LCAT deficiency in mice is associated with a diminished adrenal glucocorticoid function

In vitro studies have suggested that HDL and apoB-containing lipoproteins can provide cholesterol for synthesis of glucocorticoids. Here we assessed adrenal glucocorticoid function in LCAT knockout (KO) mice to determine the specific contribution of HDL-cholesteryl esters to adrenal glucocorticoid output in vivo. LCAT KO mice exhibit an 8-fold higher plasma free cholesterol-to-cholesteryl ester ratio (P < 0.001) and complete HDL-cholesteryl ester deficiency. ApoB-containing lipoprotein and associated triglyceride levels are increased in LCAT KO mice as compared with C57BL/6 control mice (44%; P < 0.05). Glucocorticoid-producing adrenocortical cells within the zona fasciculata in LCAT KO mice are devoid of neutral lipids. However, adrenal weights and basal corticosterone levels are not significantly changed in LCAT KO mice. In contrast, adrenals of LCAT KO mice show compensatory up-regulation of genes involved in cholesterol synthesis (HMG-CoA reductase; 516%; P < 0.001) and acquisition (LDL receptor; 385%; P < 0.001) and a marked 40–50% lower glucocorticoid response to adrenocorticotropic hormone exposure, endotoxemia, or fasting (P < 0.001 for all). In conclusion, our studies show that HDL-cholesteryl ester deficiency in LCAT KO mice is associated with a 40–50% lower adrenal glucocorticoid output. These findings further highlight the important novel role for HDL as cholesterol donor for the synthesis of glucocorticoids by the adrenals.     

Proteolysis of Apolipoprotein A-I by Secretory Phospholipase A2: A NEW LINK BETWEEN INFLAMMATION AND ATHEROSCLEROSIS*

In the acute phase of the inflammatory response, secretory phospholipase A₂ (sPLA₂) reaches its maximum levels in plasma, where it is mostly associated with high density lipoproteins (HDL). Overexpression of human sPLA₂ in transgenic mice reduces both HDL cholesterol and apolipoprotein A-I (apoA-I) plasma levels through increased HDL catabolism by an unknown mechanism. To identify unknown PLA₂-mediated activities on the molecular components of HDL, we characterized the protein and lipid products of the PLA₂ reaction with HDL. Consistent with previous studies, hydrolysis of HDL phospholipids by PLA₂ reduced the particle size without changing its protein composition. However, when HDL was destabilized in the presence of PLA₂ by the action of cholesteryl ester transfer protein or by guanidine hydrochloride treatment, a fraction of apoA-I, but no other proteins, dissociated from the particle and was rapidly cleaved. Incubation of PLA₂ with lipid-free apoA-I produced similar protein fragments in the range of 6–15 kDa, suggesting specific and direct reaction of PLA₂ with apoA-I. Mass spectrometry analysis of isolated proteolytic fragments indicated at least two major cleavage sites at the C-terminal and the central domain of apoA-I. ApoA-I proteolysis by PLA₂ was Ca²⁺-independent, implicating a different mechanism from the Ca²⁺-dependent PLA₂-mediated phospholipid hydrolysis. Inhibition of proteolysis by benzamidine suggests that the proteolytic and lipolytic activities of PLA₂ proceed through different mechanisms. Our study identifies a previously unknown proteolytic activity of PLA₂ that is specific to apoA-I and may contribute to the enhanced catabolism of apoA-I in inflammation and atherosclerosis.     

Increased plasma cholesterol esterification by LCAT reduces diet-induced atherosclerosis in SR-BI knockout mice

