processes including development, inflammation, cell migration and apoptosis. Galectin-4 is a member of tendem-repeat galectins, plays vital roles in intestinal epithelial barrier. Here, one galectin-4 gene was captured in turbot (SmLgals4) contains a 1197 bp open reading frame (ORF). In comparison to other species, SmLgals4 showed the highest similarity and identity both to large yellow croaker. The genomic structure analysis showed that SmLgals4 had conserved exons in the CRD domains compared to other vertebrate species. The syntenic analysis revealed that galectin-4 had the same neighboring genes across all the selected species, which suggested the synteny encompassing galectin-4 region during vertebrate evolution. Subsequently, SmLgals4 was widely expressed in all the examined tissues, with the highest expression level in intestine and the lowest expression level in skin. In addition, SmLgals4 was significantly down-regulated in intestine following both Gram-negative bacteria Vibrio anguillarum, and Gram-positive bacteria Streptococcus iniae immersion challenge. Finally, the rSmLgals4 showed strong binding ability to all the examined microbial ligands. Taken together, our results suggested SmLgals4 plays vital roles in fish intestinal immune responses against infection, but the detailed roles of galectin-4 in teleost are still lacking, further studies are needed to be carried out to characterize whether galectin-4 plays similar roles in teleost intestinal immunity.

Full-length model of the human galectin-4 and insights into dynamics of inter-domain communication.

Galectins are proteins involved in diverse cellular contexts due to their capacity to decipher and respond to the information encoded by ß-galactoside sugars. In particular, human galectin-4, normally expressed in the healthy gastrointestinal tract, displays differential expression in cancerous tissues and is considered a potential drug target for liver and lung cancer. Galectin-4 is a tandem-repeat galectin characterized by two carbohydrate recognition domains connected by a linker-peptide. Despite their relevance to cell function and pathogenesis, structural characterization of full-length tandem-repeat galectins has remained elusive. Here, we investigate galectin-4 using X-ray crystallography, small- and wide-angle X-ray scattering, molecular modelling, molecular dynamics simulations, and differential scanning fluorimetry assays and describe for the first time a structural model for human galectin-4. Our results provide insight into the structural role of the linker-peptide and shed light on the dynamic characteristics of the mechanism of carbohydrate recognition among tandem-repeat galectins.

Structural characterisation of human galectin-4 N-terminal carbohydrate recognition domain in complex with glycerol, lactose, 3'-sulfo-lactose, and 2'-fucosyllactose.

Galectin-4 is a tandem-repeat galectin with two distinct carbohydrate recognition domains (CRD). Galectin-4 is expressed mainly in the alimentary tract and is proposed to function as a lipid raft and adherens junction stabilizer by its glycan cross-linking capacity. Galectin-4 plays divergent roles in cancer and inflammatory conditions, either promoting or inhibiting each disease progression, depending on the specific pathological condition. The study of galectin-4's ligand-binding profile may help decipher its roles under specific conditions. Here we present the X-ray structures of human galectin-4 N-terminal CRD (galectin-4N) bound to different saccharide ligands. Galectin-4's overall fold and its core interactions to lactose are similar to other galectin CRDs. Galectin-4N recognises the sulfate cap of 3'-sulfated glycans by a weak interaction through Arg45 and two water-mediated hydrogen bonds via Trp84 and Asn49. When galectin-4N interacts with the H-antigen mimic, 2'-fucosyllactose, an interaction is formed between the ring oxygen of fucose and Arg45. The extended binding site of galectin-4N may not be well suited to the A/B-antigen determinants, α-GalNAc/α-Gal, specifically due to clashes with residue Phe47. Overall, galectin-4N favours sulfated glycans whilst galectin-4C prefers blood group determinants. However, the two CRDs of galectin-4 can, to a less extent, recognise each other's ligands.

Recombinant expression, purification and preliminary biophysical and structural studies of C-terminal carbohydrate recognition domain from human galectin-4.

