Galectin-1-binding glycoforms of haptoglobin with altered intracellular trafficking, and increase in metastatic breast cancer patients

Sera from 25 metastatic breast cancer patients and 25 healthy controls were subjected to affinity chromatography using immobilized galectin-1. Serum from the healthy subjects contained on average 1.2 mg per ml (range 0.7-2.2) galectin-1 binding glycoproteins, whereas serum from the breast cancer patients contained on average 2.2 mg/ml (range 0.8-3.9), with a higher average for large primary tumours. The major bound glycoproteins were α-2-macroglobulin, IgM and haptoglobin. Both the IgM and haptoglobin concentrations were similar in cancer compared to control sera, but the percentage bound to galectin-1 was lower for IgM and higher for haptoglobin: about 50% (range 20-80) in cancer sera and about 30% (range 25-50) in healthy sera. Galectin-1 binding and non-binding fractions were separated by affinity chromatography from pooled haptoglobin from healthy sera. The N-glycans of each fraction were analyzed by mass spectrometry, and the structural differences and galectin-1 mutants were used to identify possible galectin-1 binding sites. Galectin-1 binding and non-binding fractions were also analyzed regarding their haptoglobin function. Both were similar in forming complex with haemoglobin and mediate its uptake into alternatively activated macrophages. However, after uptake there was a dramatic difference in intracellular targeting, with the galectin-1 non-binding fraction going to a LAMP-2 positive compartment (lysosomes), while the galectin-1 binding fraction went to larger galectin-1 positive granules. In conclusion, galectin-1 detects a new type of functional biomarker for cancer: a specific type of glycoform of haptoglobin, and possibly other serum glycoproteins, with a different function after uptake into tissue cells.     

Galectin-8 modulates neutrophil function via interaction with integrin alphaM

The members of the galectin family are associated with diverse cellular events, including immune response. We investigated the effects of galectin-8 on neutrophil function. Human galectin-8 induced firm and reversible adhesion of peripheral blood neutrophils but not eosinophils to a plastic surface in a lactose-sensitive manner. Other human galectins, galectins-1, -3, and -9, showed low or negligible effects on neutrophil adhesion. Confocal microscopy revealed actin bundle formation in the presence of galectin-8. Cytochalasins inhibited both actin assembly and cell adhesion induced by galectin-8. Affinity purification of galectin-interacting proteins from solubilized neutrophil membrane revealed that N-terminal carbohydrate recognition domain (CRD) of galectin-8 bound promatrix metalloproteinase-9 (proMMP-9), and C-terminal CRD bound integrin alphaM/CD11b and proMMP-9. A mutant galectin-8 lacking the carbohydrate-binding activity of N-terminal CRD (galectin-8R69H) retained adhesion-inducing activity, but inactivation of C-terminal CRD (galectin-8R233H) abolished the activity. MMP-3-mediated processing of proMMP-9 was accelerated by galectin-8, and this effect was inhibited by lactose. Galectins-1 and -3 did not affect the processing. Superoxide production, an essential event in bactericidal function of neutrophils, was stimulated by galectin-8 to an extent comparable to that induced by fMLP. Galectin-8R69H but not galectin-8R233H could stimulate superoxide production. Taken together, these results suggest that galectin-8 is a novel factor that modulates the neutrophil function related to transendothelial migration and microbial killing.     

Toward functional glycomics by localization of tissue lectins: immunohistochemical galectin fingerprinting during diethylstilbestrol-induced kidney tumorigenesis in male Syrian hamster

The current study focused on galectins (-1, -3, -4, -7, and –8) and deliberately performed immunohistochemical fingerprinting to explore their complexity in a context of experimental renal carcinogenesis. The diethylstilbestrol (DES)-induced renal tumors in male Syrian hamster kidney (SHKT) represent a unique animal model for the study of estrogen-dependent renal malignancies. Kidney sections of DES-treated hamsters (3 days to 11 months of DES exposure) were analyzed by immunohistochemistry using a panel of non-crossreactive antibodies raised against galectins-1, -3, -4, -7, and -8. Levels of expression were quantitatively determined by using computer-assisted microscopy on immunostained tissue sections. Except for galectin-4, all above mentioned galectins were expressed in kidney tumors. Small clusters of galectin-1-positive, most likely preneoplastic cells at the corticomedullary junction were already evident 1 week after DES administration. Galectin-1 and -3 expression was apparently associated with the first steps of the neoplastic transformation, because small tumorous buds were found to be positive after 1 month of treatment. In contrast, galectins-7 and -8 were detected in large tumors and medium-sized tumors, respectively, thereby indicating an involvement in later stages of DES-induced SHKT. Galectins-1, -3, -7, and -8 were also detected by immunofluorescence staining in the HKT-1097 cell line established from SHKT, thus illustrating the stability of galectin expression in tumor cells. Our data document the presence and differential regulation of galectins in the course of renal tumorigenesis in the model of DES-induced SHKT.     

