RESUMEN
RATIONALE: Hypertension in obesity has become a major threat for public health. Omentin-1, a novel adipokine, is down-regulated in obesity. Tetrahydroxystilbene glycoside (TSG) is the main ingredient extracted from Polygonum multiflorum Thunb (PMT), a traditional Chinese medicinal herb safely used for protecting cardiovascular systems over bimillennium. This study aims to examine (i) the impact of omentin-1 downregulation on obesity-related hypertension in murine models and the underlying mechanisms; (ii) whether tetrahydroxystilbene glycoside (TSG) improved endothelial dysfunction and obesity-associated hypertension via the increase of omentin-1. METHODS: (TSG-treated) male Zucker diabetic fatty (ZDF) rats and omentin-1 knockout (OMT-/-) mice were used. In vitro, human umbilical vein endothelial cells (HUVECs) and mature adipocytes differentiated from human visceral preadipocyte (HPA-v) were maintained in a co-culture system. RESULTS: TSG was the main active component of PMT reducing systolic blood pressure and improving endothelial vasodilation. Fortnight-TSG treatment (100 mg/kg/day) increased serum omentin-1 level, also activated Akt/eNOS signaling and enhanced NO bioactivity; decreased expression of NOX2 and p22phox, suppressed production of superoxide and peroxynitrite anion. OMT-/- mice showed elevated blood pressure and impaired endothelial vasorelaxation, whereas hypotensive effect of TSG was blunted. In co-culture system, TSG incubation promoted binding of peroxisome proliferator-activated receptor-γ (PPAR-γ) and Itln-1 promoter in adipocytes, activated Akt/eNOS/NO signaling and attenuated oxidative/nitrative stress in HUVECs. Suppression of Itln-1 with siRNA significantly blocked the protective effect of TSG in vitro. CONCLUSIONS: Down-regulation of omentin-1 induces endothelial dysfunction and hypertension in obesity. TSG treatment (at least partially) increases omentin-1 via promoting binding of PPAR-γ and Itln-1 promoter in adipose tissues, subsequently exerts protective effects on endothelial function via activating Akt/eNOS/NO signaling and attenuating oxidative/nitrative stress. These results suggest that TSG could be developed as a promising anti-hypertension agent that protects against endothelial dysfunction and obesity-associated cardiovascular diseases.
Asunto(s)
Citocinas/biosíntesis , Citocinas/deficiencia , Endotelio Vascular/efectos de los fármacos , Proteínas Ligadas a GPI/biosíntesis , Proteínas Ligadas a GPI/deficiencia , Glucósidos/uso terapéutico , Hipertensión/tratamiento farmacológico , Lectinas/biosíntesis , Lectinas/deficiencia , Estilbenos/uso terapéutico , Animales , Citocinas/genética , Endotelio Vascular/metabolismo , Proteínas Ligadas a GPI/genética , Glucósidos/metabolismo , Glucósidos/farmacología , Humanos , Hipertensión/genética , Hipertensión/metabolismo , Lectinas/genética , Masculino , Ratones , Ratones Noqueados , Ratas , Ratas Zucker , Estilbenos/metabolismo , Estilbenos/farmacologíaRESUMEN
Neurokinin B, encoded by the tachykinin3 gene, plays a crucial role in regulating reproduction in mammals via KNDy neurons and interaction with GnRH. Previous work in teleost fishes has focused on hypothalamic tac3 expression for its role in reproduction, but detailed studies on extra-hypothalamic tac3 expression are limited. Here, we identified two tac3 genes in the social African cichlid fish Astatotilapia burtoni, only one of which produces a functional protein containing the signature tachykinin motif. In situ hybridization for tac3a mRNA identified cell populations throughout the brain. Numerous tac3a cells lie in several thalamic and hypothalamic nuclei, including periventricular nucleus of posterior tuberculum, lateral tuberal nucleus (NLT), and nucleus of the lateral recess (NRL). Scattered tac3-expressing cells are also present in telencephalic parts, such as ventral (Vv) and supracomissural (Vs) part of ventral telencephalon. In contrast to other teleosts, tac3 expression was absent from the pituitary. Using double-fluorescent staining, we localized tac3a-expressing cells in relation to GnRH and kisspeptin cells. Although no GnRH-tac3a colabeled cells were observed, dense GnRH fibers surround and potentially synapse with tac3a cells in the preoptic area. Only minimal (<5%) colabeling of tac3a was observed in kiss2 cells. Despite tac3a expression in many nodes of the mesolimbic reward system, it was absent from tyrosine hydroxylase (TH)-expressing cells, but tac3a cells were located in areas with dense TH fibers. The presence of tac3a-expressing cells throughout the brain, including in socially relevant brain regions, suggest more diverse functions beyond regulation of reproductive physiology that may be conserved across vertebrates.
