RESUMEN
Preeclampsia (PE) is a severe pregnancy disorder with a pathophysiology not yet completely understood and without curative therapy. The histone modifications H3K4me3 and H3K9ac, as well as galectin-2 (Gal-2), are known to be decreased in PE. To gain a better understanding of the development of PE, the influence of Gal-2 on histone modification in trophoblasts and in syncytialisation was investigated. Immunohistochemical stains of 13 PE and 13 control placentas were correlated, followed by cell culture experiments. An analysis of H3K4me3 and H3K9ac was conducted, as well as cell fusion staining with E-cadherin and ß-catenin-both after incubation with Gal-2. The expression of H3K4me3 and H3K9ac correlated significantly with the expression of Gal-2. Furthermore, we detected an increase in H3K4me3 and H3K9ac after the addition of Gal-2 to BeWo/HVT cells. Moreover, there was increased fusion of HVT cells after incubation with Gal-2. Gal-2 is associated with the histone modifications H3K4me3 and H3K9ac in trophoblasts. Furthermore, syncytialisation increased after incubation with Gal-2. Therefore, we postulate that Gal-2 stimulates syncytialisation, possibly mediated by H3K4me3 and H3K9ac. Since Gal-2, as well as H3K4me3 and H3K9ac, are decreased in PE, the induction of Gal-2 might be a promising therapeutic target.
Asunto(s)
Galectina 2 , Histonas , Preeclampsia , Trofoblastos , Fusión Celular , Femenino , Galectina 2/metabolismo , Histonas/metabolismo , Humanos , Placenta/metabolismo , Preeclampsia/metabolismo , Embarazo , Trofoblastos/metabolismoRESUMEN
There are several open questions to be answered regarding the pathophysiology of the development of preeclampsia (PE). Numerous factors are involved in its genesis, such as defective placentation, vascular impairment, and an altered immune response. The activation of the adaptive and innate immune system represents an immunologic, particularity during PE. Proinflammatory cytokines are predominantly produced, whereas immune regulatory and immune suppressive factors are diminished in PE. In the present study, we focused on the recruitment of regulatory T cells (Tregs) which are key players in processes mediating immune tolerance. To identify Tregs in the decidua, an immunohistochemical staining of FoxP3 of 32 PE and 34 control placentas was performed. A clearly reduced number of FoxP3-positive cells in the decidua of preeclamptic women could be shown in our analysis (p = 0.036). Furthermore, CCL22, a well-known Treg chemoattractant, was immunohistochemically evaluated. Interestingly, CCL22 expression was increased at the maternal-fetal interface in PE-affected pregnancies (psyncytiotrophoblast = 0.035, pdecidua = 0.004). Therefore, the hypothesis that Tregs undergo apoptosis at the materno-fetal interface during PE was generated, and verified by FoxP3/TUNEL (TdT-mediated dUTP-biotin nick end labeling) staining. Galectin-2 (Gal-2), a member of the family of carbohydrate-binding proteins, which is known to be downregulated during PE, seems to play a pivotal role in T cell apoptosis. By performing a cell culture experiment with isolated Tregs, we could identify Gal-2 as a factor that seems to prevent the apoptosis of Tregs. Our findings point to a cascade of apoptosis of Tregs at the materno-fetal interface during PE. Gal-2 might be a potential therapeutic target in PE to regulate immune tolerance.