LCAT, a plasma enzyme that esterifies cholesterol, has been proposed to play an antiatherogenic role, but animal and epidemiologic studies have yielded conflicting results. To gain insight into LCAT and the role of free cholesterol (FC) in atherosclerosis, we examined the effect of LCAT over- and underexpression in diet-induced atherosclerosis in scavenger receptor class B member I-deficient [Scarab(−/−)] mice, which have a secondary defect in cholesterol esterification. Scarab(−/−)×LCAT-null [Lcat(−/−)] mice had a decrease in HDL-cholesterol and a high plasma ratio of FC/total cholesterol (TC) (0.88 ± 0.033) and a marked increase in VLDL-cholesterol (VLDL-C) on a high-fat diet. Scarab(−/−)×LCAT-transgenic (Tg) mice had lower levels of VLDL-C and a normal plasma FC/TC ratio (0.28 ± 0.005). Plasma from Scarab(−/−)×LCAT-Tg mice also showed an increase in cholesterol esterification during in vitro cholesterol efflux, but increased esterification did not appear to affect the overall rate of cholesterol efflux or hepatic uptake of cholesterol. Scarab(−/−)×LCAT-Tg mice also displayed a 51% decrease in aortic sinus atherosclerosis compared with Scarab(−/−) mice (P < 0.05). In summary, we demonstrate that increased cholesterol esterification by LCAT is atheroprotective, most likely through its ability to increase HDL levels and decrease pro-atherogenic apoB-containing lipoprotein particles.     

LCAT deficiency does not impair amyloid metabolism in APP/PS1 mice

A key step in plasma HDL maturation from discoidal to spherical particles is the esterification of cholesterol to cholesteryl ester, which is catalyzed by LCAT. HDL-like lipoproteins in cerebrospinal fluid (CSF) are also spherical, whereas nascent lipoprotein particles secreted from astrocytes are discoidal, suggesting that LCAT may play a similar role in the CNS. In plasma, apoA-I is the main LCAT activator, while in the CNS, it is believed to be apoE. apoE is directly involved in the pathological progression of Alzheimer’s disease, including facilitating β-amyloid (Aβ) clearance from the brain, a function that requires its lipidation by ABCA1. However, whether apoE particle maturation by LCAT is also required for Aβ clearance is unknown. Here we characterized the impact of LCAT deficiency on CNS lipoprotein metabolism and amyloid pathology. Deletion of LCAT from APP/PS1 mice resulted in a pronounced decrease of apoA-I in plasma that was paralleled by decreased apoA-I levels in CSF and brain tissue, whereas apoE levels were unaffected. Furthermore, LCAT deficiency did not increase Aβ or amyloid in APP/PS1 LCAT^−/− mice. Finally, LCAT expression and plasma activity were unaffected by age or the onset of Alzheimer’s-like pathology in APP/PS1 mice. Taken together, these results suggest that apoE-containing discoidal HDLs do not require LCAT-dependent maturation to mediate efficient Aβ clearance.     

An apoA-I mimetic peptide increases LCAT activity in mice through increasing HDL concentration

Lecithin cholesterol acyltransferase (LCAT) plays a key role in the reverse cholesterol transport (RCT) process by converting cholesterol to cholesteryl ester to form mature HDL particles, which in turn deliver cholesterol back to the liver for excretion and catabolism. HDL levels in human plasma are negatively correlated with cardiovascular risk and HDL functions are believed to be more important in atheroprotection. This study investigates whether and how D-4F, an apolipoprotein A-I (apoA-I) mimetic peptide, influences LCAT activity in the completion of the RCT process. We demonstrated that the apparent rate constant value of the LCAT enzyme reaction gives a measure of LCAT activity and determined the effects of free metals and a reducing agent on LCAT activity, showing an inhibition hierarchy of Zn²⁺>Mg²⁺>Ca²⁺ and no inhibition with β-mercaptoethanol up to 10 mM. We reconstituted nano-disc particles using apoA-I or D-4F with phospholipids. These particles elicited good activity in vitro in the stimulation of cholesterol efflux from macrophages through the ATP-binding cassette transporter A1 (ABCA1). With these particles we studied the LCAT activity and demonstrated that D-4F did not activate LCAT in vitro. Furthermore, we have done in vivo experiments with apoE-null mice and demonstrated that D-4F (20 mg/kg body weight, once daily subcutaneously) increased LCAT activity and HDL level as well as apoA-I concentration at 72 hours post initial dosing. Finally, we have established a correlation between HDL concentration and LCAT activity in the D-4F treated mice.     