Asunto(s)
Encéfalo/metabolismo , Cíclidos/metabolismo , Lectinas/biosíntesis , Animales , Cíclidos/genética , Neuronas Dopaminérgicas/fisiología , Femenino , Peces/clasificación , Peces/genética , Hormona Liberadora de Gonadotropina/análisis , Hipotálamo/metabolismo , Hibridación in Situ , Kisspeptinas/análisis , Lectinas/genética , Masculino , Especificidad de Órganos , Filogenia , Reproducción/genética , Reproducción/fisiología , Recompensa , Conducta SocialRESUMEN
Lectins, one of the most important bioactive compounds, are nonimmunoglobulin proteins that can bind carbohydrates specifically. However, few reports have been published on Pleurotus ostreatus lectin at the molecular level. Hence, in this study, seven lectins were identified based on transcriptomes in four developmental stages, i.e., mycelium, primordium, young fruiting body, and mature fruiting body. The expression profiles of the lectin genes were verified by quantitative real-time PCR. Lectin2-lectin6 had the highest expression in mycelium, while lectin1 was rich in mature fruiting body, and lectin7 was in primordium. We inferred that lectin2-lectin6 may take part in cell flocculation, lectin7 was the critical gene for primordium formation, and lectinl may be involved in fruiting body maturation, respectively. By in silico analysis, all lectins were divided into three distinct groups. Lectin1-Lectin5 were about 38.5-40.7 kDa as extracellular protein and belonged to the PCL-like lectins. Lectin6 (15.4 kDa) was predicted in nucleus and belonged to fungal fruit body lectins. Lectin7 (38.5 kDa) was a member of legume-like lectins and located in the plasma membrane. This study will help us understand how lectins mediate mushroom development.
Asunto(s)
Perfilación de la Expresión Génica , Lectinas/biosíntesis , Pleurotus/crecimiento & desarrollo , Pleurotus/metabolismo , Membrana Celular/química , Núcleo Celular/química , Simulación por Computador , Cuerpos Fructíferos de los Hongos/crecimiento & desarrollo , Cuerpos Fructíferos de los Hongos/metabolismo , Lectinas/química , Peso Molecular , Micelio/crecimiento & desarrollo , Micelio/metabolismo , Pleurotus/genética , Transporte de Proteínas , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The Ym1/2 lectin is expressed abundantly in the allergic mouse lung in an IL-13-dependent manner. However, the role of Ym1/2 in the development of allergic airways disease is largely unknown. In this investigation, we show that treatment of mice with anti-Ym1/2 Ab during induction of allergic airways disease attenuated mediastinal lymph node production of IL-5 and IL-13. Ym1/2 was found to be expressed by dendritic cells (DCs) in an IL-13-dependent manner and supplementation of DC/CD4(+) T cell cocultures with Ym1/2 enhanced the ability of IL-13(-/-) DCs to stimulate the secretion of IL-5 and IL-13. Affinity chromatography identified 12/15(S)-lipoxygenase (12/15-LOX) as a Ym1/2-interacting protein and functional studies suggested that Ym1/2 promoted the ability of DCs to stimulate cytokine production by inhibiting 12/15-LOX-mediated catalysis of 12-hydroxyeicosatetraenoic acid (12(S)-HETE). Treatment of DC/CD4(+) T cell cultures with the 12/15-LOX inhibitor baicalein enhanced, whereas 12(S)-HETE inhibited the production of Th2 cytokines. Notably, delivery of 12(S)-HETE to the airways of mice significantly attenuated the development of allergic airways inflammation and the production of IL-5 and IL-13. In summary, our results suggest that production of Ym1/2 in response to IL-13 promotes Th2 cytokine production and allergic airways inflammation by inhibiting the production of 12(S)-HETE by 12/15-LOX.
