Pharmacogenetics of paraoxonases: a brief review.

The human paraoxonase (PON) gene family consists of three members, PON1, PON2, and PON3, aligned next to each other on chromosome 7. By far the most-studied member of the family is the serum paraoxonase 1 (PON1), a high-density lipoprotein-associated esterase/lactonase. Early research focused on its capability to hydrolyze toxic organophosphates, and its name derives from one of its most commonly used in vitro substrates, paraoxon. Studies in the last 2 decades have demonstrated PON1's ability to protect against atherosclerosis by hydrolyzing specific derivatives of oxidized cholesterol and/or phospholipids in oxidized low-density lipoprotein and in atherosclerotic lesions. Levels and genetic variability of PON1 influence sensitivity to specific insecticides and nerve agents, as well as the risk of cardiovascular disease. More recently, the other two members of the PON family, PON2 and PON3, have also been shown to have antioxidant properties. A major goal in present research on the paraoxonases is to identify their natural substrates and to elucidate the mechanism(s) of their catalytic activities.

Baculovirus-mediated expression and purification of human serum paraoxonase 1A.

Human paraoxonase 1 (hPON1) is a lipid-associated enzyme transported on HDL. There is considerable interest in hPON1 because of its putative antioxidative/antiatherogenic properties. We have created a recombinant baculovirus (BV) to generate hPON1A in large quantities for structure-function studies and here describe the method for production and isolation of the enzyme. A high level of recombinant hPON1 type A (rPON1A) was produced by Hi-5 insect cells (40 mg/l); a fraction ( approximately 10 mg/l) was secreted into the cell culture medium, but the majority ( approximately 30 mg/l) remained associated with the host insect cells. Cell-associated rPON1A was purified by detergent extraction (Tergitol NP-10) followed by three simple chromatography steps (DEAE-Sepharose, Sephacryl S-200, and concanavalin A). The purified enzyme bound to concanavalin A and was converted to a lower molecular mass by endoglycosidase H digestion, suggesting that rPON1A contained high-mannose N-glycan chains. There was a significant decrease in arylesterase activity (>99%) concomitant with enzymatic deglycosylation. rPON1A was dependent on Ca(2+) for arylesterase activity, exhibiting kinetic parameters similar to native hPON1A (K(m) = 3.8 +/- 2.1 vs. 3.7 +/- 2.0 mM and V(max) = 1,305 +/- 668 vs. 1,361 +/- 591 U/mg protein, rPON1A and hPON1A, respectively). Both rPON1A and hPON1A efficiently inhibited lipoxygenase-mediated peroxidation of phospholipid. In contrast to the arylesterase activity, which was sensitive to endoglycosidase H treatment, enzymatic deglycosylation did not inhibit the antioxidant activity of rPON1A. In conclusion, our BV-mediated PON1A expression system appears ideally suited for the production of relatively large quantities of rPON1A for structure-function studies.

Apolipoprotein A-I promotes the formation of phosphatidylcholine core aldehydes that are hydrolyzed by paraoxonase (PON-1) during high density lipoprotein oxidation with a peroxynitrite donor.