Plasma lecithin:cholesterol acyltransferase activity modifies the inverse relationship of C-reactive protein with HDL cholesterol in nondiabetic men

Lecithin:cholesterol acyltransferase (LCAT) is instrumental in high-density lipoprotein (HDL) maturation, but high LCAT levels do not predict low cardiovascular risk. LCAT may affect antioxidative or anti-inflammatory properties of HDL. We determined the relationship of plasma high-sensitivity C-reactive protein (CRP) with LCAT activity and evaluated whether LCAT activity modifies the decreasing effect of HDL cholesterol (HDL-C) on CRP, as an estimate of its anti-inflammatory properties. Plasma HDL-C, apolipoprotein (apo) A-I and LCAT activity (exogenous substrate method) were measured in 260 nondiabetic men without cardiovascular disease. CRP was correlated inversely with HDL-C and apo A-I, and positively with LCAT activity (P < 0.01 to 0.001). Multivariate regression analysis demonstrated that age- and smoking-adjusted plasma CRP levels were associated negatively with HDL-C (β = − 0.224, P < 0.001) and positively with LCAT activity (β = 0.119, P = 0.034), as well as with the interaction between HDL-C and LCAT activity (β = 0.123, P = 0.026). There was also an interaction between apo A-I and LCAT activity on CRP (β = 0.159, P = 0.005). These relationships remained similar after adjustment for apo B-containing lipoproteins. In conclusion, the inverse relationship of HDL-C with CRP is attenuated by LCAT activity at higher HDL-C levels. It is hypothesized that LCAT could mitigate HDL's anti-inflammatory or antioxidative properties at higher HDL-C concentrations.     

Dexamethasone,a Specific PLA2 Inhibitor,Allows Nonpathogenic Pseudomonas fluorescens to Induce Virulence Against Spodoptera exigua

Phospholipase A₂ (PLA₂) catalyzes phospholipids at sn-2 position to release arachidonic acid (20:4n-6). The arachidonic acid is further oxidized to form different eicosanoids, which play biological mediators to express cellular or humoral immune reactions in response to pathogen infection. Xeno-rahbdus and Photorhabdus, the symbiotic bacteria of entomopathogenic nematodes, have been known to inhibit PLA₂ to express their pathogenicity. This research aimed to test a hypothesis that other entomopathogenic bacteria also inhibit PLA₂ to express their pathogenicity in Spodopera exigua. Two bacterial species of Enterococcus faecalis and Pseudomonas fluorescens presumably different in ento-mopathogenicity were analyzed in their PLA₂ inhibitory activities. A pathogenic E. faecalis induced significantly immunodepression of S. exigua by inhibiting PLA₂ activity because the bacteria-infected S. exigua recovered immune reactions after the addition of arachidonic acid. However, the nonpathogenic P. fluorescens did not induce immunodepression because the addition of arachidonic acid to P. fluorescens-infected S. exigua did not further increase immune capacities while dexamethasone, a PLA₂ inhibitor, could decrease the immune activities. Injection of E. faecalis along with 10 μg of dexamethasone significantly increased pathogenicity in comparison with the bacteria alone. Moreover, the addition of dexamethasone transformed nonpathogenic P. fluorescens into pathogenic bacterium. This study suggests an evidence that PLA₂ is an inhibitory target even for entomopathogenic bacteria not related with entomopathogenic nematodes, and that the inhibition of PLA₂ determines the bacterial virulence in S. exigua.     

ApoA-IV promotes the biogenesis of apoA-IV-containing HDL particles with the participation of ABCA1 and LCAT

The objective of this study was to establish the role of apoA-IV, ABCA1, and LCAT in the biogenesis of apoA-IV-containing HDL (HDL-A-IV) using different mouse models. Adenovirus-mediated gene transfer of apoA-IV in apoA-I^−/− mice did not change plasma lipid levels. ApoA-IV floated in the HDL2/HDL3 region, promoted the formation of spherical HDL particles as determined by electron microscopy, and generated mostly α- and a few pre-β-like HDL subpopulations. Gene transfer of apoA-IV in apoA-I^−/− × apoE^−/− mice increased plasma cholesterol and triglyceride levels, and 80% of the protein was distributed in the VLDL/IDL/LDL region. This treatment likewise generated α- and pre-β-like HDL subpopulations. Spherical and α-migrating HDL particles were not detectable following gene transfer of apoA-IV in ABCA1^−/− or LCAT^−/− mice. Coexpression of apoA-IV and LCAT in apoA-I^−/− mice restored the formation of HDL-A-IV. Lipid-free apoA-IV and reconstituted HDL-A-IV promoted ABCA1 and scavenger receptor BI (SR-BI)-mediated cholesterol efflux, respectively, as efficiently as apoA-I and apoE. Our findings are consistent with a novel function of apoA-IV in the biogenesis of discrete HDL-A-IV particles with the participation of ABCA1 and LCAT, and may explain previously reported anti-inflammatory and atheroprotective properties of apoA-IV.     

