N-SMase 1 was the first discover mammalian N-SMase based on sequence homology to bacterial SMases. It is not activated by phospholipids.8 N-SMase 1 activity on sphingomyelin in vitro, overexpression in cells does not affect sphingomyelin metabolism.8,9 The role of N-SMase 1 in sphingolipid metabolism is unclear. Although a function for N-SMase 1 is not apparent, With regards to N-SMase 1, this may be particularly relevant given the different subcellular localizations of endogenous and overexpressed proteins.

N-SMase 2 is to be known to study mammalian N-SMase and has emerged as a key mediator of cellular stress-induced generation of ceramide. Several characterizations have identified a number of mechanisms for activation and regulation of N-SMase 2. These studies provide a base to investigate other N-SMase isoforms, as well as potential areas and modes for therapeutic intervention.

Human N-SMase 3 was be come to in 2006,10 relatively little work has been reported since regarding further biochemical and functional characterization. The peptide sequence only matches 7 of the 11 residues in the identified human protein and bovine homolog.11 The question if the original purified bovine and identified human proteins correspond to the same protein. N-SMase 3 shares no sequence homology with any N-SMases or any other characterized type of enzyme catalytic domain. The region comprising the catalytic domain is yet to be identified. A C-terminal transmembrane helix is predicted to embed N-SMase 3 in the membrane.10

Two conflicting reports have characterized N-SMase 3 activity. In the original identification, N-SMase 3 activity is reported to occur at neutral pH and require Mg²⁺ or Mn²⁺.10 The observed activity was slightly enhanced, approximately two-fold, by the phospholipid. In a later study, MCF-7 cells transiently and stably overexpressing N-SMase 3 did not have significant N-SMase activity over vector controls.11 We suppose to future studies will determine the underlying reasons behind this major discrepancy. N-SMase 3 suggested that may activity a role in tumor necrosis factor-α (TNF-α) mediated signaling.10,12 However, another report found that N-SMase 2 was the primary N-SMase activated by TNF-α in MCF-7 cells.11,13,14

MA-N-SMase is the most recently identified mammalian N-SMase being discovered in 201015 by sequence homology with N-SMase 2 and a zebrafish mitochondrial N-SMase.16 The subcellular localization of overexpressed MA-N-SMase protein varied with cell type, showing strong or partial co-localization with mitochondrial markers, in addition to co-localization with endoplasmic reticulum markers.15 Present in mitochondria providing a putative mechanism for activation and/or regulation of MA-N-SMase activity in vivo.15 MA-N-SMase is little known about beyond basic properties. However, the identification of a mammalian mitochondrial N-SMase presents another potential endogenous mechanism, in addition to action of ceramide synthesis, for mitochondrial ceramide generation. This is exciting considering the numerous studies linking ceramide to mitochondrial activation of apoptosis.17 Another potential role for MA-N-SMase is in fertilization. Activation of an unidentified SMase during fertilization was inferred by a corresponding decrease in sphingomyelin and increase in ceramide levels.18

TNF-α triggers the activation of the sphingomyelin-ceramide pathway through stimulation of SMases.21 TNF-α pathway is induced by the hydrolysis of sphingomyelin to ceramide, which is considered as a putative bioactive sphingolipid in cell signaling.22 TNF-α has been reported to activate acidic and N-SMases 2 and 3.10,21,23 Activation of N-SMases in response to the cytokine TNF-α was initially observed in the HL-60 cell line (leukemia cell line). The TNF-α receptor-1 (TNFR-1), ordinarily known to the p55 receptor.24,25,26,27 It is pathway a distinct region of the p55 receptor was found to mediate the increase in N-SMase activity and was the neutral N-SMase activation domain (NSAD).24 Factor associated with N-SMase (FAN-SMase) that influences N-SMase activity in response to TNF-α.24,25 Indeed, overexpression of a dominant-negative form of FAN-SMase alters TNF-α induced N-SMase 3 activation in MCF-7 (human breast cancer cell line) cells.10 In addition to the elucidation of the molecular partners, other investigations have focused on cellular factors that influence TNF-α activation of N-SMase 2. Reported that the levels of glutathione, which can inhibit neutral SMase activity, are critical for TNF-α activation. Functionally, the activation of N-SMase 2 has implications in the inflammatory response of TNF-α. HeLa cells were known that N-SMase 2 acts on the PI3K/Akt pathway to activate endothelial nitric oxide synthase (eNOS). This suggests that N-SMase 2-derived ceramide can have bioactive action by itself in response to TNF-α or can be metabolized into other lipids to induce differential responses.

Recent findings have demonstrated a functional regulation of N-SMase 2 in response to lung insults and in certain lung diseases. Initially, reported that H₂O₂ stimulation and reactive oxygen species (ROS) formation activates N-SMase 2 to mediate apoptosis in human airway epithelium, while glutathione pretreatment prevents apoptosis.