Author Report for: Draganov DI

Mammalian paraoxonases (PONs) are a unique, highly conserved family of calcium-dependent esterases consisting of PON1, PON2, and PON3. The PONs can hydrolyze the lactone ring of a range of N-acyl-L: -homoserine lactone (AHL) quorum sensing signaling molecules, rendering them inactive. This chapter describes a method that utilizes high-performance liquid chromatography analysis with UV detection for determining the rate of AHL hydrolysis in cell lysates, tissue homogenates, serum, and with purified proteins. Also described are the techniques used to prepare cell culture lysates and tissue homogenates for analysis and the use of class-specific enzyme inhibitors to determine the contribution of PONs to AHL hydrolysis in the samples.

Serum paraoxonase (PON1) is well recognized for its ability to hydrolyze arylesters, toxic oxon metabolites of organophosphate insecticides and nerve agents. PON1 is a member of gene family including also PON2 and PON3; however, the later two enzymes have very limited arylesterase and practically no organophosphatase activity. We have established that all three PONs are lactonases/lactonyzing enzymes with overlapping, but also distinct substrate specificity. Dihydrocoumarin (DHC), long chain fatty acid lactones and acylhomoserine lactones (AHLs) are hydrolyzed by all three PONs and likely represent their natural substrates. The 3D structure of PON1 is a six-bladed beta-propeller containing two Ca(2+) ions necessary for the enzyme stability and enzymatic activity. Senescence marker protein (SMP30), another putative six-bladed beta-propeller, hydrolyzes DFP, sarin and soman in the presence of Mg(2+) or Mn(2+). More recently, SMP30 was characterized as a gluconolactonase with a role in vitamin C metabolism. Bacterial phosphotriesterases (PTEs) are members of the amidohydrolase superfamily and differ in their structure from the eukaryotic organophosphatases; PTEs are (beta/alpha)(8) barrels with an active site containing two transition metal ions such as Co(2+), Mn(2+) or Zn(2+). PTE from Pseudomonas diminuta hydrolyzes paraoxon extremely efficiently; this enzyme was shown to hydrolyze also DHC and other lactones. At least 3 more bacterial lactonases, dubbed PTE-like lactonases (or PLL), have been identified to possess both lactonase and organophosphatase activities. Lactones are natural compounds, many of them with high biological activity, while organophosphates are human-made chemicals introduced in the 20th century. Thus, it is plausible that lactonase is the primary activity for which the enzymes discussed here evolved for, while the organophosphatase activity arose as a promiscuous activity during their evolution. Laboratory (directed) evolution studies provided mechanisms for their catalytic versatility and demonstrated experimentally the evolvability of promiscuous enzyme functions

Sepsis is the leading cause of death in critically ill patients. The pathophysiological mechanisms implicated in the development of sepsis and organ failure are complex and involve activation of systemic inflammatory response and coagulation together with endothelial dysfunction. Oxidative stress is a major promoter and mediator of the systemic inflammatory response. Serum PON1 has been demonstrated in multiple clinical and animal studies to protect against oxidative stress, but also to undergo inactivation upon that condition. We found decreased plasma PON1 activity in patients with sepsis compared to healthy controls or critically ill patients without sepsis; furthermore, in sepsis patients PON1 activity was lower and remained lower in the course of sepsis in the non-survivors compared to the survivors. Plasma PON1 activity was positively correlated with high-density lipoprotein cholesterol and negatively correlated with markers of lipid peroxidation. In an experimental animal model of sepsis, murine cecal ligation and puncture, the time course of plasma PON1 activity was very similar to that found in sepsis patients. Persistently low PON1 activity in plasma was associated with lethal outcome in human and murine sepsis.

Paraoxonase 1 (PON1) requires calcium for activity and is inactivated in the presence of EDTA. Because of this, studies to date have used serum or heparinized plasma for both activity and mass assays of PON1. Whole serum and EDTA plasma were analyzed by SDS-electrophoresis and Western blot using anti-PON1 monoclonal antibody 4C10. Because PON1 has one disulfide and one free cysteine residue, the samples were reduced with dithiothreitol before electrophoresis. Western blot identified a major PON1 band with a molecular mass of approximately 45 kDa and two minor bands of approximately 40 and 35 kDa in both serum and EDTA plasma. This established that PON1 is inactive, but structurally intact, in EDTA plasma and suggested that a mass assay could be developed based on SDS-electrophoresis and Western blot. Linearity was established for plasma and for a PON1 standard. Quantification was based on the major PON1 band at 45 kDa. The correlation between serum and plasma PON1 mass was 0.9553. The between-run variation was determined with a serum pool to be 7.8%. The mass of PON1 in serum was significantly correlated with arylesterase activity (r = 0.85). Thus, we have demonstrated the feasibility of measuring PON1 mass in either serum or EDTA plasma.

