Analysis of membrane topology of neutral sphingomyelinase 2

Abstract

Neutral sphingomyelinase 2 (nSMase2), which has two hydrophobic segments at its NH 2 -terminus, plays an important role in ceramide-mediated cell regulation. Here, we investigated the membrane topology of nSMase2. When a double-tagged nSMase2 at both the NH 2 and COOH termini, was overexpressed in MCF-7 cells, the signals from both tags were detected in the inner leaflet of the plasma membrane. Furthermore, insertion of a tag into the internal sequence and green fluorescent protein-fused deletion mutants revealed that the entire catalytic region of the protein was located on the cytosolic face of the membranes and each hydrophobic segment is integrated into the membranes, but unlikely to span the entire membrane. These results indicate the presence of the enzyme in the inner leaflet of plasma membrane.

An Obligate Role for Membrane-Associated Neutral Sphingomyelinase Activity in Orienting Chemotactic Migration of Human Neutrophils

“…N-SMase was virtually undetectable on the cell exterior (0.4% of total immunoreactivity; Figure 5A). Combined with the confocal images, these findings indicate that the great preponderance of N-SMase activity is associated with the cytofacial leaflet of the plasma membrane, as described previously (23) this effect precludes the possibility of N-SMase neosynthesis, suggesting instead that FMLP induces changes in the conformation or microenvironment of N-SMase that enhance the intensity of its immunolabeling. In keeping with this observation, previous work demonstrated that FMLP stimulation doubles the N-SMase activity associated with plasma membrane fractions in vitro (16).…”

Transbilayer lipid distribution in nano scale

“…It has been reported that PC in the cytoplasmic side of the plasma membrane is hydrolysed by PC-specific phospholipase C (Ramoni et al, 2004) and phospholipase D2 (PLD2) (Du et al, 2004). Sphingomyelin is also digested by intracellular neutral sphingomyelinase 2 (Tani and Hannun, 2007). In both cases, signalling lipids, including diacylglycerol, PA, ceramide and sphingosine-1-phosphate (Nagata et al, 2006) are formed.…”

Upregulation of smpd3 via BMP2 stimulation and Runx2

“…3), with weak Runx2 binding to the -562 to -557 region despite the strong transactivating power of this region. smpd3 appears to play a role in the mineralization of bone and dentine (20).…”