High density lipoprotein (HDL) is rich in polyunsaturated phospholipids that are sensitive to oxidation. However, the effect of apolipoprotein A-I and paraoxonase-1 (PON-1) on phosphatidylcholine oxidation products has not been identified. We subjected native HDL, trypsinized HDL, and HDL lipid suspensions to oxidation by the peroxynitrite donor, 3-morpholinosydnonimine. HDL had a basal level of phosphatidylcholine mono- and di-hydroperoxides that increased to a greater extent in HDL, compared with either trypsinized HDL or HDL lipid alone. Phosphatidylcholine core aldehydes, which were present in small amounts, increased 10-fold during oxidation of native HDL, compared with trypsinized HDL (p = 0.004), and 4-fold compared with HDL lipid suspensions (p = 0.0021). In addition, the content of lysophosphatidylcholine increased 300% during oxidation of native HDL, but only 80 and 25%, respectively, during oxidation of trypsinized HDL and HDL lipid suspensions. Phosphatidylcholine isoprostanes accumulated in comparable amounts during the oxidation of all three preparations. Incubation of apolipoprotein A-I with 1-palmitoyl-2-linoleoyl glycerophosphocholine proteoliposomes in the presence of 3-morpholinosydnonimine or apoAI with phosphatidylcholine hydroperoxides resulted in a significant increase in phosphatidylcholine core aldehydes with no formation of lysophosphatidylcholine. We propose that apolipoprotein A-I catalyzes a one-electron oxidation of alkoxyl radicals. Purified PON-1 hydrolyzed phosphatidylcholine core aldehydes to lysophosphatidylcholine. We conclude that, upon HDL oxidation with peroxynitrite, apolipoprotein AI increases the formation of phosphatidylcholine core aldehydes that are subsequently hydrolyzed by PON1.

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.

Serum paraoxonase (PON1) isozymes: the quantitative analysis of isozymes affecting individual sensitivity to environmental chemicals.

In a recent study on Gulf War veterans who developed delayed neurotoxicity symptoms, we found their levels of serum paraoxonase (PON1) isozyme type Q to be significantly lower than in the control, unaffected veteran group. These results were obtained in 25 ill veterans and 20 well control subjects, of which 10 were deployed and 10 were nondeployed battalion members who remained in the United States during the Gulf War. The blood samples were also assayed for serum butyrylcholinesterase in our laboratory, and more recently in Dr. C. Broomfield's laboratory for somanase and sarinase activities. The cholinesterase activities showed no significant correlation with the PON1 isozyme levels or the severity of the clinical symptoms, but the somanase and sarinase levels ran parallel to the PON1 type Q isozyme concentrations. Although there is no direct evidence that these Gulf War veterans were directly exposed to or encountered either of these nerve gases, they may have been exposed to some environmental or chemical toxin with a similar preference for hydrolysis by the PON1 type Q isozyme. The number of subjects is relatively small, but the results should encourage other investigators to examine both the individual phenotypes and the levels of PON1 isozymes in other groups exhibiting neurological symptoms.

Human serum paraoxonase (PON1) isozymes Q and R hydrolyze lactones and cyclic carbonate esters.

It is well established that human serum paraoxonase (PON1) catalyzes the hydrolysis of organophosphate insecticides and nerve agents, as well as that of a number of aromatic carboxylic acid esters. Our laboratory has recently found a new class of PON1 substrates that includes at least 30 lactones and cyclic carbonate esters. The lactone substrates vary in their ring size from 4 to 7 atoms. Substituents on the ring carbons may enhance or reduce the rate of lactone hydrolysis. An appreciable degree of stereospecificity exists with some activities differing up to 9-fold between enantiomers (i.e., S-alpha-hydroxy-gamma-butyrolactone is hydrolyzed 5 to 9 times faster than the R form). Thiolactones are hydrolyzed less efficiently, and some lactams are potent inhibitors. Four lactone-containing drugs-spironolactone, mevastatin, simvastatin, and lovastatin-have been identified as substrates for PON1. All lactone substrates are hydrolyzed by both the Q and R isozymes of human serum PON1. However, some lactone substrates are hydrolyzed faster by the Q than R isozyme, whereas others show a reverse preference. Moreover, these new substrates include homogentisic acid lactone, mevalonic acid lactone, homocysteine thiolactone, and gamma-hydroxybutyric acid lactone-all lactone forms of endogenous compounds. It is reasonable to expect that further investigations may uncover PON1 lactone substrates that are, themselves, endogenous compounds. In this article we characterize the basic enzymatic properties of PON1's newly identified hydrolytic activities with lactone and cyclic carbonate ester substrates and compare these properties with those of representative arylesters and organophosphates.

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, and PON3. 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.