Thermodynamic binding studies of bivalent oligosaccharides to galectin-1, galectin-3, and the carbohydrate recognition domain of galectin-3

Galectins are a growing family of animal lectins with common consensus sequences that bind beta-Gal and LacNAc residues. There are at present 14 members of the galectin family; however, certain galectins possess different structures as well as biological properties. Galectin-1 is a dimer of two homologous carbohydrate recognition domains (CRDs) and possesses apoptotic and proinvasive activities. Galectin-3 consists of a C-terminal CRD and an N-terminal nonlectin domain implicated in the oligomerization of the protein and is often associated with antiapoptotic activity. Because many cellular oligosaccharide receptors are multivalent, it is important to characterize the interactions of multivalent carbohydrates with galectins-1 and -3. In the present study, binding of bovine heart galectin-1 and recombinant murine galectin-3 to a series of synthetic analogs containing two LacNAc residues separated by a varying number of methylene groups, as well as biantennary analogs possessing two LacNAc residues, were examined using isothermal titration microcalorimetry (ITC) and hemagglutination inhibition measurements. The thermodynamics of binding of the multivalent carbohydrates to the C-terminal CRD domain of galectin-3 was also investigated. ITC results showed that each bivalent analog bound by both LacNAc residues to the two galectins. However, galectin-1 shows a lack of enhanced affinity for the bivalent straight chain and branched chain analogs, whereas galectin-3 shows enhanced affinity for only lacto-N-hexaose, a naturally occurring branched chain carbohydrate. The CRD domain of galectin-3 was shown to possess similar thermodynamic binding properties as the intact molecule. The results of this study have important implications for the design of carbohydrate inhibitors of the two galectins.     

The anti-angiogenic peptide anginex greatly enhances galectin-1 binding affinity for glycoproteins

Angiogenesis is a key event in cancer progression and therefore a promising target in cancer treatment. Galectin-1, a β-galactoside binding lectin, is up-regulated in the endothelium of tumors of different origin and has been shown to be the target for anginex, a powerful anti-angiogenic peptide with anti-tumor activity. Here we show that when bound to anginex, galectin-1 binds various glycoproteins with hundred- to thousand-fold higher affinity. Anginex also interacts with galectin-2, -7, -8N, and -9N but not with galectin-3, -4, or -9C.     

The role of integrin glycosylation in galectin-8-mediated trabecular meshwork cell adhesion and spreading

Primary open angle glaucoma (POAG) is a major blindness-causingdisease, characterized by elevated intraocular pressure dueto an insufficient outflow of aqueous humor. The trabecularmeshwork (TM) lining the aqueous outflow pathway modulates theaqueous outflow facility. TM cell adhesion, cell–matrixinteractions, and factors that influence Rho signaling in TMcells are thought to play a pivotal role in the regulation ofaqueous outflow. In a recent study, we demonstrated that galectin-8(Gal8) modulates the adhesion and cytoskeletal arrangement ofTM cells and that it does so through binding to β₁ integrinsand inducing Rho signaling. The current study is aimed at thecharacterization of the mechanism by which Gal8 mediates TMcell adhesion and spreading. We demonstrate here that TM cellsadhere to and spread on Gal8-coated wells but not on galectin-1(Gal1)- or galectin-3 (Gal3)-coated wells. The adhesion of TMcells to Gal8-coated wells was abolished by a competing sugar,β-lactose, but not by a noncompeting sugar, sucrose. Also,a trisaccharide, NeuAc2-3Galβ1-4GlcNAc, which binds specificallyto the N-CRD of Gal8, inhibited the spreading of TM cells toGal8-coated wells. In contrast, NeuAc2-6Galβ1-4GlcNAc whichlacks affinity for Gal8 had no effect. Affinity chromatographyof cell extracts on a Gal8-affinity column and binding experimentswith plant lectins, Maakia Amurensis and Sambucus Nigra, revealedthat ₃β₁, ₅β₁, and _vβ₁ integrins are major counterreceptorsof Gal8 in TM cells and that TM cell β₁ integrins carrypredominantly 2-3-sialylated glycans, which are high-affinityligands for Gal8 but not for Gal1 or Gal3. These data lead usto propose that Gal8 modulates TM cell adhesion and spreading,at least in part, by interacting with 2-3-sialylated glycanson β₁ integrins.     

