Homophilic Binding Properties of Galectin-3: Involvement of the Carbohydrate Recognition Domain

Abstract: Galectin-3, an animal lectin specific for β-galactosides, is composed of three different domains. The N-terminal half of the molecule (N domain) consists of a short N-terminal segment followed by glycine-, proline-, and tyrosine-rich tandem repeats. The C-terminal domain (C domain) harbors the carbohydrate recognition domain homologous to other members of the galectin family of lectins. Galectin-3 aggregates in solution, and participation of the N domain of the molecule in this process has already been demonstrated. Using a solid-phase radioligand binding assay, which allows the direct analysis of galectin-3 self-association, here we provide evidence that the carbohydrate recognition domain of the lectin is involved in carbohydrate-dependent homophilic interactions: (a) Radiolabeled galectin-3 binds to immobilized galectin-3, and the addition of unlabeled galectin-3 in solution increases the rate of binding of radiolabeled lectin; (b) binding of radiolabeled galectin-3 to immobilized galectin-3 is inhibited by the C domain; (c) binding of radiolabeled galectin-3 to immobilized galectin-3 or the C domain is inhibited by lactose but not by sucrose; and (d) the radiolabeled C domain does not bind to immobilized C domain. Taken together, these data suggest that in addition to the N domain, the homophilic interactions of galectin-3 are mediated by the C domain.     

Specific recognition and cleavage of galectin-3 by Leishmania major through species-specific polygalactose epitope

Lipophosphoglycan is a major surface molecule of Leishmania, protozoa parasites, which are the causative agents of leishmaniasis, a disease that annually afflicts millions of people worldwide. The oligosaccharide structures of lipophosphoglycan varies among species, and epitopes of these species-specific oligosaccharides are suggested to be implicated in the interaction of Leishmania with macrophages as well as species-specific tissue tropism observed in leishmaniasis. The recognition of the species-specific variation of oligosaccharides is likely to be mediated by host carbohydrate-binding proteins, lectins, but the identities of the lectins remain elusive. Galectin-3 is a mammalian soluble beta-galactoside-binding lectin and is expressed in macrophages, dendritic cells, and keratinocytes, as well as fibroblasts, all of which are present in the site of Leishmania infection. In this paper, we found that galectin-3 binds to lipophosphoglycan of Leishmania major but not to those of Leishmania donovani through L. major-specific polygalactose epitopes. Association of galectin-3 with L. major led to the cleavage of galectin-3, resulting in truncated galectin-3 containing the C-terminal lectin domain but lacking the N-terminal domain implicated in lectin oligomerization. This cleavage was inhibited by the galectin-3 antagonist lactose, as well as 1,10-ortho-phenanthroline, suggesting that galectin-3 is cleaved by zinc metalloproteases after its binding to lipophosphoglycans. The modulation of various innate immunity reactions by galectin-3 is affected by its oligomerization; therefore, we propose the L. major-specific truncation of galectin-3 may contribute to the species-specific immune responses induced by Leishmania.     

Oxidation of goat hepatic galectin-1 induces change in secondary structure

Galectin-1 requires a reducing environment for its lectin activity and the carbohydrate binding function is destroyed in oxidizing condition. In this report we provide direct evidence that the oxidation of goat hepatic galectin-1 perturbs its carbohydrate recognition domain and this could be due to changes in secondary structure of goat hepatic galectin-1. Conformational changes in goat hepatic galectin-1 due to oxidation were investigated by absorption, fluorescence and circular dichroism measurements.     

Apical sorting by galectin-3-dependent glycoprotein clustering

Epithelial cells are characterized by their polarized organization based on an apical membrane that is separated from the basolateral membrane domain by tight junctions. Maintenance of this morphology is guaranteed by highly specific sorting machinery that separates lipids and proteins into different carrier populations for the apical or basolateral cell surface. Lipid-raft-independent apical carrier vesicles harbour the beta-galactoside-binding lectin galectin-3, which interacts directly with apical cargo in a glycan-dependent manner. These glycoproteins are mistargeted to the basolateral membrane in galectin-3-depleted cells, dedicating a central role to this lectin in raft-independent sorting as apical receptor. Here, we demonstrate that high-molecular-weight clusters are exclusively formed in the presence of galectin-3. Their stability is sensitive to increased carbohydrate concentrations, and cluster formation as well as apical sorting are perturbed in glycosylation-deficient Madin-Darby canine kidney (MDCK) II cells. Together, our data suggest that glycoprotein cross-linking by galectin-3 is required for apical sorting of non-raft-associated cargo.     

