RESUMEN
Intestinal γδ T-cell receptor-bearing intraepithelial lymphocytes (γδ IELs) play a multifaceted role in maintaining mucosal homeostasis. In order to investigate the relationship between O-glycosylation and inflammation, we carried out an in-depth mass spectrometric comparison of the intestinal O-glycosylation profile of mice lacking γδ IELs (TCRδ(-/-)) and of their wild-type (WT) littermates. A total of 69 nonsulfated and 59 sulfated compositional types of O-glycans were identified in the small intestine and colon of TCRδ(-/-) and WT mice. Our results demonstrated structural differences in intestinal glycosylation in TCRδ(-/-) mice compared with WT littermates. TCRδ(-/-) colons contained a lower proportion of core-2 structures and an increased proportion of core-1 structures whereas TCRδ(-/-) small intestines had a decreased percentage of core-3 structures. The glycan antennae in TCRδ(-/-) colon and small intestine showed altered structural diversity compared with WT mice. There were significant differences in the sialylated species between the TCRδ(-/-) and WT mice with the sialylated Tn antigen found exclusively in the TCRδ(-/-)small intestine, whereas the sulfation pattern remained mostly unchanged. These findings provide novel molecular insights underpinning the role of γδ IELs in maintaining gut homeostasis.
Asunto(s)
Mucosa Intestinal/metabolismo , Polisacáridos/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Ácidos Siálicos/metabolismo , Linfocitos T/metabolismo , Animales , Secuencia de Carbohidratos , Colon/citología , Colon/inmunología , Colon/metabolismo , Femenino , Expresión Génica , Glicosilación , Inmunidad Mucosa , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Intestino Delgado/citología , Intestino Delgado/inmunología , Intestino Delgado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Especificidad de Órganos , Polisacáridos/química , Receptores de Antígenos de Linfocitos T gamma-delta/deficiencia , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Ácidos Siálicos/química , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
Mucins are the main components of the gastrointestinal mucus layer. Mucin glycosylation is critical to most intermolecular and intercellular interactions. However, due to the highly complex and heterogeneous mucin glycan structures, the encoded biological information remains largely encrypted. Here we have developed a methodology based on force spectroscopy to identify biologically accessible glycoepitopes in purified porcine gastric mucin (pPGM) and purified porcine jejunal mucin (pPJM). The binding specificity of lectins Ricinus communis agglutinin I (RCA), peanut (Arachis hypogaea) agglutinin (PNA), Maackia amurensis lectin II (MALII), and Ulex europaeus agglutinin I (UEA) was utilized in force spectroscopy measurements to quantify the affinity and spatial distribution of their cognate sugars at the molecular scale. Binding energy of 4, 1.6, and 26 aJ was determined on pPGM for RCA, PNA, and UEA. Binding was abolished by competition with free ligands, demonstrating the validity of the affinity data. The distributions of the nearest binding site separations estimated the number of binding sites in a 200-nm mucin segment to be 4 for RCA, PNA, and UEA, and 1.8 for MALII. Binding site separations were affected by partial defucosylation of pPGM. Furthermore, we showed that this new approach can resolve differences between gastric and jejunum mucins.
Asunto(s)
Mucinas Gástricas/metabolismo , Mucinas/metabolismo , Polisacáridos/metabolismo , Animales , Mucinas Gástricas/química , Mucinas Gástricas/ultraestructura , Mucosa Gástrica/metabolismo , Mucosa Intestinal/metabolismo , Lectinas/química , Lectinas/metabolismo , Lectinas/ultraestructura , Microscopía de Fuerza Atómica/métodos , Mucinas/química , Mucinas/ultraestructura , Polisacáridos/química , Polisacáridos/ultraestructura , Análisis Espectral/métodos , Porcinos , Distribución TisularRESUMEN
The O-glycosylated domains of mucins and mucin-type glycoproteins contain 50-80% of carbohydrate and possess expanded conformations. Herein, we describe a flow cytometry (FCM) method for determining the carbohydrate-binding specificities of lectins to mucin. Biotinylated mucin was immobilized on streptavidin-coated beads, and the binding specificities of the major mucin sugar chains, as determined by GC-MS and MALDI-ToF, were monitored using fluorescein-labeled lectins. The specificities of lectins toward specific biotinylated glycans were determined as controls. The advantage of flexibility, multiparametric data acquisition, speed, sensitivity, and high-throughput capability makes flow cytometry a valuable tool to study diverse interactions between glycans and proteins.
Asunto(s)
Citometría de Flujo , Mucinas/metabolismo , Lectinas de Plantas/metabolismo , Animales , Polisacáridos/metabolismo , Unión ProteicaRESUMEN
Polyamine biosynthesis in plants differs from other eukaryotes because of the contribution of genes from the cyanobacterial ancestor of the chloroplast. Plants possess an additional biosynthetic route for putrescine formation from arginine, consisting of the enzymes arginine decarboxylase, agmatine iminohydrolase and N-carbamoylputrescine amidohydrolase, derived from the cyanobacterial ancestor. They also synthesize an unusual tetraamine, thermospermine, that has important developmental roles and which is evolutionarily more ancient than spermine in plants and algae. Single-celled green algae have lost the arginine route and are dependent, like other eukaryotes, on putrescine biosynthesis from the ornithine. Some plants like Arabidopsis thaliana and the moss Physcomitrella patens have lost ornithine decarboxylase and are thus dependent on the arginine route. With its dependence on the arginine route, and the pivotal role of thermospermine in growth and development, Arabidopsis represents the most specifically plant mode of polyamine biosynthesis amongst eukaryotes. A number of plants and algae are also able to synthesize unusual polyamines such as norspermidine, norspermine and longer polyamines, and biosynthesis of these amines likely depends on novel aminopropyltransferases similar to thermospermine synthase, with relaxed substrate specificity. Plants have a rich repertoire of polyamine-based secondary metabolites, including alkaloids and hydroxycinnamic amides, and a number of polyamine-acylating enzymes have been recently characterised. With the genetic tools available for Arabidopsis and other model plants and algae, and the increasing capabilities of comparative genomics, the biological roles of polyamines can now be addressed across the plant evolutionary lineage.
