RESUMEN
One of the advantages for initial survival of inhaled fungal spores in the respiratory tract is the ability for iron acquisition via hemolytic factor-production. To examine the ability of indoor Aspergillus and Penicillium affecting hemolysis, the secreted factors during the growth of thirteen strains from eight species were characterized in vitro for their hemolytic activity (HA) and CAMP-like reaction. The hemolytic index of HA on human blood agar of Aspergillus micronesiensis, Aspergillus wentii, Aspergillus westerdijkiae, Penicillium citrinum, Penicillium copticola, Penicillium paxilli, Penicillium steckii, and Penicillium sumatrense were 1.72 ± 0.34, 1.61 ± 0.41, 1.69 ± 0.16, 1.58 ± 0.46, 3.10 ± 0.51, 1.22 ± 0.19, 2.55 ± 0.22, and 1.90 ± 0.14, respectively. The secreted factors of an Aspergillus wentii showed high HA when grown in undernourished broth at 25°C at an exponential phase and were heat sensitive. Its secreted proteins have an estimated relative molecular weight over 50 kDa. Whereas, the factors of Penicillium steckii were secreted in a similar condition at a late exponential phase but showed low HA and heat tolerance. In a CAMP-like test with sheep blood, the synergistic hemolytic reactions between most tested mold strains and Staphylococcus aureus were identified. Moreover, the enhancement of α-hemolysis of Staphylococcus aureus could occur through the interaction of Staphylococcus aureus-sphingomyelinase and CAMP-like factors secreted from Aspergillus micronesiensis. Further studies on the characterization of purified hemolytic- and CAMP-like-factors secreted from Aspergillus wentii and Aspergillus micronesiensis may lead to more understanding of their involvement of hemolysis and cytolysis for fungal survival prior to pathogenesis.
Asunto(s)
Aspergillus/fisiología , Hemólisis , Interacciones Microbianas/fisiología , Penicillium/fisiología , Esfingomielina Fosfodiesterasa/aislamiento & purificación , Microbiología del Aire , Animales , Aspergillus/aislamiento & purificación , Toxinas Bacterianas , Sangre , Proteínas Hemolisinas , Calor , Humanos , Técnicas In Vitro , Penicillium/aislamiento & purificación , Ovinos , Esfingomielina Fosfodiesterasa/metabolismo , Infecciones Estafilocócicas , Staphylococcus aureus , TailandiaRESUMEN
Amoebiasis is a worldwide health problem caused by the pathogen Entamoeba histolytica. Several virulence factors have been implicated in host invasion, immune evasion, and tissue damage. There are still new factors that remain to be elucidated and characterized. In this work, we obtained amoebic transfectants overexpressing three of the neutral sphingomyelinase enzymes encoded in the E. histolytica genome. The EhnSM3 overexpression induced an increase in hemolytic and cytotoxic activities, besides an increase in gene expression of amoebapore A, B, and C. Meanwhile the EhnSM1 and EhnSM2 overexpression caused an increase in cytopathic activity. In all the neutral sphingomyelinases overexpressing strains, the gene expression levels for cysteine proteinase 5, adhesin 112 and, heavy and light Gal/GalNAc lectin subunits were not affected. We propose that the increase of cytotoxic and lytic effect of EhnSM3 overexpressed strain can be related to the sum of the effect of EhnSM3 plus amoebapores, in a process cell contact-dependent or as mediator by inducing the gene expression of amoebapores enabling a link between EhnSM3 with the virulence phenotype in E. histolytica. Our results suggest a differential role for neutral sphingomyelinases in E. histolytica virulence.
Asunto(s)
Entamoeba histolytica/patogenicidad , Esfingomielina Fosfodiesterasa/metabolismo , Animales , Perros , Entamoeba histolytica/enzimología , Entamoeba histolytica/genética , Eritrocitos/metabolismo , Expresión Génica , Genoma de Protozoos , Hemólisis , Humanos , Células de Riñón Canino Madin Darby , Esfingomielina Fosfodiesterasa/genética , Esfingomielina Fosfodiesterasa/aislamiento & purificación , Esfingomielinas/metabolismo , Transfección , VirulenciaRESUMEN
Acid sphingomyelinase (ASMase, ASM, SMPD1) converts sphingomyelin into ceramide, modulating membrane properties and signal transduction. Inactivating mutations in ASMase cause Niemann-Pick disease, and its inhibition is also beneficial in models of depression and cancer. To gain a better understanding of this critical therapeutic target, we determined crystal structures of mammalian ASMase in various conformations. The catalytic domain adopts a calcineurin-like fold with two zinc ions and a hydrophobic track leading to the active site. Strikingly, the membrane interacting saposin domain assumes either a closed globular conformation independent from the catalytic domain, or an open conformation, which establishes an interface with the catalytic domain essential for activity. Structural mapping of Niemann-Pick mutations reveals that most of them likely destabilize the protein's fold. This study sheds light on the molecular mechanism of ASMase function, and provides a platform for the rational development of ASMase inhibitors and therapeutic use of recombinant ASMase.