Asunto(s)
Quitinasas/fisiología , Citocinas/biosíntesis , Eosinofilia/prevención & control , Mediadores de Inflamación/fisiología , Lectinas/fisiología , Inhibidores de la Lipooxigenasa , Transducción de Señal/inmunología , Células Th2/inmunología , beta-N-Acetilhexosaminidasas/fisiología , Animales , Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Células Cultivadas , Quitinasas/biosíntesis , Quitinasas/genética , Técnicas de Cocultivo , Citocinas/antagonistas & inhibidores , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/patología , Eosinofilia/enzimología , Eosinofilia/patología , Interleucina-13/deficiencia , Interleucina-13/genética , Interleucina-13/fisiología , Lectinas/biosíntesis , Lectinas/genética , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Células Th2/enzimología , Células Th2/patología , beta-N-Acetilhexosaminidasas/biosíntesis , beta-N-Acetilhexosaminidasas/genéticaRESUMEN
Intelectin (IntL), a lectin that exists on the brush border membrane of the small intestine, plays a role in the innate immune response and also acts as a receptor for lactoferrin (LF), an iron-binding glycoprotein found in milk and other secretions. Similar to human LF (hLF), bovine LF (bLF) has been shown to induce proliferation and differentiation of human enterocytes and to modulate their cytokine productions. To evaluate the interaction between human IntL (hIntL) and bLF, recombinant hIntL (rhIntL) conjugated with a tag sequence was examined for its ligand-binding capacity by using microtiter plates coated with LF or other proteins. Interestingly, rhIntL showed higher binding for bLF than hLF. It also bound pepsin hydrolysate of bLF, but to a lower degree than native bLF. A very low binding of rhIntL was observed for bovine serum albumin or transferrin. These findings suggest that hIntL acts as a receptor for bLF and its digested fragments.
Asunto(s)
Citocinas/metabolismo , Lactoferrina/metabolismo , Lectinas/metabolismo , Animales , Bovinos , Citocinas/biosíntesis , Citocinas/genética , ADN Complementario/biosíntesis , ADN Complementario/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Proteínas Ligadas a GPI , Humanos , Lectinas/biosíntesis , Lectinas/genética , Leche Humana/química , Péptidos/metabolismo , Pliegue de Proteína , Proteínas Recombinantes , Tinción con Nitrato de PlataRESUMEN
Real-time PCR was used to measure changes in transcript abundance of genes encoding important immune proteins, namely prophenoloxidase (proPO gene), beta-1,3-glucan binding protein (betaGBP gene) and a 12.2 kDa antimicrobial peptide (amp gene) in post-larval stage VI (PLVI) juveniles of the European lobster, Homarus gammarus. Gene expression was studied in both healthy PLVI and following single or repeat exposure to a range of compounds claimed to induce immune reactivity. A single acute (3-h) exposure to any of the tested stimulants did not produce a significant increase in expression of either the proPO or betaGBP genes, measured 6h after stimulation. However, there were a small sub-group of positive responders, identified mainly from betaGBP expression, within the experimental groups stimulated with either a beta-1,3-glucan or an alginate. There was also no significant increase in the expression of any of the three genes tested 24 h after repeated weekly (3-h) exposures to a either the beta-1,3-glucan or the alginate over the longer (36-day) period. The results do show that amp is expressed at an extremely high level compared to proPO or betaGBP in healthy animals and a significant correlation was found between the expression of proPO and both betaGBP and amp, irrespective of whether or not the larvae were stimulated. None of the immune stimulated compounds improved survival of PLVI challenged with the opportunistic pathogen, Listonella anguillarum, or the lobster pathogen, Aerococcus viridans var. homari. Thus, we found no evidence to support recent claims that immunity and disease resistance can be primed or promoted within a given population of crustaceans or that these animals exhibit functional immune memory to some soluble immune elicitors.
Asunto(s)
Proteínas Portadoras/genética , Catecol Oxidasa/genética , Precursores Enzimáticos/genética , Infecciones por Bacterias Gramnegativas/inmunología , Inmunidad Innata/genética , Lectinas/genética , Listonella , Nephropidae/inmunología , Animales , Proteínas Portadoras/biosíntesis , Catecol Oxidasa/biosíntesis , Precursores Enzimáticos/biosíntesis , Regulación de la Expresión Génica/efectos de los fármacos , Infecciones por Bacterias Gramnegativas/genética , Inmunidad Innata/efectos de los fármacos , Factores Inmunológicos/farmacología , Memoria Inmunológica , Lectinas/biosíntesis , Nephropidae/genéticaRESUMEN
Human (h)Langerin/CD207 is a C-type lectin of Langerhans cells (LC) that induces the formation of Birbeck granules (BG). In this study, we have cloned a cDNA-encoding mouse (m)Langerin. The predicted protein is 66% homologous to hLangerin with conservation of its particular features. The organization of human and mouse Langerin genes are similar, consisting of six exons, three of which encode the carbohydrate recognition domain. The mLangerin gene maps to chromosome 6D, syntenic to the human gene on chromosome 2p13. mLangerin protein, detected by a mAb as a 48-kDa species, is abundant in epidermal LC in situ and is down-regulated upon culture. A subset of cells also expresses mLangerin in bone marrow cultures supplemented with TGF-beta. Notably, dendritic cells in thymic medulla are mLangerin-positive. By contrast, only scattered cells express mLangerin in lymph nodes and spleen. mLangerin mRNA is also detected in some nonlymphoid tissues (e.g., lung, liver, and heart). Similarly to hLangerin, a network of BG form upon transfection of mLangerin cDNA into fibroblasts. Interestingly, substitution of a conserved residue (Phe(244) to Leu) within the carbohydrate recognition domain transforms the BG in transfectant cells into structures resembling cored tubules, previously described in mouse LC. Our findings should facilitate further characterization of mouse LC, and provide insight into a plasticity of dendritic cell organelles which may have important functional consequences.
