Ceramides increase the activity of the secretory phospholipase A2 and alter its fatty acid specificity

Abstract

Modulation of human recombinant secretory type II phospholipase A(2) activity by ceramide and cholesterol was investigated using model glycerophospholipid substrates composed of phosphatidylethanolamine and phosphatidylserine dispersed in aqueous medium. Enzyme activity was monitored by measurement of released fatty acids using capillary GC-MS. Fatty acids from the sn-2 position of the phospholipids were hydrolysed by the enzyme in proportion to the relative abundance of the phospholipid in the substrate. Addition of increasing amounts of ceramide to the substrate progressively enhanced phospholipase activity. The increased activity was accomplished largely by preferential hydrolysis of polyunsaturated fatty acids, particularly arachidonic acid, derived from phosphatidylethanolamine. The addition of sphingomyelin to the substrate glycerophospholipids inhibited phospholipase activity but its progressive substitution by ceramide, so as to mimic sphingomyelinase activity, counteracted the inhibition. The presence of cholesterol in dispersions of glycerophospholipid-substrate-containing ceramides suppressed activation of the enzyme resulting from the presence of ceramide. The molecular basis of enzyme modulation was investigated by analysis of the phase structure of the dispersed lipid substrate during temperature scans from 46 to 20 degrees C using small-angle synchrotron X-ray diffraction. These studies indicated that intermediate structures created after ceramide-dependent phase separation of hexagonal and lamellar phases represent the most susceptible form of the substrate for enzyme hydrolysis.

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

“…The stimulation was uniform for all the unsaturated fatty acids, but was significantly less for 16:0. These studies show that ceramide activates the release of unsaturated fatty acids by sPLA 2 V, similar to its previously reported effect on sPLA 2 IIa acting on PE/PS substrate [13]. These results also support our conclusion that the increased release of 16:0 in the presence of SMase C and sPLA 2 V is not due to the direct action of the PLA 2 on PC or ceramide.…”

“…This indicates either that the other components of HDL such as apoproteins and cholesterol modulate the action of ceramide, or that the site of incorporation of exogenous ceramide in HDL is different from that in PC liposomes. The results of Kumanov et al [13] indeed showed that cholesterol suppresses the effect of ceramide on sPLA 2 IIa. Nevertheless, the effect of ceramide on the fatty acid specificity of sPLA 2 V is consistently observed in defined PC liposomes.…”

“…The stimulation of activity by the long chain ceramides was attributed to the lateral phase segregation of the bilayer into gel and liquid crystalline domains, which facilitates the penetration of the enzyme into the bilayer, as well as the binding of substrate PC to the active site. Koumanov et al [13] reported that the long chain ceramide not only simulated sPLA 2 IIa, but also promoted the release of polyunsaturated fatty acid from PE/PS substrate. 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.…”

“…However, it is not known whether the fatty acid specificity of the enzyme is affected by the presence of SM or ceramide. Previous studies by Koumanov et al on sPLA 2 group IIa showed that ceramide specifically stimulated the release of polyunsaturated fatty acids from a substrate of PE/PS mixture [13]. However, as mentioned above, since this enzyme does not readily hydrolyze the lipoprotein or membrane phospholipids [5], the physiological relevance of this finding is unclear.…”

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

“…Studies from several laboratories, including ours, have shown that ceramide is an activator of secretory and cytosolic phospholipases in vitro (12)(13)(14)(15)(16). Because of its tendency to form inverted hexagonal phase structures (H II ), it disrupts membrane bilayer (12,17), and this may be the basis for its activation of phospholipase activities.…”