Asunto(s)
Mamíferos/metabolismo , Esfingomielina Fosfodiesterasa/química , Animales , Dominio Catalítico , Cristalografía por Rayos X , Pruebas de Enzimas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Humanos , Hidrólisis , Lípidos de la Membrana/metabolismo , Ratones , Modelos Moleculares , Simulación del Acoplamiento Molecular , Mutación/genética , Enfermedades de Niemann-Pick/genética , Estructura Secundaria de Proteína , Saposinas/química , Esfingomielina Fosfodiesterasa/genética , Esfingomielina Fosfodiesterasa/aislamiento & purificación , Esfingomielina Fosfodiesterasa/metabolismo , Electricidad Estática , Especificidad por SustratoRESUMEN
Sphingomyelinases (SMases) catalyze the hydrolysis of sphingomyelin to ceramide and phosphorylcholine. Sphingolipids are recognized as diverse and dynamic regulators of a multitude of cellular processes mediating cell cycle control, differentiation, stress response, cell migration, adhesion, and apoptosis. Bacterial SMases are virulence factors for several species of pathogens. Whole cell extracts of Mycobacterium tuberculosis strains H37Rv and CDC1551 were assayed using [N-methyl-14C]-sphingomyelin as substrate. Acidic Zn2+-dependent SMase activity was identified in both strains. Peak SMase activity was observed at pH 5.5. Interestingly, overall SMase activity levels from CDC1551 extracts are approximately 1/3 of those of H37Rv. The presence of exogenous SMase produced by M. tuberculosis during infection may interfere with the normal host inflammatory response thus allowing the establishment of infection and disease development. This Type C activity is different from previously identified M. tuberculosis SMases. Defining the biochemical characteristics of M. tuberculosis SMases helps to elucidate the roles that these enzymes play during infection and disease.
Las esfingomielinasas (SMasas) catalizan la hidrólisis de esfingomielina a ceramida y fosforilcolina. Los esfingolípidos son reconocidos como reguladores diversos y dinámicos de una multitud de procesos celulares que median en el control del ciclo celular, la diferenciación, la respuesta al estrés, la migración celular, la adhesión y la apoptosis. Las esfingomielinasas bacterianas son factores de virulencia reconocidos en varias especies de patógenos. En este trabajo se analizaron los extractos de células enteras de las cepas de Mycobacterium tuberculosis H37Rv y CDC1551 utilizando [N-metil-14C]-esfingomielina como sustrato. Se identificó actividad de SMasa-ácida dependiente de zinc en ambas cepas. La actividad máxima se observó a pH 5.5. Curiosamente, los niveles de actividad de SMasa generados a partir de extractos de la cepa CDC1551 son aproximadamente un tercio de los de la cepa H37Rv. La presencia de una SMasa exógena producida por M. tuberculosis durante la infección puede interferir con la respuesta inflamatoria del huésped, permitiendo así el establecimiento de la infección y el desarrollo de la enfermedad. Esta actividad tipo C es distinta de las actividades previamente reportadas para M. tuberculosis. Definir las características bioquímicas de las esfingomielinasas de M. tuberculosis ayudará a dilucidar el papel que desempeñan estas enzimas durante la infección y la enfermedad.
Asunto(s)
Esfingomielina Fosfodiesterasa/biosíntesis , Mycobacterium tuberculosis/aislamiento & purificación , Esfingomielina Fosfodiesterasa/aislamiento & purificación , Factores de Virulencia/análisis , México/epidemiologíaRESUMEN
The beta-haemolysin of Staphylococcus aureus (SA-hlb) is a secreted neutral sphingomyelinase (nSMase) implicated in the pathogenesis of infection and responsible for the characteristic in vitro 'hot-cold' haemolytic ability of the bacterium. Here, we describe the production of a biologically active N-terminally-truncated recombinant SA-hlb protein for use in in vitro assays and as a research tool. Using local isolates of S. aureus, we PCR-amplified an SA-hlb DNA sequence of 891 nucleotides, 99 nucleotides shorter than the full-length molecule, before cloning and sequencing (GenBank accession no. JN580071). The pQE.TriSystem vector (Qiagen, Germany) was used to express recombinant SA-hlb (r-SA-hlb) with a C-terminal 8xHis tag in Escherichia coli JM107 cells. Both JM107 lysate and the purified r-SA-hlb possessed hot-cold lytic activity against sheep and buffalo erythrocytes, which was abolished by incubation at ≥90 °C for 30 min or exposure to dithiothreitol, and could be neutralized by bovine immune sera. Purified r-SA-hlb was also cytotoxic to buffalo mononuclear cells and was effective as a coating antigen for indirect ELISA to screen for reactive sera. Importantly, the r-SA-hlb was suitable for use as a ß-toxin in the modified CAMP test. We conclude that the r-SA-hlb protein produced was functionally active and has numerous potential applications.
