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PubMed1998

Evidence for subsites in the galectins involved in sugar binding at the nonreducing end of the central galactose of oligosaccharide ligands: sequence analysis, homology modeling and mutagenesis studies of hamster galectin-3.

A model of the carbohydrate recognition domain CRD, residues 111-245, of hamster galectin-3 has been made using homology modeling and dynamics minimization methods. The model is based on the known x-ray structures of bovine galectin-1 and human galectin-2. The oligosaccharides NeuNAc-alpha2,3-Gal-beta1,4-Glc and GalNAc-alpha1, 3-[Fuc-alpha1,2]-Gal-beta1,4-Glc, known to be specific high-affinity ligands for galectin-3, as well as lactose recognized by all galectins were docked in the galectin-3 CRD model structure and a minimized binding conformation found in each case. These studies indicate a putative extended carbohydrate-binding subsite in the hamster galectin-3 involving Arg139, Glu230, and Ser232 for NeuNAc-alpha2,3-; Arg139 and Glu160 for fucose-alpha1,2-; and Arg139 and Ile141 for GalNAc-alpha1,3- substituents on the primary galactose. Each of these positions is variable within the whole galectin family. Two of these residues, Arg139 and Ser232, were selected for mutagenesis to probe their importance in this newly identified putative subsite. Residue 139 adopts main-chain dihedral angles characteristic of an isolated bridge structural feature, while residue 232 is the C-terminal residue of beta-strand-11, and is followed immediately by an inverse gamma-turn. A systematic series of mutant proteins have been prepared to represent the residue variation present in the aligned sequences of galectins-1, -2, and -3. Minimized docked models were generated for each mutant in complex with NeuNAc-alpha2,3-Gal-beta1,4-Glc, GalNAc-alpha1, 3-[Fuc-alpha1,2]-Gal-beta1,4- Glc, and Gal-beta1,4-Glc. Correlation of the computed protein-carbohydrate interaction energies for each lectin-oligosaccharide pair with the experimentally determined binding affinities for fetuin and asialofetuin or the relative potencies of lactose and sialyllactose in inhibiting binding to asiolofetuin is consistent with the postulated key importance of Arg139 in recognition of the extended sialylated ligand.

Amino Acid Sequence↗

PubMed2003

Biochemical and molecular characterization of galectins from zebrafish (Danio rerio): notochord-specific expression of a prototype galectin during early embryogenesis.

Galectins are a family of beta-galactoside-binding lectins that on synthesis are either translocated into the nucleus or released to the extracellular space. Their developmentally regulated expression, extracellular location, and affinity for extracellular components (such as laminin and fibronectin) suggest a role in embryonic development, but so far this has not been unequivocally established. Zebrafish constitute an ideal model for developmental studies because of their external fertilization, transparent embryos, rapid growth, and availability of a large collection of mutants. As a first step in addressing the biological roles in zebrafish embryogenesis, we identified and characterized members of the three galectin types: three protogalectins (Drgal1-L1, Drgal1-L2, Drgal1-L3), one chimera galectin (Drgal3), and one tandem-repeat galectin (Drgal9-L1). Like mammalian prototype galectin-1, Drgal1-L2 preferentially binds to N-acetyllactosamine. Genomic structure of Drgal1-L2 revealed four exons, with the exon-intron boundaries conserved with the mammalian galectin-1. Interestingly, this gene also encodes an alternatively spliced form of Drgal1-L2 that lacks eight amino acids near the carbohydrate-binding domain. Zebrafish galectins exhibited distinct patterns of temporal expression during embryo development. Drgal1-L2 is expressed postbud stage, and its expression is strikingly specific to the notochord. In contrast, Drgal1-L1 is expressed maternally in the oocytes. Drgal1-L3, Drgal3, and Drgal9-L1 are expressed both maternally and zygotically, ubiquitously in the adult tissues. The distinct temporal and spatial patterns of expression of members of the zebrafish galectin repertoire suggest that each may play distinct biological roles during early embryogenesis.

Amino Acid Sequence↗

PubMed2001

Galectin-8: a complex sub-family of galectins (Review).