Glycosylated nuclear lectin CBP70 also associated with endoplasmic reticulum and the Golgi apparatus: does the "classic pathway" of glycosylation also apply to nuclear glycoproteins?

The subcellular plurilocalization of some lectins (galectin-1, galectin-3, galectin-10, calreticulin, etc.) is an intriguing problem, implying different partners according to their localization, and involvement in a variety of cellular activities. For example, the well-known lectin, galectin-3, a lactose-binding protein, can act inside the nucleus in splicing events, and at the plasma membrane in adhesion, and it was demonstrated that galectin-3 interacts in the cytoplasm with Bcl-2, an antiapoptotic protein. Some years ago, our group isolated a nuclear lectin CBP70, capable of recognizing N-acetylglucosamine residues. This lectin, first isolated from the nucleus of HL60 cells, was also localized in the cytoplasm. It has been demonstrated that CBP70 is a glycosylated lectin, with different types of glycosylation, comparing cytoplasmic and nuclear forms. In this article, we have studied the localization of CBP70 in undifferentiated HL60 cells by electron microscopy, immunofluorescence analysis, and subcellular fractionation. The results obtained clearly demonstrated that CBP70 is a plurilocalized lectin that is found in the nucleus, at the endoplasmic reticulum, the Golgi apparatus, and mitochondria, but not at the plasma membrane. Because CBP70, a nuclear glycoprotein, was found to be associated also with the endoplasmic reticulum and the Golgi apparatus where the glycosylation take place, it raised the question: where does the glycosylation of nuclear proteins occur?     

Visualization of galectin-3 oligomerization on the surface of neutrophils and endothelial cells using fluorescence resonance energy transfer

Galectin-3, a member of the galectin family of carbohydrate binding proteins, is widely expressed, particularly in cells involved in the immune response. Galectin-3 has also been indicated to play a role in various biological activities ranging from cell repression to cell activation and adhesion and has, thus, been recognized as an immunomodulator. Whereas those activities are likely to be associated with ligand cross-linking by this lectin, galectin-3, unlike other members of the galectin family, exists as a monomer. It has consequently been proposed that oligomerization of the N-terminal domains of galectin-3 molecules, after ligand binding by the C-terminal domain, is responsible for this cross-linking. The oligomerization status of galectin-3 could, thus, control the majority of its extracellular activities. However, little is known about the actual mode of action through which galectin-3 exerts its function. In this report we present data suggesting that oligomerization of galectin-3 molecules occurs on cell surfaces with physiological concentrations of the lectin. Using galectin-3 labeled at the C terminus with Alexa 488 or Alexa 555, the oligomerization between galectin-3 molecules on cell surfaces was detected using fluorescence resonance energy transfer. We observed this fluorescence resonance energy transfer signal in different biological settings representing the different modes of action of galectin-3 that we previously proposed; that is, ligand crosslinking leading to cell activation, cell-cell interaction/adhesion, and lattice formation. Furthermore, our data suggest that galectin-3 lattices are robust and could, thus, be involved, as previously proposed, in the restriction of receptor clustering.     

NMR solution studies of hamster galectin-3 and electron microscopic visualization of surface-adsorbed complexes: evidence for interactions between the N- and C-terminal domains

Galectin-3, a beta-galactoside binding protein, contains a C-terminal carbohydrate recognition domain (CRD) and an N-terminal domain that includes several repeats of a proline-tyrosine-glycine-rich motif. Earlier work based on a crystal structure of human galectin-3 CRD, and modeling and mutagenesis studies of the closely homologous hamster galectin-3, suggested that N-terminal tail residues immediately preceding the CRD might interfere with the canonical subunit interaction site of dimeric galectin-1 and -2, explaining the monomeric status of galectin-3 in solution. Here we describe high-resolution NMR studies of hamster galectin-3 (residues 1--245) and several of its fragments. The results indicate that the recombinant N-terminal fragment Delta 126--245 (residues 1--125) is an unfolded, extended structure. However, in the intact galectin-3 and fragment Delta 1--93 (residues 94--245), N-terminal domain residues lying between positions 94 and 113 have significantly reduced mobility values compared with those expected for bulk N-terminal tail residues, consistent with an interaction of this segment with the CRD domain. In contrast to the monomeric status of galectin-3 (and fragment Delta 1--93) in solution, electron microscopy of negatively stained and rotary shadowed samples of hamster galectin-3 as well as the CRD fragment Delta 1--103 (residues 104--245) show the presence of a significant proportion (up to 30%) of oligomers. Similar imaging of the N-terminal tail fragment Delta 126--245 reveals the presence of fibrils formed by intermolecular interactions between extended polypeptide subunits. Oligomerization of substratum-adsorbed galectin-3, through N- and C-terminal domain interactions, could be relevant to the positive cooperativity observed in binding of the lectin to immobilized multiglycosylated proteins such as laminin.     

