RESUMO
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.
Assuntos
Mucinas , Polissacarídeos , Azul Alciano/química , Mucinas/metabolismo , Coloração e Rotulagem , DensitometriaRESUMO
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.
Assuntos
Galectina 2 , Mucinas , Mucinas/metabolismo , Proteínas de Fluorescência Verde/genética , Lectinas/metabolismo , Polissacarídeos/químicaRESUMO
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.
Assuntos
Reabsorção Óssea , Galectina 1 , Tirosina , Animais , Humanos , Camundongos , Reabsorção Óssea/metabolismo , Diferenciação Celular , Galectina 1/metabolismo , Galectina 1/farmacologia , Leucócitos Mononucleares , Açúcares , Tirosina/análogos & derivados , Células RAW 264.7/metabolismoRESUMO
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.
Assuntos
Escherichia coli , Galectina 2 , Camundongos , Animais , Humanos , Galectina 2/química , Escherichia coli/genética , Escherichia coli/metabolismo , Códon de Terminação/metabolismo , Proteínas Recombinantes/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
We previously reported that lysophosphatidylinositol (LPI) functions as an endogenous agonist of GPR55, a novel cannabinoid receptor. However, the physiological roles of LPI-GPR55 have not yet been elucidated in detail. In the present study, we found that LPI induced morphological changes in GPR55-expressing HEK293 cells. LPI induced the cell rounding of GPR55-expressing HEK293 cells but not of empty-vector-transfected cells. LPI also induced the activation of small GTP-binding protein RhoA and increased stress fiber formation in GPR55-expressing HEK293 cells. The inhibition of RhoA and Rho kinase ROCK by the C3 exoenzyme and the ROCK inhibitor reduced LPI-induced cell rounding and stress fiber formation. These results clearly indicated that the LPI-induced morphological changes and the assembly of the cytoskeletons were mediated through the GPR55-RhoA-ROCK pathway.
Assuntos
Receptores Acoplados a Proteínas G , Quinases Associadas a rho , Células HEK293 , Humanos , Lisofosfolipídeos/metabolismo , Receptores de Canabinoides/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Fibras de Estresse/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
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.
Assuntos
Diferenciação Celular/efeitos dos fármacos , N-Acetilglucosaminiltransferases/metabolismo , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Piranos/farmacologia , Tiazóis/farmacologia , Animais , Diferenciação Celular/fisiologia , Feminino , Glicosilação/efeitos dos fármacos , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Masculino , Camundongos , Células RAW 264.7RESUMO
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.
Assuntos
Galactosídeos/farmacologia , Galectina 2/farmacologia , Helicobacter pylori/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Animais , Infecções por Helicobacter , Helicobacter pylori/crescimento & desenvolvimento , Humanos , Masculino , CamundongosRESUMO
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.
Assuntos
Cisteína/análogos & derivados , Galectina 2/genética , Proteínas Recombinantes/química , S-Nitrosotióis/análise , Cisteína/análise , Cisteína/química , Cisteína/metabolismo , Galectina 2/química , Galectina 2/metabolismo , Humanos , Modelos Moleculares , Óxido Nítrico/metabolismo , Conformação Proteica , Engenharia de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/metabolismoRESUMO
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.
Assuntos
Galectina 2/química , Trato Gastrointestinal/química , Mucina-5AC/química , Animais , Humanos , Lactose , Camundongos , Muco , EstômagoRESUMO
Interferon-stimulated gene 15 (ISG15) encodes a protein that is most upregulated by type I interferon stimulation and, upon activation, is conjugated to various target proteins in a process known as ISGylation. ISGylation has been shown to have roles in various biological phenomena such as viral infection and cancer. To gain further insight into the function of ISGylation, it would be useful to be able to identify ISGylated proteins. Here, we describe a method for the identification of proteins modified with ISG15. This method involves the generation of stable ISG15-transfectant cells, followed by affinity purification, and then identification of the ISGylated proteins by mass spectrometry.
