Sphingomyelinase D, a novel probe for cellular sphingomyelin

Abstract

Sphingomyelin (SM) and free cholesterol (FC) are concentrated in the plasma membranes of eukaryotes; however, the physiological significance of their association is unclear. A common tool for studying the role of membrane SM is digestion with bacterial sphingomyelinase (SMase) C, which hydrolyzes SM to ceramide. However, it is not known whether the observed effects of SMase C treatment are due to the loss of SM per se or to the signaling effects of ceramide. In this study, we tested SMase D from Corynebacterium pseudotuberculosis, which hydrolyzes SM to ceramide phosphate, as an alternative probe. This enzyme specifically hydrolyzed SM in fibroblasts without causing accumulation of ceramide. Several studies have shown that the bulk of cellular free cholesterol (FC) and sphingomyelin (SM) are localized in the plasma membrane (1-3). These two lipids interact strongly with each other in the membrane through hydrogen bonding and van der Waal's interactions (4, 5). It is therefore not surprising that each influences the intracellular transport and metabolism of the other. For example, depletion of membrane SM by enzymatic degradation leads to a rapid translocation of FC from the plasma membrane to intracellular sites, where it is esterified to cholesteryl ester (CE) by the enzyme acyl-CoA:cholesterol acyltransferase (ACAT) (2, 5, 6). Degradation of membrane SM also dramatically increases the oxidizability of membrane cholesterol by bacterial cholesterol oxidase (7), and blocks the proteolysis of sterol regulatory element binding protein, thereby inhibiting cholesterol biosynthesis (8). Conversely, exogenous addition of SM to human skin fibroblasts stimulates the biosynthesis of cholesterol (9). Ohvo, Olsio, and Slotte (10) reported that SM depletion of fibroblasts results in increased efflux of membrane FC to 2-hydroxypropyl-␤ -cyclodextrin (HPCD). In all these studies, the depletion of membrane SM was achieved by enzymatic degradation using bacterial sphingomyelinase (SMase) C, which hydrolyzes SM to ceramide and phosphorylcholine. Since ceramide is a strong mediator of signal transduction reactions (11-13), it is not clear whether the various effects observed following SMase C treatment are due to the disruption of the SM-cholesterol interaction and the consequent loss of cholesterol from the membrane, or rather to the effects of ceramide on cellular metabolism. A study in Chinese hamster ovary (CHO) cells showed that short-chain ceramides are strong inhibitors of ACAT activity (14). However, natural long-chain ceramides had no effect on the enzyme activity. C2 ceramides have also been reported to inhibit the synthesis of phosphatidylcholine (PC), sphingolipids, and cholesterol in baby hamster kidney cells, whereas the elevation of natural ceramides in the cells had very little effect on lipid synthesis (15). It has been suggested that the generation of ceramide disrupts the membrane bilayer because of the formation of hexagonal II phases (12). Such local phase transitions may cause profound changes in

Role of sphingomyelin and ceramide in the regulation of the activity and fatty acid specificity of group V secretory phospholipase A2

“…All ceramides and SMase C (S. aureus, 174 units/mg) were purchased from Sigma Chemical Co (St. Louis, MO). SMase D (Corynbacterium pseudotuberculosis) was purified from transfected E.Coli as described previously [15]. The SMase activities were assayed using the Amplex Red SMase Kit from Invitrogen Inc. Ceramide phosphate was prepared by the action of SMase D on egg SM and PE, as described before [16].…”

Sphingolipids and cellular cholesterol homeostasis. Effect of ceramide on cholesterol trafficking and HMG CoA reductase activity

“…DMAPP (1S,2R)-D-erythro-2-(Nmyristoylamino)-1-phenyl-1-propanol) was purchased from Biomol (Plymouth Meeting, PA, USA). SMase D was purified from E.Coli transfected with the C. pseudotuberculosis SMase D by affinity chromatography, as described earlier [6]. The transfected E. Coli were obtained from Dr. Stephen Billington, University of Arizona.…”

“…Several studies showed that hydrolysis of plasma membrane SM by bacterial SMase C (which degrades it to ceramide) results in a rapid translocation of plasma membrane cholesterol to endoplasmic reticulum (ER), as evident from the stimulation of acyl CoA: cholesterol acyltransferase (ACAT) reaction [4,5]. Our previous studies in human foreskin fibroblasts showed that unlike the effect of SMase C, treatment of the cells with SMase D (which degrades SM to ceramide phosphate) results in relatively modest stimulation of ACAT, although the extent of SM hydrolysis by the two enzymes was comparable [6]. This raised the possibility that the cellular response to SMase C treatment with respect to cholesterol trafficking is at least partly due to the generation of ceramide rather than the loss of SM alone from the PM.…”

“…ACAT activity in intact cells was measured by the conversion of labeled cholesterol to CE as described previously [6]. Briefly, the cells were labeled in 6 well (35 mm) plates with [4-14 C] cholesterol by incubation with 0.1 µCi of labeled cholesterol complexed with 20% 2-hydroxypropyl β-cyclodextrin (HPCD) (final concentration of HPCD, 0.05%) for 10 min at room temperature.…”

“…Therefore, the movement of cholesterol from PM to ER is measured indirectly by determining the activation of ACAT activity, which is regulated by the ER cholesterol levels [4,6]. However, exogenous ceramide is known to inhibit this enzyme both in cell-free systems and in intact cells [6,16], and therefore, we employed the inhibition of HMG CoA reductase (HMGCR) activity as a measure of ER cholesterol levels in response to altered ceramide levels in the cells. This enzyme has also been shown to respond rapidly to variations in plasma membrane concentrations [17] [18], showing that its inhibition can be used as an alternate measure of ER cholesterol.…”