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.
Departamento de Biología, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Guanajuato, Mexico.
Claudia Leticia
CL
Mendoza-Macías
Minerva Paola
MP
Barrios-Ceballos
Fernando
F
Anaya-Velázquez
Kumiko
K
Nakada-Tsukui
Tomoyoshi
T
Nozaki
Felipe
F
Padilla-Vaca
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