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.

Human serum paraoxonase (PON 1) is inactivated by oxidized low density lipoprotein and preserved by antioxidants.

Human serum paraoxonase (PON1) can protect low density lipoprotein (LDL) from oxidation induced by either copper ion or by the free radical generator azo bis amidinopropane hydrochloride (AAPH). During LDL oxidation in both of these systems, a time-dependent inactivation of PON arylesterase activity was observed. Oxidized LDL (Ox-LDL) produced by lipoprotein incubation with either copper ion or with AAPH, indeed inactivated PON arylesterase activity by up to 47% or 58%, respectively. Three possible mechanisms for PON inactivation during LDL oxidation were considered and investigated: copper ion binding to PON, free radical attack on PON, and/or the effect of lipoprotein-associated peroxides on the enzyme. As both residual copper ion and AAPH are present in the Ox-LDL preparations and could independently inactivate the enzyme, the effect of minimally oxidized (Ox-LDL produced by LDL storage in the air) on PON activity was also examined. Oxidized LDL, as well as oxidized palmitoyl arachidonoyl phosphatidylcholine (PAPC), lysophosphatidylcholine (LPC, which is produced during LDL oxidation by phospholipase A2-like activity), and oxidized cholesteryl arachidonate (Ox-CA), were all potent inactivators of PON arylesterase activity (PON activity was inhibited by 35%-61%). PON treatment with Ox-LDL (but not with native LDL), or with oxidized lipids, inhibited its arylesterase activity and also reduced the ability of the enzyme to protect LDL against oxidation. PON Arylesterase activity however was not inhibited when PON was pretreated with the sulfhydryl blocking agent, p-hydroxymercurybenzoate (PHMB). Similarly, on using recombinant PON in which the enzyme's only free sulfhydryl group at the position of cysteine-284 was mutated, no inactivation of the enzyme arylesterase activity by Ox-LDL could be shown. These results suggest that Ox-LDL inactivation of PON involves the interaction of oxidized lipids in Ox-LDL with the PON's free sulfhydryl group. Antioxidants such as the flavonoids glabridin or quercetin, when present during LDL oxidation in the presence of PON, reduced the amount of lipoprotein-associated lipid peroxides and preserved PON activities, including its ability to hydrolyze Ox-LDL cholesteryl linoleate hydroperoxides. We conclude that PON's ability to protect LDL against oxidation is accompanied by inactivation of the enzyme. PON inactivation results from an interaction between the enzyme free sulfhydryl group and oxidized lipids such as oxidized phospholipids, oxidized cholesteryl ester or lysophosphatidylcholine, which are formed during LDL oxidation. The action of antioxidants and PON on LDL during its oxidation can be of special benefit against atherosclerosis since these agents reduce the accumulation of Ox-LDL by a dual effect: i.e. prevention of its formation, and removal of Ox-LDL associated oxidized lipids which are generated during LDL oxidation.

Paraoxonase active site required for protection against LDL oxidation involves its free sulfhydryl group and is different from that required for its arylesterase/paraoxonase activities: selective action of human paraoxonase allozymes Q and R.

Human serum paraoxonase (PON 1) exists in 2 major polymorphic forms (Q and R), which differ in the amino acid at position 191 (glutamine and arginine, respectively). These PON allozymes hydrolyze organophosphates and aromatic esters, and both also protect LDL from copper ion-induced oxidation. We have compared purified serum PONs of both forms and evaluated their effects on LDL oxidation, in respect to their arylesterase/paraoxonase activities. Copper ion-induced LDL oxidation, measured by the production of peroxides and aldehydes after 4 hours of incubation, were reduced up to 61% and 58%, respectively, by PON Q, but only up to 46% and 38%, respectively, by an equivalent concentration of PON R. These phenomena were PON-concentration dependent. Recombinant PON Q and PON R demonstrated similar patterns to that shown for the purified serum allozymes. PON Q and PON R differences in protection of LDL against oxidation were further evaluated in the presence of glutathione peroxidase (GPx). GPx (0.1 U/mL) alone reduced copper ion-induced LDL oxidation by 20% after 4 hours of incubation. The addition of PON R to the above system resulted in an additive inhibitory effect on LDL oxidation, whereas PON Q had no such additive effect. The 2 PON allozymes also differed by their ability to inhibit initiation, as well as propagation, of LDL oxidation. PON Q was more efficient in blocking LDL oxidation if added when oxidation was initiated, whereas PON R was more potent when added 1 hour after the initiation of LDL oxidation. These data suggest that the 2 allozymes act on different substrates. Both PON allozymes were also able to reduce the oxidation of phospholipids and cholesteryl ester. PON Q arylesterase activity was reduced after 4 hours of LDL oxidation by only 28%, whereas the arylesterase activity of PON R was reduced by up to 55%. Inactivation of the calcium-dependent PON arylesterase activity by using the metal chelator EDTA, or by calcium ion removal on a Chelex column, did not alter PON's ability to inhibit LDL oxidation. However, blockage of the PON free sulfhydryl group at position 283 with p-hydroxymercuribenzoate inhibited both its arylesterase activity and its protection of LDL from oxidation. Recombinant PON mutants in which the PON free sulfhydryl group was replaced by either alanine or serine were no longer able to protect against LDL oxidation, even though they retained paraoxonase and arylesterase activities. Overall, these studies demonstrate that PON's arylesterase/paraoxonase activities and the protection against LDL oxidation do not involve the active site on the enzyme in exactly the same way, and PON's ability to protect LDL from oxidation requires the cysteine residue at position 283.