Complex N-glycans are the major ligands for galectin-1, -3, and -8 on Chinese hamster ovary cells

Galectins are implicated in a large variety of biological functions, many of which depend on their carbohydrate-binding ability. Fifteen members of the family have been identified in vertebrates based on binding to galactose (Gal) that is mediated by one or two, evolutionarily conserved, carbohydrate-recognition domains (CRDs). Variations in glycan structures expressed on glycoconjugates at the cell surface may, therefore, affect galectin binding and functions. To identify roles for different glycans in the binding of the three types of mammalian galectins to cells, we performed fluorescence cytometry at 4 degrees C with recombinant rat galectin-1, human galectin-3, and three forms of human galectin-8, to Chinese hamster ovary (CHO) cells and 12 different CHO glycosylation mutants. All galectin species bound to parent CHO cells and binding was inhibited >90% by 0.2 M lactose. Galectin-8 isoforms with either a long or a short inter-CRD linker bound similarly to CHO cells. However, a truncated form of galectin-8 containing only the N-terminal CRD bound only weakly to CHO cells and the C-terminal galectin-8 CRD exhibited extremely low binding. Binding of the galectins to the different CHO glycosylation mutants revealed that complex N-glycans are the major ligands for each galectin except the N-terminal CRD of galectins-8, and also identified some fine differences in glycan recognition. Interestingly, increased binding of galectin-1 at 4 degrees C correlated with increased propidium iodide (PI) uptake, whereas galectin-3 or -8 binding did not induce permeability to PI. The CHO glycosylation mutants with various repertoires of cell surface glycans are a useful tool for investigating galectin-cell interactions as they present complex and simple glycans in a natural mixture of multivalent protein and lipid glycoconjugates anchored in a cell membrane.     

Galectin-2 at the enterocyte brush border of the small intestine

The brush border of pig small intestine is a local hotspot for β-galactoside-recognizing lectins, as evidenced by its prominent labeling with fluorescent lectin PNA. Previously, galectins 3-4, intelectin, and lectin-like anti-glycosyl antibodies have been localized to this important body boundary. Together with the membrane glycolipids these lectins form stable lipid raft microdomains that also harbour several of the major digestive microvillar enzymes. In the present work, we identified a lactose-sensitive 14-kDa protein enriched in a microvillar detergent resistant fraction as galectin-2. Its release from closed, right-side-out microvillar membrane vesicles shows that at least some of the galectin-2 resides at the lumenal surface of the brush border, indicating that it plays a role in the organization/stabilization of the lipid raft domains. Galectin-2 was released more effectively from the membrane by lactose than was galectin-4, and surprisingly, it was also released by the noncanonical disaccharides sucrose and maltose. Furthermore, unlike galectin-4, galectin-2 was preferentially coimmunoisolated with sucrase-isomaltase rather than with aminopeptidase N. Together, these results show that the galectins are not simply redundant proteins competing for the same ligands but rather act in concert to ensure an optimal cross-linking of membrane glycolipids and glycoproteins. In this way, they offer a maximal protection of the brush border against exposure to bile, pancreatic enzymes and pathogens.     

Crystal structure of the galectin-9 N-terminal carbohydrate recognition domain from Mus musculus reveals the basic mechanism of carbohydrate recognition

The galectins are a family of beta-galactoside-binding animal lectins with a conserved carbohydrate recognition domain (CRD). They have a high affinity for small beta-galactosides, but binding specificity for complex glycoconjugates varies considerably within the family. The ligand recognition is essential for their proper function, and the structures of several galectins have suggested their mechanism of carbohydrate binding. Galectin-9 has two tandem CRDs with a short linker, and we report the crystal structures of mouse galectin-9 N-terminal CRD (NCRD) in the absence and the presence of four ligand complexes. All structures form the same dimer, which is quite different from the canonical 2-fold symmetric dimer seen for galectin-1 and -2. The beta-galactoside recognition mechanism in the galectin-9 NCRD is highly conserved among other galectins. In the apo form structure, water molecules mimic the ligand hydrogen-bond network. The galectin-9 NCRD can bind both N-acetyllactosamine (Galbeta1-4GlcNAc) and T-antigen (Galbeta1-3GalNAc) with the proper location of Arg-64. Moreover, the structure of the N-acetyllactosamine dimer (Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc) complex shows a unique binding mode of galectin-9. Finally, surface plasmon resonance assay showed that the galectin-9 NCRD forms a homophilic dimer not only in the crystal but also in solution.     

