Cloning and characterization of the mammalian brain-specific, Mg ²⁺ -dependent neutral sphingomyelinase

Abstract

The enzymatic breakdown of sphingomyelin by sphingomyelinases is considered the major source of the second messenger ceramide. Studies on the contribution of the various described acidic and neutral sphingomyelinases to the signaling pool of ceramide have been hampered by the lack of molecular data on the neutral sphingomyelinases (nSMases). We recently identified a mammalian nSMase, an integral membrane protein with remote similarity to bacterial sphingomyelinases. However, its ubiquitous expression pattern is in contrast to previous findings that sphingomyelinase activity is found mainly in brain tissues. By using an improved database search method, combined with phylogenetic analysis, we identified a second mammalian nSMase (nSMase2) with predominant expression in the brain. The sphingomyelinase activity of nSMase2 has a neutral pH optimum, depends on Mg 2؉ ions, and is activated by unsaturated fatty acids and phosphatidylserine. Immunofluorescence reveals a neuron-specific punctate perinuclear staining, which colocalizes with a Golgi marker in a number of cell lines. The likely identity of nSMase2 with cca1, a rat protein involved in contact inhibition of 3Y1 fibroblasts, suggests a role for this enzyme in cell cycle arrest. Both mammalian nSMases are members of a superfamily of Mg 2؉ -dependent phosphohydrolases, which also contains nucleases, inositol phosphatases, and bacterial toxins.sequence analysis ͉ generalized profiles ͉ Ccr4 ͉ cytolethal distending toxin ͉ three-dimensional structure

Integration of cytokine biology and lipid metabolism in stroke

“…The PS relocation also causes flipping of SM from outer to inner leaflet and hydrolysis by a cytosolic SMase (66)(67)(68) to generate ceramide with subsequent activation of death cascade mechanisms.…”

Identification of Novel Anionic Phospholipid Binding Domains in Neutral Sphingomyelinase 2 with Selective Binding Preference

“…Thus, the identification of PA/PS-interacting proteins as well as their binding domains is of interest, and the information will provide further insight into the lipid interaction and regulation of these effectors. Previous studies have shown that nSMase2 activity can be stimulated by APLs (6,31). However, as nSMase2 does not contain a previously characterized phospholipid binding domain, it is unclear if and where APLs interact with this enzyme.…”

“…Recently, considerable progress has been made regarding the specific roles of the identified N-SMase isoforms, particularly neutral sphingomyelinase 2 (nSMase2), in signal transduction and a variety of cell processes (11). Mammalian nSMase2 was first identified in 2000 based on similarity to bacterial SMase (6). The human nSMase2 protein is a membrane-bound 655 amino acid protein consisting of a C-terminal catalytic domain, two N-terminal hydrophobic segments (HSs), and a 200-residue collagen-like domain between the N and C termini (6).…”

“…Mammalian nSMase2 was first identified in 2000 based on similarity to bacterial SMase (6). The human nSMase2 protein is a membrane-bound 655 amino acid protein consisting of a C-terminal catalytic domain, two N-terminal hydrophobic segments (HSs), and a 200-residue collagen-like domain between the N and C termini (6). More recently, nSMase2 was also found to be palmitoylated via thioester bonds at two major sites, one in the N terminus and the other in the catalytic region of the enzyme.…”

“…The hydrolysis of sphingomyelin (SM) 2 is a major pathway for ceramide production, and this reaction is catalyzed by the sphingomyelinases (SMases). Currently, six different SMases have been identified and grouped based upon their catalytic pH optima, including an acid SMase, an alkaline SMase and four neutral SMases (N-SMases) (5)(6)(7)(8). Activation of N-SMases has been implicated in various biological responses, such as growth arrest, inflammation and aging (9,10).…”