Phase composition of lipoprotein SM/cholesterol/PtdCho affects FA specificity of sPLA2s

Abstract

We have previously reported preferential release of polyunsaturated FAs during hydrolysis of lipoprotein phosphatidylcholine (PtdCho) by group X secretory phospholipase A 2 (sPLA 2 ) and preferential release of oligounsaturated FAs during hydrolysis of lipoprotein PtdCho by group V sPLA 2 , but the mechanism of this selectivity has remained unknown. We now show that the rate and specificity of hydrolysis are affected by relative increases in endogenous SM and free cholesterol (FC) during the lipase digestion. The highest preference for arachidonate release from LDL and HDL by group X sPLA 2 was observed for residual SM/PtdCho molar ratio of 1.2 and 0.4, compared with the respective starting ratios of 0.4 and 0.2, as measured by liquid chromatography/electrospray ionization-mass spectrometry. Group V sPLA 2 showed preferential release of linoleate from LDL and HDL at SM/PtdCho ratio 1.5 and 0.6, respectively. We have attributed the change in FA specificity to segregation of molecular species of PtdCho and of sPLA 2 s between disordered and ordered SM/FC/ PtdCho lipid phases. The increases in SM and FC during digestion with group IIA sPLA 2 were more limited, and a preferential hydrolysis of any FAs was not observed. A preferential release of linoleate during hydrolysis of phosphatidylcholine (PtdCho) by group V secretory phospholipase A 2 (sPLA 2 ) was first reported in liposomal (1-3) and later in lipoprotein (4-6) incubations, but the mechanism of this selectivity has remained unknown. Early work (1, 2) attributed the selective enzyme activity to a difference in the physical state of substrate between the sonicate and the natural membranes that may contain a more optimum mixture of phospholipids. Alternatively, a preferential release of arachidonate by group V sPLA 2 exogenously added to cell cultures has been attributed to its combined action with cytosolic PLA 2 (7). Subsequent work with liposomes suggested a special role for ceramides (3) in regulation of group V sPLA 2 hydrolysis of plasma PtdCho, which, however, was difficult to reconcile with the apparent absence of sphingomyelinase activity from plasma lipoproteins (6,8,9). Likewise, the apparent preferential release of PtdCho arachidonate by group X sPLA 2 during liposomal (10, 11), lipoprotein (4-6), and cellular (10, 12) incubations has remained unexplained. In contrast, group IIA sPLA 2 apparently attacked liposomal and lipoprotein PtdCho without FA discrimination, although at a significantly lower rate than group V and group X sPLA 2 s (3,5,9,10,12). Therefore, the true differences in the activity and apparent FA specificity of PtdCho hydrolysis by the sPLA 2 s have remained obscure.In other studies, a preferential release of arachidonate by group IIA sPLA 2 from phosphatidylethanolamine/ phosphatidylserine (PtdEtn/PtdSer) micelles was observed (13,14) in the presence of added SM/ceramide and was attributed to phase separation and exclusion of polyunsaturates from the ordered liquid phase formed under these conditions. The inhibitory effect...

In Vitro Anti-Plasmodium falciparum Properties of the Full Set of Human Secreted Phospholipases A 2

“…We previously demonstrated that the main mediators of parasite death by bee venom sPLA 2 -hydrolyzed VLDLs are PUFAs, especially AA, suggesting that the ability to produce PUFAs might be an important feature of the sPLA 2 anti-Plasmodium activity. It is known that hGX sPLA 2 preferentially hydrolyzes PC with polyenoic fatty acid species, including AA (41,42,(46)(47)(48), and that hGIIF sPLA 2 exhibits preference for PC species with AA (44), whereas hGV sPLA 2 preferentially attacks oligoenoic PL species and mostly releases saturated and monounsaturated fatty acids (41,42,(46)(47)(48). hGIII sPLA 2 did not seem to exhibit any PL preference (44).…”

“…This hypothesis was also supported by studies showing that human sPLA 2 s exhibit different selectivities toward lipoprotein-PC species with different fatty acids at the sn-2 position. Indeed, it was shown that hGX sPLA 2 preferentially hydrolyzes PC with linoleic acid (LA; 18:2) and arachidonic acid (20:4) at the sn-2 position, whereas hGV sPLA 2 prefers PC with LA and oleic acid (OA; 18:1) (41,44,46,47). Furthermore, hGIII exhibited no preference toward PC species from LDL and HDL, whereas hGIIF sPLA 2 attacks PC species with AA preferentially (44).…”

Biology of Secretory Phospholipase A2

“…Nevertheless, GV and GX sPLA 2 have been shown to be quite potent in mobilizing arachidonic acid and stimulating eicosanoid synthesis in various cell types [51,52], and thus may play an important role in inflammation and inflammatory diseases, as discussed in more detail in a subsequent section of this review. Interestingly, membrane sphingomyelin/ceramide content has been shown to modulate both the activity and arachidonic acid selectivity of GV and GX sPLA 2 [46,[53][54][55], suggesting a novel mechanism whereby sphingolipids may regulate sPLA 2 -induced inflammatory responses. The exact mechanism by which sphingomyelin/ceramide modulates sPLA 2 activity is not clearly understood, although the change in fatty acid specificity has been attributed to segregation of PC and sPLA 2 between disordered and ordered sphingomyelin/free cholesterol/PC lipid phases [53].…”

“…Interestingly, membrane sphingomyelin/ceramide content has been shown to modulate both the activity and arachidonic acid selectivity of GV and GX sPLA 2 [46,[53][54][55], suggesting a novel mechanism whereby sphingolipids may regulate sPLA 2 -induced inflammatory responses. The exact mechanism by which sphingomyelin/ceramide modulates sPLA 2 activity is not clearly understood, although the change in fatty acid specificity has been attributed to segregation of PC and sPLA 2 between disordered and ordered sphingomyelin/free cholesterol/PC lipid phases [53].…”

The Capacity of Group V sPLA 2 to Increase Atherogenicity of ApoE −/− and LDLR −/− Mouse LDL In Vitro Predicts its Atherogenic Role In Vivo

“…Secondly, lipoprotein phospholipid (PL) content is altered in apoE -/- mice, such that the SM to PC ratio is relatively high due to increased SM production and decreased SM degradation13. Since previous in vitro data demonstrate that GV sPLA 2 hydrolysis of PC in liposomes and human LDL is inhibited by SM 6, 14-16, we speculated that particles from apoE -/- mice may be relatively poor substrates for GV sPLA 2 hydrolysis.…”