Sphingomyelin Inhibits the Lecithin-Cholesterol Acyltransferase Reaction with Reconstituted High Density Lipoproteins by Decreasing Enzyme Binding

Abstract

Lecithin-cholesterol acyltransferase (LCAT) catalyzes the formation of cholesterol esters on high density lipoproteins (HDL) and plays a critical role in reverse cholesterol transport. Sphingomyelin, an important constituent of HDL, may regulate the activity of LCAT at any of the key steps of the enzymatic reaction: binding of LCAT to the interface, activation by apo A-I, or inhibition at the catalytic site. In order to clarify the role of sphingomyelin in the regulation of the LCAT reaction and its effects on the structure of apolipoprotein A-I, we prepared reconstituted HDL (rHDL) containing egg phosphatidylcholine, cholesterol, apolipoprotein A-I, and up to 22 mol % sphingomyelin. Because the interfacial properties of substrate particles can dramatically affect LCAT binding and kinetics, we also prepared and analyzed proteoliposome substrates having the same components as the rHDL, except for a 4-fold higher ratio of phospholipid to apolipoprotein A-I. The reaction kinetics of LCAT with the rHDL particles revealed no significant change in the apparent V max but showed a concentration-dependent increase in slope of the reciprocal plots and in the apparent K m values with sphingomyelin content. The dissociation constant (K d ) for LCAT with these particles increased linearly with sphingomyelin content up to 22 mol %, changing in parallel with the apparent K m values. No structural changes of apolipoprotein A-I were detected in the particles with increasing content of sphingomyelin, but fluorescence results with lipophilic probes revealed that significant changes in the acyl chain, backbone, and head group regions of the lipid bilayer of the particles are introduced by the addition of sphingomyelin. On the other hand, the proteoliposome substrates also had increased K d values for LCAT at high sphingomyelin contents but compared with the rHDL particles had a 6 -10-fold lower affinity for LCAT binding and exhibited kinetics consistent with competitive inhibition by sphingomyelin at the active site. These results show conclusively that the dominant mechanism for the inhibition of LCAT activity with rHDL particles by sphingomyelin is the impaired binding of the enzyme to the interface. The results also underscore the significant differences in the enzyme reaction kinetics with different substrate particles. Lecithin-cholesterol acyltransferase (LCAT)1 plays a critical role in the maintenance of cholesterol homeostasis. LCAT participates in the reverse cholesterol transport pathway, maintaining a gradient for the diffusion of free cholesterol from peripheral tissues into high density lipoproteins (HDL) by catalyzing cholesterol ester (CE) formation from HDL surface phosphatidylcholine (PC) and cholesterol. As a result of LCAT activity, cholesterol is removed from peripheral cell membranes and, as CE, is ultimately removed and metabolized by the liver.Several factors contribute to the maintenance of cellular cholesterol homeostasis, including the activity of HMG-CoA reductase and acyl-CoA acyltransferase, which are r...

Alteration in Apolipoprotein A-I 22-Mer Repeat Order Results in a Decrease in Lecithin:Cholesterol Acyltransferase Reactivity

“…This difference was primarily due to a 5-6-fold increase in the apparent K m for 10F6 apoA-I complexes. These results suggest that the reduced binding affinity of LCAT to DMPC 10F6 apoA-I complexes was responsible for the large decrease in catalytic efficiency (38). Thus, we conclude that substitution of a single 22-mer repeat within the 143-165 domain resulted in a substantial reduction in LCAT binding to the recombinant substrate and ultimately in a lower catalytic efficiency.…”

Endotoxin and Cytokines Increase Hepatic Sphingolipid Biosynthesis and Produce Lipoproteins Enriched in Ceramides and Sphingomyelin

“…Sphingomyelin also inhibits the activity of lecithin-cholesterol acyltransferase . 45,46 Thus, the host response to infection and inflammatory stimuli is accompanied by several changes that could increase the atherogenicity of lipoprotein particles.…”

“…Sphingomyelin is the substrate for the formation of ceramide by sphingomyelinases in the arterial wall and macrophages. 43 Sphingomyelin inhibits the activity of lecithin-cholesterol acyltransferase, 45,46 which could decrease the reverse-cholesterol transport pathway, thereby increasing the risk of atherogenesis. Sphingomyelin also slows the clearance of triglyceride-rich lipoproteins, 47 which could result in an accumulation of VLDL and chylomicron remnant particles that are atherogenic.…”

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

“…Furthermore, ceramide was reported to specifically stimulate the release of unsaturated fatty acids from phospholipids by sPLA 2 IIa (13), suggesting that it facilitates the interaction of lipolytic enzymes with specific phospholipid species. The possible effect of ceramide on the activity and specificity of LCAT, an enzyme that is essentially a modified PLA 2 , has not been investigated, although SM, its precursor, has been shown to inhibit LCAT activity (18)(19)(20)(21). In this study, we addressed the effect of SM and its metabolites, ceramide and ceramide phosphate, on the activity and fatty acid specificity of human LCAT.…”