Human serum Paraoxonase/Arylesterase's retained hydrophobic N-terminal leader sequence associates with HDLs by binding phospholipids : apolipoprotein A-I stabilizes activity.

In serum, human paraoxonase/arylesterase (PON1) is found exclusively associated with high density lipoprotein (HDL) and contributes to its antiatherogenic properties by inhibiting low density lipoprotein (LDL) oxidation. Difficulties in purifying PON1 from apolipoprotein A-I (apoA-I) suggested that PON1's association with HDL may occur through a direct binding between these 2 proteins. An unusual property of PON1 is that the mature protein retains its hydrophobic N-terminal signal sequence. By expressing in vitro a mutant PON1 with a cleavable N-terminus, we demonstrate that PON1 associates with lipoproteins through its N-terminus by binding phospholipids directly rather than binding apoA-I. Nonetheless, apoA-I stabilized arylesterase activity more than did phospholipid alone, apoA-II, or apoE. Consequently, we studied the role of apoA-I in PON1 expression and HDL association in mice genetically deficient in apoA-I. Though present in HDL fractions at decreased levels, PON1 arylesterase activity was less stable than in control mice. Furthermore, PON1 could be competitively removed from HDL by phospholipids, suggesting that PON1's retained N-terminal peptide allows transfer of the enzyme between phospholipid surfaces. Thus, our data suggest that PON1 is stabilized by apoA-I, and its binding to HDL and physiological distribution are dependent on the direct binding of the retained hydrophobic N-terminus to phospholipids optimally presented in association with apoA-I.

Evidence that several conserved histidine residues are required for hydrolytic activity of human paraoxonase/arylesterase.

Recent evidence has been acquired that implicates an important role for several histidine residues in the hydrolytic mechanisms of human paraoxonase/arylesterase (PON1). Following titration with diethylpyrocarbonate (DEPC), both human serum and recombinant human type Q PON1 were inhibited in respect to their hydrolytic activity in a dose-responsive manner. Human PON1 treated with varying concentrations lost hydrolytic activity, and with each histidine modified, there was an exponential drop in hydrolytic activity. The reaction was followed spectrophotometrically at 244 nm. Recombinant wild-type and C283A PON1 enzymes inhibited with DEPC and subsequently treated with hydroxylamine had partial restoration of activity. The C283A mutant lacks a free sulfhydryl group, indicating that its inactivation is due to histidine specific modification. The dose response and time course of inactivation as well as the extent of reactivation by hydroxylamine were similar for both the wild-type and mutant recombinant enzymes. Mutants of PON1 containing an asparagine substituted for each of several conserved histidine residues lost hydrolytic activity for each single substitution. The mutants of PON1 constructed and assayed for arylesterase activity were H114N, H133N, and H284N. Each single aminoacid substitution rendered the enzyme catalytically inactive. These two pieces of evidence implicate an important role for several histidine residues in the hydrolytic mechanism of PON1. Although it is unusual for a calcium dependent enzyme to require histidines for its catalytic activity, acquired data suggest such a circumstance.

Properties of the retained N-terminal hydrophobic leader sequence in human serum paraoxonase/arylesterase.

Human serum paraoxonase/arylesterase (PON1) is HDL-associated and appears to protect low density lipoproteins (LDL) from oxidation. Mature PON1 retains its N-terminal hydrophobic signal sequence, which may be needed for binding to HDL. By site-directed mutagenesis, we created a mutant PON1 (A19A20) with a cleavable N-terminus to determine if this peptide mediated binding to lipoproteins. As a model system, we studied binding of mutant and wild type PON1s to lipoproteins in fetal bovine serum-containing expression medium and found that the wild type recombinant enzyme associated with lipoproteins whereas the A19A20 mutant did not. These results show that the N-terminus is required for binding to either apolipoproteins or phospholipids. Furthermore, we showed that wild type enzyme can bind to phospholipids directly without apolipoproteins. To determine if lipid binding is a requirement for PON1's protection against LDL oxidation, we used a copper ion-induced oxidation system and found that the wild type enzyme and A19A20 mutant showed similar reductions in both peroxide and aldehyde formation. We conclude that PON1 depends upon its N-terminal hydrophobic peptide for its association with serum lipoproteins.