Asunto(s)
Antígenos de Superficie/aislamiento & purificación , Células Dendríticas/química , Células de Langerhans/química , Tejido Linfoide/química , Lectinas de Unión a Manosa , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Anticuerpos Monoclonales/química , Antígenos CD/biosíntesis , Antígenos CD/genética , Antígenos CD/inmunología , Antígenos CD/aislamiento & purificación , Antígenos de Superficie/biosíntesis , Antígenos de Superficie/genética , Antígenos de Superficie/inmunología , Secuencia de Bases , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Línea Celular , Células Cultivadas , Medios de Cultivo/farmacología , Gránulos Citoplasmáticos/genética , Gránulos Citoplasmáticos/metabolismo , ADN Complementario/aislamiento & purificación , Células Dendríticas/inmunología , Humanos , Células de Langerhans/inmunología , Lectinas/biosíntesis , Lectinas/genética , Lectinas/inmunología , Lectinas/aislamiento & purificación , Lectinas Tipo C , Leucina/genética , Tejido Linfoide/citología , Tejido Linfoide/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microtúbulos/genética , Microtúbulos/metabolismo , Datos de Secuencia Molecular , Especificidad de Órganos/genética , Especificidad de Órganos/inmunología , Fenilalanina/genética , ARN Mensajero/metabolismo , Transfección , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
DC-SIGN is a C-type lectin, expressed on a dendritic cell subset. It is able to bind ICAM3 and HIV gp120 in a calcium-dependent manner. Here we report the genomic organization of DC-SIGN and map it to chromosome 19p13 adjacent to the C-type lectin CD23 (FcepsilonRII). We also report a novel, closely linked gene, DC-SIGNR, which shows 73% identity to DC-SIGN at the nucleic acid level and a similar genomic organization. Proteins encoded by both genes have tracts of repeats of 23 aa, predicted to form a coiled coil neck region. They also possess motifs that are known to bind mannose in a calcium-dependent fashion. We show concomitant expression of the two genes in endometrium, placenta, and stimulated KG1 cells (phenotypically similar to monocyte-derived dendritic cells). The existence of a DC-SIGN-related gene calls for reinterpretation of the HIV data to consider possible DC-SIGN/DC-SIGNR hetero-oligomerization.
Asunto(s)
Moléculas de Adhesión Celular , Cromosomas Humanos Par 19/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Lectinas Tipo C , Lectinas/genética , Familia de Multigenes/inmunología , Receptores de Superficie Celular/genética , Receptores de IgE/genética , Secuencia de Aminoácidos , Línea Celular , Mapeo Cromosómico , Clonación Molecular , ADN Complementario/química , ADN Complementario/aislamiento & purificación , Exones , Expresión Génica/inmunología , Ligamiento Genético , Humanos , Intrones , Lectinas/biosíntesis , Lectinas/química , Datos de Secuencia Molecular , Especificidad de Órganos/genética , Especificidad de Órganos/inmunología , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/química , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
A lipopolysaccharide-specific lectin, immulectin-2, was isolated from plasma of the tobacco hornworm, Manduca sexta. Immulectin-2 has specificity for xylose, glucose, lipopolysaccharide, and mannan. A cDNA clone encoding immulectin-2 was isolated from an Escherichia coli-induced M. sexta larval fat body cDNA library. The cDNA is 1253 base pairs long, with an open reading frame of 981 base pairs, encoding a 327-residue polypeptide. Immulectin-2 is a member of the C-type lectin superfamily. It consists of two carbohydrate recognition domains, which is similar to the organization of M. sexta immulectin-1. Immulectin-2 was present at a constitutively low level in plasma of control larvae and increased 3-4-fold after injection of Gram-negative bacteria or lipopolysaccharide. Immulectin-2 mRNA was detected in fat body of control larvae, and its level increased dramatically after injection of E. coli. The concentration of immulectin-2 in plasma did not change significantly after injection of Gram-positive bacteria or yeast, even though its mRNA level was increased by these treatments. Compared with immulectin-1, immulectin-2 has a more restricted specificity for binding to Gram-negative bacteria. Immulectin-2 at low physiological concentrations agglutinated E. coli in a calcium-dependent manner. It also bound to immobilized lipopolysaccharide from E. coli. Binding of immulectin-2 to lipopolysaccharide stimulated phenol oxidase activation in plasma. The properties of immulectin-2 are consistent with its function as a pattern recognition receptor for detection and defense against Gram-negative bacterial infection in M. sexta.