Purification and Characterization of a Low-Molecular-Weight Phospholipase A2 from Developing Seeds of Elm

Phospholipase A₂ (PLA₂) was purified about 180,000 times compared with the starting soluble-protein extract from developing elm (Ulmus glabra) seeds. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified fraction showed a single protein band with a mobility that corresponded to 15 kD, from which activity could be recovered. When analyzed by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry, the enzyme had a deduced mass of 13,900 D. A 53-amino acid-long N-terminal sequence was determined and aligned with other sequences, giving 62% identity to the deduced amino acid sequence of some rice (Oryza sativa) expressed sequence tag clones. The purified enzyme had an alkaline pH optimum and required Ca²⁺ for activity. It was unusually stable with regard to heat, acidity, and organic solvents but was sensitive to disulfide bond-reducing agents. The enzyme is a true PLA₂, neither hydrolyzing the sn-1 position of phosphatidylcholine nor having any activity toward lysophosphatidylcholine or diacylglycerol. The biochemical data and amino acid sequence alignments indicate that the enzyme is related to the well-characterized family of animal secretory PLA₂s and, to our knowledge, is the first plant enzyme of this type to be described.     

Effects of weight loss,induced by gastric bypass surgery,on HDL remodeling in obese women

Plasma lipoproteins and glucose homeostasis were evaluated after marked weight loss before and over 12 months following Roux-en-Y gastric-bypass (RYGBP) surgery in 19 morbidly obese women. Standard lipids, remnant-lipoprotein cholesterol (RLP-C); HDL-triglyceride (TG); apolipoproteins (apo) A-I, A-II, E, and A-I-containing HDL subpopulations; lecithin-cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein (CETP) mass and activity; plasma glucose and insulin levels were measured before and at 1, 3, 6, and 12 months after GBP surgery. Baseline concentrations of TG, RLP-C, glucose, and insulin were significantly higher in obese than in normal-weight, age-matched women, whereas HDL cholesterol (HDL-C), apoA-I, apoA-II, α-1 and α-2 levels were significantly lower. Over 1 year, significant decreases of body mass index, glucose, insulin, TG, RLP-C, HDL-TG, and preβ-1 levels were observed with significant increases of HDL-C and α-1 levels (all P < 0.05). Changes of fat mass were correlated with those of LDL cholesterol (P = 0.018) and LCAT mass (P = 0.011), but not with CETP mass (P = 0.265). Changes of fasting plasma glucose concentrations were inversely correlated with those of CETP mass (P = 0.005) and α-1 level (P = 0.004). Changes of fasting plasma insulin concentrations were positively correlated with those of LCAT mass (P = 0.043) and inversely with changes of α-1 (P = 0.03) and α-2 (P = 0.05) concentrations. These results demonstrate beneficial changes in HDL remodeling following substantial weight loss induced by RYGBP surgery and that these changes are associated with improvement of glucose homeostasis in these patients.     

Infrared characterization of conformational differences in the lamellar phases of 1,3-dipalmitoyl-sn-glycero-2-phosphocholine