Purification,Characterization, Sequencing and Biological Chemistry of Galectin-1 Purified from <Emphasis Type="Italic">Capra hircus</Emphasis> (goat) Heart

A soluble β-galactoside binding 14.5 kDa lectin was purified from the heart of Capra hircus. Its metal independent nature, preferential affinity for β-d-lactose and 90–94% homology with carbohydrate recognition domain of previously reported galectin-1 confirmed its inclusion in galectin-1 subfamily. The secondary structures of the deduced amino acid sequences were generally conserved with previously reported Gal-1. Exposure of the purified protein to varying temperature and pH, oxidant, thiol blocking reagents, denaturants and detergents resulted in significant changes in UV (ultraviolet), fluorescence, CD (circular dichroism) and FTIR (fourier transform infra red) spectra, thus strongly emphasizing the vitality of regular secondary structure of galectins for maintaining their active conformation. Bioinformatics studies corroborated the results obtained in wet lab. Our findings based on physico-chemical properties, oxidative inactivation and structural analysis of the goat heart galectin-1 suggests significant implications in potential biological and clinical applications.     

Galectin-3: an open-ended story

Galectins, an ancient lectin family, are characterized by specific binding of beta-galactosides through evolutionary conserved sequence elements of carbohydrate-recognition domain (CRD). A structurally unique member of the family is galectin-3; in addition to the CRD it contains a proline- and glycine-rich N-terminal domain (ND) through which is able to form oligomers. Galectin-3 is widely spread among different types of cells and tissues, found intracellularly in nucleus and cytoplasm or secreted via non-classical pathway outside of cell, thus being found on the cell surface or in the extracellular space. Through specific interactions with a variety of intra- and extracellular proteins galectin-3 affects numerous biological processes and seems to be involved in different physiological and pathophysiological conditions, such as development, immune reactions, and neoplastic transformation and metastasis. The review attempts to summarize the existing information on structural, biochemical and intriguing functional properties of galectin-3.     

Physicochemical Properties and Amino Acid Sequence of Sheep Brain Galectin-1

A beta-galactoside-specific soluble 14-kD lectin from sheep brain was isolated, sequenced, and compared with similar galectins from other species. Percent identities of amino acid sequence and the carbohydrate recognition domain (CRD) revealed that the isolated galectin shares all the absolutely preserved and critical residues of the mammalian galectin-1 subfamily. The isolated sheep brain galectin (SBG) showed more than 90% amino acid sequence (92%) and carbohydrate recognition domain identity (96%) with human brain galectin-1. Conformational changes were found induced by interaction of the protein with its specific disaccharide and oxidizing agent (hydrogen peroxide). Upon oxidation a drastic change in the environment of aromatic residues and conformation of the galectin was observed with the loss of hemagglutination activity, while no significant change was observed upon addition of D-lactose (Gal(beta1-4)Glc) in the far-UV and near-UV spectra, suggesting no significant modification in the secondary as well as tertiary structures of sheep brain galectin. But the functional integrity of the CRD is found to be affected in the presence of oxidizing agent, indicating intramolecular disulfide bonds and requirement of complete polypeptide chain for functional integrity of the carbohydrate recognition domain.     

Molecular dynamics simulations of galectin-1-oligosaccharide complexes reveal the molecular basis for ligand diversity

Galectin-1 is a member of a protein family historically characterized by its ability to bind carbohydrates containing a terminal galactosyl residue. Galectin-1 is found in a variety of mammalian tissues as a homodimer of 14.5-kDa subunits. A number of developmental and regulatory processes have been attributed to the ability of galectin-1 to bind a variety of oligosaccharides containing the Gal-beta-(1,4)-GlcNAc (LacNAc(II)) sequence. To probe the origin of this permissive binding, solvated molecular dynamics (MD) simulations of several representative galectin-1-ligand complexes have been performed. Simulations of structurally defined complexes have validated the computational approach and expanded upon data obtained from X-ray crystallography and surface plasmon resonance measurements. The MD results indicate that a set of anchoring interactions between the galectin-1 carbohydrate recognition domain (CRD) and the LacNAc core are maintained for a diverse set of ligands and that substituents at the nonreducing terminus of the oligosaccharide extend into the remainder of a characteristic surface groove. The anionic nature of ligands exhibiting relatively high affinities for galectin-1 implicates electrostatic interactions in ligand selectivity, which is confirmed by a generalized Born analysis of the complexes. The results suggest that the search for a single endogenous ligand or function for this lectin may be inappropriate and instead support a more general role for galectin-1, in which the lectin is able to crosslink heterogeneous oligosaccharides displayed on a variety of cell surfaces. Such binding promiscuity provides an explanation for the variety of adhesion phenomena mediated by galectin-1.     