Asunto(s)
Escherichia coli/fisiología , Proteínas de Insectos , Lectinas Tipo C , Lectinas/biosíntesis , Lipopolisacáridos/farmacología , Manduca/metabolismo , Pruebas de Aglutinación , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario , Electroforesis en Gel de Poliacrilamida , Lectinas/genética , Lectinas/aislamiento & purificación , Lectinas/metabolismo , Ligandos , Manduca/microbiología , Datos de Secuencia Molecular , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de AminoácidoRESUMEN
Bauhinia purpurea lectin (BPA) is one of the beta-galactose-binding leguminous lectins. Leguminous lectins contain a long metal-binding loop, part of which determines their carbohydrate-binding specificities. Random mutations were introduced into a portion of the cDNA coding BPA that corresponds to the carbohydrate-binding loop of the lectin. An library of the mutant lectin expressed on the surface of lambda foo phages was screened by the panning method. Several phage clones with an affinity for mannose or N-acetylglucosamine were isolated. These results indicate the possibility of making artificial lectins (so-called "cyborg lectins") with distinct and desired carbohydrate-binding specificities.
Asunto(s)
Fabaceae/genética , Lectinas/síntesis química , Lectinas/genética , Proteínas de Plantas/síntesis química , Proteínas de Plantas/genética , Plantas Medicinales , Acetilglucosamina/análogos & derivados , Acetilglucosamina/genética , Acetilglucosamina/metabolismo , Secuencia de Aminoácidos , Bacteriófago lambda/genética , Bacteriófago lambda/metabolismo , Sitios de Unión/genética , Fabaceae/química , Vectores Genéticos/síntesis química , Lectinas/biosíntesis , Lectinas/metabolismo , Manosa/genética , Manosa/metabolismo , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Biblioteca de Péptidos , Lectinas de Plantas , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/metabolismo , Ingeniería de Proteínas , Albúmina Sérica/genética , Albúmina Sérica/metabolismo , Albúmina Sérica Bovina/genética , Albúmina Sérica Bovina/metabolismo , Virión/genética , Virión/metabolismoRESUMEN
Phytohemagglutinin (Phaseolus vulgaris agglutinin; PHA; E- and L-forms) and snowdrop lectin (Galanthus nivalis agglutinin; GNA) were expressed in Pichia pastoris using native signal peptides, or the Saccharomyces alpha-factor preprosequence, to direct proteins into the secretory pathway. PHA and GNA were present as soluble, functional proteins in culture supernatants when expressed from constructs containing the alpha-factor preprosequence. The recombinant lectins, purified by affinity chromatography, agglutinated rabbit erythrocytes at concentrations similar to the respective native lectins. However, incomplete processing of the signal sequence resulted in PHA-E, PHA-L and GNA with heterogenous N-termini, with the majority of the protein containing N-terminal extensions derived from the alpha-factor prosequence. Polypeptides in which most of the alpha-factor prosequence was present were also glycosylated. Inclusion of Glu-Ala repeats at the C-terminal end of the alpha-factor preprosequence led to efficient processing N-terminal to the Glu-Ala sequence, but inefficient removal of the repeats themselves, resulting in polypeptides with heterogenous N-termini still containing N-terminal extensions. In contrast, PHA expressed with the native signal peptide was secreted, correctly processed, and also fully functional. No expression of GNA from a construct containing the native GNA signal peptide was observed. The PHA-E signal peptide directed correct processing and secretion of both GNA and green fluorescent protein (GFP) when used in expression constructs, and is suggested to have general utility for synthesis of correctly processed proteins in Pichia.
