Regulation of the Activity and Fatty Acid Specificity of Lecithin-Cholesterol Acyltransferase by Sphingomyelin and Its Metabolites, Ceramide and Ceramide Phosphate

Abstract

Sphingomyelin (SM), the second most abundant phospholipid in plasma lipoproteins, was previously shown to be a physiological inhibitor of the lecithin-cholesterol acyltransferase (LCAT) reaction. In this study, we investigated the effects of its metabolites, ceramide and ceramide phosphate, on the activity and fatty acid specificity of LCAT in vitro. Treatment of SM-containing substrate with SMase C, which hydrolyzes SM to ceramide, abolished the inhibitory effect of SM, whereas treatment with SMase D, which hydrolyzes it to ceramide phosphate, increased the inhibition. Although incorporation of ceramide into the substrate in the absence of SM activated the LCAT reaction only modestly, its co-incorporation with SM neutralized the inhibitory effect of SM. Ceramide phosphate, on the other hand, inhibited the LCAT reaction more strongly than SM. The effects of the sphingolipids were similar on the phospholipase A and cholesterol esterification reactions of the enzyme, indicating that they regulate the binding of phosphatidylcholine (PC) to the active site, rather than the esterification step. Ceramide incorporation into the substrate stimulated the synthesis of unsaturated cholesteryl esters at the expense of saturated esters. However these effects on fatty acid specificity disappeared when the PC substrates were incorporated into an inert diether PC matrix, suggesting that ceramide increases the availability of polyunsaturated PCs to the enzyme by altering the macromolecular structure of the substrate particle. Since the plasma ceramide levels are increased during inflammation, these results indicate that the activity and fatty acid specificity of LCAT may be altered during the inflammatory response. Abbreviations usedApo : apolipoprotein; CE: cholesteryl ester; FC: free (unesterified) cholesterol; HDL: high density lipoproteins; LCAT: lecithin-cholesterol acyltransferase; LDL: low density lipoproteins; PC: phosphatidylcholine; PLA: phospholipase A; SM: sphingomyelin; SMase: sphingomyelinase Sphingomyelin (SM) is one of the most abundant phospholipids in cell membranes and lipoproteins, constituting up to 30% of certain lipoprotein fractions (1,2). Although its role as a structural component of membranes, and as a precursor of signaling molecules has been well recognized, its function in plasma lipoproteins has received less attention. Recent studies show that plasma SM may be an independent risk factor for atherosclerosis (3), and that a reduction in SM levels by myriocin treatment reduces atherosclerosis in apo E deficient mice (4,5). While the exact mechanism of the proatherogenic effect of SM is unknown, one proposed mechanism involves the generation of ceramide in LDL by the action of arterial SMase, which causes † Supported by a grant from National Institutes of Health, HL 68585. increased retention, aggregation and oxidation of LDL, with subsequent uptake by the scavenger receptors of the macrophage (3). The formation of large amounts of ceramide in the plasma compartment is unlikely because of the...

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

“…4). We have previously reported a similar inhibition of LCAT activity by ceramide phosphate, whereas ceramide activated this enzyme activity also [16].…”

“…It is of interest to note that ceramide phosphate, which has been reported to directly interact with, and activate cytosolic PLA 2 [35], is actually inhibitory to sPLA 2 V (Fig. 7) as well as to LCAT [16]. The cytosolic PLA 2 , however differs significantly from the secretory enzymes both structurally and in its calcium requirements, and therefore the differential effect of ceramide phosphate is not surprising.…”

“…They proposed that the polyunsaturated phospholipids are specifically excluded from the ceramide-rich lamellar phase, and this results in their increased susceptibility to PLA 2 attack. Recent studies from our laboratory showed that LCAT, a specialized phospholipase A that esterifies cholesterol in plasma, is also activated by ceramide, and that the synthesis of unsaturated cholesteryl esters by the enzyme is specifically stimulated [16]. In addition, we found that group X sPLA 2 , an enzyme that is more active in the release of arachidonate from inflammatory cells [9], is inhibited by SM, and activated by ceramide, and alters its the fatty acid specificity in presence of ceramide (Singh and Subbaiah, unpublished data).…”

“…In addition, we found that group X sPLA 2 , an enzyme that is more active in the release of arachidonate from inflammatory cells [9], is inhibited by SM, and activated by ceramide, and alters its the fatty acid specificity in presence of ceramide (Singh and Subbaiah, unpublished data). The influence of ceramide on the fatty acid specificity of LCAT was minimized when the fluidity differences among the PC substrates was eliminated by incorporating the PCs into an inert common matrix [16], suggesting that ceramide affects the physical presentation of the substrate PC to the enzyme, rather than directly interacting with the enzyme. The results from the present study also show that the preferential activation of 20:4 release by ceramide was not apparent when the ceramide was incorporated into normal HDL (Fig.…”

“…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].…”