Entamoeba histolytica: Molecular cloning and characterization of a novel neutral sphingomyelinase |
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| Authors: | Claudia Leticia Mendoza-Macías Fernando Anaya-Velázquez Tomoyoshi Nozaki |
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| Institution: | a Departamento de Biología, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Guanajuato 36050, Mexico
b Department of Parasitology, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo 162-8640, Japan |
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| Abstract: | 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. Mg2+-independent nSMase activity in the soluble fraction of the trophozoites was stimulated by Mn2+ and partially inhibited by Zn2+. nSMase activity of the recombinant protein EhnSM1, increased 4.5-fold in the presence of 0.5 mM Mn2+, and abolished by 5 mM Zn2+. 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 35-37 kDa 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. |
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