Galectins are animal lectins, that can specifically bind beta-galactosides. Twelve galectins have been described in vertebrates, belonging to three different groups: prototype, tandem-repeat and chimeric. These proteins seem to be involved in cellular interactions and neoplastic transformations. We present an overview of a particular galectin member: galectin-8. This galectin, which has been intensively studied over the last six years, presents a particular type of gene regulation. It is widely expressed in tumoral tissues and seems to be involved in integrin-like cell interactions. Studies show that the LGALS8 gene encodes for almost seven mRNAs by alternative splicing pathways and various polyadenylation sites. These mRNAs could encode for six isoforms of galectin-8, of which three belong to the tandem-repeat galectin group (with two carbohydrate binding domains) and the three others to the prototype group (one carbohydrate binding domain). All these isoforms seem to be differentially expressed in various tumoral cells. This untypical galectin-8 subfamily seems to have a complex expression regulation, that could be involved in cancer phenomena.

Alternative Splicing↗

PubMedDate not supplied

Association of galectin-1- but not galectin-3-dependent parameters with proliferation activity in human neuroblastomas and small cell lung carcinomas.

BACKGROUND: Galectins, a family of animal lectins binding beta-galactosides, are involved in growth regulation of diverse cell types in vitro, even harboring the potential to act as biphasic modulators with cell type selectivity. Owing to this capacity they might affect tumor growth when expression is adapted adequately. MATERIALS AND METHODS: To determine galectin-1-/-3- related features in routinely-fixed sections of two tumor types with poor prognosis (neuroblastoma and small cell lung carcinoma), immuno- and lectin histochemistry with specific antibodies and labeled galectins was performed. RESULTS: In comparison to previously studied tissue culture models, galectin-3 was frequently present, documenting occurrence of discrepancies between tumor models and clinical material for this protein. Cytoplasmic staining with galectin-1 and its antibody coincides with the proliferative activity of positive tumor cells determined by the MIB-1 monoclonal antibody. No statistical correlation was seen for galectin-3. CONCLUSION: These results encourage further cell biological studies to assess a regulatory role of galectin-1 on cell growth in vitro as a model for interfering with tumor proliferation by modulating expression of this type of endogenous effector(s).

Antigens, Differentiation↗

PubMed2003

Specific recognition of Leishmania major poly-beta-galactosyl epitopes by galectin-9: possible implication of galectin-9 in interaction between L. major and host cells.

Leishmania parasites are the causative agents of leishmaniasis, manifesting itself in a species-specific manner. The glycan epitopes on the parasite are suggested to be involved in the Leishmania pathogenesis. One of such established species-unique glycan structures is the poly-beta-galactosyl epitope (Galbeta1-3)n found on L. major, which can develop cutaneous infections with strong inflammatory responses. Interestingly, the polygalactosyl epitope is also suggested to be involved in the development of the parasites in its host vector, sand fly. Thus, the recognition of the galactosyl epitope by lectins expressed in host or sand fly should be implicated in the species-specific manifestations of leishmaniasis and in the parasite life cycle, respectively. We recently reported that one host beta-galactoside-binding protein, galectin-3, can distinguish L. major from the other species through its binding to the poly-beta-galactosyl epitope, proposing a role for galectin-3 as an immunomodulator that could influence the L. major-specific immune responses in leishmaniasis. Here we report that galectin-9 can also recognize L. major by binding to the L. major-specific polygalactosyl epitope. Frontal affinity analysis with different lengths of poly-beta-galactosyllactose revealed that the galectin-9 affinity for polygalactose was enhanced in proportion to the number of Galbeta1-3 units present. Even though both galectins have comparable affinities toward the polygalactosyl epitopes, only galectin-9 can promote the interaction between L. major and macrophages, suggesting distinctive roles for the galectins in the L. major-specific development of leishmaniasis in the host.

Animals↗

PubMed1999

galectin-1 and galectin-3 expression in human bladder transitional-cell carcinomas.

