Characterization and subcellular localization of murine and human magnesium-dependent neutral sphingomyelinase.
Sphingomyelinases (SMases) catalyze the hydrolysis of sphingomyelin, an essential lipid constituent of the plasma membrane, lysosomal membranes, endoplasmic reticulum, and the Golgi membrane stacks of mammalian cells. In this study, we report the biochemical and functional characterization and subcellular localization of magnesium-dependent nSMase1 from overexpressing human embryonic kidney (HEK293) cells. Site-directed mutagenesis of conserved residues probably involved in the enzymatic sphingomyelin cleavage as well as the removal of one or both putative transmembrane domains lead to the complete loss of enzymatic activity of human nSMase1 expressed in HEK293 cells. Polyclonal antibodies raised against recombinant mammalian nSMase1 immunoprecipitated and inactivated the enzyme in membrane extracts of overexpressing HEK293 cells and different murine tissues. Cell fractionation combined with immunoprecipitation studies localized the nSMase1 protein predominantly in the microsomal fraction. The enzyme colocalized with marker proteins of the endoplasmic reticulum and the Golgi apparatus in immunocytochemistry. Anti-nSMase1 antibodies did not affect the nSMase activity in the plasma membrane fraction and membrane extracts from murine brain. Our study leads to the conclusion that nSMase1 is one of at least two mammalian neutral sphingomyelinases with different subcellular localization, tissue specificity, and enzymatic properties.
Tomiuk
S
S
Laboratory of Molecular Neuroscience, Institute of Biochemistry, Faculty of Medicine, University of Cologne, D-50931 Cologne, Germany.
Zumbansen
M
M
Stoffel
W
W
D000818:Animals; D015152:Blotting, Northern; D001921:Brain / Q000201:enzymology; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D018871:Endoplasmic Reticulum, Smooth / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D008862:Microsomes, Liver / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational; D013108:Sphingomyelin Phosphodiesterase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014018:Tissue Distribution; D014162:Transfection
D004791:Enzyme Inhibitors; D013108:Sphingomyelin Phosphodiesterase; D008274:Magnesium