Asunto(s)
Decidua/inmunología , Regulación hacia Abajo , Galectina 2/metabolismo , Preeclampsia/metabolismo , Linfocitos T Reguladores/citología , Adolescente , Adulto , Apoptosis , Estudios de Casos y Controles , Células Cultivadas , Quimiocina CCL22/metabolismo , Femenino , Factores de Transcripción Forkhead/metabolismo , Humanos , Edad Materna , Embarazo , Linfocitos T Reguladores/metabolismo , Regulación hacia Arriba , Adulto JovenRESUMEN
Gestational diabetes mellitus (GDM) is the most common pregnancy-associated metabolic disorder that negatively impacts on the health of both mothers and their offspring in the long-term. The molecular mechanisms involved are not fully understood. As in other states of insulin resistance, a disproportionate immune response in GDM leads to a state of chronic low-grade inflammation. Galectin-2 exerts regulatory effects on different immune cells. This study investigated galectin-2 expression in the placenta of 40 GDM patients and 40 controls, in a sex-specific manner. Immunohistochemistry was used for semi-quantitative analysis of expression strength. The phenotypes of galectin-2 expressing cells were characterized through double immunofluorescence. We found a significant up-regulation of galectin-2 in the fetal syncytiotrophoblast, as well as in the maternal decidua of GDM placentas. Double staining showed a strong galectin-2 expression in extra villous trophoblast cells and fetal endothelial cells in GDM. These findings present the first systematic investigation of galectin-2 in GDM. The findings contribute to the emerging understanding of the role of immunomodulation and inflammation in GDM and of galectin-2 itself. This might also have implications for the long-term cardiovascular health of the offspring.
Asunto(s)
Diabetes Gestacional/metabolismo , Galectina 2/metabolismo , Placenta/metabolismo , Placenta/patología , Adulto , Colon/patología , Células Endoteliales/metabolismo , Femenino , Feto/metabolismo , Galectina 2/genética , Regulación de la Expresión Génica , Humanos , Inflamación , Resistencia a la Insulina , Masculino , Embarazo , Complicaciones del Embarazo/metabolismo , Trofoblastos/metabolismo , Trofoblastos/patologíaRESUMEN
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.
Asunto(s)
Cisteína/análogos & derivados , Galectina 2/genética , Proteínas Recombinantes/química , S-Nitrosotioles/análisis , Cisteína/análisis , Cisteína/química , Cisteína/metabolismo , Galectina 2/química , Galectina 2/metabolismo , Humanos , Modelos Moleculares , Óxido Nítrico/metabolismo , Conformación Proteica , Ingeniería de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/metabolismoRESUMEN
Maintenance of cell surface residency and function of glycoproteins by lectins are essential for regulating cellular functions. Galectins are ß-galactoside-binding lectins and form a galectin-lattice, which regulates stability, clustering, membrane sub-domain localization and endocytosis of plasmalemmal glycoproteins. We have previously reported that galectin-2 (Gal-2) forms a complex with cationic amino acid transporter 3 (CAT3) in pancreatic ß cells, although the biological significance of the molecular interaction between Gal-2 and CAT3 has not been elucidated. In this study, we demonstrated that the structure of N-glycan of CAT3 was either tetra- or tri-antennary branch structure carrying ß-galactosides, which works as galectin-ligands. Indeed, CAT3 bound to Gal-2 using ß-galactoside epitope. Moreover, the disruption of the glycan-mediated bindings between galectins and CAT3 significantly reduced cell surface expression levels of CAT3. The reduced cell surface residency of CAT3 attenuated the cellular arginine uptake activities and subsequently reduced nitric oxide production, and thus impaired the arginine-stimulated insulin secretion of pancreatic ß cells. These results indicate that galectin-lattice stabilizes CAT3 by preventing endocytosis to sustain the arginine-stimulated insulin secretion of pancreatic ß cells. This provides a novel cell biological insight into the endocrinological mechanism of nutrition metabolism and homeostasis.
