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

Abstract

The sequence similarity with bacterial neutral sphingomyelinase (NSM) resulted in the isolation of putative mammalian counterparts and, subsequently, identification of similar molecules in a number of other eukaryotic organisms. Based on sequence similarities and previous characterization of the mammalian enzymes, we have chemically modified specific residues and performed site directed mutagenesis in order to identify critical catalytic residues and determinants for membrane localization. Modification of histidine residues and the substrate protection experiments demonstrated the presence of reactive histidine residues within the active site. Site directed mutagenesis suggested an essential role in catalysis for two histidine residues (His136 and His272) which are conserved in all sequences. Mutations of two additional histidines (His138 and His151), conserved only in eukaryotes, resulted in reduced NSM activity. In addition to sphingomyelin, the enzyme also hydrolyzed lysophosphatidylcholine.Exposure to an oxidizing environment or modification of cysteine residues using several specific compounds also inactivated the enzyme. Site-directed mutagenesis of eight cysteine residues and gel-shift analysis demonstrated that these residues did not participate in the catalytic reaction and suggested the involvement of cysteines in the formation/breakage of disulfide bonds which could underlie the reversible inactivation by the oxidizing compounds.Cellular localization studies of a series of deletion mutants expressed as GFP-fusion proteins, demonstrated that the transmembrane region contains determinants for the ER localization.by guest on

Neutral Sphingomyelinase 1 Deficiency in the Mouse Causes No Lipid Storage Disease

“…We found that the nSMase 1 protein is located in the endoplasmic reticulum (35), which was confirmed by other laboratories (15,24). This subcellular localization makes a signaling function of this isoform unlikely, which was underscored by studies unable to confirm the influence of overexpressed nSMase 1 in tumor necrosis factor alpha-induced signaling pathways (34) and on Fas-induced apoptosis (33).…”

“…These lysolipids could also be hydrolyzed by His-tagged purified mouse nSMase 1 enzyme in the in vitro assay but at a rate reduced more than 100-fold from that of SPM (data not shown; see Discussion). A role for our cloned nSMase 1 as a lysophospholipase C has been proposed recently (13,24,28).…”

Mammalian Neutral Sphingomyelinases: Regulation and Roles in Cell Signaling Responses

“…Consistent with this, nSMase1 is also an integral membrane protein with two putative transmembrane domains at the C-terminus. Analysis of N-SMase activity in vitro revealed that nSMase1 was Mg 2+ -dependent (Tomiuk et al, 1998) and mutagenesis studies identified two histidine residues, His-136 and His-272, essential for catalysis (Rodrigues-Lima et al, 2000). Enzymatic activity required reducing agents and was reversibly inhibited by reactive oxygen species (ROS) and oxidized glutathione (GSH) (Fensome et al, 2000).…”

The Redox State of the Glutathione/Glutathione Disulfide Couple Mediates Intracellular Arginase Activation in HCT-116 Colon Cancer Cells

“…The data shown are the means ± SEM of duplicate samples of three separate assays erythrocyte arginase activity in vitro by driving the Fenton reaction [20]. Also, previous evidence demonstrates that the reversible activation/inhibition of some enzymes seems to be regulated by reducing/oxidizing agents through the reversible breakage or formation of disulfide bonds [30]. Therefore, it is very likely that the GSH redox couple could play a role in the regulation of arginase activity, although there is no evidence to support a direct physiologic effect of GSH or GSH redox oxidation on arginase.…”