Method for Preparing Recombinant Galectin-2 Protein without Escherichia coli-Specific Post-translational Modifications.

Galectin-2 (Gal-2) is an animal lectin with specificity for ß-galactosides. It is predominantly expressed and suggested to play a protective function in the gastrointestinal tract; therefore, it can be used as a protein drug. Recombinant proteins have been expressed using Escherichia coli and used to study the function of Gal-2. The recombinant human Gal-2 (hGal-2) protein purified via affinity chromatography after being expressed in E. coli was not completely homogeneous. Mass spectrometry confirmed that some recombinant Gal-2 were phosphogluconoylated. In contrast, the recombinant mouse Gal-2 (mGal-2) protein purified using affinity chromatography after being expressed in E. coli contained a different form of Gal-2 with a larger molecular weight. This was due to mistranslating the original mGal-2 stop codon TGA to tryptophan (TGG). In this report, to obtain a homogeneous Gal-2 protein for further studies, we attempted the following methods: for hGal-2, 1) replacement of the lysine (Lys) residues, which was easily phosphogluconoylated with arginine (Arg) residues, and 2) addition of histidine (His)-tag on the N-terminus of the recombinant protein and cleavage with protease after expression; for mGal-2, 3) changing the stop codon from TGA to TAA, which is commonly used in E. coli. We obtained an almost homogeneous recombinant Gal-2 protein (human and mouse). These results have important implications for using Gal-2 as a protein drug.

Osteoclast Differentiation Is Suppressed by Increased O-GlcNAcylation Due to Thiamet G Treatment.

Osteoclasts are the only bone-resorbing cells in organisms and understanding their differentiation mechanism is crucial for the treatment of osteoporosis. In the present study, we investigated the effect of Thiamet G, an O-GlcNAcase specific inhibitor, on osteoclastogenic differentiation. Thiamet G treatment increased global O-GlcNAcylation in murine RAW264 cells and suppressed receptor activator of nuclear factor-κB ligand (RANKL)-dependent formation in tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells, thereby suppressing the upregulation of osteoclast specific genes. Meanwhile, knockdown of O-linked N-acetylglucosamine (O-GlcNAc) transferase promoted the formation TRAP-positive multinuclear cells. Thiamet G treatment also suppressed RANKL and macrophage colony-stimulating factor (M-CSF) dependent osteoclast formation and bone-resorbing activity in mouse primary bone marrow cells and human peripheral blood mononuclear cells. These results indicate that the promotion of O-GlcNAc modification specifically suppresses osteoclast formation and its activity and suggest that chemicals affecting O-GlcNAc modification might potentially be useful in the prevention or treatment of osteoporosis in future.

Potential Interaction between Galectin-2 and MUC5AC in Mouse Gastric Mucus.

Galectins are a group of animal lectins characterized by their specificity for ß-galactosides. Of these, galectin-2 (Gal-2) is predominantly expressed in the gastrointestinal tract. In the current study, we used a mouse gastric mucous fraction to investigate whether Gal-2 is secreted from epithelial cells and identify its potential ligands in gastric mucus. Gal-2 was detected in the mouse gastric mucous fraction and could be eluted from it by the addition of lactose. Affinity chromatography using recombinant mouse galectin-2 (mGal-2)-immobilized adsorbent and subsequent LC-MS/MS identified MUC5AC, one of the major gastric mucin glycoproteins, as a potential ligand of mGal-2. Furthermore, MUC5AC was detected in the mouse gastric mucous fraction by Western blotting, and recombinant mGal-2 was adsorbed to this fraction in a carbohydrate-dependent manner. These results suggested that Gal-2 and MUC5AC in mouse gastric mucus interact in a ß-galactoside-dependent manner, resulting in a stronger barrier structure protecting the mucosal surface.

Galectin-2 Has Bactericidal Effects against Helicobacter pylori in a ß-galactoside-Dependent Manner.

Helicobacter pylori is associated with the onset of gastritis, peptic ulcers, and gastric cancer. Galectins are a family of ß-galactoside-binding proteins involved in diverse biological phenomena. Galectin-2 (Gal-2), a member of the galectin family, is predominantly expressed in the gastrointestinal tract. Although some galectin family proteins are involved in immunoreaction, the role of Gal-2 against H. pylori infection remains unclear. In this study, the effects of Gal-2 on H. pylori morphology and survival were examined. Gal-2 induced H. pylori aggregation depending on ß-galactoside and demonstrated a bactericidal effect. Immunohistochemical staining of the gastric tissue indicated that Gal-2 existed in the gastric mucus, as well as mucosa. These results suggested that Gal-2 plays a role in innate immunity against H. pylori infection in gastric mucus.