Asunto(s)
Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Galectina 2/metabolismo , Secreción de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Transducción de Señal/inmunología , Sistemas de Transporte de Aminoácidos Básicos/inmunología , Animales , Anticuerpos/inmunología , Arginina/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Endocitosis/inmunología , Epítopos/metabolismo , Galactósidos/metabolismo , Galectina 2/inmunología , Lactosa/farmacología , Ligandos , Ratones , Óxido Nítrico/biosíntesis , Polisacáridos/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Galectins are among the most abundant excretory/secretory (ES) products of filarial worms, but their role in filarial biology is poorly understood. Galectin-2 (Lec-2), a major component of Brugia malayi extracellular vesicles, is released by filarial worms, and was recently identified in the serum of persons with loiasis. We therefore sought to clone and characterize Lec-2, and to develop reagents to examine its potential as a biomarker and its role in parasite biology. We cloned and expressed recombinant B. malayi Lec-2 (rBmLec-2), generated a Lec-2-specific monoclonal antibody (4B4), and used it to confirm the presence of Lec-2 in B. malayi ES products and whole worm lysate. We show that Lec-2 is absent in B. malayi oocytes, and increases in concentration as embryos mature. Recombinant BmLec-2 hemagglutinates rabbit red blood cells at concentrations less than 1 µg/mL, and this is abrogated by single amino acid substitutions in the predicted carbohydrate recognition domains. rBmLec-2 binds multiple LacNAc oligosaccharides on a mammalian carbohydrate array. Sera from 17/23 (78 %) persons with microfilaremic loiasis and 4/10 (40 %) persons with bancroftian filariasis had detectable antibody to Lec-2 by western blot. Our studies confirm the functionality of BmLec-2 and indicate anti-Lec-2 antibody responses are common in persons with filariasis. These studies set the stage for further examination of the role of Lec-2 in filarial biology and in filarial-host interactions.
Asunto(s)
Anticuerpos Antihelmínticos/sangre , Brugia Malayi , Galectina 2 , Polisacáridos/metabolismo , Animales , Antígenos Helmínticos/inmunología , Biomarcadores , Western Blotting , Brugia Malayi/inmunología , Brugia Malayi/metabolismo , Filariasis Linfática , Filariasis , Galectina 2/biosíntesis , Galectina 2/genética , Galectina 2/inmunología , Galectina 2/metabolismo , Hemaglutinación , Interacciones Huésped-Parásitos , Loiasis , Mamíferos , Proteínas RecombinantesRESUMEN
Background In the presence of arterial stenosis, collateral artery growth (arteriogenesis) can alleviate ischemia and preserve tissue function. In patients with poorly developed collateral arteries, Gal-2 (galectin 2) expression is increased. In vivo administration of Gal-2 inhibits arteriogenesis. Blocking of Gal-2 potentially stimulates arteriogenesis. This study aims to investigate the effect of Gal-2 inhibition on arteriogenesis and macrophage polarization using specific single-domain antibodies. Methods and Results Llamas were immunized with Gal-2 to develop anti-Gal-2 antibodies. Binding of Gal-2 to monocytes and binding inhibition of antibodies were quantified. To test arteriogenesis in vivo, Western diet-fed LDLR.(low-density lipoprotein receptor)-null Leiden mice underwent femoral artery ligation and received treatment with llama antibodies 2H8 or 2C10 or with vehicle. Perfusion restoration was measured with laser Doppler imaging. In the hind limb, arterioles and macrophage subtypes were characterized by histology, together with aortic atherosclerosis. Llama-derived antibodies 2H8 and 2C10 strongly inhibited the binding of Gal-2 to monocytes (93% and 99%, respectively). Treatment with these antibodies significantly increased perfusion restoration at 14 days (relative to sham, vehicle: 41.3±2.7%; 2H8: 53.1±3.4%, P=0.016; 2C10: 52.0±3.8%, P=0.049). In mice treated with 2H8 or 2C10, the mean arteriolar diameter was larger compared with control (vehicle: 17.25±4.97 µm; 2H8: 17.71±5.01 µm; 2C10: 17.84±4.98 µm; P<0.001). Perivascular macrophages showed a higher fraction of the M2 phenotype in both antibody-treated animals (vehicle: 0.49±0.24; 2H8: 0.73±0.15, P=0.007; 2C10: 0.75±0.18, P=0.006). In vitro antibody treatment decreased the expression of M1-associated cytokines compared with control (P<0.05 for each). Atherosclerotic lesion size was comparable between groups (overall P=0.59). Conclusions Inhibition of Gal-2 induces a proarteriogenic M2 phenotype in macrophages, improves collateral artery growth, and increases perfusion restoration in a murine hind limb model.