Fourier transform infrared spectroscopy was used to characterize the lamellar phases of 1,3-dipalmitoyl-sn-glycero-2-phosphocholine (1,3-DPPC), a positional isomer of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (1,2-DPPC). The molecule exists in three distinct phases over the temperature interval 0–70°C. In the low-temperature (L_c) phase, the spectra are indicative of acyl chains packed in an orthorhombic subcell, while the carbonyl groups and phosphate ester at the head group show evidence of only partial hydration. The transition from the low-temperature (L_c) phase to the intermediate-temperature (Lβ) phase at 25°C corresponds to a temperature-induced head-group hydration in which the hydration of the phosphate and carbonyl ester groups results in the reorganization of the hydrocarbon chain-packing subcell from orthorhombic to hexagonal. The transition from the intermediate (L_β) to the high-temperature (L_α) phase at 37°C is a gel-to-liquid-crystalline phase transition analogous to the 41.5°C transition of 1,2-DPPC. The spectra of the acyl-chain carbonyl groups show evidence of significant differences in molecular conformation at the carbonyl esters in the L_c phase. In the L_β and L_α phases, the carbonyl band contour becomes much more symmetric. However, two components are clearly present in the spectra indicating that the sn-1 and sn-3 carbonyls experience slightly different environments. The observed differences are likely due to a preferred conformation of the phosphocholine group relative to the glycerol backbone. Indications from the infrared spectra of differences in the structure of the C=O groups provide a possible explanation for the selection of the sn-1 chain of 1,3-DPPC by phospholipase A₂ on the basis of a preferred head group conformation.     

Progesterone-induced Acrosome Exocytosis Requires Sequential Involvement of Calcium-independent Phospholipase A2β (iPLA2β) and Group X Secreted Phospholipase A2 (sPLA2)

Phospholipase A₂ (PLA₂) activity has been shown to be involved in the sperm acrosome reaction (AR), but the molecular identity of PLA₂ isoforms has remained elusive. Here, we have tested the role of two intracellular (iPLA₂β and cytosolic PLA₂α) and one secreted (group X) PLA₂s in spontaneous and progesterone (P4)-induced AR by using a set of specific inhibitors and knock-out mice. iPLA₂β is critical for spontaneous AR, whereas both iPLA₂β and group X secreted PLA₂ are involved in P4-induced AR. Cytosolic PLA₂α is dispensable in both types of AR. P4-induced AR spreads over 30 min in the mouse, and kinetic analyses suggest the presence of different sperm subpopulations, using distinct PLA₂ pathways to achieve AR. At low P4 concentration (2 μm), sperm undergoing early AR (0–5 min post-P4) rely on iPLA₂β, whereas sperm undergoing late AR (20–30 min post-P4) rely on group X secreted PLA₂. Moreover, the role of PLA₂s in AR depends on P4 concentration, with the PLA₂s being key actors at low physiological P4 concentrations (≤2 μm) but not at higher P4 concentrations (∼10 μm).     

The essentiality of B chain in stabilizing the structure of the A chain in β1-bungarotoxin fromBungarus multicinctus venom

The dynamic of Trp residue inΒ ₁-bungarotoxin (gb ₁-Bgt), the A chain ofΒ ₁-Bgt and phospholipase A₂ (PLA₂) was assessed by fluorescence measurement. Acrylamide quenching studies showed that the exposure degree of the Trp in PLA₂ is higher than the Trp inΒ ₁-Bgt. The Trp ofΒ ₁-Bgt had a higher accessibility for iodide, reflecting that the basic nature of the B chain might exert an attractive electrostatic force for iodide and increase the susceptibility of Trp in the A chain to iodide. Removal of the B chain ofΒ ₁-Bgt did not significantly affect the exposure degree of Trp in the A chain. Alternatively, the polarity of the environment around the Trp and the hydrophobic character of ANS and substrate binding sites in the separated A chain changed. Measurement of Trp fluorescence with increasing temperature showed that the stability of structure ofΒ ₁-Bgt was higher than those of the separated A chain and PLA₂. These results suggest that the B chain might interact with the A chain and stabilize the conformation of the A chain inΒ ₁-Bgt.     

Depletion in LpA-I:A-II particles enhances HDL-mediated endothelial protection in familial LCAT deficiency