Galectin-1 and galectin-3 are galactoside-binding proteins involved in different steps of tumor progression and potential targets for therapy. We have investigated the expression of these galectins in 38 human bladder transitional-cell carcinomas of different histological grade and clinical stage and in 5 normal urothelium samples. Galectin-1 mRNA levels were highly increased in most high-grade tumors compared with normal bladder or low-grade tumors. Western blot and immuno-histochemical analysis of normal and neoplastic tissues revealed a higher content of galectin-1 in tumors. Galectin-3 mRNA levels were also increased in most tumors compared with normal urothelium, but levels were comparable among tumors of different histological grade.

Adult↗

PubMed1999

Galectin-1 and galectin-3 expression in human prostate tissue and prostate cancer.

Galectin-1 and galectin-3, two beta-galactoside-binding proteins, have been suggested to play a role in the development and progression of cancer. We have studied the expression of these molecules in normal human prostate tissue and prostate adenocarcinoma. Immunohistochemistry was used to examine formalin-fixed, paraffin-embedded sections of seven normal human prostates, eight cases of prostatic intraepithelial neoplasia (PIN), 20 primary adenocarcinomas of the prostate, and 12 prostate cancer metastases. Galectin-1 was expressed in most cases of all four histologic types. In contrast, galectin-3 expression was significantly decreased in primary carcinoma and metastatic disease compared with normal and premalignant tissue. Galectin-3 expression in primary tumors tended to be less than that of surrounding normal glands. We conclude that loss of galectin-3 expression may be associated with the progression of prostate cancer.

Adenocarcinoma↗

PubMed2000

Human epidermal Langerhans cells are selectively recognized by galectin-3 but not by galectin-1.

Langerhans cells are dendritic antigen-presenting cells residing predominantly in the epidermis. Since endogenous galactoside-binding lectins with the jelly-roll motif (galectins) are known to trigger cellular responses, including mediator release, we investigated by lectin histochemistry the cells' capacity to bind two common members of this family, i.e. galectin-1 and -3. Actually, surrounding keratinocytes express a high level of galectin-3, and these cells can be considered as donors of this lectin to Langerhans cells. Employing biotinylated galectin-1 and -3, and concomitantly an antibody against CD1a as a second marker, to visualize the position of Langerhans cells in the human epidermis, the expression of galectin-3-reactive glycoligands in contrast to the lack of binding of galectin-1 was observed. Although the functional consequences of this selectivity are unclear, these results reveal an example for differential cellular reactivity towards two related endogenous lectins.

Adult↗

PubMed2006

Regulatory mechanisms of galectin-9 and eotaxin-3 synthesis in epidermal keratinocytes: possible involvement of galectin-9 in dermal eosinophilia of Th1-polarized skin inflammation.

BACKGROUND: Skin eosinophilia is a common feature of allergic skin diseases, but it is unclear how epidermal and dermal eosinophil infiltration is controlled. To investigate regulation of localization of eosinophils in skin, we examined the regulatory mechanisms of expression of eosinophil-specific chemoattractants in dermal fibroblasts and epidermal keratinocytes. METHODS: We analyzed production of eotaxin, eotaxin-2, eotaxin-3 and galectin-9 by dermal fibroblasts and epidermal keratinocytes in response to several stimuli in vitro. RESULTS: Dermal fibroblasts produced eotaxin and eotaxin-3 in response to stimulation by interleukin (IL)-4 and/or tumor necrosis factor-alpha. Similarly, IL-4 stimulated epidermal keratinocytes to secrete eotaxin-3. However, we did not detect eotaxin mRNA expression or protein secretion by keratinocytes stimulated in vitro. Interferon (IFN)-gamma induced galectin-9 expression on dermal fibroblasts. Conversely, expression of galectin-9 on epidermal keratinocytes was dose-dependently inhibited by IFN-gamma. The immunohistochemical assays revealed that dermal fibroblasts (but not epidermal keratinocytes) in the lesional skin of psoriasis vulgaris (a Th1-polarized disease) express significant levels of galectin-9. CONCLUSION: Eotaxin-3 contributes to dermal and epidermal eosinophil infiltration in Th2-polarized skin inflammation in which IL-4 is produced. In contrast, IFN-gamma-dominated inflammation appears to mediate eosinophil extravasation into the dermis and eosinophil adhesion to dermal fibroblasts via galectin-9 in association with decreased chemoattractant activity of epidermal galectin-9. The present results reveal a novel mechanism of dermal eosinophilia in IFN-gamma-mediated skin inflammation, and reflect concerted chemoattractant production involving dermal and/or epidermal eosinophilia during changes in the local cytokine profile.