Asunto(s)
Toxinas Bacterianas/aislamiento & purificación , Proteínas Hemolisinas/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Esfingomielina Fosfodiesterasa/aislamiento & purificación , Animales , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Secuencia de Bases , Productos Biológicos/química , Productos Biológicos/aislamiento & purificación , Productos Biológicos/metabolismo , Productos Biológicos/uso terapéutico , Búfalos , Bovinos , Clonación Molecular , Codón sin Sentido , Ensayo de Inmunoadsorción Enzimática/métodos , Eritrocitos/efectos de los fármacos , Proteínas Hemolisinas/química , Proteínas Hemolisinas/genética , Hemólisis/efectos de los fármacos , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Análisis de Secuencia de ADN , Ovinos , Esfingomielina Fosfodiesterasa/química , Esfingomielina Fosfodiesterasa/genética , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismoRESUMEN
Glycosylphosphatidylinositol (GPI) anchoring is a post-translational modification widely observed among eukaryotic membrane proteins. GPI anchors are attached to proteins via the carboxy-terminus in the outer leaflet of the cell membrane, where GPI-anchored proteins (GPI-APs) perform important functions as coreceptors and enzymes. Precursors of GPI-APs (Pre-GPI-APs) contain a C-terminal hydrophobic sequence that is involved in cleavage of the signal sequence from the protein and addition of the GPI anchor by the transamidase complex. In order to confirm that a given protein contains a GPI anchor, it is essential to identify the C-terminal peptide containing the GPI-anchor modification site (ω-site). Previously, efficient identification of GPI-anchored C-terminal peptides by mass spectrometry has been difficult, in part because of complex structure of the GPI-anchor moiety. We developed a method to experimentally identify GPI-APs and their ω-sites. In this method, a part of GPI-anchor moieties are removed from GPI-anchored peptides using phosphatidylinositol-specific phospholipase C (PI-PLC) and aqueous hydrogen fluoride (HF), and peptide sequence is then determined by mass spectrometry. Using this method, we successfully identified 10 GPI-APs and 12 ω-sites in the cultured ovarian adenocarcinoma cells, demonstrating that this method is useful for identifying efficiently GPI-APs.
Asunto(s)
Proteínas Ligadas a GPI/química , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Línea Celular Tumoral , Proteínas Ligadas a GPI/aislamiento & purificación , Proteínas Ligadas a GPI/metabolismo , Glicosilfosfatidilinositoles/química , Glicosilfosfatidilinositoles/metabolismo , Humanos , Microdominios de Membrana/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Fragmentos de Péptidos/química , Mapeo Peptídico , Procesamiento Proteico-Postraduccional , Proteómica , Esfingomielina Fosfodiesterasa/química , Esfingomielina Fosfodiesterasa/aislamiento & purificación , Esfingomielina Fosfodiesterasa/metabolismoRESUMEN
Trichomoniasis is one of the most common acute sexually transmitted curable diseases, and it is disseminated worldwide generating more than 170 million cases annually. Trichomonas vaginalis is the parasite that causes trichomoniasis and has the ability to destroy cell monolayers of the vaginal mucosa in vitro. Sphingomyelinases (SMase) are enzymes that catalyze the hydrolysis of sphingomyelin into ceramide and phosphorylcholine. Ceramide appears to be a second messenger lipid in programmed apoptosis, cell differentiation, and cell proliferation. Sphingomyelinase is probably a major source of ceramide in cells. Signal transduction mediated by ceramide leads cells to produce cytokine induced apoptosis during several inflammatory responses. SMase are also relevant toxins in several microorganisms. The main objective of this research is to identify SMase activity of T. vaginalis in the total extract (TE), P30, and S30 subfractions from brooked trophozoites. It was found that these fractions of T. vaginalis have SMase activity, which comes principally from P30 subfraction and was mainly type C. Enzymatic activity of SMase increased linearly with time and is pH dependent with two peaks by pH 5.5 and pH 7.5. The addition of manganese to the reaction mixture increased the SMase activity by 1.97.