Galectin-2 suppresses nematode development by binding to the invertebrate-specific galactoseß1-4fucose glyco-epitope.

Galactoseß1-4Fucose (GalFuc) is a unique disaccharide found in invertebrates including nematodes. A fungal galectin CGL2 suppresses nematode development by recognizing the galactoseß1-4fucose epitope. The Caenorhabditis elegans galectin LEC-6 recognizes it as an endogenous ligand and the Glu67 residue of LEC-6 is responsible for this interaction. We found that mammalian galectin-2 (Gal-2) also has a comparable glutamate residue, Glu52. In the present study, we investigated the potential nematode-suppressing activity of Gal-2 using C. elegans as a model and focusing on Gal-2 binding to the GalFuc epitope. Gal-2 suppressed C. elegans development whereas its E52D mutant (Glu52 substituted by Asp), galectin-1 and galectin-3 had little effect on C. elegans growth. Lectin-staining using fluorescently-labeled Gal-2 revealed that, like CGL2, it specifically binds to the C. elegans intestine. Natural C. elegans glycoconjugates were specifically bound by immobilized Gal-2. Western blotting with anti-GalFuc antibody showed that the bound glycoconjugates had the GalFuc epitope. Frontal affinity chromatography with pyridylamine-labeled C. elegans N-glycans disclosed that Gal-2 (but not its E52D mutant) recognizes the GalFuc epitope. Gal-2 also binds to the GalFuc-bearing glycoconjugates of Ascaris and the GalFuc epitope is present in the parasitic nematodes Nippostrongylus brasiliensis and Brugia pahangi. These results indicate that Gal-2 suppresses C. elegans development by binding to its GalFuc epitope. The findings also imply that Gal-2 may prevent infestations of various parasitic nematodes bearing the GalFuc epitope.

Glucosamine Suppresses Osteoclast Differentiation through the Modulation of Glycosylation Including O-GlcNAcylation.

Osteoclasts represent the only bone resorbing cells in an organism. In this study, we investigated the effect of glucosamine (GlcN), a nutrient used to prevent joint pain and bone loss, on the osteoclastogenesis of murine macrophage-like RAW264 cells. GlcN supplementation suppressed the upregulation of osteoclast-specific genes (tartrate-resistant acid phosphatase (TRAP), cathepsin K, matrix metallopeptidase 9, and nuclear factor of activated T cell c1 (NFATc1)), receptor activator of nuclear factor-κB ligand (RANKL)-dependent upregulation of TRAP enzyme activity, and the formation of TRAP-positive multinuclear cells more effectively than N-acetylglucosamine (GlcNAc), which we have previously shown to inhibit osteoclast differentiation. To clarify the mechanism by which GlcN suppresses osteoclastogenesis, we further investigated the effect of GlcN on O-GlcNAcylation by Western blotting and on other types of glycosylation by lectin blotting. We found that, upon addition of GlcN, the O-GlcNAcylation of cellular proteins was increased whereas α2,6-linked sialic acid modification was decreased. Therefore, these glycan modifications in cellular proteins may contribute to the suppression of osteoclastogenesis.

Identification of Galectin-2-Mucin Interaction and Possible Formation of a High Molecular Weight Lattice.

Galectins comprise a group of animal lectins characterized by their specificity for ß-galactosides. Galectin-2 (Gal-2) is predominantly expressed in the gastrointestinal tract and has been identified as one of the main gastric mucosal proteins that are proposed to have a protective role in the stomach. As Gal-2 is known to form homodimers in solution, this may result in crosslinking of macromolecules with the sugar structures recognized by Gal-2. In this study, we report that Gal-2 could interact with mucin, an important component of gastric mucosa, in a ß-galactoside-dependent manner. Furthermore, Gal-2 and mucin could form an insoluble precipitate, potentially through the crosslinking of mucins via Gal-2 and the formation of a lattice, resulting in a large insoluble complex. Therefore, we suggest that Gal-2 plays a role in the gastric mucosa by strengthening the barrier structure through crosslinking the mucins on the mucosal surface.

S-nitrosylation of mouse galectin-2 prevents oxidative inactivation by hydrogen peroxide.