The aim of this study was to evaluate the vasoprotective effects of HDL isolated from carriers of LCAT deficiency, which are characterized by a selective depletion of LpA-I:A-II particles and predominance of preβ migrating HDL. HDLs were isolated from LCAT-deficient carriers and tested in vitro for their capacity to promote NO production and to inhibit vascular cell adhesion molecule-1 (VCAM-1) expression in cultured endothelial cells. HDLs from carriers were more effective than control HDLs in promoting eNOS activation with a gene-dose-dependent effect (P_Trend = 0.048). As a consequence, NO production induced by HDL from carriers was significantly higher than that promoted by control HDL (1.63 ± 0.24-fold vs. 1.34 ± 0.07-fold, P= 0.031). HDLs from carriers were also more effective than control HDLs in inhibiting the expression of VCAM-1 (homozygotes, 65.0 ± 8.6%; heterozygotes, 53.1 ± 7.2%; controls, 44.4 ± 4.1%; P_Trend = 0.0003). The increased efficiency of carrier HDL was likely due to the depletion in LpA-I:A-II particles. The in vitro findings might explain why carriers of LCAT deficiency showed flow-mediated vasodilation and plasma-soluble cell adhesion molecule concentrations comparable to controls, despite low HDL-cholesterol levels. These results indicate that selective depletion of apoA-II-containing HDL, as observed in carriers of LCAT deficiency, leads to an increased capacity of HDL to stimulate endothelial NO production, suggesting that changes in HDL apolipoprotein composition may be the target of therapeutic interventions designed to improve HDL functionality.     

The essentiality of His-47 and the N-terminal region for the binding of 8-anilinonaphthalene-1-sulfonate with Taiwan cobra phospholipase A2

The binding of the apolar fluorescent dye 8-anilinonaphthalene-1-sulfonate (ANS) toNaja naja atra phospholipase A₂ (PLA₂) as well as the enhancement of ANS fluorescence of the PLA₂-ANS complex decreased with increasing pH in a pH range from 3 to 9. These pH-dependent curves can be well interpreted as the perturbation of an ionizable group with pK value of 5.8, which was assigned as His-47 in the active site of PLA₂. The ionizable group with pK 5.8 was no longer observed after methylation of His-47, supporting the idea that thepH dependence of ANS binding arose from an electrostatic interaction between His-47 and the bound ANS. Removal of the N-terminal octapeptide of PLA₂ caused a precipitous drop in the capability of PLA₂ for binding with ANS and enhancing ANS fluorescence, reflecting that the integrity of the N-terminal region was essential for maintaining the hydrophobic character of the ANS-binding site. However, the nonpolarity of the ANS-binding site in the N-terminus-removed derivative was still partially retained at lowpH, but was completely lost at highpH. Evidently, the N-terminal region plays a more crucial role in ANS binding at highpH than at lowpH. These results indicate that hydrophobic interaction as well as electrostatic interaction are involved in the binding of ANS to PLA₂, and that the relative contributions of both interactions in ANS fluorescence enhancement may be different under differentpH.     

apoE3[K146N/R147W] acts as a dominant negative apoE form that prevents remnant clearance and inhibits the biogenesis of HDL

The K146N/R147W substitutions in apoE3 were described in patients with a dominant form of type III hyperlipoproteinemia. The effects of these mutations on the in vivo functions of apoE were studied by adenovirus-mediated gene transfer in different mouse models. Expression of the apoE3[K146N/R147W] mutant in apoE-deficient (apoE^−/−) or apoA-I-deficient (apoA-I^−/−)×apoE^−/− mice exacerbated the hypercholesterolemia and increased plasma apoE and triglyceride levels. In apoE^−/− mice, the apoE3[K146N/R147W] mutant displaced apoA-I from the VLDL/LDL/HDL region and caused the accumulation of discoidal apoE-containing HDL. The WT apoE3 cleared the cholesterol of apoE^−/− mice without induction of hypertriglyceridemia and promoted formation of spherical HDL. A unique property of the truncated apoE3[K146N/R147W]202 mutant, compared with similarly truncated apoE forms, is that it did not correct the hypercholesterolemia. The contribution of LPL and LCAT in the induction of the dyslipidemia was studied. Treatment of apoE^−/− mice with apoE3[K146N/R147W] and LPL corrected the hypertriglyceridemia, but did not prevent the formation of discoidal HDL. Treatment with LCAT corrected hypertriglyceridemia and generated spherical HDL. The combined data indicate that the K146N/R147W substitutions convert the full-length and the truncated apoE3[K146N/R147W] mutant into a dominant negative ligand that prevents receptor-mediated remnant clearance, exacerbates the dyslipidemia, and inhibits the biogenesis of HDL.     