Asunto(s)
Esfingomielina Fosfodiesterasa/biosíntesis , Tricomoniasis/enzimología , Trichomonas vaginalis/enzimología , Apoptosis/genética , Ceramidas/química , Femenino , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Fosforilcolina/química , Transducción de Señal , Esfingomielina Fosfodiesterasa/aislamiento & purificación , Esfingomielinas/química , Tricomoniasis/genética , Tricomoniasis/parasitología , Trichomonas vaginalis/patogenicidadRESUMEN
The hemolytic phospholipase C/sphingomyelinase PlcH from the opportunistic pathogen Pseudomonas aeruginosa represents the founding member of a growing family of virulence factors identified in a wide range of bacterial and fungal pathogens. In P. aeruginosa PlcH is co-expressed with a 17 kDa chaperone (PlcR2) and secreted as a fully folded heterodimer (PlcHR2) of approximately 95 kDa, by the twin arginine translocase (TAT) via the cytoplasmic membrane and through the outer membrane, by the Xcp (TypeII) secretory system. PlcHR2 has been shown to be an important virulence factor in model P. aeruginosa infections and is selectively cytotoxic, at picomolar concentrations to mammalian endothelial cells. Here we report how the various challenges starting from protein overexpression in the native organism P. aeruginosa, the use of detergents in the crystallization and data collection using the most advanced µ-focus synchrotron beam lines were overcome. Native diffraction data of this heterodimeric protein complex were collected up to a resolution of 4Å, whereas needle-shaped crystals of l-selenomethionine substituted PlcHR2 with a maximum diameter of 10 micron were used to collect data sets with a maximum resolution of 2.75Å.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Pseudomonas aeruginosa/enzimología , Fosfolipasas de Tipo C/genética , Fosfolipasas de Tipo C/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Cristalización , Cristalografía por Rayos X , Pseudomonas aeruginosa/metabolismo , Selenometionina/química , Esfingomielina Fosfodiesterasa/genética , Esfingomielina Fosfodiesterasa/aislamiento & purificación , Esfingomielina Fosfodiesterasa/metabolismo , Fosfolipasas de Tipo C/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/aislamiento & purificación , Factores de Virulencia/metabolismoRESUMEN
Sphingomyelinase C (SMC) of the actinomycete, Streptomycesgriseocarneus NBRC13471, was constitutively expressed to high levels using Streptomyces lividans host and thereafter was extracellularly secreted into the cell culture. Purified SMC had a high specific activity (approximately 550-950 U/mg) and was obtained in high yields (approximately 120 mg/L of culture). SMC activity was enhanced by MgCl(2), and the maximum activity (542±25 U/mg) was observed in the presence of 1.5 mol/L (M) MgCl(2). Dynamic light scattering analysis proved that the highest specific SMC activity was obtained with the smallest mixed micelles of sphingomyelin (SM) and Triton X-100. The turnover rate (k(cat)), K(m) and k(cat)/K(m) values for SM were 346 s(-1), 0.458 mM, and 756 mM(-1)s(-1), respectively, in the presence of 1M MgCl(2). The k(cat) was strongly influenced by the MgCl(2) concentration. By contrast, the K(m) value was independent of the MgCl(2) concentration and was almost constant. Circular dichroism spectroscopy indicated that MgCl(2) did not cause local structural changes in SMC. From these results, we concluded that the SMC activity enhancement by MgCl(2) was caused by the increased specific surface area of the mixed micelles composed of substrate, SM, and Triton X-100.
Asunto(s)
Cloruro de Magnesio/farmacología , Proteínas Recombinantes/metabolismo , Esfingomielina Fosfodiesterasa/metabolismo , Streptomyces/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Dicroismo Circular , Clonación Molecular , Medios de Cultivo/metabolismo , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Pruebas de Enzimas , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Cinética , Cloruro de Magnesio/metabolismo , Micelas , Octoxinol/metabolismo , Plásmidos/genética , Plásmidos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Espectrofotometría Atómica , Esfingomielina Fosfodiesterasa/genética , Esfingomielina Fosfodiesterasa/aislamiento & purificación , Esfingomielinas/metabolismo , Streptomyces/efectos de los fármacos , Streptomyces/genética , Especificidad por Sustrato , Transformación BacterianaRESUMEN
Ceramide is produced by sphingomyelinase (SMase) and it plays a key role in cellular responses such as apoptosis. In this study, we report the purification and characterization of neutral SMase2 (nSMase2) from bovine brain tissue. Triton X-100 extracts of bovine brain membranes were purified in nine steps, including sequential chromatography. The specific activity of purified nSMase increased 8183-fold over the brain membrane fraction. Purified nSMase showed similarities to nSMase2, which had been purified and cloned previously. Interestingly, purified nSMase2 was Ca2+-dependent and could be activated by micromolar concentrations of Ca2+ under Mg2+-free conditions. Ceramide generation was dependent upon the calcium ionophore A23187 and was observed in nSMase2-over-expressing COS-7 cells. This generation was suppressed by GW4869, an nSMase2 inhibitor, but not to fumonisin B(1), an inhibitor of the de novo ceramide synthesis pathway. The present study demonstrates the Ca2+-dependent activation of nSMase2.