Galectin-4 (Gal4), a tandem-repeat type galectin, is expressed in healthy epithelium of the gastrointestinal tract. Altered levels of Gal4 expression are associated with different types of cancer, suggesting its usage as a diagnostic marker as well as target for drug development. The functional data available for this class of proteins suggest that the wide spectrum of cellular activities reported for Gal4 relies on distinct glycan specificity and structural characteristics of its two carbohydrate recognition domains. In the present work, two independent constructs for recombinant expression of the C-terminal domain of human galectin-4 (hGal4-CRD2) were developed. His6-tagged and untagged recombinant proteins were overexpressed in Escherichia coli, and purified by affinity chromatography followed by gel filtration. Correct folding and activity of hGal4-CRD2 were assessed by circular dichroism and fluorescence spectroscopies, respectively. Diffraction quality crystals were obtained by vapor-diffusion sitting drop setup and the crystal structure of CRD2 was solved by molecular replacement techniques at 1.78 Å resolution. Our work describes the development of important experimental tools that will allow further studies in order to correlate structure and binding properties of hGal4-CRD2 and human galectin-4 functional activities.

Structural characterization of human galectin-4 C-terminal domain: elucidating the molecular basis for recognition of glycosphingolipids, sulfated saccharides and blood group antigens.

Human galectin-4 is a lectin that is expressed mainly in the gastrointestinal tract and exhibits metastasis-promoting roles in some cancers. Its tandem-repeat nature exhibits two distinct carbohydrate recognition domains allowing crosslinking by simultaneous binding to sulfated and non-sulfated (but not sialylated) glycosphingolipids and glycoproteins, facilitating stabilization of lipid rafts. Critically, galectin-4 exerts favourable or unfavourable effects depending upon the cancer. Here we report the first X-ray crystallographic structural information on human galectin-4, specifically the C-terminal carbohydrate recognition domain of human (galectin-4C) in complex with lactose, lactose-3'-sulfate, 2'-fucosyllactose, lacto-N-tetraose and lacto-N-neotetraose. These structures enable elucidation of galectin-4C binding fine-specificity towards sulfated and non-sulfated lacto- and neolacto-series sphingolipids as well as to human blood group antigens. Analysis of the lactose-3'-sulfate complex structure shows that galectin-4C does not recognize the sulfate group using any specific amino acid, but binds the ligand nonetheless. Complex structures with lacto-N-tetraose and lacto-N-neotetraose displayed differences in binding interactions exhibited by the non-reducing-end galactose. That of lacto-N-tetraose points outward from the protein surface whereas that of lacto-N-neotetraose interacts directly with the protein. Recognition patterns of human galectin-4C towards lacto- and neolacto-series glycosphingolipids are similar to those of human galectin-3; however, detailed scrutiny revealed differences stemming from the extended binding site that offer distinction in ligand profiles of these two galectins. Structural characterization of the complex with 2'-fucosyllactose, a carbohydrate with similarity to the H antigen, and molecular dynamics studies highlight structural features that allow specific recognition of A and B antigens, whilst a lack of interaction with the 2'-fucose of blood group antigens was revealed. DATABASE ACCESSION CODES: 4YLZ, 4YM0, 4YM1, 4YM2, 4YM3.

Expression of mucin 1 possessing a 3'-sulfated core1 in recurrent and metastatic breast cancer.

Breast cancer is the most frequent cancer threatening the lives of women between the ages of 30 and 64. The cancer antigen 15-3 assay (CA15-3) has been widely used for the detection of breast cancer recurrence; however, its sensitivity and specificity are inadequate. We previously found that the breast cancer cell line YMBS secretes mucin 1 possessing 3'-sulfated core1 (3Score1-MUC1) into the medium. Therefore, we here evaluated whether 3Score1-MUC1 is secreted into the blood streams of breast cancer patients, and whether it can serve as an improved breast cancer marker. We developed a lectin-sandwich immunoassay, called Gal4/MUC1, using a 3'-sulfated core1-specific galectin-4 and a MUC1 monoclonal antibody. Using the Gal4/MUC1 assay method, we found that 3Score1-MUC1 was profoundly expressed in the blood streams of patients with recurrent and/or metastatic breast cancer. The positive ratio of the Gal4/MUC1 assay was higher than that of the CA15-3 assay in both primary (n = 240) and relapsed (n = 43) patients, especially in the latter of which the positive ratio of Gal4/MUC1 was 86%. whereas that of CA15-3 was 47%. Furthermore, serum Gal4/MUC1 levels could more sensitively reflect the recurrence of primary breast cancer patients after surgery. Therefore, the Gal4/MUC1 assay should be an excellent alternative to the CA15-3 tumor marker for tracking the recurrence and metastasis of breast cancer.

Combining glycocluster synthesis with protein engineering: an approach to probe into the significance of linker length in a tandem-repeat-type lectin (galectin-4).