Asunto(s)
Anticuerpos/farmacología , Aterosclerosis/metabolismo , Circulación Colateral/fisiología , Arteria Femoral/metabolismo , Galectina 2/antagonistas & inhibidores , Miembro Posterior/irrigación sanguínea , Animales , Aterosclerosis/patología , Aterosclerosis/fisiopatología , Modelos Animales de Enfermedad , Femenino , Arteria Femoral/fisiopatología , Galectina 2/metabolismo , Humanos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de SeñalRESUMEN
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.
Asunto(s)
Caenorhabditis elegans/crecimiento & desarrollo , Disacáridos/química , Epítopos/química , Galectina 2/metabolismo , Animales , Ascaris suum/crecimiento & desarrollo , Ascaris suum/metabolismo , Sitios de Unión , Biomphalaria , Caenorhabditis elegans/metabolismo , Disacáridos/metabolismo , Epítopos/metabolismo , Galectina 2/química , Células HeLa , Humanos , Ratones , Ratones Endogámicos ICRRESUMEN
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.
Asunto(s)
Galectina 2/química , Espectroscopía de Resonancia Magnética/métodos , Óxido Nítrico/química , S-Nitrosotioles/química , Animales , Cisteína/química , Cisteína/metabolismo , Galectina 2/metabolismo , Peróxido de Hidrógeno/farmacología , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Modelos Moleculares , Óxido Nítrico/metabolismo , Oxidantes/farmacología , Oxidación-Reducción/efectos de los fármacos , Agregado de Proteínas/efectos de los fármacos , Conformación Proteica , S-Nitrosotioles/metabolismoRESUMEN
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.
Asunto(s)
Galectina 2/química , Galectina 2/metabolismo , Mucinas/química , Mucinas/metabolismo , Animales , Células Epiteliales/metabolismo , Galectina 2/genética , Mucosa Gástrica/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Lactosa/química , Lactosa/metabolismo , Peso Molecular , Plásmidos , Multimerización de Proteína , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , PorcinosRESUMEN
A ß-galactoside binding lectin, designated as galectin-2, was identified and characterized from rock bream Oplegnathus fasciatus (OfGal-2). The cDNA of OfGal-2 comprised of 692 bp with a coding sequence of 396 bp, encoding a putative polypeptide of 131 amino acids. Gene structure analysis of OfGal-2 revealed a four exon-three intron organization. A single carbohydrate-binding domain containing all seven important residues for carbohydrate binding was located in the third exon, which formed a carbohydrate-binding pocket. Homology screening and sequence analysis demonstrated that OfGal-2 is an evolutionarily conserved proto-type galectin. OfGal-2 transcripts were detected in several healthy fish tissues, with the highest level observed in the intestine, followed by the liver. The expression of OfGal-2 was elevated upon the injection of various mitogenic stimulants and pathogens in a time-dependent manner. Upregulated expression in the liver after tissue injury suggested its role as a damage-associated molecular pattern. Recombinant OfGal-2 protein had hemagglutinating potential and possessed affinity towards lactose and galactose. Moreover, the recombinant protein agglutinated and bound potential pathogenic bacteria and a ciliate. The results of this study indicate that the galectin-2 from rock bream has a potential role in immunity, particularly in the recognition of invading pathogens.