Asunto(s)
Lectinas/biosíntesis , Lectinas de Unión a Manosa , Fitohemaglutininas/biosíntesis , Proteínas de Plantas , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/biosíntesis , Secuencia de Aminoácidos , Cromatografía de Afinidad , Galanthus , Glicoproteínas/biosíntesis , Glicoproteínas/genética , Glicosilación , Lectinas/genética , Factor de Apareamiento , Datos de Secuencia Molecular , Péptidos/genética , Fitohemaglutininas/genética , Pichia/genética , Lectinas de Plantas , Señales de Clasificación de Proteína , Análisis de Secuencia de ProteínaRESUMEN
Mass spectrometric methods were used to investigate the proteolytic processing and glycopeptide structures of three seed defensive proteins from Phaseolus vulgaris. The proteins were the alpha-amylase inhibitors alphaAI-1 and alphaAI-2 and arcelin-5, all of which are related to the seed lectins, PHA-E and PHA-L. The mass data showed that the proteolytic cleavage required for activation of the amylase inhibitors is followed by loss of the terminal Asn residue in alphaAI-1, and in all three proteins, seven or more residues were clipped from the C-termini, in the manner of the seed lectins. In most instances, individual glycoforms could be assigned at each Asn site, due to the unique masses of the plant glycopeptides. It was found that alphaAI-1 and alphaAI-2 differed significantly in their glycosylation patterns, despite their high sequence homology. These data complement the previous X-ray studies of the alpha1-amylase inhibitor and arcelin, where many of the C-terminal residues and glycopeptide residues could not be observed.
Asunto(s)
Fabaceae/metabolismo , Glicoproteínas/biosíntesis , Lectinas/biosíntesis , Proteínas de Plantas/biosíntesis , Plantas Medicinales , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Inhibidores Enzimáticos , Fabaceae/genética , Glicoproteínas/genética , Péptidos y Proteínas de Señalización Intercelular , Lectinas/genética , Datos de Secuencia Molecular , Lectinas de Plantas , Proteínas de Plantas/genética , alfa-Amilasas/antagonistas & inhibidoresRESUMEN
Post-translational processing of concanavalin A (Con A) is complex, involving deglycosylation, proteolytic cleavage on the carboxy group side of asparagine residues and formation of a peptide bond de novo. This has been studied with the 125I-labelled Con A glycoprotein precursor as a substrate for processing in vitro. Extracts of immature jackbean cotyledons and the commercially available purified preparation of asparaginylendo-peptidase were able to catalyse the above processes. The processing resulted in the conversion of the 33.5 kDa inactive glycoprotein precursor into an active lectin. Processing activity was maximal at approx. pH 5.5. Evidence to support processing at authentic sites was obtained by observation of the release of 125I at positions in the sequence where tyrosine residues were present.
Asunto(s)
Concanavalina A/metabolismo , Péptidos/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Secuencia de Aminoácidos , Cromatografía de Afinidad , Cisteína Endopeptidasas/metabolismo , Electroforesis en Gel de Poliacrilamida , Fabaceae/enzimología , Fabaceae/metabolismo , Glicoproteínas/metabolismo , Concentración de Iones de Hidrógeno , Lectinas/biosíntesis , Datos de Secuencia Molecular , Compuestos Organofosforados/metabolismo , Lectinas de Plantas , Proteínas de Plantas/metabolismo , Plantas MedicinalesRESUMEN
Griffonia simplicifolia II, an N-acetylglucosamine-specific legume lectin, has insecticidal activity when fed to the cowpea weevil, Callosobruchus maculatus (F.). A cDNA clone encoding G. simplicifolia II was isolated from a leaf cDNA library, sequenced, and expressed in a bacterial expression system. The recombinant protein exhibited N-acetylglucosamine-binding and insecticidal activity against cowpea weevil, indicating that glycosylation and multimeric structure are not required for these properties. These results support the hypothesis that genes of the legume lectin gene family encode proteins that function in plant defense against herbivores.
Asunto(s)
Acetilglucosamina , Fabaceae/genética , Genes de Plantas , Insecticidas/farmacología , Lectinas/genética , Plantas Medicinales , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario/genética , Escherichia coli/genética , Regulación del Desarrollo de la Expresión Génica , Biblioteca de Genes , Glicosilación , Insectos/efectos de los fármacos , Lectinas/biosíntesis , Lectinas/química , Datos de Secuencia Molecular , Familia de Multigenes , Hojas de la Planta/química , Lectinas de Plantas , Conformación Proteica , Proteínas Recombinantes/biosíntesis , Semillas/química , Análisis de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Distribución TisularRESUMEN
Secretion of inflammatory mediators by rat mast cells (line RBL-2H3) was earlier shown to be inhibited upon clustering a membrane glycoprotein by monoclonal antibody G63. This glycoprotein, named mast cell function-associated antigen (MAFA), was also shown to interfere with the coupling cascade of the type 1 Fc epsilon receptor upstream to phospholipase C gamma 1 activation by protein-tyrosine kinases. Here we report that the MAFA is expressed as both a monomer and a homodimer. Expression cloning of its cDNA shows that it contains a single open reading frame, encoding a 188-amino acid-long type II integral membrane protein. The 114 C-terminal amino acids display sequence homology with the carbohydrate-binding domain of calcium-dependent animal lectins, many of which have immunological functions. The cytoplasmic tail of MAFA contains a YXXL (YSTL) motif, which is conserved among related C-type lectins and is an essential element in the immunoreceptor tyrosine-based activation motifs. Finally, changes in the MAFA tyrosyl- and seryl-phosphorylation levels are observed in response to monoclonal antibody G63 binding, antigenic stimulation, and a combination of both treatments.
