Relationships between paraoxon and 2-coumaranone hydrolytic activities in sera genotyped for PON1 Q192R polymorphism☆
Introduction
Paraoxonase (serum paraoxonase/arylesterase, EC 3.1.1.2/EC 3.1.8.1; PON1) is a 45-kDa mammalian serum enzyme associated to high-density lipoproteins (HDL) [1], [2]. Paraoxonase 1 (PON1) belongs to the A-esterase class [3], that inhibits low density lipoprotein (LDL) oxidation [4] and requires calcium ions for both activity and stability. PON1 is a member of the paraoxonase gene family, that in humans and mice includes PON1, paraoxonase 2 (PON2) and paraoxonase 3 (PON3) [5]. Paraoxonase activity is only present in PON1 [6]. Human PON1 has two common polymorphic sites: one located at aminoacid 55 (L/M) [7], and another one at residue 192 (Q/R) [8], the latter significantly affecting the catalytic efficiency of PON1, in a substrate-dependent manner [8]. As a matter of fact PON1 hydrolyzes a broad range of substrates and has been traditionally described as paraoxonase/arylesterase. However, it recently became apparent that PON1 is in fact a lactonase with lipophylic lactones constituting its primary substrates. The metabolic conversion of homocysteine (Hcy) to its thiolactone and the reactivity of the latter compound toward proteins, leading to protein damage, have been considered contributing factors in atherosclerosis [9. Thus, it has been suggested that PON1 may act as a protecting agent that reduces protein homocysteinylation through its specific enzymatic activity that hydrolyzes homocysteine thiolactone (HTL) [9].
Substrate specificity studies have demonstrated that HTL is likely to be a natural substrate of this enzyme. However, PON1 can also hydrolyze non-natural substrates, such as phenyl acetate and the organophospate compound paraoxon [10]. Interestingly, the previously described phospholipase A2 (PLA2) activity of PON1, has been recently attributed to a platelet-activating factor-acetylhydrolase (PAF-AH), that co-purifies together with the PON1 enzyme from HDL [11]. These molecules are probably hydrolyzed at different sites of PON1, in fact this enzyme is likely to possess multiple binding sites for phenyl acetate or paraoxon [10]. In addition, it has been suggested that the latter substrates may be non-competitive inhibitors of thiolactonase activity. Moreover, it has been reported that purified PON1 can also hydrolyze other lactones and cyclic carbonate esters, depending on polymorphism. In particular, a lactone substrate, 2-coumaranone, has been shown to be hydrolyzed more efficiently by the Q than the R form of the enzyme, whereas the paraoxonase activity show a reverse preference [12].
It has been also reported that the inhibition of erythrocytes membrane oxidation [13] and of LDL peroxidation due to HDL could be related to the activity of PON1 [14]. Previous studies evaluated the clinical significance of Arg 192 (R) polymorphism of PON1 in subjects with elevated plasma HDL levels, showing that this allele is associated with a significantly increased risk of carotid atherosclerosis and/or lack of protection from HDL carrying this allelic variant [15]. These results have been associated to a reduced ability of the Arg 192 form of the enzyme to utilize lipid peroxides as substrate, implying that it exerts a less effective antioxidant activity on lipoproteins [16].
The aim of this work was i) to set-up a method for a direct evaluation in human serum of paraoxonase and arylesterase activity, and of 2-coumaranone hydrolysis; and ii) to establish a possible correlation with Q192R genotype polymorphism. The method used here to assess lactonase activity was applied to whole serum, and suggests the suitability of the analysis of 2-coumaranone hydrolysis and paraoxonase activity as markers related to Q/R polymorphism, and possibly to the risk of atherosclerosis.
Section snippets
Chemicals
Phenyl acetate, paraoxon, 2-coumaranone and all chemicals were of purest grade and purchased from Sigma-Aldrich (Milwaukee, WI).
Human sera
Human blood samples were obtained from 101 healthy volunteers (white subjects, age 43.52 ± 12.21) supplied from the Internal Medicine Department of University of Tor Vergata, Rome, Italy, as approved by the Institutional Human Investigation Review Board. Blood was collected in vacutainer tubes and allowed to clot for 2 h. Finally, sera were recovered after centrifugation
Results
One hundred and one human serum samples were analyzed with regard to arylesterase, paraoxonase and 2-coumarananone hydrolase activities. It has been demonstrated that the arylesterase activity of PON is unrelated to Q/R polymorphism and it is only positively correlated with PON1 concentration [19]. However, the PON1 concentration can be different in healthy subjects and individuals with angiographically verified coronary disease [19]. On this basis, we calculated both paraoxonase and lactonase
Discussion
Serum paraoxonase (PON1), the HDL-associated enzyme that inhibits LDL oxidation [21], has been shown to exhibit homocysteine thiolactonase activity, thus playing a role in the degradation of pro-atherogenic homocysteine metabolites [10]. It has been proposed that PON1 may provide protection against the risk of atherosclerosis [15], by reducing oxidized lipid levels. In particular, the polymorphisms in the PON1 (Q192R) gene have been associated with this risk, since the Q type PON1 is more
Acknowledgments
This work was supported by the MIUR program: “Study of biochemical and genetic determinants of cardiovascular risk in relation to difference in food habits” (to R.G-S.)
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Cited by (2)
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This paper is dedicated to Prof. Corradino Motti sorrowfully missed on May 1, 2001.