Asunto(s)
Infecciones Bacterianas/inmunología , Proteínas de Peces/metabolismo , Galectina 2/metabolismo , Intestinos/fisiología , Tejido Linfoide/fisiología , Perciformes/inmunología , Receptores de Reconocimiento de Patrones/metabolismo , Proteínas Recombinantes/metabolismo , Virosis/inmunología , Animales , Clonación Molecular , Proteínas de Peces/genética , Galectina 2/genética , Regulación de la Expresión Génica , Estructuras Genéticas , Tejido Linfoide/microbiología , Tejido Linfoide/virología , Filogenia , Receptores de Reconocimiento de Patrones/genética , Proteínas Recombinantes/genética , TranscriptomaRESUMEN
Galectin-2 (Gal-2) plays a role in cancer, myocardial infarction, immune response, and gastrointestinal tract diseases. The only reported crystal structure of Gal-2 shows that it is a dimer in which the monomer subunits have almost identical structures, each binding with one molecule of lactose. In this study, we crystallized Gal-2 under new conditions that produced three crystal structures. In each Gal-2 dimer structure, lactose was shown to be bound to only one of the carbohydrate recognition domain subunits. In solution studies, the thermal shift assay demonstrated that inequivalent monomer subunits in the Gal-2 dimer become equivalent upon ligand binding. In addition, galectin-mediated erythrocyte agglutination assays using lactose and larger complex polysaccharides as inhibitors showed the structural differences between Gal-1 and Gal-2. Overall, our results reveal some novel aspects to the structural differentiation in Gal-2 and expand the potential for different types of molecular interactions that may be specific to this lectin.
Asunto(s)
Galectina 2/química , Lactosa/química , Péptidos/química , Aminoácidos/química , Aminoácidos/genética , Aminoácidos/metabolismo , Sitios de Unión/genética , Cristalografía por Rayos X , Galectina 2/genética , Galectina 2/metabolismo , Hemaglutinación/efectos de los fármacos , Pruebas de Hemaglutinación , Humanos , Lactosa/metabolismo , Modelos Moleculares , Péptidos/metabolismo , Polisacáridos/farmacología , Unión Proteica , Conformación Proteica , Dominios Proteicos , Multimerización de ProteínaRESUMEN
Galectins are galactose binding proteins and, in addition, factors for a wide range of pathologies in pregnancy. We have analyzed the expression of prototype (gal-1, -2, -7, -10) and chimera-type (gal-3) galectins in the placenta in cases of spontaneous abortions (SPA) and recurrent abortions (RA) in the first trimester. Fifteen placental samples from healthy pregnancies were used as a control group. Nine placentas were examined for spontaneous abortions, and 12 placentas for recurrent abortions. For differentiation and evaluation of different cell types of galectin-expression in the decidua, immunofluorescence was used. For all investigated prototype galectins (gal-1, -2, -7, -10) in SPA and RA placenta trophoblast cells the expression is significantly decreased. In the decidua/extravillous trophoblast only gal-2 expression was significantly lowered, which could be connected to its role in angiogenesis. In trophoblasts in first-trimester placentas and in cases of SPA and RA, prototype galectins are altered in the same way. We suspect prototype galectins have a similar function in placental tissue because of their common biochemical structure. Expression of galectin 3 as a chimera type galectin was not found to be significantly altered in abortive placentas.
Asunto(s)
Aborto Espontáneo/patología , Galectinas/metabolismo , Placenta/metabolismo , Aborto Espontáneo/metabolismo , Adulto , Estudios de Casos y Controles , Demografía , Femenino , Galectina 2/metabolismo , Galectina 3/metabolismo , Humanos , Microscopía Fluorescente , Placenta/patología , Embarazo , Primer Trimestre del Embarazo , Trofoblastos/metabolismo , Trofoblastos/patologíaRESUMEN
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.