Asunto(s)
Lectinas Tipo C , Lectinas/fisiología , Mastocitos/fisiología , Glicoproteínas de Membrana/fisiología , Transducción de Señal , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Secuencia de Bases , Línea Celular , Chlorocebus aethiops , Clonación Molecular , ADN Complementario , Electroforesis en Gel de Poliacrilamida , Humanos , Lectinas/biosíntesis , Lectinas/química , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/química , Ratones , Datos de Secuencia Molecular , Peso Molecular , Sistemas de Lectura Abierta , Ratas , Receptores Inmunológicos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Transactivadores , TransfecciónRESUMEN
Peptide-23 is a 16-kilodalton protein secreted by rat pituitary cells that was first identified because it was regulated by GRF and somatostatin in a similar fashion to GH. Cloning of peptide-23 complementary DNA revealed that it is identical to pancreatitis-associated protein (PAP) and a member of the c-lectin gene family. We examined the expression of peptide-23/PAP and a structurally related protein, pancreatic stone protein (PSP/reg), in the rat gastrointestinal tract. Here we report age-related changes in the expression and GRF regulation of peptide-23. Both peptide-23/PAP messenger RNA (mRNA) and PSP/reg mRNA were virtually undetectable in the small intestine of newborn and 1- and 2-week-old rats. A dramatic increase in the expression of both genes was seen at the time of weaning in the third week postpartum. The abundance of both of these mRNA decreases after 3 and 6 months of age. Peptide-23/PAP mRNA is most abundant in the ileum, whereas PSP/reg is maximally expressed in the pancreas and duodenum. Human GRF analog pellets were implanted sc into adult male rats for 2 weeks to study the chronic effects of GRF on the expression of these genes. Both peptide-23/PAP and PSP/reg mRNA levels in duodenum and jejunum were increased in these rats compared with levels in control rats. However, no increase in peptide-23/PAP mRNA in response to GRF treatment was seen in the ileum, where the level of expression of this gene is very high, and GRF had no effect on peptide-23/PSP expression in the heart, pituitary, or hypothalamus, where expression is normally undetectable. In situ hybridization was used to localize peptide-23/PSP in the small intestine and pancreas of GRF-treated rats. An increase in peptide-23/PAP mRNA was restricted to acinar cells close to islets, whereas little expression was seen in acinar cells distant from islets, suggesting that either peptide-23/PAP may have some paracrine action on the islets, or alternatively, an islet-derived factor may function as a paracrine modulator of peptide-23/PAP expression. These data demonstrate that GRF modulates peptide-23/PAP expression in the gastrointestinal tract in a similar fashion to that previously reported for pituitary cells in primary culture.
