RESUMO
Besides bulk amounts of SM, mammalian cells produce small quantities of the SM analog ceramide phosphoethanolamine (CPE). Little is known about the biological role of CPE or enzymes responsible for CPE production. Heterologous expression studies revealed that SM synthase (SMS)2 is a bifunctional enzyme producing both SM and CPE, whereas SMS-related protein (SMSr) serves as monofunctional CPE synthase. Acute disruption of SMSr catalytic activity in cultured cells causes a rise in endoplasmic reticulum (ER) ceramides, fragmentation of ER exit sites, and induction of mitochondrial apoptosis. To address the relevance of CPE biosynthesis in vivo, we analyzed the tissue-specific distribution of CPE in mice and generated mouse lines lacking SMSr and SMS2 catalytic activity. We found that CPE levels were >300-fold lower than SM in all tissues examined. Unexpectedly, combined inactivation of SMSr and SMS2 significantly reduced, but did not eliminate, tissue-specific CPE pools and had no obvious impact on mouse development or fertility. While SMSr is widely expressed and serves as the principal CPE synthase in the brain, blocking its catalytic activity did not affect ceramide levels or secretory pathway integrity in the brain or any other tissue. Our data provide a first inventory of CPE species and CPE-biosynthetic enzymes in mammals.
Assuntos
Biocatálise , Esfingomielinas/biossíntese , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Animais , Encéfalo/citologia , Encéfalo/enzimologia , Encéfalo/metabolismo , Domínio Catalítico , Sobrevivência Celular , Ativação Enzimática , Éxons/genética , Deleção de Genes , Regulação Enzimológica da Expressão Gênica , Fígado/citologia , Fígado/enzimologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Mutação Puntual , Transporte Proteico , Esfingomielinas/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transferases (Outros Grupos de Fosfato Substituídos)/deficiência , Transferases (Outros Grupos de Fosfato Substituídos)/genéticaRESUMO
The N-acyl chain length of ceramides is determined by the specificity of different ceramide synthases (CerS). The CerS family in mammals consists of six members with different substrate specificities and expression patterns. We have generated and characterized a mouse line harboring an enzymatically inactive ceramide synthase 6 (CerS6KO) gene and lacz reporter cDNA coding for ß-galactosidase directed by the CerS6 promoter. These mice display a decrease in C16:0 containing sphingolipids. Relative to wild type tissues the amount of C16:0 containing sphingomyelin in kidney is â¼35%, whereas we find a reduction of C16:0 ceramide content in the small intestine to about 25%. The CerS6KO mice show behavioral abnormalities including a clasping abnormality of their hind limbs and a habituation deficit. LacZ reporter expression in the brain reveals CerS6 expression in hippocampus, cortex, and the Purkinje cell layer of the cerebellum. Using newly developed antibodies that specifically recognize the CerS6 protein we show that the endogenous CerS6 protein is N-glycosylated and expressed in several tissues of mice, mainly kidney, small and large intestine, and brain.
Assuntos
Comportamento Animal , Esfingolipídeos/metabolismo , Esfingosina N-Aciltransferase/metabolismo , Animais , Ansiedade/patologia , Ansiedade/fisiopatologia , Encéfalo/metabolismo , Encéfalo/patologia , Ativação Enzimática , Ensaios Enzimáticos , Comportamento Exploratório , Imunofluorescência , Glicosilação , Células HEK293 , Habituação Psicofisiológica , Humanos , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Glomérulos Renais/ultraestrutura , Espectrometria de Massas , Aprendizagem em Labirinto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade de Órgãos , Fenótipo , Esfingolipídeos/química , Esfingosina N-Aciltransferase/deficiência , beta-Galactosidase/metabolismoRESUMO
Ceramide synthase 1 (CerS1) catalyzes the synthesis of C18 ceramide and is mainly expressed in the brain. Custom-made antibodies to a peptide from the C-terminal region of the mouse CerS1 protein yielded specific immunosignals in neurons but no other cell types of wild type brain, but the CerS1 protein was not detected in CerS1-deficient mouse brains. To elucidate the biological function of CerS1-derived sphingolipids in the brain, we generated CerS1-deficient mice by introducing a targeted mutation into the coding region of the cers1 gene. General deficiency of CerS1 in mice caused a foliation defect, progressive shrinkage, and neuronal apoptosis in the cerebellum. Mass spectrometric analyses revealed up to 60% decreased levels of gangliosides in cerebellum and forebrain. Expression of myelin-associated glycoprotein was also decreased by about 60% in cerebellum and forebrain, suggesting that interaction and stabilization of oligodendrocytic myelin-associated glycoprotein by neuronal gangliosides is due to the C18 acyl membrane anchor of CerS1-derived precursor ceramides. A behavioral analysis of CerS1-deficient mice yielded functional deficits including impaired exploration of novel objects, locomotion, and motor coordination. Our results reveal an essential function of CerS1-derived ceramide in the regulation of cerebellar development and neurodevelopmentally regulated behavior.
Assuntos
Cerebelo/metabolismo , Gangliosídeos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Glicoproteína Associada a Mielina/biossíntese , Oligodendroglia/metabolismo , Oxirredutases/metabolismo , Animais , Apoptose/fisiologia , Linhagem Celular , Ceramidas/genética , Ceramidas/metabolismo , Cerebelo/citologia , Cerebelo/embriologia , Gangliosídeos/genética , Camundongos , Camundongos Mutantes , Glicoproteína Associada a Mielina/genética , Neurônios/citologia , Neurônios/metabolismo , Oligodendroglia/citologia , Oxirredutases/genética , Prosencéfalo/citologia , Prosencéfalo/embriologiaRESUMO
Ceramide synthase 2 (CerS2) catalyzes the synthesis of dihydroceramides from dihydrosphingosine and very long fatty acyl (C22-C24)-CoAs. CerS2-deficient (gene trap) mice were reported to exhibit myelin and behavioral abnormalities, associated with the expression of CerS2 in oligodendrocytes and neurons based on expression of lacZ reporter cDNA instead of the cers2 gene in these mice. In order to clarify the cell-type-specific expression of CerS2 protein, we have raised antibodies that specifically recognize the glycosylated and non-glycosylated CerS2 protein in wild-type but not in CerS2-deficient mouse tissues. In early postnatal, juvenile and adult mouse brain, the new antibodies detect CerS2 protein only in oligodendrocytes but not in neurons, suggesting that the gene trap vector in CerS2-deficient mice led to ectopic expression of the lacZ reporter gene in neurons. In liver, the CerS2 protein is expressed in hepatocytes but not in Ito cells or Kupffer cells. We conclude that the behavioral abnormalities observed in CerS2-deficient mice originate primarily in oligodendrocytes and not in neurons. The identification of specific cell types in which CerS2 protein is expressed is prerequisite to further mechanistic characterization of phenotypic abnormalities exhibited by CerS2-deficient mice. The amount of CerS2 protein detected in different tissues by immunoblot analyses does not strictly correspond to the activity of the CerS2 enzyme. Disproportional results are likely due to post-translational regulation of the CerS2 protein.