Asunto(s)
Galectina 2/química , Peróxido de Hidrógeno/química , Sustitución de Aminoácidos , Animales , Galectina 2/genética , Galectina 2/metabolismo , Peróxido de Hidrógeno/metabolismo , Ratones , Mutación Missense , Oxidación-ReducciónRESUMEN
Low-temperature plasma is useful for the care of wounded skin. It accelerates wound healing. However, the mechanism of this effect has not been fully elucidated yet. Galectin-1 is reported to accelerate wound healing via the Smad signaling pathway. In the present study to clarify whether or not galectins were expressed during the process of wound healing in the plasma-treated skin, we examined the effect of low-temperature plasma on galectin expression in the healing skin. We compared the effects of low-temperature plasma on the expression of galectin-1, -2, and -3 in the healing skin with those of electrocoagulation conducted with a high-frequency electrical coagulator. Immediately after the start of low-temperature plasma treatment following the incision made in the skin, a membrane-like structure was formed on the surface of the wound. Immunoelectron microscopy showed that these galectins were localized in the membrane-like structure of the plasma-treated skin. The expressions of these galectins were increased by the low-temperature plasma treatment, whereas they were inhibited by the electrocoagulation. These results suggest that galectins were involved in the wound healing of low-temperature plasma-treated skin. Galectins will thus be good markers for further examination of the effects of low-temperature plasma on the healing of wounded skin.
Asunto(s)
Galectina 1/metabolismo , Galectina 2/metabolismo , Galectina 3/metabolismo , Gases em Plasma/uso terapéutico , Cicatrización de Heridas , Animales , Frío , Electrocoagulación , Femenino , Regulación de la Expresión Génica , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Microscopía Inmunoelectrónica , Transducción de Señal , Piel/metabolismo , Piel/patología , Proteínas Smad/metabolismoRESUMEN
INTRODUCTION: Galectin 2 (gal-2) belongs to the proto type group and consists of two homologous carbohydrate recognition domains (CRDs) resulting in multiple sugar binding sites. The expression of gal-2 has been shown to be involved in processes of angiogenesis and inflammation but was not analyzed before in preeclamptic (PE) placentas. Therefore the aim of this study was an analysis of expression and localization of gal-2 in placentas from patients suffering from PE. METHODS: Placental tissues were obtained from 14 women following a normal course of pregnancy and 13 women with PE. Expression of gal-2 was evaluated with immunohistochemistry and a semi quantitative score. Gal-2 mRNA expression was quantified in placental tissue using real time TaqMan PCR. Identification of gal-2 expressing cells in the decidua was achieved by double immunofluorescence microscopy. RESULTS: Expression of gal-2 is downregulated in the syncytiotrophoblast of preeclamptic placentas. Downregulation of gal-2 could also be identified in the decidua of PE patients. These findings on protein level could be supported by the results of TaqMan PCR. Gal-2 is downregulated in PE placentas on mRNA level. Finally, gal-2 expressing cells could be identified as extravillous trophoblast cells in both normal pregnancy and PE. DISCUSSION: Gal-2 was identified as an inhibitor of arteriogenesis in a murine model supposedly via modulation of the monocyte/macrophage population. In PE, both the formation of spiral arteries as well as influx of macrophages are dramatically changed. Therefore we might speculate that disturbed transformation of spiral arteries in PE might correlate with the downregulated gal-2 expression by the trophoblast.
Asunto(s)
Regulación hacia Abajo , Galectina 2/metabolismo , Regulación del Desarrollo de la Expresión Génica , Placenta/metabolismo , Preeclampsia/metabolismo , ARN Mensajero/metabolismo , Adulto , Decidua/metabolismo , Decidua/patología , Femenino , Galectina 2/genética , Humanos , Inmunohistoquímica , Especificidad de Órganos , Placenta/patología , Preeclampsia/patología , Embarazo , Tercer Trimestre del Embarazo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trofoblastos/metabolismo , Trofoblastos/patología , Adulto JovenRESUMEN
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.