Ganglioside GM1/Galectin-Dependent Growth Regulation in Human Neuroblastoma Cells: Special Properties of Bivalent Galectin-4 and Significance of Linker Length for Ligand Selection

Orchestrated upregulation of cell surface presentation of ganglioside GM1 and homodimeric galectin-1 is the molecular basis for growth regulation of human neuroblastoma (SK-N-MC) cells. Further study led to the discovery of competitive inhibition by galectin-3, prompting us to test tandem-repeat-type galectin-4 (two different lectin domains connected by a 42-amino-acid linker). This lectin bound to cells at comparably high affinity without involvement of the ganglioside, as disclosed by assays in the presence of cholera toxin B-subunit or galectin-1 and blocking glucosylceramide synthesis. Notably, when tested separately, binding of both lectin domains showed partial sensitivity to the bacterial agglutinin. Despite its ability for cross-linking surface association of galectin-4 did not affect proliferation, in contrast to homodimeric galectins. The truncation of linker length from 42 to 16 amino acids altered binding properties to let partial sensitivity to the bacterial lectin emerge. Cross-competition between parental and engineered proteins did not exceed 40%. No effect on cell growth was detected. This study reveals complete functional divergence between galectins differing in the spatial mode of lectin-site presentation and dependence of reactivity to distinct counter-receptor(s) on linker length. Due to the documented presence of galectin-4 in the nervous system and its affinity for sulfatide these in vitro results indicate the potential for a distinct functionality profile of this lectin in vivo, giving further research direction.     

Purification, characterization, structural analysis and protein chemistry of a buffalo heart galectin-1

A soluble β-galactoside-binding lectin was purified by gel filtration chromatography from Bubalus bubalis heart. Its metal-independent nature, molecular weight of 14.5 kDa, preferential affinity for β-d-lactose, and 87–92% identity with carbohydrate recognition domain of previously reported galectin-1 confirmed its inclusion in galectin-1 subfamily. Stokes radii determination using gel filtration under reducing and non-reducing conditions revealed its homo-dimeric nature, further confirming its Gal-1 nomenclature. The purified lectin was found to be the most stable mammalian heart galectin purified till date, suggesting its preferential use in various recognition studies. Treatment of the purified lectin with oxidizing agent, thiol blocking reagents, denaturants, and detergents resulted in significant changes in UV–VIS, fluorescence, CD and FTIR spectra, which strongly emphasized the important aspect of regular secondary structure of galectins for the maintenance of their active conformation. Reduction of the activity of the purified lectin after oxidation by H₂O₂, with remarkable fluorescence quenching, may suggest potential role for galectin-1 in free radical-induced, oxidative stress-mediated cardiovascular disorders. The predictions of bioinformatics studies were found to be in accordance with the results obtained in wet lab.     

Galectin-9 regulates T helper cell function independently of Tim-3

β-Galactoside-binding lectin 9 (galectin-9) is a tandem repeat-type member of the galectin family. It was initially characterized as an eosinophil chemoattractant and an inducer of apoptosis in thymocytes. Subsequently, galectin-9 was identified as a ligand for transmembrane immunoglobulin mucin domain 3 (Tim-3), a type I glycoprotein induced on T cells during chronic inflammation. Work in autoimmune diseases and chronic viral infections have led to the current hypothesis that the function of Tim-3 is to limit immune responses. However, it is still not known to what degree these effects are due to the galectin-9/Tim-3 interaction. In this study, we show that galectin-9 is not limited to the role of a pro-apoptotic agent, but that it can also induce the production of pro-inflammatory cytokines from T helper cells. This effect is dose-dependent and does not require Tim-3. These findings suggest that the effects of galectin-9 on T cells are more complex than previously thought and are mediated by additional receptors apart from Tim-3.     