Animals↗

PubMed1997

Quantitation and histochemical localization of galectin-1 and galectin-1-reactive glycoconjugates in fetal development of bovine organs.

The display of cellular oligosaccharide chains is known to undergo marked developmental changes, as monitored histochemically with plant lectins. In conjunction with endogenous lectins respective ligand structures may have a functional role during fetal development. The assumption of a recognitive, functionally productive interplay prompts the study of the expression of a tissue lectin and of lectin-reactive glycoconjugates concomitantly. Focusing on common beta-galactosides as constituents of oligosaccharide chains and the predominant member of the family of galectins in mammals, namely galectin-1, the question therefore is addressed as to whether expression of lectin and lectin-reactive glycoconjugates exhibits alterations, assessed in three morphologically defined fetal stages and in adult bovine organs. Using a sandwich ELISA, the level of the rather ubiquitous galectin-1 is mostly increased in adult organs relative to respective fetal stages, except for the case of kidney. This developmental course is seen rather seldom, when the amounts of lectin-reactive glycoproteins or glycolipids are quantitated in solid-phase assays after tissue homogenization. Western blotting, combined with probing by labeled galectin-1, discloses primarily quantitative changes in the reactivity of individual glycoproteins. Performing the same assays on extract aliquots with a plant agglutinin, namely the galactoside-binding mistletoe lectin, whose fine specificity is different from galectin-1, its reduced extent of binding in solid-phase assays and the disparate profile of lectin-reactive glycoproteins reveal a non-uniform developmental alteration within the group of structural variants of beta-galactosides. Although sample preparation can affect ligand preservation and/or presentation and thus restricts the comparability of biochemical and histochemical results, especially for soluble reactants, the histochemical studies on frozen and paraffin-embedded sections of bovine heart, kidney and liver demonstrate that the localization of the galectin and of lectin-reactive epitopes can show a similar distribution, as seen in liver and heart, with organ-typical quantitative changes of a rather similar staining profile (heart, kidney) or notable changes in the spatial distribution (liver) in the course of development. This report emphasizes the potential value of combined monitoring of the lectin and its potential in vivo ligands to contribute to eventually unravel organ-related function(s) of a tissue lectin.

Animals↗

PubMed2026

Functions of tandem-repeat galectins and domain coordination governs galectin-4 activity in grass carp (Ctenopharyngodon idella).

Galectins are β-galactoside-binding lectins that play essential roles in innate immunity. Among them, tandem-repeat galectins (TrGals), typically composed of two distinct carbohydrate-recognition domains (CRDs) connected by a linker peptide, are well established as key regulators of pathogen recognition and host defense in mammals. However, their structural diversity and immunological functions in teleost fish remain poorly understood. In this study, five TrGals (Gal-4, Gal-8a, Gal-8b, Gal-9, and Gal-9like) were identified in grass carp. Sequence and structural analysis revealed that Gal-8a/b, Gal-9, and Gal-9like possess the canonical two-CRD architecture, whereas Gal-4 uniquely contains four highly similar tandem-repeat domains. All five TrGals were broadly expressed across examined tissues, with predominant expression in the liver. Upon Aeromonas hydrophila infection, Gal-4, Gal-8a, Gal-8b, and Gal-9 were rapidly up-regulated at early time points (3-6 h). To elucidate the functional significance of CRD number, recombinant full-length CiGal-4 (CiGal4-full) and three truncated variants containing one, two, or three CRDs (CiGal4-1CRD, CiGal4-2CRD, and CiGal4-3CRD) were generated and systematically characterized. All recombinant proteins contained the conserved β-sheet structure typical of galectin CRDs. Functional assays revealed that CiGal4-full displayed the strongest growth-inhibitory activity against all tested bacteria, whereas CiGal4-1CRD showed the weakest effect. Notably, CiGal4-2CRD exhibited the most potent bactericidal activity, surpassing the full-length protein, while CiGal4-3CRD showed no further enhancement. CiGal4-full and CiGal4-2CRD showed superior carbohydrate-binding activities compared with the other variants. Collectively, these results reveal that CRD copy number alone does not linearly determine galectin function. Instead, domain organization and conformational coordination are critical for optimizing antimicrobial activity. This study provides new insights into the structure-function relationships and evolutionary diversification of galectins in teleosts and highlights their potential as novel antimicrobial and immunomodulatory agents in aquaculture.