Asunto(s)
Evolución Biológica , Chlorophyta/metabolismo , Enzimas/metabolismo , Plantas/metabolismo , Poliaminas/metabolismo , Arabidopsis/metabolismo , Vías Biosintéticas , Bryopsida/metabolismoRESUMEN
Polyamines are small flexible organic polycations found in almost all cells. They likely existed in the last universal common ancestor of all extant life, and yet relatively little is understood about their biological function, especially in bacteria and archaea. Unlike eukaryotes, where the predominant polyamine is spermidine, bacteria may contain instead an alternative polyamine, sym-homospermidine. We demonstrate that homospermidine synthase (HSS) has evolved vertically, primarily in the alpha-Proteobacteria, but enzymatically active, diverse HSS orthologues have spread by horizontal gene transfer to other bacteria, bacteriophage, archaea, eukaryotes, and viruses. By expressing diverse HSS orthologues in Escherichia coli, we demonstrate in vivo the production of co-products diaminopropane and N(1)-aminobutylcadaverine, in addition to sym-homospermidine. We show that sym-homospermidine is required for normal growth of the alpha-proteobacterium Rhizobium leguminosarum. However, sym-homospermidine can be replaced, for growth restoration, by the structural analogues spermidine and sym-norspermidine, suggesting that the symmetrical or unsymmetrical form and carbon backbone length are not critical for polyamine function in growth. We found that the HSS enzyme evolved from the alternative spermidine biosynthetic pathway enzyme carboxyspermidine dehydrogenase. The structure of HSS is related to lysine metabolic enzymes, and HSS and carboxyspermidine dehydrogenase evolved from the aspartate family of pathways. Finally, we show that other bacterial phyla such as Cyanobacteria and some alpha-Proteobacteria synthesize sym-homospermidine by an HSS-independent pathway, very probably based on deoxyhypusine synthase orthologues, similar to the alternative homospermidine synthase found in some plants. Thus, bacteria can contain alternative biosynthetic pathways for both spermidine and sym-norspermidine and distinct alternative pathways for sym-homospermidine.
Asunto(s)
Transferasas Alquil y Aril/metabolismo , Evolución Biológica , Vías Biosintéticas , Poliaminas/metabolismo , Espermidina/análogos & derivados , Bacterias , Cromatografía Líquida de Alta Presión , Modelos Moleculares , Filogenia , Espermidina/metabolismoRESUMEN
Hydroxycinnamic acid amides are a class of secondary metabolites distributed widely in plants. We have identified two sinapoyl spermidine derivatives, N-((4'-O-glycosyl)-sinapoyl),N'-sinapoylspermidine and N,N'-disinapoylspermidine, which comprise the two major polyamine conjugates that accumulate in Arabidopsis thaliana seed. Using metabolic profiling of knockout mutants to elucidate the functions of members of the BAHD acyltransferase family in Arabidopsis, we have also identified two genes encoding spermidine disinapoyl transferase (SDT) and spermidine dicoumaroyl transferase (SCT) activities. At2g23510, which is expressed mainly in seeds, encodes a spermidine sinapoyl CoA acyltransferase (SDT) that is required for the production of disinapoyl spermidine and its glucoside in Arabidopsis seed. The structurally related BAHD enzyme encoded by At2g25150 is expressed specifically in roots and has spermidine coumaroyl CoA acyltransferase (SCT) activity both in vitro and in vivo.
Asunto(s)
Aciltransferasas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Semillas/metabolismo , Espermidina/biosíntesis , Aciltransferasas/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , ADN Bacteriano/genética , Regulación de la Expresión Génica de las Plantas , Técnicas de Inactivación de Genes , Prueba de Complementación Genética , Metaboloma , Mutagénesis Insercional , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , ARN de Planta/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Semillas/genéticaRESUMEN
Members of the BAHD family of plant acyl transferases are very versatile catalytically, and are thought to be able to evolve new substrate specificities rapidly. Acylation of anthocyanins occurs in many plant species and affects anthocyanin stability and light absorption in solution. The versatility of BAHD acyl transferases makes it difficult to identify genes encoding enzymes with defined substrate specificities on the basis of structural homology to genes of known catalytic function alone. Consequently, we have used a modification to standard functional genomics strategies, incorporating co-expression profiling with anthocyanin accumulation, to identify genes encoding three anthocyanin acyl transferases from Arabidopsis thaliana. We show that the activities of these enzymes influence the stability of anthocyanins at neutral pH, and some acylations also affect the anthocyanin absorption maxima. These properties make the BAHD acyl transferases suitable tools for engineering anthocyanins for an improved range of biotechnological applications.