PLA2 activity in Tetrahymena pyriformis. Effects of inhibitors and stimulators

Phospholipase A₂ (PLA₂) is an enzyme which participates in signalling mechanisms cleaving arachidonate from sn-2 position of glycerophospholipids. In this study we have verified the existence of a PLA₂-like activity in the free living protozoan, Tetrahymena pyriformis GL. This activity is Ca²⁺-independent, EDTA (10 mM) has no effect on its activity. Quinacrine (0.1 mM) and 4-bromophenacyl bromide (BPB; 0.1 mM) inhibited, melittin (20 μg/ml significantly stimulated the PLA₂ activity and the release of free arachidonic acid (AA) from 1-acyl 2-¹⁴C-arachidonyl-3-phosphatidylethanolamine substrate. Melittin stimulated PLA₂ hyperactivity is Ca²⁺-dependent. There was no considerable alteration in the PLA₂ activity by stimulation of the activity by tyrosine kinase (with vanadate, H₂0₂), phospholipase C (PLC) (with phorbol 12, 13-dibutyrate) or G-proteins (with NaF, AlF₄ thus in Tetrahymena PLA₂ activity seems to be independent of these—in Tetrahymena (also functioning)—signalling pathways. Treatment with quinacrine and BPB leads to decreased synthesis and disturbed breakdown of phospholipids and phosphoinositides. These findings suggest that PLA₂ activity is in connection with the phospholipid metabolism of Tetrahymena.     

Allosteric regulation by membranes and hydrophobic subsites in phospholipase A2 enzymes determine their substrate specificity

Lipids play critical roles in several major chronic diseases of our times, including those that involve inflammatory sequelae such as metabolic syndrome including obesity, insulin sensitivity, and cardiovascular diseases. However, defining the substrate specificity of enzymes of lipid metabolism is a challenging task. For example, phospholipase A₂ (PLA₂) enzymes constitute a superfamily of degradative, biosynthetic, and signaling enzymes that all act stereospecifically to hydrolyze and release the fatty acids of membrane phospholipids. This review focuses on how membranes interact allosterically with enzymes to regulate cell signaling and metabolic pathways leading to inflammation and other diseases. Our group has developed “substrate lipidomics” to quantify the substrate phospholipid specificity of each PLA₂ and coupled this with molecular dynamics simulations to reveal that enzyme specificity is linked to specific hydrophobic binding subsites for membrane phospholipid substrates. We have also defined unexpected headgroup and acyl chain specificity for each of the major human PLA₂ enzymes, which explains the observed specificity at a structural level. Finally, we discovered that a unique hydrophobic binding site—and not each enzyme’s catalytic residues or polar headgroup binding site—predominantly determines enzyme specificity. We also discuss how PLA₂s release specific fatty acids after allosteric enzyme association with membranes and extraction of the phospholipid substrate, which can be blocked by stereospecific inhibitors. After decades of work, we can now correlate PLA₂ specificity and inhibition potency with molecular structure and physiological function.     

Structural and phylogenetic basis for the classification of group III phospholipase A2

Secretory phospholipase A₂ (PLA₂) catalyses the hydrolysis of the sn-2 position of glycerophospholipids to liberate arachidonic acid, a precursor of eicosanoids, that are known mediators of inflammation. The group III PLA₂ enzymes are present in a wide array of organisms across many species with completely different functions. A detailed understanding of the structure and evolutionary proximity amongst the enzymes was carried out for a meaningful classification of this group. Fifty protein sequences from different species of the group were considered for a detailed sequence, structural and phylogenetic studies. In addition to the conservation of calcium binding motif and the catalytic histidine, the sequences exhibit specific ‘amino acid signatures’. Structural analysis reveals that these enzymes have a conserved globular structure with species specific variations seen at the active site, calcium binding loop, hydrophobic channel, the C-terminal domain and the quaternary conformational state. Character and distance based phylogenetic analysis of these sequences are in accordance with the structural features. The outcomes of the structural and phylogenetic analysis lays a convincing platform for the classification the group III PLA₂s into (1A) venomous insects; (IB) non-venomous insects; (II) mammals; (IIIA) gila monsters; (IIIB) reptiles, amphibians, fishes, sea anemones and liver fluke, and (IV) scorpions. This classification also helps to understand structure-function relationship, enzyme-substrate specificity and designing of potent inhibitors against the drug target isoforms.