Complementarity in lectin-glycan interactions in situ is assumed to involve spatial features in both the lectin and the glycan, giving a functional meaning to structural aspects of the lectin beyond its carbohydrate-binding site. In combining protein engineering with glycocluster synthesis, it is shown that the natural linker length of a tandem-repeat-type human lectin (galectin-4) determines binding properties in two binding assays (using surface-presented glycoprotein and cell surface assays). The types of glycocluster tested included bivalent lactosides based on tertiary amides of terephthalic, isophthalic, 2,6-naphthalic and oxalic acids as well as bivalent H(type 2) trisaccharides grafted on secondary/tertiary terephthalamides and two triazole-linker-containing cores. The presented data reveal a marked change in susceptibility to the test compounds when turning the tandem-repeat-type to a proto-type-like display. The testing of glycoclusters is suggested as a general strategy to help to delineate the significance of distinct structural features of lectins beyond their contact sites to the glycan.

Phosphorylation and externalization of galectin-4 is controlled by Src family kinases.

Galectin-4 is a cytosolic protein that lacks a signal sequence but is externalized and binds to 3-O-sulfated glycoconjugates extracellularly. The mechanism of subcellular localization and externalization of galectin-4 has not yet been determined. A preliminary experiment using pervanadate (PV) showed that galectin-4 is tyrosine-phosphorylated in cells and suggested that Src kinases are involved. Cell transfection with galectin-4 and active Src plasmids showed that galectin-4 can be tyrosine phosphorylated by members of the Src kinase family. The C-terminal peptide YVQI of galectin-4 was found to play an important role in its tyrosine phosphorylation, and the SH2 domains of Src and SHP2 were found to bind to this peptide. Immunofluorescence analysis showed that galectin-4 and phosphorylated proteins were intensely stained in the area of membrane protrusions of PV-treated or Src-activated cells. Furthermore, MUC1 derived from NUGC-4 cells was observed to bind to galectin-4, and externalization of the bound molecules from the cell to the medium increased in the hyperphosphorylated condition. Study of the transfection of the mutant galectin-4 which lacks the C-terminal peptide revealed that the phosphorylation status is important for externalization of galectin-4. These results suggest that externalization of galectin-4 can be regulated by signaling molecules and that it may function intracellularly as an adaptor protein serving to modulate the trafficking of glycoproteins.

Expression patterns suggest that despite considerable functional redundancy, galectin-4 and -6 play distinct roles in normal and damaged mouse digestive tract.

The galectin-4 protein is mostly expressed in the digestive tract and is associated with lipid raft stabilization, protein apical trafficking, wound healing, and inflammation. While most mammalian species, including humans, have a single Lgals4 gene, some mice have two paralogues: Lgals4 and Lgals6. So far, their significant similarities have hindered the analysis of their respective expression and function. We took advantage of two antibodies that discriminate between the galectin-4 and galectin-6 proteins to document their patterns of expression in the normal and the dextran sodium sulfate (DSS)-damaged digestive tract in the mouse. In the normal digestive tract, their pattern of expression from tongue to colon is quite similar, which suggests functional redundancy. However, the presence of galectin-4, but not galectin-6, in the lamina propria of the DSS-damaged colon, its association with luminal colonic bacteria, and differences in subcellular localization of these proteins suggest that they also have distinct roles in the normal and the damaged mouse digestive tract. Our results provide a rare example of ancestral and derived functions evolving after tandem gene duplication.

Comparative study of the glycan specificities of cell-bound human tandem-repeat-type galectin-4, -8 and -9.

