Entamoeba histolytica: molecular cloning and characterization of a novel neutral sphingomyelinase.
A novel neutral sphingomyelinase (nSMase) was characterized in Entamoeba histolytica trophozoites. SMase, a sphingomyelin-specific form of phospholipase C, catalyzes the hydrolysis of sphingomyelin to ceramide and phosphorylcholine. Three amebic putative nSMase genes were found to be actively transcribed. Mg(2+)-independent nSMase activity in the soluble fraction of the trophozoites was stimulated by Mn(2+) and partially inhibited by Zn(2+). nSMase activity of the recombinant protein EhnSM1, increased 4.5-fold in the presence of 0.5mM Mn(2+), and abolished by 5mM Zn(2+). A dose-dependent inhibition of rEhnSM1 was observed with scyphostatin, a specific inhibitor of nSMases. The EhnSM1 and EhnSM3 were detected in the soluble fraction of the amebic lysate as 35-37kDa proteins by western blot analysis. Immunofluorescence assay showed that the overexpressed HA-tagged EhnSM1 and EhnSM3 were localized to the cytosol. The biological role of these novel E. histolytica nSMases described in this work remains to be determined.
Mendoza-Macías
Claudia Leticia
CL
Departamento de Biología, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Guanajuato, Mexico.
Barrios-Ceballos
Minerva Paola
MP
Anaya-Velázquez
Fernando
F
Nakada-Tsukui
Kumiko
K
Nozaki
Tomoyoshi
T
Padilla-Vaca
Felipe
F
D000577:Amides / Q000494:pharmacology; D000595:Amino Acid Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004748:Entamoeba histolytica / Q000201:enzymology* / Q000235:genetics*; D019084:Fluorescent Antibody Technique, Indirect; D008969:Molecular Sequence Data; D011753:Pyrones / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D013108:Sphingomyelin Phosphodiesterase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D053058:Trophozoites / Q000201:enzymology
D000577:Amides; D011753:Pyrones; D011994:Recombinant Proteins; C121123:scyphostatin; D013108:Sphingomyelin Phosphodiesterase