Asunto(s)
Química Encefálica , Encéfalo/metabolismo , Calcio/metabolismo , Esfingomielina Fosfodiesterasa/aislamiento & purificación , Esfingomielina Fosfodiesterasa/metabolismo , Compuestos de Anilina/farmacología , Animales , Compuestos de Bencilideno/farmacología , Encéfalo/efectos de los fármacos , Química Encefálica/efectos de los fármacos , Células COS , Calcimicina/farmacología , Bovinos , Ceramidas/metabolismo , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Líquido Intracelular/efectos de los fármacos , Líquido Intracelular/metabolismo , Ionóforos/farmacología , Octoxinol/farmacología , Ácido Palmítico/metabolismo , Tensoactivos/farmacología , Transfección/métodos , Tritio/metabolismo , Fosfolipasas de Tipo C/metabolismoRESUMEN
The acid sphingomyelinase is an emerging drug target, especially for inflammatory lung diseases. Presently, there are no directly-acting potent inhibitors available for cell-based studies. The potent inhibitor phosphatidylinositol-3,5-bisphosphate (PtdIns3,5P2) is not only unsuited for cell culture studies, but also does not provide hints for further structural improvements. In the SAR study described here, we replaced the inositolphosphate moiety by a carbohydrate derivative and the phosphatidic acid residue by an alkylsulfone ester. The resulting compound is more active than its parent compound and offers new means for further structural modification.
Asunto(s)
Carbohidratos/farmacología , Inhibidores Enzimáticos/farmacología , Esfingomielina Fosfodiesterasa/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Carbohidratos/síntesis química , Carbohidratos/química , Línea Celular , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Conformación Molecular , Ratas , Esfingomielina Fosfodiesterasa/aislamiento & purificación , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Sphingomyelinase C (SMC) was purified to homogeneity from the culture supernatant of Streptomyces griseocarneus NBRC13471. The purified enzyme appeared as a single band of 38 kDa by using an electropherogram trace. The molecular mass of the enzyme as determined by MALDI-TOF MS was 32,102 Da, indicating that SMC is monomeric in nature. Under experimental conditions, the highest enzyme activity was found at pH 9.0 and 50-55 degrees C, and the enzyme was stable from pH 5 to 10 and up to 37 degrees C. The SMC activity requires Mg(2+) or Mn(2+) and the order of potency to enhance the activity was Zn(2+)> or =Mn(2+)>Cu(2+)> or =Fe(2+). Phenylmethylsulfonyl fluoride and EDTA inhibited the enzyme activity, showing that SMC belongs to a group of metalloenzymes and a class of serine hydrolases. The enzyme activity was inhibited by DTT, but not by mercaptoethanol and iodoacetamide. SDS inhibited the enzyme activity; by contrast, Triton X-100 stimulated the activity. The N-terminal and internal amino-acid sequences were determined as H(2)N-APAAATPSLK, AREIAAAGFFQGND, and NTVVQETSAP. The gene encoding SMC consisted of 1020 bp encoding a signal peptide of 42 amino acids and a mature protein of 297 amino acids with a calculated molecular mass of 32,125 Da. The conserved region of DNase I-like family enzymes and the amino acid residues that are highly conserved in the active center of other bacterial SMCs were also found in the deduced amino acid sequence of S. griseocarneus SMC.
