RESUMO
Ceramidase plays an important role in regulating the metabolism of sphingolipids, such as ceramide, sphingosine (SPH), and sphingosine-1-phosphate (S1P), by controlling the hydrolysis of ceramide. Here we report the cloning and biochemical characterization of a neutral ceramidase from the red flour beetle Tribolium castaneum which is an important storage pest. The Tribolium castaneum neutral ceramidase (Tncer) is a protein of 696 amino acids. It shares a high degree of similarity in protein sequence to neutral ceramidases from various species. Tncer mRNA levels are higher in the adult stage than in pre-adult stages, and they are higher in the reproductive organs than in head, thorax, and midgut. The mature ovary has higher mRNA levels than the immature ovary. Tncer is localized to the plasma membrane. It uses various ceramides (D-erythro-C(6), C(12), C(16), C(18:1), and C(24:1)-ceramide) as substrates and has an abroad pH optimum for its in vitro activity. Tncer has an optimal temperature of 37 °C for its in vitro activity. Its activity is inhibited by Fe(2+). These results suggest that Tncer has distinct biochemical properties from neutral ceramidases from other species.
Assuntos
Proteínas de Insetos/genética , Ceramidase Neutra/genética , Tribolium/genética , Sequência de Aminoácidos , Animais , Western Blotting , Linhagem Celular , Membrana Celular/enzimologia , Ceramidas/metabolismo , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Concentração de Íons de Hidrogênio , Proteínas de Insetos/metabolismo , Masculino , Dados de Sequência Molecular , Ceramidase Neutra/classificação , Ceramidase Neutra/metabolismo , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Tribolium/enzimologia , Tribolium/crescimento & desenvolvimentoRESUMO
Ceramidases are key enzymes in the regulation of the cellular levels of ceramide, sphingosine and sphingosine-1-phosphate. This study first reports on the molecular cloning, sequencing and expression profile of the gene encoding the wheat neutral ceramidase designated as Ta-CDase. A full length wheat Ta-CDase gene is obtained by rapid amplification of cDNA ends (RACE) based on the sequence of the WSRC36 fragment from an incompatible suppression subtractive hybridization (SSH) cDNA library of wheat leaves infected by Puccinia striiformis f. sp. tritici. The open reading frame (ORF) of 2,839 nucleotides encodes a polypeptide of 785 amino acids with a predicted isoelectric point (pI) of 6.398. The protein conserved domain search indicates that the polypeptide contains the signature of ceramidase, signal peptide sequence and transmembrane region. A phylogenetic analysis reveals that a high degree of relatedness exists among wheat Ta-CDase and ceramidases from other plant species at the amino acid level, while its relationship to that of animals and pathogens is more distant. The expression profile of the Ta-CDase shows a very strong expression of transcripts only at 48 h post inoculation (hpi), while expression level is low at other time points. Southern blot analyses showed that Ta-CDase is a multi-copy gene and located on wheat chromosome 4D and 5A.