The pathogenic bacterium Pseudomonas aeruginosa causes serious infections in immunocompromised patients. N-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL) is a key component of P. aeruginosa's quorum-sensing system and regulates the expression of many virulence factors. 3OC12-HSL was previously shown to be hydrolytically inactivated by the paraoxonase (PON) family of calcium-dependent esterases, consisting of PON1, PON2, and PON3. Here we determined the specific activities of purified human PONs for 3OC12-HSL hydrolysis, including the common PON1 polymorphic forms, and found they were in the following order: PON2 >> PON1(192R) > PON1(192Q) > PON3. PON2 exhibited a high specific activity of 7.6 +/- 0.4 micromols/min/mg at 10 microM 3OC12-HSL, making it the best PON2 substrate identified to date. By use of class-specific inhibitors, approximately 85 and 95% of the 3OC12-HSL lactonase activity were attributable to PON1 in mouse and human sera, respectively. In mouse liver homogenates, the activity was metal dependent, with magnesium- and manganese-dependent lactonase activities comprising 10 to 15% of the calcium-dependent activity. In mouse lung homogenates, all of the activity was calcium dependent. The calcium-dependent activities were irreversibly inhibited by extended EDTA treatment, implicating PONs as the major enzymes inactivating 3OC12-HSL. In human HepG2 and EA.hy 926 cell lysates, the 3OC12-HSL lactonase activity closely paralleled the PON2 protein levels after PON2 knockdown by small interfering RNA treatment of the cells. These findings suggest that PONs, particularly PON2, could be an important mechanism by which 3OC12-HSL is inactivated in mammals.

Mammalian paraoxonases (PONs 1, 2 and 3) are a highly conserved family of esterases, with uncertain physiological functions and natural substrates. Here we characterize the ability of purified recombinant human PONs to hydrolyze estrogen esters, a class of compounds previously not known to be PON substrates. PONs hydrolyzed estrogen mono- and diesters at position 3 of the steroid A-ring. Diesters were better substrates for the PONs and were very efficiently hydrolyzed, particularly by PON3. Esters at position 17 were not cleaved by the PONs unless an adjacent double bound was present. Purified human serum butyryl cholinesterase also hydrolyzed estrogen esters, however it preferably hydrolyzed the mono-esters. The ability of the PONs' to effectively hydrolyze a variety of estrogen esters provides further insight into the structure of their active sites and suggests that natural compounds with aromatic ester groups might be relevant substrates for the PONs.

The paraoxonase (PON) gene family in humans has three members, PON1, PON2, and PON3. Their physiological role(s) and natural substrates are uncertain. We developed a baculovirus-mediated expression system, suitable for all three human PONs, and optimized procedures for their purification. The recombinant PONs are glycosylated with high-mannose-type sugars, which are important for protein stability but are not essential for their enzymatic activities. Enzymatic characterization of the purified PONs has revealed them to be lactonases/lactonizing enzymes, with some overlapping substrates (e.g., aromatic lactones), but also to have distinctive substrate specificities. All three PONs metabolized very efficiently 5-hydroxy-eicosatetraenoic acid 1,5-lactone and 4-hydroxy-docosahexaenoic acid, which are products of both enzymatic and nonenzymatic oxidation of arachidonic acid and docosahexaenoic acid, respectively, and may represent the PONs' endogenous substrates. Organophosphates are hydrolyzed almost exclusively by PON1, whereas bulky drug substrates such as lovastatin and spironolactone are hydrolyzed only by PON3. Of special interest is the ability of the human PONs, especially PON2, to hydrolyze and thereby inactivate N-acyl-homoserine lactones, which are quorum-sensing signals of pathogenic bacteria. None of the recombinant PONs protected low density lipoprotein against copper-induced oxidation in vitro.

Purified serum paraoxonase (PON1) had been shown to attenuate the oxidation of LDL in vitro. We critically reevaluated the antioxidant properties of serum PON1 in the in vitro assays initiated with copper or the free radical generator 2,2'-azobis-2-amidinopropane hydrochloride (AAPH). The antioxidant activity of different purified PON1 preparations did not correlate with their arylesterase (AE), lactonase, or phospholipase A2 activities or with the amounts of detergent or protein. Dialysis of three of these preparations resulted in a 30-40% loss of their AE activities but in a complete loss of their antioxidant activities. We also followed the distribution of the antioxidant activity during human serum PON1 purification by two purification methods. The antioxidant activity of the anion-exchange chromatography fractions did not copurify with PON1 using either method and could largely be accounted for by the "antioxidant" activity of the detergent present. In conclusion, using the copper or AAPH in vitro assays, no PON1-mediated antioxidant activity was detected, suggesting that the removal of PON1 from its natural environment may impair its antioxidative activity and that this assay with highly purified PON1 may be an inappropriate method with which to study the antioxidative properties of the enzyme.

