Estrogen esters as substrates for human paraoxonases.

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.

Human paraoxonases (PON1, PON2, and PON3) are lactonases with overlapping and distinct substrate specificities.

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 human serum PON1 does not protect LDL against oxidation in the in vitro assays initiated with copper or AAPH.

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.

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.

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.

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.

Current progress on esterases: from molecular structure to function.

This article reports on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the April 2001 Experimental Biology meeting. Current developments in molecular-based studies into the structure and function of cholinesterases, carboxylesterases, and paraoxonases are described. This article covers mechanisms of regulation of gene expression of the various esterases by developmental factors and xenobiotics, as well as the interplay between physiological and chemical regulation of enzyme activity.

Multiple substrates for paraoxonase-1 during oxidation of phosphatidylcholine by peroxynitrite.

Paraoxonase (PON-1) is a high-density lipoprotein (HDL)-bound enzyme with activity toward multiple substrates. It hydrolyzes organic phosphate and aromatic carboxylic acid esters. It also inhibits accumulation of oxidized phospholipids in plasma lipoproteins by a mechanism yet to be determined. Therefore, we subjected apolipoprotein A-I proteoliposomes containing either 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine or 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine to oxidation by a peroxynitrite generator, SIN-1, in the presence and absence of purified PON-1. PON-1 modified the proportion of oxidation products without affecting the overall extent of PC oxidation. However, in the presence of PON-1, phosphatidylcholine isoprostanes were hydrolyzed to lysophosphatidylcholine. In addition, PON-1 hydrolyzed the phosphatidylcholine core aldehydes 1-palmitoyl-2-(9-oxo)nonanoyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-(5-oxo)valeroyl-sn-glycero-3-phosphocholine to lysophosphatidylcholine. This hydrolysis was not affected by pefabloc, a serine esterase inhibitor. There was no detectable release of linoleate, arachidonate, or their hydroperoxy or hydroxy derivatives in the presence of PON-1. We conclude that PON-1 minimizes the accumulation of phosphatidylcholine oxidation products by the hydrolysis of phosphatidylcholine isoprostanes and core aldehydes to lysophosphatidylcholine with a serine esterase-independent mechanism.