Dragomir I. Draganov

Human Serum Paraoxonases (PON1) Q and R Selectively Decrease Lipid Peroxides in Human Coronary and Carotid Atherosclerotic Lesions PON1 Esterase and Peroxidase-Like Activities

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.

Rabbit serum paraoxonase 3 (PON3) is a high density lipoprotein-associated lactonase and protects low density lipoprotein against oxidation.

The paraoxonase gene family contains at least three members: PON1, PON2, andPON3. The physiological roles of the corresponding gene products are still uncertain. Until recently, only the serum paraoxonase/arylesterase (PON1) had been purified and characterized. Here we report the purification, cloning, and characterization of rabbit serum PON3. PON3 is a 40-kDa protein associated with the high density lipoprotein fraction of serum. In contrast to PON1, PON3 has very limited arylesterase and no paraoxonase activities but rapidly hydrolyzes lactones such as statin prodrugs (e.g.lovastatin). These differences facilitated the complete separation of PON3 from PON1 during purification. PON3 hydrolyzes aromatic lactones and 5- or 6-member ring lactones with aliphatic substituents but not simple lactones or those with polar substituents. We cloned PON3 from total rabbit liver RNA and expressed it in mammalian 293T/17 cells. The recombinant PON3 has the same apparent molecular mass and substrate specificity as the enzyme purified from serum. Rabbit serum PON3 is more efficient than rabbit PON1 in protecting low density lipoprotein from copper-induced oxidation. This is the first report that identifies a second PON enzyme in mammalian serum and the first to describe an enzymatic activity for PON3.

Mouse macrophage paraoxonase 2 activity is increased whereas cellular paraoxonase 3 activity is decreased under oxidative stress.

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.

Lactonase and lactonizing activities of human serum paraoxonase (PON1) and rabbit serum PON3

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.

Dominant Role of Paraoxonases in Inactivation of the Pseudomonas aeruginosa Quorum-Sensing Signal N-(3-Oxododecanoyl)-L-Homoserine Lactone

ABSTRACT 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. aeruginosas 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 ≫ PON1192R > PON1192Q > PON3. PON2 exhibited a high specific activity of 7.6 ± 0.4 μmols/min/mg at 10 μM 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.

Development of an immunoblot assay with infrared fluorescence to quantify paraoxonase 1 in serum and plasma

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 ∼45 kDa and two minor bands of ∼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.

Paraoxonase-1 reduces monocyte chemotaxis and adhesion to endothelial cells due to oxidation of palmitoyl, linoleoyl glycerophosphorylcholine

OBJECTIVE High-density lipoprotein (HDL) is postulated to protect against the development of atherosclerosis, in part, by inhibiting the oxidation of low density lipoprotein (LDL) in the sub-endothelial space and thus inhibiting activation of the endothelium. The HDL-associated enzyme, paraoxonase-1, is proposed to be a major protective factor. However, HDL is also prone to oxidation when exposed to peroxynitrite and may therefore, once oxidized, have properties similar to oxidized LDL. METHODS AND RESULTS We exposed human HDL to the peroxynitrite donor 3-morpholinosydnonimine and incubated oxidized HDL with human umbilical vein endothelial cells (HUVECs). Oxidized HDL increased monocyte binding (P<0.001) and enhanced chemotaxis (P<0.001). The major oxidized phospholipids were 1-palmitoyl (stearoyl)-2-[9-oxo]nanoyl(azelaoyl)-sn-glycero-phosphocholine, derived from linoleate-containing phosphatidylcholines, and 1-palmitoyl(stearoyl)-2-[5-oxo]valeroyl(glutaroyl)-sn-glycero-phosphocholine, derived from arachidonate-containing phosphatidylcholines. Incubation of HUVECs with synthetically prepared 1-palmitoyl-2-[9-oxo]nanoyl(azelaoyl)-sn-glycero-phosphocholine, or 1-palmitoyl-2-[5-oxo]valeroyl(glutaroyl)-sn-glycero-phosphocholine increased binding of monocytes (P<0.001) and chemotaxis (P<0.001). Purified paraoxonase-1 reduced monocyte adhesion and chemotaxis (P<0.001). CONCLUSIONS (i) HDL can be a source of oxidatively-derived bioactive phospholipids; (ii) the fragmented phospholipids with a 9-carbon aldehyde or acid are as effective as a 5-carbon aldehyde or acid at inducing monocyte adhesion and chemotaxis; and (iii) paraoxonase-1 is effective at reducing the activity of these phospholipid oxidation products.

Paraoxonase-1 and clopidogrel efficacy

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PON1 and oxidative stress in human sepsis and an animal model of sepsis

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.

Rabbits possess a serum paraoxonase polymorphism similar to the human Q192R.

Serum paraoxonase (PON1) is a high-density lipoprotein (HDL)-associated enzyme that hydrolyses aromatic esters, organophosphates and lactones and can protect low-density lipoprotein (LDL) against oxidation. These properties are influenced by a well-characterized polymorphism (Q192R) in human PON1. We now report the identification and characterization of a phenotypically similar, but genetically distinct polymorphism in rabbit PON1. This polymorphism in rabbits was detected by phenotyping sera obtained from 16 inbred rabbit strains and 20 outbred New Zealand White rabbits by paraoxonase/arylesterase activity. The genetic basis of the rabbit polymorphism was determined by DNA sequencing and found to reside in a region distinct from the human Q192R and M55L polymorphisms. Three variant nucleotides within exon 4 (corresponding to P82S, K93E and S1O1G) were found to segregate with the observed rabbit PON1 phenotypes (rPON1A and rPON1B). The rPON1A and rPON1B proteins were purified and compared to the two human isoforms (192Q and 192R). The human and rabbit PON1s displayed similar characteristics with respect to physical properties and substrate specificity. However, rPON1A and rPON1B hydrolysed a variety of substrates at different rates. The rPON1A was also at least three-fold more efficient at protecting LDL from oxidation than rPON1B. Our characterization of a rabbit PON1 polymorphism provides useful insights into important functional residues in PON1. In addition, due to the observed similarities between the rabbit and human polymorphisms, the rabbit may serve as a good model to examine the effect of human PON1 polymorphisms in disease development.