Adhesion/growth-regulatory galectins (gals) exert their functionality by the cis/trans-cross-linking of distinct glycans after initial one-point binding. In order to define the specificity of ensuing association events leading to cross-linking, we recently established a cell-based assay using fluorescent glycoconjugates as flow cytometry probes and tested it on two human gals (gal-1 and -3). Here we present a systematic study of tandem-repeat-type gal-4, -8 and -9 loaded on Raji cells resulting in the following key insights: (i) all three gals bound to oligolactosamines; (ii) binding to ligands with Galß1-3GlcNAc or Galß1-3GalNAc as basic motifs was commonly better than that to canonical Galß1-4GlcNAc; (iii) all three gals bound to 3'-O-sulfated and 3'-sialylated disaccharides mentioned above better than that to parental neutral forms and (iv) histo-blood group ABH antigens were the highest affinity ligands in both the cell and the solid-phase assay. Fine specificity differences were revealed as follows: (i) gal-8 and -9, but not gal-4, bound to disaccharide Galß1-3GlcNAc; (ii) increase in binding due to negatively charged substituents was marked only in the case of gal-4 and (iii) gal-4 and -8 bound preferably to histo-blood group A glycans, whereas gal-9 targeted B-type glycans. Experiments with single carbohydrate recognition domains (CRDs) of gal-4 showed that the C-CRD preferably bound to ABH glycans, whereas the N-CRD associated with oligolactosamines. In summary, the comparative analysis disclosed the characteristic profiles of glycan reactivity for the accessible CRD of cell-bound gals. These results indicate the distinct sets of functionality for these three members of the same subgroup of human gals.

An integrated computational analysis of the structure, dynamics, and ligand binding interactions of the human galectin network.

Galectins, a family of evolutionarily conserved animal lectins, have been shown to modulate signaling processes leading to inflammation, apoptosis, immunoregulation, and angiogenesis through their ability to interact with poly-N-acetyllactosamine-enriched glycoconjugates. To date 16 human galectin carbohydrate recognition domains have been established by sequence analysis and found to be expressed in several tissues. Given the divergent functions of these lectins, it is of vital importance to understand common and differential features in order to search for specific inhibitors of individual members of the human galectin family. In this work we performed an integrated computational analysis of all individual members of the human galectin family. In the first place, we have built homology-based models for galectin-4 and -12 N-terminus, placental protein 13 (PP13) and PP13-like protein for which no experimental structural information is available. We have then performed classical molecular dynamics simulations of the whole 15 members family in free and ligand-bound states to analyze protein and protein-ligand interaction dynamics. Our results show that all galectins adopt the same fold, and the carbohydrate recognition domains are very similar with structural differences located in specific loops. These differences are reflected in the dynamics characteristics, where mobility differences translate into entropy values which significantly influence their ligand affinity. Thus, ligand selectivity appears to be modulated by subtle differences in the monosaccharide binding sites. Taken together, our results may contribute to the understanding, at a molecular level, of the structural and dynamical determinants that distinguish individual human galectins.

Structure of the mouse galectin-4 N-terminal carbohydrate-recognition domain reveals the mechanism of oligosaccharide recognition.

Galectin-4, a member of the tandem-repeat subfamily of galectins, participates in cell-membrane interactions and plays an important role in cell adhesion and modulation of immunity and malignity. The oligosaccharide specificity of the mouse galectin-4 carbohydrate-recognition domains (CRDs) has been reported previously. In this work, the structure and binding properties of the N-terminal domain CRD1 were further investigated and the crystal structure of CRD1 in complex with lactose was determined at 2.1 Šresolution. The lactose-binding affinity was characterized by fluorescence measurements and two lactose-binding sites were identified: a high-affinity site with a K(d) value in the micromolar range (K(d1) = 600 ± 70 µM) and a low-affinity site with K(d2) = 28 ± 10 mM.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the N-terminal carbohydrate-recognition domain of human galectin-4.

Galectin-4 is a tandem-repeat-type galectin that is expressed in the epithelium of the alimentary tract from the tongue to the large intestine. Additionally, strong expression of galectin-4 can also be induced in cancers in other tissues, including the breast and liver. In order to explore its potential as a target for anticancer drug design, elucidation of the structural basis of the carbohydrate-binding specificities of galectin-4 has been focused on. As an initial step, the N-terminal carbohydrate-recognition domain of human galectin-4 (hGal4-CRD-1) has been successfully crystallized using the vapour-diffusion technique, a complete data set has been collected to 2.2 A resolution and the structure has been solved by the molecular-replacement technique. The crystals belonged to space group P6(1)22, with unit-cell parameters a = b = 71.25, c = 108.66 A. The asymmetric unit contained one molecule of hGal4-CRD-1, with a V(M) value of 2.34 A(3) Da(-1) and a solvent content of 47.51%.

Crystallization and preliminary X-ray diffraction analysis of mouse galectin-4 N-terminal carbohydrate recognition domain in complex with lactose.