Agrocybe cylindracea lectin is a member of the galectin family

The complete amino acid sequence of Agrocybe cylindracea lectin was determined from the peptides obtained by chemical cleavages and enzymatic hydrolyses. The sequence shows 19.1% and 36.8% identity with those of human galectin-1 and Coprinus lectin-1, a fungal galectin, respectively. Seven residues, which are commonly found in carbohydrate recognizing domain (CRD) of galectins, were conserved. However, several insertions in the sequence, compared with those of human galectin-1 and Coprinus lectin-1, suggest that -strands S2, F3, and S4 and the loop structures between -strands F2 & S3 and F5 & S2 are different from those of galectins reported so far.     

Purification and Characterization of a Human Brain Galectin-1 Ligand

Abstract: Our previous studies have characterized an endogenous lectin from human brain identified as galectin-1. A soluble ligand of galectin-1 was purified from human brain by affinity chromatography and preparative electrophoresis. The purified ligand (termed HBGp82, for human brain galectin-1-binding polypeptide of 82,000 daltons) has an apparent molecular mass of 82 kDa and is glycosylated by N -linked biantennary complex structures. HBGp82 was partially characterized by microsequencing of peptide fragments. Similar peptides were found in a heat shock of protein of 90,000 daltons, hsp90. However, comparison of apparent molecular weights and matrix-assisted laser desorption mass spectrometry clearly showed that HBGp82 differs to some degree from hsp90.     

Presentation of galectin-1 by extracellular matrix triggers T cell death

Apoptotic elimination of T cells at sites of inflammation or infiltration into tumors limits an effective immune response. T cell apoptosis can be initiated by a variety of triggers, including galectin-1, a soluble, secreted lectin that binds to oligosaccharide ligands on cell surface glycoproteins, or to oligosaccharide ligands on extracellular matrix glycoproteins in tissue stroma. Although galectin-1 has no transmembrane domain and is secreted from cells that make it, it is not clear if galectin-1 functions as a soluble death trigger in vivo. We examined the ability of stromal cells secreting galectin-1 to kill T cells. Although the stromal cells synthesized abundant galectin-1, the majority of the galectin-1 remained bound to the cell surface, and stromal cell-associated galectin-1 killed bound T cells. In contrast, insufficient amounts of functional galectin-1 were released from the stromal cells into the media to kill T cells in the absence of contact with stromal cells. However, when stromal cells were grown on Matrigel, a mixture of extracellular matrix proteins, or on permeable membranes above Matrigel, secreted galectin-1 bound to Matrigel and killed T cells without stromal cell contact. Ten-fold less galectin-1 on Matrigel was sufficient to kill adherent T cells compared with soluble galectin-1. These results demonstrate that galectin-1 in extracellular matrix is able to directly kill susceptible T cells. Because increased galectin-1 deposition in tumor stroma occurs with tumor progression in various types of cancer, galectin-1 in stroma may act locally in the apoptotic elimination of infiltrating T cells during an immune response.     

Nuclear localisation of wild type and mutant galectin-3 in transfected cells

Galectin-3, a member of a family of carbohydrate-binding proteins, is present generally in the cytoplasm of cells. However, galectin 3 can also be located in nuclei under certain conditions although it lacks any known nuclear localisation signal and the mechanism by which the protein is sequestered in nuclei is unknown. Here we describe that Cos-7 cells or rabbit smooth muscle Rb-1 cells transfected with cDNA encoding hamster galectin-3 sequester the protein in nuclei whereas untransfected BHK cells expressing the endogenous hamster lectin or transfected BHK cells over-expressing the protein, do not. Confocal immunofluorescence microscopy of Cos-7 cells or rabbit smooth muscle Rb-1 cells transfected with cDNAs encoding mutants of hamster galectin-3 containing N-terminal or internal deletions shows that nuclear localisation does not require the first 103 amino acid residues of the protein. Further deletion of residues 104-110 dramatically prevents sequestration in nuclei. However, the sequence A104PTGALT110 by itself is not obligatory for nuclear localisation and can be substituted by other unrelated sequences. A truncated galectin-3 protein, that is blocked in nuclear expression, retains carbohydrate-binding activity, making less likely the possibility that severe N-terminal truncations of galectin-3 induce mis-folding leading to aggregation and cytoplasmic sequestration and an incidental effect on nuclear trafficking. These studies indicate that nuclear import and retention of galectin-3 is a property of the CRD domain and is independent of N-terminal domains that others have shown to contain binding domains for various nuclear components.