Mayumi Tamura

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

Galectins are a group of animal lectins characterized by their specificity for β-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.

Mammalian galectins bind Galactoseβ1–4Fucose disaccharide, a unique structural component of protostomial N-type glycoproteins

Galactoseβ1-4Fucose (Galβ1-4Fuc) 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 Galβ1-4Fuc compared to Galβ1-4GlcNAc, 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 Galβ1-4Fuc disaccharide. Further, we assessed the inhibitory effect of Galβ1-4Fuc, Galβ1-4Glc, and Gal on the interaction between hGal-1 and its model ligand glycan, and found that Galβ1-4Fuc is the most effective. Although the biological significance of galectin-Galβ1-4Fuc interaction is obscure, it might be possible that Galβ1-4Fuc disaccharide is recognized as a non-self-glycan antigen. Furthermore, Galβ1-4Fuc could be a promising seed compound for the synthesis of novel galectin inhibitors.

Caenorhabditis elegans proteins captured by immobilized Galβ1-4Fuc disaccharide units: assignment of 3 annexins

Galβ1-4Fuc is a key structural motif in Caenorhabditis elegans glycans and is responsible for interaction with C. elegans galectins. In animals of the clade Protostomia, this unit seems to have important roles in glycan-protein interactions and corresponds to the Galβ1-4GlcNAc unit in vertebrates. Therefore, we prepared an affinity adsorbent having immobilized Galβ1-4Fuc in order to capture carbohydrate-binding proteins of C. elegans, which interact with this disaccharide unit. Adsorbed C. elegans proteins were eluted with ethylenediaminetetraacetic acid (EDTA) and followed by lactose (Galβ1-4Glc), digested with trypsin, and were then subjected to proteomic analysis using LC-MS/MS. Three annexins, namely NEX-1, -2, and -3, were assigned in the EDTA-eluted fraction. Whereas, galectins, namely LEC-1, -2, -4, -6, -9, -10, and DC2.3a, were assigned in the lactose-eluted fraction. The affinity of annexins for Galβ1-4Fuc was further confirmed by adsorption of recombinant NEX-1, -2, and -3 proteins to the Galβ1-4Fuc column in the presence of Ca(2+). Furthermore, frontal affinity chromatography analysis using an immobilized NEX-1 column showed that NEX-1 has an affinity for Galβ1-4Fuc, but no affinity toward Galβ1-3Fuc and Galβ1-4GlcNAc. We would hypothesize that the recognition of the Galβ1-4Fuc disaccharide unit is involved in some biological processes in C. elegans and other species of the Protostomia clade.

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 β-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-β1,4-fucose (Galβ1-4Fuc) 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 Galβ1-4Fuc unit. Frontal affinity chromatography analysis indicated that the affinities of E67D and E67A mutants for Galβ1-4Fuc are lower than that of wild-type LEC-6. Furthermore, the affinities of Glu67 mutants for an endogenous oligosaccharide, which contains a Galβ1-4Fuc 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.

Crosslinking of N-acetyllactosamine-containing glycoproteins to galectin-1 with an introduced cysteine using a photoactivatable sulfhydryl reagent

Relatively weak interactions between galectins and their potential ligands can hinder identification of physiological lectin ligands using conventional methods such as affinity purification. We have employed a combination of cysteine mutagenesis with chemical crosslinking using a photoactivatable sulfhydryl reagent benzophenone-4-maleimide to obtain a covalent complex between human galectin-1 and the model glycoprotein ligands asialofetuin and laminin which contain an N-acetyllactosamine structure. A crosslinked product was obtained only when galectin-1 with an introduced cysteine interacted with these glycoproteins via their carbohydrate moiety. This procedure should be useful for the detection of important, and as yet unidentified, ligands for galectins which cannot be currently detected because of their relatively weak interaction.

ISG15 Regulates RANKL-Induced Osteoclastogenic Differentiation of RAW264 Cells

Interferon-stimulated gene 15 kDa (ISG15) is a protein upregulated by interferon-β 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.

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 β-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 β-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.

N-acetylglucosamine suppresses osteoclastogenesis in part through the promotion of O-GlcNAcylation

Osteoclasts are the only cells in an organism capable of resorbing bone. These cells differentiate from monocyte/macrophage lineage cells upon stimulation by receptor activator of NF-κB ligand (RANKL). On the other hand, osteoclastogenesis is reportedly suppressed by glucose via the downregulation of NF-κB activity through suppression of reactive oxygen species generation. To examine whether other sugars might also affect osteoclast development, we compared the effects of monomeric sugars (glucose, galactose, N-acetylglucosamine (GlcNAc), and N-acetylgalactosamine (GalNAc)) on the osteoclastogenesis of murine RAW264 cells. Our results demonstrated that, in addition to glucose, both GlcNAc and GalNAc, which each have little effect on the generation of reactive oxygen species, suppress osteoclastogenesis. We hypothesized that GlcNAc might affect osteoclastogenesis through the upregulation of O-GlcNAcylation and showed that GlcNAc increases global O-GlcNAcylation, thereby suppressing the RANKL-dependent phosphorylation of NF-κB p65. Furthermore, an inhibitor of N-acetyl-β-D-glucosaminidase, O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino N-phenylcarbamate (PUGNAc), which also increases O-GlcNAcylation, suppressed the osteoclastogenesis of RAW264 cells and that of human peripheral blood mononuclear cells. Together, these data suggest that GlcNAc suppresses osteoclast differentiation in part through the promotion of O-GlcNAcylation.

Structural mechanisms for the S‐nitrosylation‐derived protection of mouse galectin‐2 from oxidation‐induced inactivation revealed by NMR

Galectin‐2 (Gal‐2) is a lectin thought to play protective roles in the gastrointestinal tract. Oxidation of mouse Gal‐2 (mGal‐2) by hydrogen peroxide (H2O2) results in the loss of sugar‐binding activity, whereas S‐nitrosylation of mGal‐2, which does not change its sugar‐binding profile, has been shown to protect the protein from H2O2‐induced inactivation. One of the two cysteine residues, C57, has been identified as being responsible for controlling H2O2‐induced inactivation; however, the underlying molecular mechanism has not been elucidated. We performed structural analyses of mGal‐2 using nuclear magnetic resonance (NMR) and found that residues near C57 experienced significant chemical shift changes following S‐nitrosylation, and that S‐nitrosylation slowed the H2O2‐induced aggregation of mGal‐2. We also revealed that S‐nitrosylation improves the thermal stability of mGal‐2 and that the solvent accessibility and/or local dynamics of residues near C57 and the local dynamics of the core‐forming residues in mGal‐2 are reduced by S‐nitrosylation. Structural models of Gal‐2 indicated that C57 is located in a hydrophobic pocket that can be plugged by S‐nitrosylation, which was supported by the NMR experiments. Based on these results, we propose two structural mechanisms by which S‐nitrosylation protects mGal‐2 from H2O2‐induced aggregation without changing its sugar‐binding profile: (a) stabilization of the hydrophobic pocket around C57 that prevents oxidation‐induced destabilization of the pocket, and (b) prevention of oxidation of C57 during the transiently unfolded state of the protein, in which the residue is exposed to H2O2.

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.