Asunto(s)
Esfingomielina Fosfodiesterasa/genética , Streptomyces/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Cationes Bivalentes/metabolismo , Clonación Molecular , Cartilla de ADN , Ácido Edético/farmacología , Inhibidores Enzimáticos/farmacología , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Peso Molecular , Fluoruro de Fenilmetilsulfonilo/farmacología , Plásmidos , Reacción en Cadena de la Polimerasa , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Esfingomielina Fosfodiesterasa/química , Esfingomielina Fosfodiesterasa/aislamiento & purificación , Esfingomielina Fosfodiesterasa/metabolismo , Streptomyces/genética , Especificidad por Sustrato , TermodinámicaRESUMEN
Sphingolipids are important signaling molecules in many biological processes, but little is known regarding their physiological roles in the mitochondrion. We focused on the biochemical characters of a novel sphingomyelinase (SMase) and its function in mitochondrial ceramide generation in zebrafish embryonic cells. The cloned SMase cDNA encoded a polypeptide of 545 amino acid residues (putative molecular weight, 61,300) containing a mitochondrial localization signal (MLS) and a predicted transmembrane domain. The mature endogenous enzyme was predicted to have a molecular weight of 57,000, and matrix-assisted laser de sorption ionization time-of-flight mass spectrometry analysis indicated that the N-terminal amino acid residue of the mature enzyme was Ala-36. The purified enzyme optimally hydrolyzed [(14)C]sphingomyelin in the presence of 10 mm Mg(2+) at pH 7.5. In HEK293 cells that overexpressed SMase cDNA, the enzyme was localized to the mitochondrial fraction, whereas mutant proteins lacking MLS or both the MLS and the transmembrane domain were absent from the mitochondrial fraction. Endogenous SMase protein co-localized with a mitochondrial cytostaining marker. Using a protease protection assay, we found that SMase was distributed throughout the intermembrane space and/or the inner membrane of the mitochondrion. Furthermore, the overexpression of SMase in HEK293 cells induced ceramide generation and sphingomyelin hydrolysis in the mitochondrial fraction. Antisense phosphorothioate oligonucleotide-induced knockdown repressed ceramide generation and sphingomyelin hydrolysis in the mitochondrial fraction in zebrafish embryonic cells. These observations indicate that SMase catalyzes the hydrolysis of sphingomyelin and generates ceramide in mitochondria in fish cells.
Asunto(s)
Mitocondrias/enzimología , Esfingomielina Fosfodiesterasa/análisis , Esfingomielina Fosfodiesterasa/metabolismo , Esfingomielinas/metabolismo , Pez Cebra/metabolismo , Secuencia de Aminoácidos , Animales , Bacillus cereus/enzimología , Bacillus cereus/genética , Proteínas Bacterianas/genética , Línea Celular , Ceramidas/análisis , Ceramidas/metabolismo , Clonación Molecular , Regulación de la Expresión Génica , Humanos , Ratones , Mitocondrias/química , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Mutación , Alineación de Secuencia , Esfingomielina Fosfodiesterasa/genética , Esfingomielina Fosfodiesterasa/aislamiento & purificación , Esfingomielinas/análisis , Pez Cebra/embriologíaRESUMEN
Alkaline sphingomyelinase (Alk-SMase) is a key enzyme in the intestinal tract for digestion of dietary sphingomyelin (SM), which generates lipid messengers with cell-cycle regulating effects. The enzyme is significantly decreased in ulcerative colitis and colon cancer. Based on this information, we wanted to investigate whether the enzyme had preventive effects against murine colitis. We report herein a method to express a biologically active Alk-SMase from Pichia pastoris yeast cells. By using the expressed enzyme to treat a rat colitis model induced by dextran sulfate sodium, we found that intrarectal instillation of Alk-SMase once daily for 1 week significantly reduced the inflammation score and protected the colonic epithelium from inflammatory destruction. We found a tendency for decreased tumor necrosis factor (TNF)-alpha expression in the Alk-SMase-treated group. This study, for the first time, provides a method to produce the enzyme and shows the potential applicability of the enzyme in the treatment of inflammatory bowel diseases.
Asunto(s)
Colitis/enzimología , Esfingomielina Fosfodiesterasa/metabolismo , Administración Rectal , Animales , Colitis/patología , Colon/patología , Sulfato de Dextran/efectos adversos , Modelos Animales de Enfermedad , Femenino , Inflamación/prevención & control , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Pichia/citología , Proyectos Piloto , Reacción en Cadena de la Polimerasa , Ratas , Ratas Sprague-Dawley , Esfingomielina Fosfodiesterasa/administración & dosificación , Esfingomielina Fosfodiesterasa/aislamiento & purificación , Esfingomielina Fosfodiesterasa/farmacología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
There is strong evidence indicating a role for ceramide as a second messenger in processes such as apoptosis, cell growth and differentiation, and cellular responses to stress. Ceramide formation from the hydrolysis of sphingomyelin is considered to be a major pathway of stress-induced ceramide production with magnesium-dependent neutral sphingomyelinase (N-SMase) identified as a prime candidate in this pathway. The recent cloning of a mammalian N-SMase-nSMase2- and generation of nSMase2 knockout/mutant mice have now provided vital tools with which to further study the regulation and roles of this enzyme in both a physiological and pathological context. In the present review, we summarize current knowledge on N-SMase relating this to what is known about nSMase2. We also discuss the future areas of nSMase2 research important for molecular understanding of this enzyme and its physiological roles.