Galectins are a group of animal lectins characterized by their specificity for ß-galactosides. Galectin-2 (Gal-2) is predominantly expressed in the gastrointestinal tract. A proteomic analysis identified Gal-2 as a protein that was S-nitrosylated when mouse gastric mucosal lysates were reacted with S-nitrosoglutathione, a physiologically relevant S-nitrosylating agent. In the present study, recombinant mouse (m)Gal-2 was S-nitrosylated using nitrosocysteine (CysNO), which had no effect on the sugar-binding specificity and dimerization capacity of the protein. On the other hand, mGal-2 oxidation by H2O2 resulted in the loss of sugar-binding ability, while S-nitrosylation prevented H2O2-inducted inactivation, presumably by protecting the Cys residue(s) in the protein. These results suggest that S-nitrosylation by nitric oxides protect Gal-2 from oxidative stress in the gastrointestinal tract.

Purification of galectin-1 mutants using an immobilized Galactoseß1-4Fucose affinity adsorbent.

Galectins are a family of lectins characterized by their carbohydrate recognition domains containing eight conserved amino acid residues, which allows the binding of galectin to ß-galactoside sugars such as Galß1-4GlcNAc. Since galectin-glycan interactions occur extracellularly, recombinant galectins are often used for the functional analysis of these interactions. Although it is relatively easy to purify galectins via affinity to Galß1-4GlcNAc using affinity adsorbents such as asialofetuin-Sepharose, it could be difficult to do so with mutated galectins, which may have reduced affinity towards their endogenous ligands. However, this is not the case with Caenorhabditis elegans galectin LEC-6; binding to its endogenous recognition unit Galß1-4Fuc, a unique disaccharide found only in invertebrates, is not necessarily affected by point mutations of the eight well-conserved amino acids. In this study, we constructed mutants of mouse galectin-1 carrying substitutions of each of the eight conserved amino acid residues (H44F, N46D, R48H, V59A, N61D, W68F, E71Q, and R73H) and examined their affinity for Galß1-4GlcNAc and Galß1-4Fuc. These mutants, except W68F, had very low affinity for asialofetuin-Sepharose; however, most of them (with the exception of H44F and R48H) could be purified using Galß1-4Fuc-Sepharose. The affinity of the purified mutant galectins for glycans containing Galß1-4Fuc or Galß1-4GlcNAc moieties was quantitatively examined by frontal affinity chromatography, and the results indicated that the mutants retained the affinity only for Galß1-4Fuc. Given that other mammalian galectins are known to bind Galß1-4Fuc, our data suggest that immobilized Galß1-4Fuc ligands could be generally used for easy one-step affinity purification of mutant galectins.

ISG15 regulates RANKL-induced osteoclastogenic differentiation of RAW264 cells.

Interferon-stimulated gene 15 kDa (ISG15) is a protein upregulated by interferon-ß that negatively regulates osteoclastogenesis. We investigated the role of ISG15 in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenic differentiation of murine RAW264 cells. RANKL stimulation induced ISG15 expression in RAW264 cells at both the mRNA and protein levels. Overexpression of ISG15 in RAW264 cells resulted in suppression of cell fusion in RANKL-stimulated cells as well as the reduced expression of ATP6v0d2, a gene essential for cell fusion in osteoclastogenic differentiation. These results suggest that ISG15 suppresses RANKL-induced osteoclastogenesis, at least in part, through inhibition of ATP6v0d2 expression.

Crosslinking of Cys-mutated human galectin-1 to the model glycoprotein ligands asialofetuin and laminin by using a photoactivatable bifunctional reagent.

Galectins are a group of animal lectins characterized by their specificity for ß-galactosides. In our previous study, we showed that a human galectin-1 (hGal-1) mutant, in which a cysteine residue was introduced at Lys(28), forms a covalently cross-linked complex with the model glycoprotein ligands asialofetuin and laminin by using the photoactivatable sulfhydryl reagent benzophenone-4-maleimide (BPM). In the present study, we used several hGal-1 mutants in which single cysteine residues were introduced at different positions and examined their ability to form a covalent complex with asialofetuin or laminin by using BPM. We found that the efficiency of formation of the cross-linked products differed depending on the positions of the cysteine introduced and also on the ligand used for crosslinking. Therefore, by using different cysteine hGal-1 mutants, the chances of isolating different ligands for hGal-1 should increase depending on the systems and cells used.

Quantitation of Mucin by Densitometry of an Alcian Blue-Stained Membrane.

It is a challenging task to quantify mucin using conventional protein quantification methods due to the large number of glycans attached to the peptide, which make up approximately 50-90% of its molecular weight. To address this issue, we propose a simple quantification method that involves spotting mucins onto a membrane and staining them with Alcian blue.