Characterization of a soluble mouse liver enzyme capable of hydrolyzing diisopropyl phosphorofluoridate.

A novel mouse liver soluble fraction DFPase which has organophosphatase activities with sarin, soman and tabun, was purified and characterized. However, it lacks paraoxonase and arylesterase activities with paraoxon and phenyl acetate, respectively. This DFPase closely resembles and may be identical with the one purified by Little et al. in 1989 from the soluble fraction of rat liver, based on its substrate specificity, size (approximately 39 kDa) and its stimulation by several metal ions, namely magnesium, manganese and cobalt. Sequencing of our purified mouse liver DFPase showed it to be identical in its amino acid sequence with the recently identified senescence marker protein-30 (SMP-30) by Fujita et al. in 1996. Other senescence marker proteins possessing high structural homology with the mouse SMP-30 have also been found and sequenced from human and rat livers. There is no structural homology between the senescence marker protein family and the group of mammalian paraoxonases. Thus, it is clear that there are at least two distinct, unrelated families of mammalian liver enzymes that share DFPase activity.

On the physiological role(s) of the paraoxonases.

In recent years several lines of evidence have indicated that serum paraoxonase (PON1), and perhaps other mammalian paraoxonases, act as important guardians against cellular damage from toxic agents, such as organophosphates, oxidized lipids in the plasma low density lipoproteins (LDL), and against bacterial endotoxins. For some of these protective activities but not all, PON1 requires calcium ion. The catalyzed chemical reactions generally seem to be hydrolytic, but for some types of protection this may not be so. Several other metals have very high affinity for PON1 and may displace calcium. Replacement or substitution of calcium by other metals could extend the range of catalytic properties and the substrate specificity of the paraoxonases, as it does for the mammalian DFPases. Although this Third International Meeting on Esterases Reacting with Organophosphorus Compounds focuses on the organophosphatase activities of paraoxonase and related enzymes, it is important to also briefly review some of the current directions in several laboratories searching for additional functions of the paraoxonases to extend our understanding of the properties of this family of enzymes which now seem to have both physiological and toxicological importance.

Association of low PON1 type Q (type A) arylesterase activity with neurologic symptom complexes in Gulf War veterans.

Previously Haley et al. described six possible syndromes identified by factor analysis of symptoms in Gulf War veterans and demonstrated that veterans with these symptom complexes were more neurologically impaired than age-sex-education-matched well controls. They also uncovered strong associations (relative risks 4-8) suggesting that these symptom complexes were related to wartime exposure to combinations of organophosphate pesticides, chemical nerve agents, high concentration DEET insect repellant, and symptoms of advanced acute toxicity after taking pyridostigmine. Here we have shown that compared to controls, ill veterans with the neurologic symptom complexes were more likely to have the R allele (heterozygous QR or homozygous R) than to be homozygous Q for the paraoxonase/arylesterase 1 (PON1) gene. Moreover, low activity of the PON1 type Q (Gln192, formerly designated type A) arylesterase allozyme distinguished ill veterans from controls better than just the PON1 genotype or the activity levels of the type R (Arg192, formerly designated type B) arylesterase allozyme, total arylesterase, total paraoxonase, or butyrylcholinesterase. A history of advanced acute toxicity after taking pyridostigmine was also correlated with low PON1 type Q arylesterase activity. Type Q is the allozyme of paraoxonase/arylesterase that most efficiently hydrolyzes several organophosphates including sarin, soman, and diazinon. These findings further support the proposal that neurologic symptoms in some Gulf War veterans were caused by environmental chemical exposures.

High density associated enzymes: their role in vascular biology.