Asunto(s)
Isoenzimas/metabolismo , Esfingomielina Fosfodiesterasa/metabolismo , Animales , Clonación Molecular , Isoenzimas/genética , Isoenzimas/aislamiento & purificación , Ratones , Ratones Noqueados , Esfingomielina Fosfodiesterasa/genética , Esfingomielina Fosfodiesterasa/aislamiento & purificaciónRESUMEN
Methanol extracts of domestic plants of Korea were evaluated as a potential inhibitor of neutral pH optimum and membrane-associated 60 kDa sphingomyelinase (N-SMase) activity. In this study, we partially purified N-SMase from bovine brain membranes using ammonium sulfate. It was purified approximately 163-fold by the sequential use of DE52, Butyl-Toyopearl, DEAE-Cellulose, and Phenyl-5PW column chromatographies. The purified N-SMase activity was assayed in the presence of the plant extracts of three hundreds species. Based on the in vitro assay, three plant extracts significantly inhibited the N-SMase activity in a time- and concentration-dependent manner. To further examine the inhibitory pattern, a Dixon plot was constructed for each of the plant extracts. The extracts of Abies nephrolepis, Acer tegmentosum, and Ginkgo biloba revealed a competitive inhibition with the inhibition constant (Ki) of 11.9 microg/ mL, 9.4 microg/mL, and 12.9 microg/mL, respectively. These extracts also inhibited in a dose-dependent manner the production of ceramide induced by serum deprivation in human neuroblastoma cell line SH-SY5Y.
Asunto(s)
Abies , Acer , Encéfalo/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Ginkgo biloba , Proteínas de la Membrana/antagonistas & inhibidores , Extractos Vegetales/farmacología , Esfingomielina Fosfodiesterasa/antagonistas & inhibidores , Animales , Unión Competitiva , Encéfalo/enzimología , Bovinos , Línea Celular Tumoral , Ceramidas/biosíntesis , Medio de Cultivo Libre de Suero/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Corea (Geográfico) , Magnesio , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Corteza de la Planta , Esfingomielina Fosfodiesterasa/aislamiento & purificación , Esfingomielina Fosfodiesterasa/metabolismo , Esfingomielinas/metabolismo , Factores de TiempoRESUMEN
OBJECTIVES: Intestinal alkaline sphingomyelinase, by exerting a major role in dietary sphingomyelin digestion, is responsible for the generation of messengers able to trigger the rapid turnover and apoptosis in intestinal epithelial cells. Markedly reduced mucosal alkaline sphingomyelinase activity has been associated with human colorectal neoplasms. The aim of this study was to analyze the alkaline sphingomyelinase activity in feces from healthy subjects and colorectal adenocarcinoma patients and to correlate it with the enzyme activity in intestinal tissues. MATERIALS AND METHODS: The enzyme activity was measured both in the intestinal samples from 12 healthy controls and 51 patients with colorectal adenocarcinoma (tumoral and paratumoral tissue) and in the fecal samples of 34 healthy subjects and 29 patients with adenocarcinoma. The relation between sphingomyelinase activity and Dukes' stage, cell differentiation degree, age, and gender was also analyzed. RESULTS: Alkaline sphingomyelinase was significantly decreased (P < 0.001; mean reduction >90%) in tumoral intestinal mucosa of patients compared with controls independently of Dukes' stage and tumor differentiation grade. Interestingly, the enzyme activity in histologically normal paratumoral tissues was statistically lower than control samples (P < 0.001). As occurs in neoplastic tissues, a relevant mean reduction (P < 0.0001; almost 90%) of alkaline sphingomyelinase was revealed in stool samples from tumor patients when compared with controls. CONCLUSION: These findings may have implications for cancer biology and perhaps also for the design of clinical test, thus suggesting that the fecal sphingomyelinase activity could really reflect the human intestinal mucosa enzyme level and could represent a new marker for human colorectal adenocarcinoma, mainly taking into account its early appearance in intestinal neoplasms.