OBJECTIVE: To determine whether paraoxonases (PONs) are expressed in macrophages and to analyze the oxidative stress effect on their expression and activities. METHODS AND
RESULTS: We demonstrated the presence (mRNA, protein, activity) of PON2 and PON3 but not PON1 in murine macrophages, whereas in human macrophages, only PON2 was expressed. Under oxidative stress as present in mouse peritoneal macrophages (MPMs) from apoE-deficient (E0) mice as well as in C57BL6 mice, MPMs that were incubated with buthionine sulfoximine, with angiotensin II, with 7-ketocholesterol, or with oxidized phosphatidylcholine, PON2 mRNA levels and lactonase activity toward dihydrocoumarin significantly increased (by 50% to 130%). In contrast, PON3 lactonase activity toward lovastatin was markedly reduced (by 29% to 57%) compared with control cells. The supplementation of E0 mice with dietary antioxidants (vitamin E, pomegranate juice) significantly increased macrophage PON3 activity (by 23% to 40%), suggesting that oxidative stress was the cause for the reduced macrophage PON3 activity. Incubation of purified PON2 or PON3 with E0 mice MPMs resulted in reduced cellular lipid peroxides content by 14% to 19% and inhibition of cell-mediated LDL oxidation by 32% to 39%.
CONCLUSIONS: Increased macrophage PON2 expression under oxidative stress could represent a selective cellular response to reduce oxidative burden, which may lead to attenuation of macrophage foam cell formation.

Human paraoxonase (PON1) was previously shown to hydrolyze over 30 different lactones (cyclic esters). In the present study purified human PON1 was found to catalyze the reverse reaction (lactonization) of a broad range of hydroxy acids. Hydroxy acid lactonization or lactone hydrolysis is catalyzed until equilibrium between the open and closed forms is reached. Lactonization by PON1 was calcium-dependent, had a pH optimum of 5.5-6 and could be stimulated with dilauroylphosphatidylcholine. Rabbit serum PON3 and a serine esterase in mouse plasma, presumably a carboxylesterase, also catalyzed hydroxy acid lactonization. Two endogenous oxidized unsaturated fatty acids, (+/-)4-hydroxy-5E,7Z,10Z,13Z,16Z,19Z-docosahexaenoic acid (4-HDoHE) and (+/-)5-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-HETE) lactone, were very efficiently lactonized and hydrolyzed, respectively, by PON1. Human and mouse plasma samples also catalyzed 4-HDoHE lactonization and 5-HETE lactone hydrolysis. Studies with the PON1 inhibitor EDTA and the serine esterase inhibitor phenylmethylsulfonylfluoride suggest that about 80-95% of both activities can be attributed to PON1 in the human samples. In the mouse sample, PON1 accounted for about 30% of the 4-HDoHE lactonizing activity and 72% of the 5-HETE lactonase activity. Our results demonstrate that PON1 can lactonize the hydroxy acid form of its lactone substrates and that reversible hydrolysis of lactones may be a property of lactonases that is not generally considered. Also, the high activity of PON1 towards 4-HDoHE and 5-HETE lactone suggests that oxidized eicosanoids and docosanoids may be important physiological substrates for PON1.

BACKGROUND: Human serum paraoxonase (PON1) exists in two polymorphic forms: one that differs in the amino acid at position 192 (glutamine and arginine, Q and R, respectively) and the second one that differs in the amino acid at position 55 (methionine and leucine, M and L, respectively). PON1 protects LDL from oxidation, and during LDL oxidation, PON1 is inactivated. METHODS AND
RESULTS: The present study compared PON1 isoforms Q and R for their effect on lipid peroxide content in human coronary and carotid lesions. After 24 hours of incubation with PON1Q or PON1R (10 arylesterase units/mL), lipid peroxides content in both coronary and carotid lesion homogenates (0.1 g/mL) was reduced up to 27% and 16%, respectively. The above incubation was associated with inactivation of PON1Q and PON1R by 15% and 45%, respectively. Lesion cholesteryl linoleate hydroperoxides and cholesteryl linoleate hydroxides were hydrolyzed by PON1 to yield linoleic acid hydroperoxides and linoleic acid hydroxides. Furthermore, lesion and pure linoleic acid hydroperoxides were reduced to yield linoleic acid hydroxides. These results thus indicate that PON1 demonstrates esterase-like and peroxidase-like activities. Recombinant PON1 mutants in which the PON1-free sulfhydryl group at cysteine-284 was replaced with either alanine or serine were no longer able to reduce lipid peroxide content in carotid lesions.
CONCLUSIONS: We conclude that PON1 may be antiatherogenic because it hydrolyzes lipid peroxides in human atherosclerotic lesions.