Functional characterization of an eosinophil-specific galectin,ovine galectin-14

Across mammalian species, human galectin-10 and ovine galectin-14 are unique in their expression in eosinophils and their release into lung and gastrointestinal tissues following allergen or parasite challenge. Recombinant galectin-14 is active in carbohydrate binding assays and has been used in this study to unravel the function of this major eosinophil constituent. In vitro cultures revealed that galectin-14 is spontaneously released by eosinophils isolated from allergen-stimulated mammary gland lavage, but not by resting peripheral blood eosinophils. Galectin-14 secretion from peripheral blood eosinophils can be induced by the same stimuli that induce eosinophil degranulation. Flow cytometric analysis showed that recombinant galectin-14 can bind in vitro to eosinophils, neutrophils and activated lymphocytes. Glycan array screening indicated that galectin-14 recognizes terminal N-acetyllactosamine residues which can be modified with α1-2-fucosylation and, uniquely for a galectin, prefers α2- over α2-sialylation. Galectin-14 showed the greatest affinity for lacto-N-neotetraose, an immunomodulatory oligosaccharide expressed by helminths. Galectin-14 binds specifically to laminin in vitro, and to mucus and mucus producing cells on lung and intestinal tissue sections. In vivo, galectin-14 is abundantly present in mucus scrapings collected from either lungs or gastrointestinal tract following allergen or parasite challenge, respectively. These results suggest that in vivo secretion of eosinophil galectins may be specifically induced at epithelial surfaces after recruitment of eosinophils by allergic stimuli, and that eosinophil galectins may be involved in promoting adhesion and changing mucus properties during parasite infection and allergies.     

Galectin-8-N-domain recognition mechanism for sialylated and sulfated glycans

Galectin-8 has much higher affinity for 3'-O-sulfated or 3'-O-sialylated glycoconjugates and a Lewis X-containing glycan than for oligosaccharides terminating in Galβ1→3/4GlcNAc, and this specificity is mainly attributed to the N-terminal carbohydrate recognition domain (N-domain, CRD) (Ideo, H., Seko, A., Ishizuka, I., and Yamashita, K. (2003) Glycobiology 13, 713-723). In this study, we elucidated the crystal structures of the human galectin-8-N-domain (-8N) in the absence or presence of 4 ligands. The apo molecule forms a dimer, which is different from the canonical 2-fold symmetric dimer observed for galectin-1 and -2. In a galectin-8N-lactose complex, the lactose-recognizing amino acids are highly conserved among the galectins. However, Arg(45), Gln(47), Arg(59), and the long loop region between the S3 and S4 β-strands are unique to galectin-8N. These amino acids directly or indirectly interact with the sulfate or sialic acid moieties of 3'-sialyl- and 3'-sulfolactose complexed with galectin-8N. Furthermore, in the LNF-III-galectin-8N complex, van der Waals interactions occur between the α1-3-branched fucose and galactose and between galactose and Tyr(141), and these interactions increase the affinity toward galectin-8N. Based on the findings of these x-ray crystallographic analyses, a mutagenesis study using surface plasmon resonance showed that Arg(45), Gln(47), and Arg(59) of galectin-8N are indispensable and coordinately contribute to the strong binding of galectins-8N to sialylated and sulfated oligosaccharides. Arg(59) is the most critical amino acid for binding in the S3-S4 loop region.     

Human galectin-2: novel inducer of T cell apoptosis with distinct profile of caspase activation

Galectin-2 is structurally closely related to galectin-1, but has a distinct expression profile primarily confined to the gastrointestinal tract. Prominent differences in the proximal promoter regions between galectins-2 and -1 concern Sp1-, hepatocyte NF-3, and T cell-specific factor-1 binding sites. Of note, these sequence elements are positioned equally in the respective regions for human and rat galectins-2. Labeled galectin-2 binds to T cells in a beta-galactoside-specific manner. In contrast to galectin-1, the glycoproteins CD3 and CD7 are not ligands, while the shared affinity to beta1 integrin (or a closely associated glycoprotein) accounts for a substantial extent of cell surface binding. The carbohydrate-dependent binding of galectin-2 induces apoptosis in activated T cells. Fluorogenic substrate and inhibitor assays reveal involvement of caspases-3 and -9, in accordance with cleavage of the DNA fragmentation factor. Enhanced cytochrome c release, disruption of the mitochondrial membrane potential, and an increase of the Bax/Bcl-2 ratio by opposite regulation of expression of both proteins add to the evidence that the intrinsic apoptotic pathway is triggered. Cell cycle distribution and expression of regulatory proteins remained unaffected. Notably, galectins-1 and -7 reduce cyclin B1 expression, defining functional differences between the structurally closely related galectins. Cytokine secretion of activated T cells was significantly shifted to the Th2 profile. Our study thus classifies galectin-2 as proapoptotic effector for activated T cells, raising a therapeutic perspective. Of importance for understanding the complex galectin network, it teaches the lesson that selection of cell surface ligands, route of signaling, and effects on regulators of cell cycle progression are markedly different between structurally closely related galectins.     