Asunto(s)
Adenocarcinoma/enzimología , Neoplasias Colorrectales/enzimología , Heces/enzimología , Esfingomielina Fosfodiesterasa/metabolismo , Adenocarcinoma/diagnóstico , Adenocarcinoma/patología , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Esfingomielina Fosfodiesterasa/aislamiento & purificaciónRESUMEN
Loxosceles adelaida spiders (Araneae, Sicariidae) are found near and inside the caves in the Parque Estadual Turistico do Alto Ribeira (PETAR), Sao Paulo, Brazil, which are visited by thousands of tourists every year. Several Loxosceles species are a public health problem in many regions of the world, by causing severe dermonecrosis and/or complement dependent haemolysis upon envenomation. The aim of this study was to characterize the biochemical and biological properties of L. adelaida venom and evaluate the toxic potential of envenomation by this non-synanthropic Loxosceles species. The biological activities of the L. adelaida venom was compared to that of Loxosceles gaucho, a synanthropic species of medical importance in Brazil. L. adelaida venom showed a similar potential to induce haemolysis, dermonecrosis and lethality as L. gaucho venom. L. adelaida crude venom was purified, yielding a 31 kDa component endowed with haemolytic and dermonecrotic activities. In conclusion, we show here that the troglophile Loxosceles species, L. adelaida, commonly found in the complex of caves from PETAR, is potentially able to cause envenomation with the same gravity of those produced by synanthropic species.
Asunto(s)
Hemólisis/efectos de los fármacos , Piel/patología , Esfingomielina Fosfodiesterasa/toxicidad , Venenos de Araña/química , Arañas/química , Animales , Western Blotting , Brasil , Cromatografía Líquida de Alta Presión , Electroforesis en Gel Bidimensional , Ensayo de Inmunoadsorción Enzimática , Eritrocitos/efectos de los fármacos , Citometría de Flujo , Dosificación Letal Mediana , Ratones , Necrosis/inducido químicamente , Conejos , Especificidad de la Especie , Esfingomielina Fosfodiesterasa/aislamiento & purificación , Venenos de Araña/enzimología , Arañas/enzimologíaAsunto(s)
Enfermedades Autoinmunes del Sistema Nervioso/complicaciones , Trastornos Mentales/complicaciones , Trastorno Obsesivo Compulsivo/complicaciones , Infecciones Estreptocócicas/complicaciones , Toxinas Bacterianas/aislamiento & purificación , Bacteriocinas , Niño , Proteínas Hemolisinas , Humanos , Masculino , Péptidos/aislamiento & purificación , Esfingomielina Fosfodiesterasa/aislamiento & purificación , Infecciones Estreptocócicas/microbiologíaRESUMEN
Leptospira interrogans causes a variety of clinical syndromes in animals and humans. Although much information has accumulated on the importance of leptospiral lipopolysaccharide in protective antibody responses, relatively little is known about proteins that participate in immune responses. Identification of those proteins induced only in the host is particularly difficult. Using a novel double-antibody screen designed to identify clones in a gene library of L. interrogans serovar Pomona expressing host-inducible proteins, we have characterized a gene (lk75.3) encoding a sphingomyelinase-like preprotein of 648 amino acids with cytotoxic activity for equine pulmonary endothelial cells and weak hemolytic activity for equine and rabbit erythrocytes. lk73.5 was found as a single gene copy in all serovars of L. interrogans but not in other Leptospira spp. except L. inadai. The open reading frame (ORF) for Lk73.5 is followed by another partially homologous sequence containing an ORF (sph-like 2) for a 28.7-kDa peptide. Lk73.5 and Sph-like 2 share 95.1 and 97.7% amino acid identity with putative sphingomyelinases Sph2 and Sph1 (N terminus) from L. interrogans serovar Lai (S.-X. Ren, G. Fu, X.-G. Jiangk, R. Zeng, Y.-G. Miao, H. Xu, Y.-X. Zhang, H. Xiong, G. Lu, L.-F. Lu, H.-Q. Jiang, J. Jia, Y.-F. Tu, J.-X. Jiang, W.-Y. Gu, Y.-Q. Zhang, Z. Cai, H.-H. Sheng, H.-F. Yin, Y. Zhang, G.-F. Zhu, M. Wank, H.-L. Huangk, Z. Qian, S.-Y. Wang, Wei Ma, Z.-J. Yao, Y. Shen, B.-Q. Qiang, Q.-C. Xia, X.-K. Guo, A. Danchinq, I. S. Girons, R. L. Somerville, Y.-M. Wen, M.-H. Shik, Z. Chen, J.-G. Xuk, and G.-P. Zhao, Nature 422:88-893, 2003). Substantial homologies to sphingomyelinases from other leptospiras and other bacteria are also present. Lk73.5 was not detected in leptospiras cultured at 30 or 37 degrees C. The recombinant protein reacted strongly with sera from recently infected mares but not with sera from horses vaccinated with commercial pentavalent bacterin. The host-inducible immunogenic Lk73.5 should have value in distinguishing vaccine from infection immune response.