Galectin-4 is thought to play a role in the process of tumour conversion of cells of the alimentary tract and the breast tissue; however, its exact function remains unknown. With the aim of elucidating the structural basis of mouse galectin-4 (mGal-4) binding specificity, we have undertaken X-ray analysis of the N-terminal domain, CRD1, of mGal-4 in complex with lactose (the basic building block of known galectin-4 carbohydrate ligands). Crystals of CRD1 in complex with lactose were obtained using vapour-diffusion techniques. The crystals belong to tetragonal space group P42(1)2 with unit-cell parameters a = 91.1, b = 91.16, c = 57.10 A and preliminary X-ray diffraction data were collected to 3.2 A resolution. An optimized crystallization procedure and cryocooling protocol allowed us to extend resolution to 2.1 A. Structure refinement is currently under way; the initial electron-density maps clearly show non-protein electron density in the vicinity of the carbohydrate binding site, indicating the presence of one lactose molecule. The structure will help to improve understanding of the binding specificity and function of the potential colon cancer marker galectin-4.

Protein subtype-targeting through ligand epimerization: talose-selectivity of galectin-4 and galectin-8.

A series of O2 and O3-derivatized methyl beta-d-talopyranosides were synthesized and evaluated in vitro as inhibitors of the galactose-binding galectin-1, -2, -3, -4 (N- and C-terminal domains), 8 (N-terminal domain), and 9 (N-terminal domain). Galectin-4C and 8N were found to prefer the d-talopyranose configuration to the natural ligand d-galactopyranose configuration. Derivatization at talose O2 and/or O3 provided selective submillimolar inhibitors for these two galectins.

Recognition mechanism of galectin-4 for cholesterol 3-sulfate.

Galectin-4 binds to glycosphingolipids carrying 3-O-sulfated Gal residues, and it co-localizes on the cell surface of human colonic adenocarcinoma cells with glycosphingolipids carrying SO(-)(3)-->3Galbeta1-->3(GalNAc) residues (Ideo, H., Seko, A., and Yamashita, K. (2005) J. Biol. Chem. 280, 4730-4737). In the present study, it was found that galectin-4 also binds to cholesterol 3-sulfate, which has no beta-galactoside moiety. This characteristic of galectin-4 is unique within the galectin family. The site-directed mutated galectin-4-R45A had diminished binding ability toward cholesterol 3-sulfate, suggesting that Arg(45) of galectin-4 is indispensable for cholesterol 3-sulfate recognition. Gel filtration and chemical cross-linking experiments revealed that some galectin-4 exists as dimers, and this multivalency seemed to enhance its avidity for cholesterol 3-sulfate binding. Cholesterol 3-sulfate and sulfatide co-existed with galectin-4 in detergent-insoluble fractions of porcine esophagus and intestine, respectively. These results suggested that not only sulfated glycosphingolipids but also cholesterol 3-sulfate are endogenous ligands for galectin-4 in vivo.

Galectins bind to the multivalent glycoprotein asialofetuin with enhanced affinities and a gradient of decreasing binding constants.

Our previous isothermal titration microcalorimetry (ITC) studies of the binding of synthetic multivalent carbohydrates to the Man/Glc-specific lectins concanavalin A (ConA) and Dioclea grandiflora lectin (DGL) showed negative binding cooperativity that was due to the carbohydrate ligands and not the proteins [Dam, T. K., et al. (2002) Biochemistry 41, 1351-1358]. The negative cooperativity was associated with the decreasing functional valence of the carbohydrates upon progressive binding of their epitopes. The present study also shows negative cooperativity in the ITC binding data of asialofetuin (ASF), a glycoprotein that possesses nine LacNAc epitopes, to galectin-1, -2, -3, -4, -5, and -7, and truncated, monomer versions of galectin-3 and -5, which are members of a family of animal lectins. Although the observed K(a) values for binding of ASF to the galectins and two truncated forms are only 50-80-fold greater than that of LacNAc, analysis of the data in terms of the relationship between the observed macroscopic free energy of binding and the decreasing microscopic free energies of binding of the epitopes shows that the first LacNAc epitope of ASF binds with approximately 6000-fold higher affinity than the last epitope. Thus, the microscopic binding constants of the galectins for the first epitope(s) of ASF are in the nanomolar range, with a gradient of decreasing binding constants of the remaining epitopes. The results indicate that the above galectins bind with fractional, high affinities to multivalent glycoproteins such as ASF, independent of the quaternary structures of the galectins. These findings have important implications for the binding of galectins to multivalent carbohydrate receptors.