Asunto(s)
Cisteína/análogos & derivados , Galectina 2/metabolismo , Peróxido de Hidrógeno/metabolismo , S-Nitrosotioles/metabolismo , Animales , Cisteína/metabolismo , Galectina 2/química , Lactosa/metabolismo , Ratones , Óxido Nítrico/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
The comparatively small number of members of the family of adhesion/growth-regulatory galectins in chicken predestines this system as an attractive model to study the divergence of these lectins after gene duplication. Expression profiling of the three homodimeric (prototype) chicken galectins (CG-1A, CG-1B and CG-2) has raised evidence of distinct functionalities, explaining the interest in a detailed crystallographic analysis of CG-2. As revealed here, marked differences are found in the ligand-binding site and in the contact pattern within the homodimer interface, underlying a characteristic orientation of the two subunits. Notably, a distinctive trimer of dimers that is unique in all galectin crystal structures reported to date forms the core unit of the crystallographic assembly. Combination with spectroscopic and thermodynamic measurements, and comparisons with CG-1A and CG-1B, identify differential changes in the circular-dichroism spectra in the presence of lactose, reflecting the far-reaching impact of the ligand on hydrodynamic behaviour, and inter-galectin differences in both the entropy and the enthalpy of binding. This structural information is a salient step to complete the analysis of the full set of galectins from this model organism.
Asunto(s)
Galectina 2/química , Galectinas/química , Animales , Pollos , Cristalografía por Rayos X , Galectina 1/química , Galectina 2/metabolismo , Galectinas/metabolismo , Humanos , Ligandos , Modelos Químicos , Unión Proteica , Multimerización de Proteína , Alineación de Secuencia , Relación Estructura-ActividadRESUMEN
The emerging insights into the physiological significance of endogenous lectins prompted us to characterize the effect of monosubstitution with poly(ethylene glycol) (PEG; 5 kDa) on a human lectin. As role model, we used a member of the galectin family, that is, galectin-2, the Cys57Met (single-site) mutant and its monoPEGylated derivative. The activities of these three proteins were comparatively studied by biochemical, cell biological, and histochemical methods, using surface-immobilized glycoproteins, different types of cells presenting gangliosides or (glyco)proteins as counterreceptors in vitro and tissue sections. PEGylation led to decreases in affinity/signal intensity with context dependence. The introduction of the mutation, too, can influence reactivity. Assays on haemagglutination and inhibition of cell proliferation underscored that mutational engineering and substitution can (but must not necessarily) affect this protein's activity. Serum clearance in rats was markedly retarded by PEGylation. Overall, the bulky substitution, spatially comparable to N-glycans, can markedly reduce binding of the galectin to physiological binding sites.
Asunto(s)
Galectina 2/química , Galectina 2/genética , Sustitución de Aminoácidos , Animales , Línea Celular , Galectina 2/metabolismo , Humanos , Ligandos , Masculino , Glicoproteínas de Membrana/metabolismo , Tasa de Depuración Metabólica , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Polietilenglicoles/química , Unión Proteica , Ratas , Receptores Mitogénicos/metabolismoRESUMEN
CD146 is a highly glycosylated junctional adhesion molecule, expressed on human vascular endothelial cells and involved in the control of vessel integrity. Galectin-1 is a lectin produced by vascular cells that can binds N- and O-linked oligosaccharides of cell membrane glycoproteins. Because both CD146 and Galectin-1 are involved in modulation of cell apoptosis, we hypothesized that Galectin-1 could interact with CD146, leading to functional consequences in endothelial cell apoptosis. We first characterized CD146 glycosylations and showed that it is mainly composed of N-glycans able to establish interactions with Galectin-1. We demonstrated a sugar-dependent binding of recombinant CD146 to Galectin-1 using both ELISA and Biacore assays. This interaction is direct, with a K(D) of 3.10(-7) M, and specific as CD146 binds to Galectin-1 and not to Galectin-2. Moreover, co-immunoprecipitation experiments showed that Galectin-1 interacts with endogenous CD146 that is highly expressed by HUVEC. We observed a Galectin-1-induced HUVEC apoptosis in a dose-dependent manner as demonstrated by Annexin-V/7AAD staining. Interestingly, both down-regulation of CD146 cell surface expression using siRNA and antibody-mediated blockade of CD146 increase this apoptosis. Altogether, our results identify Galectin-1 as a novel ligand for CD146 and this interaction protects, in vitro, endothelial cells against apoptosis induced by Galectin-1.