Animals↗

PubMed1999

Endogenous galectins and effect of galectin hapten inhibitors on the differentiation of the chick mesonephros.

Galectins are galactoside-binding lectins. In the mesonephros of the chick embryo, the 16-kDa galectin is abundant in the glomerular and tubular basement membranes where it colocalizes with fibronectin and laminin. To test whether galectin-glycoprotein interactions could play a role in mesonephric development, the effects of the galectin hapten inhibitors thiodigalactoside (TDG) and lactose on the differentiation of the cultured mesonephros were investigated. When compared to control saccharide-free or maltose-treated cultures, mesonephroi cultured in the presence of TDG and lactose exhibited defects in tissue organization. These included a distorted tubule shape, pseudo-stratification of the tubular epithelium, and detachment of glomerular podocytes from the basement membrane. The presence of molecular differentiation markers in the developing mesonephros was investigated. In vivo, expression of the epithelial-specific cell adhesion molecule E-cadherin is restricted to differentiated tubular epithelial cells, whereas the intermediate filament protein vimentin is present in mesonephrogenic mesenchyme and is undetectable in tubular epithelial cells. In mesonephroi cultured in the absence of sugars or in the presence of maltose, the expression pattern of these two marker molecules resembles that found in the mesonephros in vivo. In contrast, in the mesonephroi cultured in the presence of TDG and lactose, the epithelial tubular cells expressing E-cadherin also express vimentin. Re-expression of vimentin in the tubular epithelial cells could indicate a partial reversal to a mesenchymal phenotype. Results suggest that galectin-glycoprotein interactions in the basement membrane are important in the maintenance of the renal epithelial phenotype. Dev Dyn 1999;215:248-263.

Animals↗

PubMed2004

Galectin-3/AGE-receptor 3 knockout mice show accelerated AGE-induced glomerular injury: evidence for a protective role of galectin-3 as an AGE receptor.

We previously showed that mice lacking galectin-3/AGE-receptor 3 develop accelerated diabetic glomerulopathy. To further investigate the role of galectin-3/AGE-receptor function in the pathogenesis of diabetic renal disease, galectin-3 knockout (KO) and coeval wild-type (WT) mice were injected for 3 months with 30 microg/day of N(epsilon)-carboxymethyllysine (CML)-modified or unmodified mouse serum albumin (MSA). Despite receiving equal doses of CML, KO had higher circulating and renal AGE levels and showed more marked renal functional and structural changes than WT mice, with significantly higher proteinuria, albuminuria, glomerular, and mesangial area and glomerular sclerosis index. Renal 4-hydroxy-2-nonenal content and NFkappaB activation were also more pronounced in KO-CML vs. WT-CML. Kidney mRNA levels of fibronectin, laminin, collagen IV, and TGF-beta were up-regulated, whereas those of matrix metalloproteinase-2 and -14 were down-regulated, again more markedly in KO-CML than WT-CML mice. Basal and CML-induced RAGE and 80K-H mRNA levels were higher in KO vs. WT mice. MSA injection did not produce any significant effect in both genotypes. The association of galectin-3 ablation with enhanced susceptibility to AGE-induced renal disease, increased AGE levels and signaling, and altered AGE-receptor pattern indicates that galectin-3 is operating in vivo as an AGE receptor to afford protection toward AGE-dependent tissue injury.

Aldehydes↗

PubMed2006

Role of galectin-3 in mast cell functions: galectin-3-deficient mast cells exhibit impaired mediator release and defective JNK expression.

