Characterization and Subcellular Localization of Murine and Human Magnesium-dependent Neutral Sphingomyelinase

Abstract

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.

Structural Requirements for Catalysis and Membrane Targeting of Mammalian Enzymes with Neutral Sphingomyelinase and Lysophospholipid Phospholipase C Activities

“…1). This region is predicted to incorporate two transmembrane domains (residues 325-346 and 353-375 in mouse sequence); this is consistent with mammalian enzymes being integral membrane proteins (8,10). Despite some similarities, there are other specific features of different sequences.…”

“…Mutational analysis of the corresponding residues in bacterial NSM and DNAse I had a similar impact on activity of these enzymes (4). It has also been reported (10) that the His272Asn mutation in the human enzyme resulted in a loss of NSM activity, as determined in transiently transfected HEK 293 cells.…”

“…In neurogranin, a PKC substrate which functions through an interaction with calmodulin, the formation of S-S bridges in the presence of NO donors (or other oxidants) results in the loss of the interaction with the regulatory proteins (21). Since NSM/lysoPLC resides in the ER (8,10), it is difficult to consider the redox environment and its possible changes without knowing the membrane topology of the enzyme. Even if the catalytic part is luminally oriented and exposed to the oxidizing environment, the protein could still be kept in the reduced state as described, for example, for the cholera toxin (33).…”

“…have recognized ER retention signals (29), they were found in this compartment (8,10). Therefore, to determine the region of mouse NSM/LysoPLC required for the ER localization, a series of deletion mutants from the N-and C-terminus were made (Fig.…”

“…gluthatione and reactive oxygen species (8). However, the function of this enzyme in signalling and its activity towards cellular SM has not been clearly demonstrated and would require further studies (9,10). Nonetheless, using an antisense strategy, Tonnetti et al (11) showed that the cloned enzyme could be involved in ceramide-mediated apoptosis triggered by TCR activation.…”