Assuntos
Citocinas/metabolismo , Ubiquitinas/metabolismo , Animais , Citocinas/química , Citocinas/genética , Citocinas/isolamento & purificação , Humanos , Camundongos , Peptídeos/química , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Células RAW 264.7 , Proteínas Recombinantes de Fusão , Ubiquitinas/química , Ubiquitinas/genética , Ubiquitinas/isolamento & purificaçãoRESUMO
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.
Assuntos
Caenorhabditis elegans/crescimento & desenvolvimento , Dissacarídeos/química , Epitopos/química , Galectina 2/metabolismo , Animais , Ascaris suum/crescimento & desenvolvimento , Ascaris suum/metabolismo , Sítios de Ligação , Biomphalaria , Caenorhabditis elegans/metabolismo , Dissacarídeos/metabolismo , Epitopos/metabolismo , Galectina 2/química , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos ICRRESUMO
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 (H2 O2 ) 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 H2 O2 -induced inactivation. One of the two cysteine residues, C57, has been identified as being responsible for controlling H2 O2 -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 H2 O2 -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 H2 O2 -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 H2 O2 . DATABASE: Nuclear magnetic resonance assignments for non-S-nitrosylated mGal-2 and S-nitrosylated mGal-2 have been deposited in the BioMagResBank (http://www.bmrb.wisc.edu/) under ID code 27237 for non-S-nitrosylated mGal-2 and ID code 27238 for S-nitrosylated mGal-2.
Assuntos
Galectina 2/química , Espectroscopia de Ressonância Magnética/métodos , Óxido Nítrico/química , S-Nitrosotióis/química , Animais , Cisteína/química , Cisteína/metabolismo , Galectina 2/metabolismo , Peróxido de Hidrogênio/farmacologia , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Modelos Moleculares , Óxido Nítrico/metabolismo , Oxidantes/farmacologia , Oxirredução/efeitos dos fármacos , Agregados Proteicos/efeitos dos fármacos , Conformação Proteica , S-Nitrosotióis/metabolismoRESUMO
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.
Assuntos
Galectina 2/química , Galectina 2/metabolismo , Mucinas/química , Mucinas/metabolismo , Animais , Células Epiteliais/metabolismo , Galectina 2/genética , Mucosa Gástrica/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Lactose/química , Lactose/metabolismo , Peso Molecular , Plasmídeos , Multimerização Proteica , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SuínosRESUMO
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.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Glucosamina/farmacologia , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Acilação , Animais , Reabsorção Óssea/metabolismo , Linhagem Celular , Glicosilação , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Osteoclastos/metabolismo , Ligante RANK/metabolismo , Fosfatase Ácida Resistente a Tartarato/metabolismo , Regulação para CimaRESUMO
Galectins are a group of animal lectins characterized by their specificity for ß-galactosides. Mouse galectin-2 (mGal-2) is predominantly expressed in the gastrointestinal tract and has been identified as one of the main gastric mucosal proteins that are uniquely sensitive to S-nitrosylation. We have previously reported that oxidation of mGal-2 by hydrogen peroxide (H2O2) resulted in the loss of sugar-binding ability, whereas pre-treatment of mGal-2 with S-nitrosocysteine prevented H2O2-induced inactivation. In this study, we used point-mutated recombinant mGal-2 proteins to study which of the two highly conserved Cys residues in mGal-2 must be S-nitrosylated for protection against oxidative inactivation. Mutation of Cys57 to a Met residue (C57M) did not result in lectin inactivation following H2O2 treatment, whereas Cys75 mutation to Ser (C75S) led to significantly reduced lectin activity, as is the case for wild-type mGal-2. However, pre-treatment of the C75S mutant with S-nitrosocysteine protected the protein from H2O2-induced inactivation. Therefore, Cys57 is suggested to be responsible for oxidative inactivation of the mGal-2 protein, and protection of the sulfhydryl group of the Cys57 in mGal-2 by S-nitrosylation is likely important for maintaining mGal-2 protein function in an oxidative environment such as the gastrointestinal tract.