Analysis of the Interaction Between Mucin and Green Fluorescent Protein (GFP)-Tagged Galectin-2 Using a 96-Well Plate.

Due to a significant proportion of glycans binding to the peptide (constituting approximately 50-90% of the molecular weight), analyzing the interaction between the entire mucin molecule and its recognition protein (lectin) can be challenging. To address this, we propose a semiquantitative approach for measuring the interaction between mucin and lectin, which involves immobilizing mucin in a 96-well plate and subsequently adding lectin tagged with green fluorescent protein.

Reduced form of Galectin-1 Suppresses Osteoclastic Differentiation of Human Peripheral Blood Mononuclear Cells and Murine RAW264 Cells In Vitro.

Galectin-1 (Gal-1) is an evolutionarily conserved sugar-binding protein found in intra- and extracellular spaces. Extracellularly, it binds to glycoconjugates with ß-galactoside(s) and functions in various biological phenomena, including immunity, cancer, and differentiation. Under extracellular oxidative conditions, Gal-1 undergoes oxidative inactivation, losing its sugar-binding ability, although it exhibits sugar-independent functions. An age-related decrease in serum Gal-1 levels correlates with decreasing bone mass, and Gal-1 knockout promotes osteoclastic bone resorption and suppresses bone formation. However, the effect of extracellular Gal-1 on osteoclast differentiation remains unclear. Herein, we investigated the effects of extracellular Gal-1 on osteoclastogenesis in human peripheral blood mononuclear cells (PBMCs) and mouse macrophage RAW264 cells. Recombinant Gal-1 suppressed the macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand-dependent osteoclast formation, actin ring formation, and bone-resorption activity of human PBMCs. Similar results were obtained for RAW264 cells. Gal-1 knockdown increased osteoclast-like cell formation, suggesting that it affected differentiation in an autocrine-like manner. Oxidized Gal-1 slightly affected differentiation, and in the presence of lactose, the differentiation inhibitory effect of galectin-1 was not observed. These findings suggest that extracellular Gal-1 inhibits osteoclast differentiation in a ß-galactoside-dependent manner, and an age-related decrease in serum Gal-1 levels may contribute to reduced osteoclast activity and decreasing bone mass.

Structural basis of preferential binding of fucose-containing saccharide by the Caenorhabditis elegans galectin LEC-6.

Galectins are a group of lectins that can bind carbohydrate chains containing ß-galactoside units. LEC-6, a member of galectins of Caenorhabditis elegans, binds fucose-containing saccharides. We solved the crystal structure of LEC-6 in complex with galactose-ß1,4-fucose (Galß1-4Fuc) at 1.5 Å resolution. The overall structure of the protein and the identities of the amino-acid residues binding to the disaccharide are similar to those of other galectins. However, further structural analysis and multiple sequence alignment between LEC-6 and other galectins indicate that a glutamic acid residue (Glu67) is important for the preferential binding between LEC-6 and the fucose moiety of the Galß1-4Fuc unit. Frontal affinity chromatography analysis indicated that the affinities of E67D and E67A mutants for Galß1-4Fuc are lower than that of wild-type LEC-6. Furthermore, the affinities of Glu67 mutants for an endogenous oligosaccharide, which contains a Galß1-4Fuc unit, are drastically reduced relative to that of the wild-type protein. We conclude that the Glu67 in the oligosaccharide-binding site assists the recognition of the fucose moiety by LEC-6.

Mammalian galectins bind galactoseß1-4fucose disaccharide, a unique structural component of protostomial N-type glycoproteins.

Galactoseß1-4Fucose (Galß1-4Fuc) is a unique disaccharide exclusively found in N-glycans of protostomia, and is recognized by some galectins of Caenorhabditis elegans and Coprinopsis cinerea. In the present study, we investigated whether mammalian galectins also bind such a disaccharide. We examined sugar-binding ability of human galectin-1 (hGal-1) and found that hGal-1 preferentially binds Galß1-4Fuc compared to Galß1-4GlcNAc, which is its endogenous recognition unit. We also tested other human and mouse galectins, i.e., hGal-3, and -9 and mGal-1, 2, 3, 4, 8, and 9. All of them also showed substantial affinity to Galß1-4Fuc disaccharide. Further, we assessed the inhibitory effect of Galß1-4Fuc, Galß1-4Glc, and Gal on the interaction between hGal-1 and its model ligand glycan, and found that Galß1-4Fuc is the most effective. Although the biological significance of galectin-Galß1-4Fuc interaction is obscure, it might be possible that Galß1-4Fuc disaccharide is recognized as a non-self-glycan antigen. Furthermore, Galß1-4Fuc could be a promising seed compound for the synthesis of novel galectin inhibitors.

