Identification and Characterization of Murine Mitochondria-associated Neutral Sphingomyelinase (MA-nSMase), the Mammalian Sphingomyelin Phosphodiesterase 5

Abstract

Sphingolipids play important roles in regulating cellular responses. Although mitochondria contain sphingolipids, direct regulation of their levels in mitochondria or mitochondria-associated membranes is mostly unclear. Neutral SMase (N-SMase) isoforms, which catalyze hydrolysis of sphingomyelin (SM) to ceramide and phosphocholine, have been found in the mitochondria of yeast and zebrafish, yet their existence in mammalian mitochondria remains unknown. Here, we have identified and cloned a cDNA based on nSMase homologous sequences. This cDNA encodes a novel protein of 483 amino acids that displays significant homology to nSMase2 and possesses the same catalytic core residues as members of the extended N-SMase family. A transiently expressed V5-tagged protein co-localized with both mitochondria and endoplasmic reticulum markers in MCF-7 and HEK293 cells; accordingly, the enzyme is referred to as mitochondria-associated nSMase (MAnSMase). MA-nSMase was highly expressed in testis, pancreas, epididymis, and brain. MA-nSMase had an absolute requirement for cations such as Mg 2؉ and Mn 2؉ and activation by the anionic phospholipids, especially phosphatidylserine and the mitochondrial cardiolipin. Importantly, overexpression of MA-nSMase in HEK293 cells significantly increased in vitro N-SMase activity and also modulated the levels of SM and ceramide, indicating that the identified cDNA encodes a functional SMase. Thus, these studies identify and characterize, for the first time, a mammalian MA-nSMase. The characterization of MA-nSMase described here will contribute to our understanding of pathways regulated by sphingolipid metabolites, particularly with reference to the mitochondria and associated organelles.

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

“…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).…”

Ceramide and mitochondrial function in aging oocytes: joggling a new hypothesis and old players

“…On the basis of molecular cloning and confocal microscopy data, this activity was ascribed to mitochondria (El Bawab et al 2000); and it was demonstrated in purified mitochondria (Bionda et al 2004). A recent report suggests that ceramide could be also generated by novel mitochondrial neutral SMase hydrolyzing SM (Wu et al 2010). Given the existence and the likelihood for each one of these various pathways to occur in the mitochondria, continued research efforts are required to better understand the mechanisms of mitochondrial ceramide generation and utilization, along with their influence on mitochondrial functions.…”

“…Mitochondria are important intracellular compartments of sphingolipid metabolism (Novgorodov & Gudz 2009), and several sphingolipid-metabolizing enzymes were found to be associated with mitochondria; so far the list includes neutral ceramidase (El Bawab et al 2000), novel neutral SMase (Wu et al 2010, Clarke et al 2011, and (dihydro) CerS, a key enzyme in de novo ceramide synthesis (Bionda et al 2004, Yu et al 2007. The existence of these compartment-specific pathways clearly suggests high specialization of these pathways, which in turn suggests specific mechanisms of Ceramide and mitochondrial function in aging oocytes regulation and, equally as likely, distinct functions and mechanisms of action of their lipid products.…”

Mitochondrial Ceramide in Stroke

“…Furthermore, it appears that oxidative stress could induce nSMase2 trafficking to the plasma membrane, whereas antioxidant (glutathione) directed its translocation to the perinuclear region (Levy et al, 2006). MA-nSMase is found within the mitochondria and associated membranes (Wu et al, 2010). The physiological role of neutral SMase isoforms may be dictated by their immediate environment in the specific intracellular compartment.…”