Assuntos
Galectina 2/química , Peróxido de Hidrogênio/química , Substituição de Aminoácidos , Animais , Galectina 2/genética , Galectina 2/metabolismo , Peróxido de Hidrogênio/metabolismo , Camundongos , Mutação de Sentido Incorreto , OxirreduçãoRESUMO
Galactoseß1-4fucose (Galß1-4Fuc), a unique disaccharide unit found only on the N-glycans of Protostomia, has been intensively studied, particularly in Nematoda. Galß1-4Fuc attached to the 6-OH of the innermost GlcNAc of N-glycans has been identified as an endogenous target recognized by Caenorhabditis elegans galectin LEC-6 and might function as an endogenous ligand for other galectins as well. Interactions between galectins and N-glycans might be subject to fine-tuning through modifications of the penultimate GlcNAc and the Galß1-4Fuc unit. Similar fine-tuning is also observable in vertebrate galectins, although their major recognition unit is a Galß1-4GlcNAc. In Protostomia, it can be postulated that glycan-binding proteins and their ligands have coevolved; however, epitopes such as Galß1-4Fuc were then hijacked as targets by other organisms. Fungal (Coprinopsis cinerea) galectin 2, CGL2, binds the Galß1-4Fuc on C. elegans glycans to exert its nematotoxicity. Some human and mouse galectins bind to synthesized Galß1-4Fuc; as some parasitic nematodes express this motif, its recognition by mammalian galectins could hypothetically be involved in host defense, similar to fungal CGL2. In this review, we discuss the Galß1-4Fuc unit in Protostomia as a possible equivalent for the Galß1-4GlcNAc unit in vertebrates and a potential non-self glycomarker useful for pathogen recognition.
Assuntos
Dissacarídeos/análise , Galectinas/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Sequência de Aminoácidos , Animais , Vias Biossintéticas , Caenorhabditis elegans/química , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Dissacarídeos/metabolismo , Galectinas/química , Invertebrados/química , Invertebrados/metabolismo , Simulação de Acoplamento Molecular , Alinhamento de SequênciaRESUMO
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-ß-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.
RESUMO
Galactoseß1-4fucose (Galß1-4Fuc) is a unique disaccharide unit that has been found only in the N-glycans of protostomia. We demonstrated that this unit has a role as an endogenous ligand for Caenorhabditis elegans galectins. This unit is also recognized by fungal and mammalian galectins possibly as a non-self glycomarker. In order to clarify its biological function, we made a polyclonal antibody using (Galß1-4Fuc)n-BSA as the antigen, which was prepared by crosslinking Galß1-4Fuc-O-(CH2)2-SH and BSA. The binding specificity of the antibody was analyzed by frontal affinity chromatography, and it was confirmed that it recognizes naturally occurring N-glycans containing the Galß1-4Fuc unit linked to the reducing-end GlcNAc via α1-6 linkage. By western blotting analysis, the antibody was also found to bind to (Galß1-4Fuc)n-BSA but not to BSA or asialofetuin, which has N-glycan chains containing Galß1-4GlcNAc. Western blotting experiments also revealed presence of stained proteins in crude extracts of C. elegans, the parasitic nematode Ascaris suum, and the allergenic mite Dermatophagoides pteronyssinus, while those from Drosophila melanogaster, Mus musculus, and the allergenic mites Dermatophagoides farinae and Tyrophagus putrescentiae were negative. This antibody should be a very useful tool for research on the distribution of the Galß1-4Fuc disaccharide unit in glycans in a wide range of organisms.
Assuntos
Anticorpos/isolamento & purificação , Ascaris suum/imunologia , Dissacarídeos/imunologia , Epitopos/imunologia , Ácaros/imunologia , Animais , Anticorpos/metabolismo , Caenorhabditis elegans , Cromatografia de Afinidade , Dissacarídeos/metabolismo , Drosophila melanogaster , Epitopos/metabolismo , Camundongos Endogâmicos C57BL , CoelhosRESUMO
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.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/química , Cromatografia de Afinidade/métodos , Dissacarídeos/química , Fucose/química , Galactose/química , Galectinas , Mutação , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/isolamento & purificação , Galectinas/química , Galectinas/genética , Galectinas/isolamento & purificação , CamundongosRESUMO
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.