Quantitative evaluation of lectin-reactive glycoforms of α(1)-acid glycoprotein using lectin affinity capillary electrophoresis with fluorescence detection.

α(1)-Acid glycoprotein (AGP) was previously shown to be a marker candidate of disease progression and prognosis of patients with malignancies by analysis of its glycoforms via lectins. Herein, affinity capillary electrophoresis of fluorescein-labeled AGP using lectins with the aid of laser-induced fluorescence detection was developed for quantitative evaluation of the fractional ratios of concanavalin A-reactive or Aleuria aurantia lectin-reactive AGP. Labeled AGP was applied at the anodic end of a fused-silica capillary (50 µm id, 360 µm od, 27 cm long) coated with linear polyacryloyl-ß-alanyl-ß-alanine, and electrophoresis was carried out for about 10 min in 60 mM 3-morpholinopropane-1-sulfonic acid-NaOH buffer (pH 7.35). Addition of the lectins to the anode buffer resulted in the separation of lectin-reactive glycoform peaks from lectin-non-reactive glycoform peaks. Quantification of the peak area of each group revealed that the percent of lectin-reactive AGP is independent of a labeling ratio ranging from 0.4 to 1.5 mol fluorescein/mol AGP, i.e. the standard deviation of 0.5% for an average of 59.9% (n=3). In combination with a facile procedure for micro-purification of AGP from serum, the present procedure, marking the reactivity of AGP with lectins, should be useful in determining the prognosis for a large number of patients with malignancies.

Cross-link formation between mutant galectins of Caenorhabditis elegans with a substituted cysteine residue and asialofetuin via a photoactivatable bifunctional reagent.

LEC-1 is the first tandem repeat-type galectin isolated from an animal system; this galectin has two carbohydrate recognition domains in a single polypeptide chain. Because its two lectin domains have different sugar-binding profiles, these domains are thought to interact with different carbohydrate ligands. In our previous study, we showed that a mutant of LEC-1 in which a cysteine residue was introduced at a unique position in the N-terminal lectin domain (Nh) can be cross-linked with a model glycoprotein ligand, bovine asialofetuin, by using a bifunctional photoactivatable cross-linking reagent, benzophenone-4-maleimide. In the present work, we applied the same procedure to the C-terminal lectin domain (Ch) of LEC-1. Cross-linked products were formed in the cases of two mutants in which a cysteine residue was introduced at Lys¹77 and Ser²68, respectively. This method is very useful for capturing and assigning endogenous ligand glycoconjugates with relatively low affinities to each carbohydrate recognition domain of the whole tandem repeat-type galectin molecule.

Side chain orientation of the amino acid substituted by a cysteine residue is important for successful crosslinking of galectin to its glycoprotein ligand using a photoactivatable sulfhydryl reagent.

We have employed a combination of cysteine mutagenesis and chemical crosslinking using a photoactivatable sulfhydryl reagent, benzophenone-4-maleimide, to obtain a covalent complex between human galectin-1 and a model glycoprotein ligand, asialofetuin. We previously obtained a crosslinked product when Lys(28) of the cysteine-less form of human galectin-1 was mutated to cysteine. To investigate whether substituting either of the two flanking amino acid residues in the same ß-strand, Ala(27) and Ser(29), to cysteine could result in crosslinking to the bound asialofetuin, two cysteine-containing mutants were generated. Although both the mutants adsorbed to asialofetuin-agarose and were eluted with 0.1 M lactose, confirming their ability to interact with asialofetuin, these mutants did not crosslink to the bound glycoprotein ligand following treatment with benzophenone-4-maleimide. Therefore the orientation of the side chain of the introduced cysteine residue apparently plays an important role in the crosslinking reaction.

Expression, S-Nitrosylation, and Measurement of S-Nitrosylation Ratio of Recombinant Galectin-2.

S-nitrosylation, which involves the coupling of an NO group to the reactive thiol of Cys residue(s) in a polypeptide, is an important posttranslational modification detected in a variety of proteins. Here, we present the S-nitrosylation of recombinant galectin-2 (Gal-2) using S-nitrosocysteine and the measurement of the molecular ratio of S-nitrosylation of Cys residues in the Gal-2 protein.