Galectin-3 is a member of the beta-galactoside-binding animal lectin family expressed in various cell types, including mast cells. To determine the role of galectin-3 in the function of mast cells, we studied bone marrow-derived mast cells (BMMC) from wild-type (gal3(+/+)) and galectin-3-deficient (gal3(-/-)) mice. Cells from the two genotypes showed comparable expression of IgE receptor and c-Kit. However, upon activation by FcepsilonRI cross-linkage, gal3(-/-) BMMC secreted a significantly lower amount of histamine as well as the cytokine IL-4, compared with gal3(+/+) BMMC. In addition, we found significantly reduced passive cutaneous anaphylaxis reactions in gal3(-/-) mice compared with gal3(+/+) mice. These results indicate that there is a defect in the response of mast cells in gal3(-/-) mice. Unexpectedly, we found that gal3(-/-) BMMC contained a dramatically lower basal level of JNK1 protein compared with gal3(+/+) BMMC, which is probably responsible for the lower IL-4 production. The decreased JNK1 level in gal3(-/-) BMMC is accompanied by a lower JNK1 mRNA level, suggesting that galectin-3 regulates the transcription of the JNK gene or processing of its RNA. All together, these results point to an important role of galectin-3 in mast cell biology.

Anaphylaxis↗

PubMed2005

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

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

Base Sequence↗

PubMed2004

Galectin-1(L11A) predicted from a computed galectin-1 farnesyl-binding pocket selectively inhibits Ras-GTP.

Ras biological activity necessitates membrane anchorage that depends on the Ras farnesyl moiety and is strengthened by Ras/galectin-1 interactions. We identified a hydrophobic pocket in galectin-1, analogous to the Cdc42 geranylgeranyl-binding cavity in RhoGDI, possessing homologous isoprenoid-binding residues, including the critical L11, whose RhoGDI L77 homologue changes dramatically on Cdc42 binding. By substituting L11A, we obtained a dominant interfering galectin-1 that possessed normal carbohydrate-binding capacity but inhibited H-Ras GTP-loading and extracellular signal-regulated kinase activation, dislodged H-Ras(G12V) from the cell membrane, and attenuated H-Ras(G12V) fibroblast transformation and PC12-cell neurite outgrowth. Thus, independently of carbohydrate binding, galectin-1 cooperates with Ras, whereas galectin-1(L11A) inhibits it.

Amino Acid Sequence↗

PubMed2001

Identification of an internal gene to the human Galectin-3 gene with two different overlapping reading frames that do not encode Galectin-3.

We previously reported that alternative transcripts were initiated within the second intron of the human Galectin-3 gene (LGALS3). We now demonstrate that these transcripts arise from an internal gene embedded within LGALS3 and named galig (Galectin-3 internal gene). Tissue-specific expression of galig was assayed by screening of several human tissues. Contrary to LGALS3, galig appears to be tightly regulated and principally activated in leukocytes from peripheral blood. Cloning and characterization of galig transcripts revealed that they contain two out-of-frame overlapping open-reading frames (ORFs). Transfection of expression vectors encoding enhanced green fluorescent protein (EGFP) chimeras indicated that both ORFs could be translated in proteins unrelated to Galectin-3. The ORF1 polypeptide targets EGFP to cytosol and nucleus whereas ORF2 targets EGFP to mitochondria. These results revealed the exceptional genetic organization of the LGALS3 locus.

Amino Acid Sequence↗

PubMed1999

Recombinant expression of a galectin from the sheep gastrointestinal parasite Teladorsagia circumcincta: its use in isolating galectin-glycoconjugates.

Galectins, or beta-galactoside-binding lectins, are a family of proteins that have been described in vertebrates and, more recently, invertebrates, including nematode parasites. A tandem repeat-type galectin from the sheep gastrointestinal parasite, Teladorsagia circumcincta, has previously been isolated and the cDNA cloned. This molecule and each of its domains were expressed as fusion proteins with glutathione S-transferase (GST). The full-length molecule and the C-terminal domain were expressed in soluble form and the purified fusion proteins demonstrated a capacity to bind beta-galactoside sugars, with the greatest preference for lactose. The full-length fusion protein was used to successfully isolate potential galectin-glycoconjugates from within the parasite and from sheep serum.

Amino Acid Sequence↗