Enzymes associated with circulating HDL include lecithin: cholesterol acyl transferase, phospholipid transfer protein, cholesterol ester transfer protein, paraoxonase 1 and platelet activating factor acetylhydrolase. Together with lipoprotein lipase and hepatic lipase these enzymes produce important lipoprotein remodeling and modulate their structure and function and therefore their role in artery wall metabolism.

Calcium binding by human and rabbit serum paraoxonases. Structural stability and enzymatic activity.

Equilibrium dialysis and Scatchard plots were used to establish that human and rabbit paraoxonases both have two calcium binding sites. Independent-site and stepwise constant analyses were used to calculate a higher affinity site (Kd1) of 3.6 +/- 0.9 x 10(-7) M for human A paraoxonase, and 1.4 +/- 0.5 x 10(-8) M for rabbit paraoxonase, and a lower affinity site (Kd2) of 6.6 +/- 1.2 x 10(-6) M for human A paraoxonase, and 5.3 +/- 0.94 x 10(-6) M for rabbit paraoxonase. In both species, the higher affinity sites were found to be essential to maintain hydrolytic activity; complete removal of calcium led to irreversible inactivation. The lower affinity sites were required for catalytic activity, and their binding of calcium was reversible. Experimentally estimated values of Kd2 based on the concentration of calcium required to obtain half the maximum enzymatic activity were 3 microM for human A and B paraoxonases, and also in the order of 3 microM for rabbit paraoxonase, using three different substrates. Calcium was the only metal found that protects against denaturation and also confers hydrolytic activity with these two mammalian paraoxonases.

Paraoxonase inhibits high-density lipoprotein oxidation and preserves its functions. A possible peroxidative role for paraoxonase.

HDL levels are inversely related to the risk of developing atherosclerosis. In serum, paraoxonase (PON) is associated with HDL, and was shown to inhibit LDL oxidation. Whether PON also protects HDL from oxidation is unknown, and was determined in the present study. In humans, we found serum HDL PON activity and HDL susceptibility to oxidation to be inversely correlated (r2 = 0.77, n = 15). Supplementing human HDL with purified PON inhibited copper-induced HDL oxidation in a concentration-dependent manner. Adding PON to HDL prolonged the oxidation lag phase and reduced HDL peroxide and aldehyde formation by up to 95%. This inhibitory effect was most pronounced when PON was added before oxidation initiation. When purified PON was added to whole serum, essentially all of it became HDL-associated. The PON-enriched HDL was more resistant to copper ion-induced oxidation than was control HDL. Compared with control HDL, HDL from PON-treated serum showed a 66% prolongation in the lag phase of its oxidation, and up to a 40% reduction in peroxide and aldehyde content. In contrast, in the presence of various PON inhibitors, HDL oxidation induced by either copper ions or by a free radical generating system was markedly enhanced. As PON inhibited HDL oxidation, two major functions of HDL were assessed: macrophage cholesterol efflux, and LDL protection from oxidation. Compared with oxidized untreated HDL, oxidized PON-treated HDL caused a 45% increase in cellular cholesterol efflux from J-774 A.1 macrophages. Both HDL-associated PON and purified PON were potent inhibitors of LDL oxidation. Searching for a possible mechanism for PON-induced inhibition of HDL oxidation revealed PON (2 paraoxonase U/ml)-mediated hydrolysis of lipid peroxides (by 19%) and of cholesteryl linoleate hydroperoxides (by 90%) in oxidized HDL. HDL-associated PON, as well as purified PON, were also able to substantially hydrolyze (up to 25%) hydrogen peroxide (H2O2), a major reactive oxygen species produced under oxidative stress during atherogenesis. Finally, we analyzed serum PON activity in the atherosclerotic apolipoprotein E-deficient mice during aging and development of atherosclerotic lesions. With age, serum lipid peroxidation and lesion size increased, whereas serum PON activity decreased. We thus conclude that HDL-associated PON possesses peroxidase-like activity that can contribute to the protective effect of PON against lipoprotein oxidation. The presence of PON in HDL may thus be a major contributor to the antiatherogenicity of this lipoprotein.