Asunto(s)
Envejecimiento/metabolismo , Antígenos de Neoplasias , Biomarcadores de Tumor , Proteínas de Unión al Calcio/biosíntesis , Sistema Digestivo/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Expresión Génica , Hormona Liberadora de Hormona del Crecimiento/farmacología , Hipotálamo/metabolismo , Lectinas Tipo C , Biosíntesis de Proteínas , Animales , Animales Recién Nacidos , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Sistema Digestivo/crecimiento & desarrollo , Corazón/crecimiento & desarrollo , Hipotálamo/crecimiento & desarrollo , Lectinas/biosíntesis , Litostatina , Masculino , Datos de Secuencia Molecular , Miocardio/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Especificidad de Órganos , Páncreas/crecimiento & desarrollo , Páncreas/metabolismo , Proteínas Asociadas a Pancreatitis , Hipófisis/crecimiento & desarrollo , Hipófisis/metabolismo , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Valores de ReferenciaAsunto(s)
Proteínas de Unión al Calcio/biosíntesis , Calcio/metabolismo , Regulación Neoplásica de la Expresión Génica , Ribonucleoproteínas/biosíntesis , Terpenos/farmacología , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Calreticulina , ADN Complementario , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Cinética , Lectinas/biosíntesis , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Tapsigargina , Factores de Tiempo , Transcripción GenéticaRESUMEN
A cDNA encoding a novel member of the S-lectin family has been cloned from human epidermis. The 14-kDa protein of pI7 predicted by the 136-amino-acid open reading frame of the sequence was called galectin-7 according to the presently accepted nomenclature. A GST fusion protein authentified the lactose-binding properties expected for a member of this lectin subfamily. Galectin-7 was identified on two-dimensional gels of keratinocyte protein extracts. Differential and in situ hybridizations indicate that this lectin is specifically expressed in keratinocytes. It is expressed at all stages of epidermal differentiation (i.e., in basal and suprabasal layers). It is moderately repressed by retinoic acid, a behavior contrasting with those of other keratinocyte markers sensitive to this agent, which, either basal, are induced, or suprabasal, are repressed. This effect of retinoic acid on a keratinocyte cell type marker such as galectin-7 is more reminiscent of its metaplasiogenic effect in vivo than of its inhibitory effect on terminal epidermal differentiation in vitro. This interpretation is supported by the fact that in chick epidermis a 14-kDa S-lectin is suppressed during retinoic acid-induced mucous metaplasia.
Asunto(s)
Galectinas , Queratinocitos/metabolismo , Lectinas/biosíntesis , Tretinoina/farmacología , Secuencia de Aminoácidos , Secuencia de Bases , Diferenciación Celular , Células Cultivadas , ADN Complementario , Humanos , Queratinocitos/citología , Lectinas/genética , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/biosíntesisRESUMEN
Alterations of the hormonal status may influence diverse cell features relevant to intra- and intercellular communication. We studied histochemically the expression of glycans and endogenous sugar-binding proteins (endolectins) in the mouse endometrium during the estrous cycle. The avidin-biotin-peroxidase complex (ABC) technique was used on paraffin sections with a panel of biotinylated lectins and neoglycoproteins. Stage-specific changes were observed with Galanthus nivalis agglutinin (GNA), Ulex europaeus-I agglutinin (UEA-1), Sambucus nigra agglutinin (SNA), Ricinus communis agglutinin (RCA-I) and Glycine max agglutinin (SBA) in both the luminal and the glandular epithelium, but to a lesser extent in the latter. The stromal constituents also exhibited modifications in lectin-binding profiles in cyclic mice. In addition to the analysis of cell glycans endogenous carbohydrate-binding sites were studied using synthetic probes, biotinylated neoglycoproteins. These tools made it possible to conclude that hormonal status can affect the expression of endolectins. We have attempted to correlate the glycohistochemical findings with recently published data obtained biochemically. The results indicate that phase-specific alterations in lectin-binding glycoproteins and endolectins occur in the endometrium of mice associated with their short reproductive cycle.
Asunto(s)
Endometrio/metabolismo , Estro/fisiología , Glicoconjugados/biosíntesis , Lectinas/biosíntesis , Animales , Secuencia de Carbohidratos , Femenino , Galanthus , Histocitoquímica , Técnicas para Inmunoenzimas , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Adhesión en Parafina , Lectinas de PlantasRESUMEN
The DB58 lectin of the stems and leaves of Dolichos biflorus is a heterodimer composed of two closely related subunits, alpha and beta. These subunits were dissociated from one another in urea and isolated by high-performance anion-exchange chromatography. Steric exclusion chromatography of the isolated subunits in 6 M guanidine hydrochloride showed molecular weights of 30,900 and 29,800 for the alpha and beta subunits, respectively. The subunits have very similar amino acid compositions and are glycosylated at each of their two N-glycosylation consensus sites. Each of the subunits had weak carbohydrate binding activity. Reverse-phase chromatography of tryptic digests of the subunits showed identical peptide maps with the exception of peaks identified as COOH-terminal peptides. Analyses of these peptides, COOH-terminal amino acid analyses, and the small differences in amino acid composition between the 2 subunits establish that the beta subunit differs from the alpha subunit by the absence of 11 or 12 amino acids from its COOH terminus. This structural difference, combined with information from previous biosynthetic studies, establishes that the beta subunit is derived from the alpha subunit by posttranslational proteolytic modification at the COOH terminus. The heterogeneity in the extent of truncation suggests that this conversion occurs by sequential removal of amino acids rather than by endoproteolytic cleavage. The possible physiological significance of this modification is discussed.