RESUMO
The O-GlcNAc transferase Eogt modifies EGF repeats in proteins that transit the secretory pathway, including Dumpy and Notch. In this paper, we show that the Notch ligands Delta and Serrate are also substrates of Eogt, that mutation of a putative UDP-GlcNAc binding DXD motif greatly reduces enzyme activity, and that Eogt and the cytoplasmic O-GlcNAc transferase Ogt have distinct substrates in Drosophila larvae. Loss of Eogt is larval lethal and disrupts Dumpy functions, but does not obviously perturb Notch signaling. To identify novel genetic interactions with eogt, we investigated dominant modification of wing blister formation caused by knock-down of eogt. Unexpectedly, heterozygosity for several members of the canonical Notch signaling pathway suppressed wing blister formation. And importantly, extensive genetic interactions with mutants in pyrimidine metabolism were identified. Removal of pyrimidine synthesis alleles suppressed wing blister formation, while removal of uracil catabolism alleles was synthetic lethal with eogt knock-down. Therefore, Eogt may regulate protein functions by O-GlcNAc modification of their EGF repeats, and cellular metabolism by affecting pyrimidine synthesis and catabolism. We propose that eogt knock-down in the wing leads to metabolic and signaling perturbations that increase cytosolic uracil levels, thereby causing wing blister formation.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Pirimidinas/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Western Blotting , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Técnicas de Silenciamento de Genes , Teste de Complementação Genética , Células HEK293 , Humanos , Larva/genética , Larva/metabolismo , Dados de Sequência Molecular , Mutação , N-Acetilglucosaminiltransferases/genética , Ligação Proteica , Receptores Notch/genética , Homologia de Sequência de Aminoácidos , Uracila/metabolismo , Asas de Animais/metabolismoRESUMO
Mucin type O-glycosylation is a widespread modification of eukaryotic proteins. The transfer of N-acetylgalactosamine to selected serine or threonine residues is catalyzed by a family of polypeptide N-acetylgalactosaminyltransferases localized in the Golgi apparatus. The most abundant elongation of O-glycans is the addition of a beta1-3 linked galactose by the core-1 beta1-3 galactosyltransferase (core-1 beta3GalT), thereby building the T-antigen or core-1 structure Gal(beta1-3)GalNAc(alpha1-O). We have isolated four Drosophila melanogaster cDNAs encoding proteins structurally similar to the human core-1 beta3GalT enzyme and expressed them as FLAG-tagged proteins in Sf9 insect cells. The identity of these D. melanogasterbeta3GalT enzymes with a core-1 beta3GalT activity was confirmed by utilization of MUC5AC mucin derived O-glycopeptide acceptors. In addition to the core-1 beta3GalT activity toward O-glycoprotein substrates, one member of this enzyme family showed a strong activity towards glycolipid acceptors, thereby building the core-1 terminated Nz6 glycosphingolipid. Transcripts of the embryonically expressed core-1 beta3GalTs were found in the maternally deposited mRNA, in salivary glands and in the amnioserosa. The presence of multiple core-1 beta3GalT genes in D. melanogaster suggests an increased complexity of core-1 O-glycan expression, which is possibly related to multiple developmental and physiological functions attributable to this class of glycans.
Assuntos
Drosophila melanogaster/enzimologia , Galactosiltransferases/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromatografia Líquida de Alta Pressão , DNA Complementar/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Genes de Insetos , Humanos , Hibridização In Situ , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização por Electrospray , Especificidade por SubstratoRESUMO
Crystal (Cry) proteins made by the bacterium Bacillus thuringiensis are pore-forming toxins that specifically target insects and nematodes and are used around the world to kill insect pests. To better understand how pore-forming toxins interact with their host, we have screened for Caenorhabditis elegans mutants that resist Cry protein intoxication. We find that Cry toxin resistance involves the loss of two glycosyltransferase genes, bre-2 and bre-4. These glycosyltransferases function in the intestine to confer susceptibility to toxin. Furthermore, they are required for the interaction of active toxin with intestinal cells, suggesting they make an oligosaccharide receptor for toxin. Similarly, the bre-3 resistance gene is also required for toxin interaction with intestinal cells. Cloning of the bre-3 gene indicates it is the C. elegans homologue of the Drosophila egghead (egh) gene. This identification is striking given that the previously identified bre-5 has homology to Drosophila brainiac (brn) and that egh-brn likely function as consecutive glycosyltransferases in Drosophila epithelial cells. We find that, like in Drosophila, bre-3 and bre-5 act in a single pathway in C. elegans. bre-2 and bre-4 are also part of this pathway, thereby extending it. Consistent with its homology to brn, we demonstrate that C. elegans bre-5 rescues the Drosophila brn mutant and that BRE-5 encodes the dominant UDP-GlcNAc:Man GlcNAc transferase activity in C. elegans. Resistance to Cry toxins has uncovered a four component glycosylation pathway that is functionally conserved between nematodes and insects and that provides the basis of the dominant mechanism of resistance in C. elegans.
Assuntos
Proteínas de Bactérias/farmacologia , Toxinas Bacterianas , Proteínas de Caenorhabditis elegans/fisiologia , Proteínas de Drosophila , Endotoxinas/farmacologia , Glicosilação , Glicosiltransferases/fisiologia , Sequência de Aminoácidos , Animais , Toxinas de Bacillus thuringiensis , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/química , Clonagem Molecular , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Drosophila , Drosophila melanogaster , Endocitose , Teste de Complementação Genética , Glicosiltransferases/química , Proteínas Hemolisinas , Mucosa Intestinal/metabolismo , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Modelos Biológicos , Dados de Sequência Molecular , Mutação , N-Acetilglucosaminiltransferases/metabolismo , Oligossacarídeos/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Mutations at the Drosophila melanogaster brainiac locus lead to defective formation of the follicular epithelium during oogenesis and to neural hyperplasia. The brainiac gene encodes a type II transmembrane protein structurally similar to mammalian beta1,3-glycosyltransferases. We have cloned the brainiac gene from D. melanogaster genomic DNA and expressed it as a FLAG-tagged recombinant protein in Sf9 insect cells. Glycosyltransferase assays showed that brainiac is capable of transferring N-acetylglucosamine (GlcNAc) to beta-linked mannose (Man), with a marked preference for the disaccharide Man(beta1,4)Glc, the core of arthro-series glycolipids. The activity of brainiac toward arthro-series glycolipids was confirmed by showing that the enzyme efficiently utilized glycolipids from insects as acceptors whereas it did not with glycolipids from mammalian cells. Methylation analysis of the brainiac reaction product revealed a beta1,3 linkage between GlcNAc and Man, proving that brainiac is a beta1,3GlcNAc-transferase. Human beta1,3GlcNAc-transferases structurally related to brainiac were unable to transfer GlcNAc to Man(beta1,4)Glc-based acceptor substrates and failed to rescue a homozygous lethal brainiac allele, indicating that these proteins are paralogous and not orthologous to brainiac.