Galectin-12, an Adipose-expressed Galectin-like Molecule Possessing Apoptosis-inducing Activity.

Galectins constitute a family of proteins that bind to beta-galactoside residues and have diverse physiological functions. Here we report on the identification of a galectin-like molecule, galectin-12, in a human adipose tissue cDNA library. The protein contained two potential carbohydrate-recognition domains with the second carbohydrate-recognition domain being less conserved compared with other galectins. In vitro translated galectin-12 bound to a lactosyl-agarose column far less efficiently than galectin-8. Galectin-12 mRNA was predominantly expressed in adipose tissue of human and mouse and in differentiated 3T3-L1 adipocytes. Caloric restriction and treatment of obese animals with troglitazone increased galectin-12 mRNA levels and decreased the average size of the cells in adipose tissue. The induction of galectin-12 expression by the thiazolidinedione, troglitazone, was paralleled by an increase in the number of apoptotic cells in adipose tissue. Immunocytochemical analysis revealed that galectin-12 was localized in the nucleus of adipocytes, and transfection with galectin-12 cDNA induced apoptosis of COS-1 cells. These results suggest that galectin-12, an adipose-expressed galectin-like molecule, may participate in the apoptosis of adipocytes.     

The N-terminal carbohydrate recognition domain of galectin-8 recognizes specific glycosphingolipids with high affinity

Galectin-8 is a member of the galectin family and has two tandem repeated carbohydrate recognition domains (CRDs). We determined the binding specificities of galectin-8 and its two CRDs for oligosaccharides and glycosphingolipids using ELISA and surface plasmon resonance assays. Galectin-8 had much higher affinity for 3'-O-sulfated or 3'-O-sialylated lactose and a Lewis x-containing glycan than for oligosaccharides terminating in Galbeta1-->3/4GlcNAc. This specificity was mainly attributed to the N-terminal CRD (N-domain), whereas the C-terminal CRD (C-domain) had only weak affinity for a blood group A glycan. The N-domain bound not only to oligosaccharides but also to glycosphingolipids including sulfatide (SM4 s), SM3, sialyl Lc4Cer, SB1a, GD1a, GM3, and sialyl nLc4Cer, suggesting that the N-domain recognizes a 3-O-sulfated or 3-O-sialylated Gal residue. The substitution of the C-3 of the Gal residue in lactose or N-acetyllactosamine with sulfate increased the degree of recognition by galectin-8 more potently than substitution with sialic acid. This is the first demonstration that galectin-8 binds to specific sulfated or sialylated glycosphingolipids with high affinity (KD approximately 10-8-10-9 M). When the Gln47 residue of the N-domain was converted to Ala47, the specific affinity for sulfated or sialylated glycans was selectively lost, indicating that this Gln47 plays important roles for binding to Neu5Acalpha2-->3Gal or SO3--->3Gal residues. The binding ability of galectin-8 to membrane-associated GM3 was confirmed using CHO cells, which predominantly express GM3. Binding of CHO cells to the mutein was significantly lower than to the N-domain.     

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.     

Expression of galectin-1 and galectin-3 in human fetal thyroid gland.

High levels of expression of galectin-1 and galectin-3, the beta-galactoside-binding proteins, have been recently described in malignant thyroid tumors but not in adenomas nor in normal thyroid tissue. However, there are no data about the expression of these galectins during fetal thyroid development. In this study we analyzed immunohistochemically the presence of galectin-1 and galectin-3 in human fetal thyroid glands (16-37 weeks of gestation). Weak to moderate cytoplasmic staining for galectin-1 was observed in follicular cells of all fetal thyroids. Galectin-3 could not be detected in thyroid follicular cells of any fetal thyroid investigated. Both galectins were detected in stromal tissue, but staining for galectin-1 was more intense. The absence of galectin-3 in thyroid cells during fetal development suggests that galectin-3 is expressed de novo during malignant transformation of thyroid epithelium, and that galectin-1 could be considered an oncofetal antigen. The results obtained indicated potential roles for galectin-1 and galectin-3 during the investigated period of human fetal thyroid gland development. Both galectins might participate in developmental processes regarding stromal fetal thyroid tissue organization, whereas galectin-1 might have a function in thyroid epithelium maturation.