Paraoxonase protection of LDL against peroxidation is independent of its esterase activity towards paraoxon and is unaffected by the Q→R genetic polymorphism

Abstract

High density lipoprotein (HDL)-associated paraoxonase (PON) seems to play a major role in the protection of low density lipoprotein (LDL) against peroxidation by HDL, and the partly purified enzyme exerts a dose-dependent protective effect. A common polymorphism of the human gene (192 Q→R) modulates paraoxonase activity but purified enzyme from either genotype is equally effective against LDL peroxidation. The inhibition of Cu2+-induced LDL peroxidation by HDL was monitored by lipid peroxide assay and change in LDL electrophoretic mobility. We show that HDL from type 2 diabetic patients with the QQ or RR genotype (n = 12 for each) reduce, to the same extent, both peroxide production (by 60.6 ± 20.0 and 63.9 ± 23.5%) and relative change in mobility (61.3 ± 21.8 and 61.4 ± 26.5%) despite a 6-fold difference in paraoxonase activity (47.4 ± 4.4 vs. 299.7 ± 23.7 U/l, P < 0.0001). Protection was, however, related to paraoxonase activity, but with a different efficiency in each group corresponding to a better protection per unit of enzyme in the QQ genotype group. Inactivation of PON activity by heating (56°C, 10 min) or by EDTA was totally without effect on protection, which remained correlated with the paraoxonase activity measured prior to inactivation. In summary, these results suggest that the protein bearing both paraoxonase and arylesterase activities also possesses a third thermostable property, closely associated with paraoxon hydrolysis activity and unaffected by PON genetic variability.—Cao, H., A. Girard-Globa, F. Berthezene, and P. Moulin. Paraoxonase protection of LDL against peroxidation is independent of its esterase activity towards paraoxon and is unaffected by the Q→R genetic polymorphism. J. Lipid Res. 1999. 40: 133–139. High density lipoprotein (HDL)-associated paraoxonase (PON) seems to play a major role in the protection of low density lipoprotein (LDL) against peroxidation by HDL, and the partly purified enzyme exerts a dose-dependent protective effect. A common polymorphism of the human gene (192 Q→R) modulates paraoxonase activity but purified enzyme from either genotype is equally effective against LDL peroxidation. The inhibition of Cu2+-induced LDL peroxidation by HDL was monitored by lipid peroxide assay and change in LDL electrophoretic mobility. We show that HDL from type 2 diabetic patients with the QQ or RR genotype (n = 12 for each) reduce, to the same extent, both peroxide production (by 60.6 ± 20.0 and 63.9 ± 23.5%) and relative change in mobility (61.3 ± 21.8 and 61.4 ± 26.5%) despite a 6-fold difference in paraoxonase activity (47.4 ± 4.4 vs. 299.7 ± 23.7 U/l, P < 0.0001). Protection was, however, related to paraoxonase activity, but with a different efficiency in each group corresponding to a better protection per unit of enzyme in the QQ genotype group. Inactivation of PON activity by heating (56°C, 10 min) or by EDTA was totally without effect on protection, which remained correlated with the paraoxonase activity measured prior to inactivation. In summary, these results suggest that the protein bearing both paraoxonase and arylesterase activities also possesses a third thermostable property, closely associated with paraoxon hydrolysis activity and unaffected by PON genetic variability. —Cao, H., A. Girard-Globa, F. Berthezene, and P. Moulin. Paraoxonase protection of LDL against peroxidation is independent of its esterase activity towards paraoxon and is unaffected by the Q→R genetic polymorphism. J. Lipid Res. 1999. 40: 133–139. Oxidation of LDL is considered an efficient atherogenic process (1Steinberg D. 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Human and rabbit paraoxonase: purification, cloning, sequencing, mapping and role of polymorphism in organophosphate detoxification.Chem. Biol. Interact. 1993; 87: 35-48Google Scholar), an HDL-bound ester hydrolase to which is attributed a significant part of the protective properties displayed by HDL against LDL oxidation (9Mackness M.I. Arrol S. Durrington P.N. Paraoxonase prevents accumulation of lipoperoxide in low-density lipoprotein.FEBS. 1991; 286: 152-154Google Scholar, 10Mackness M.I. Arrol S. Abbott C. Durrington P.N. Protection of low-density lipoprotein against oxidative modification by high-density lipoprotein associated paraoxonase.Atherosclerosis. 1993; 104: 129-135Google Scholar, 11Parthasarathy S. Barnet J. Gong L.G. High-density lipoprotein inhibits the oxidative modification of low-density lipoprotein.Biochim. Biophys. Acta. 1990; 1044: 275-283Google Scholar, 12Suc I. Escargueil-Blanc I. Troly M. Salvayre R. Nègre-Salvayre A. HDL and apoA prevent cell death of endothelial cell induced by oxidized LDL.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 2158-2166Google Scholar). PON-containing HDL have proved capable of preventing the oxidative modification of LDL whether obtained by chemical interventions such as incubation in the presence of copper (9Mackness M.I. Arrol S. Durrington P.N. Paraoxonase prevents accumulation of lipoperoxide in low-density lipoprotein.FEBS. 1991; 286: 152-154Google Scholar, 10Mackness M.I. Arrol S. Abbott C. Durrington P.N. Protection of low-density lipoprotein against oxidative modification by high-density lipoprotein associated paraoxonase.Atherosclerosis. 1993; 104: 129-135Google Scholar) or by more biological means such as interaction with vascular cells (13Watson A.D. Berliner J.A. Hama S.Y. La Du B.N. Faull K.F. Fogelman A.M. Navab M. Protective effect of high density lipoprotein associated paraoxonase: inhibition of the biological activity of minimally oxidized low density lipoprotein.J. Clin. Invest. 1995; 96: 2882-2891Google Scholar, 14Van Lenten B.J. Hama S.Y. de Beer F.C. Stafforini D.M. Mclntyre T.M. Prescott S.M. La Du B.N. Fogelman A.M. Navab M. Anti-inflammatory HDL becomes pro-inflammatory during the acute phase response. Loss of protective effect of HDL against LDL oxidation in aortic wall cell cultures.J. Clin. Invest. 1995; 96: 2758-2767Google Scholar). Paraoxon hydrolysis activity (PON p.ase) varies widely among individuals. Part of this variability finds it source in the polymorphism of PON1 gene (15Humbert R. Adler D.A. Disteche C.M. Hassett C. Omiecinski C.J. Furlong C.E. The molecular basis of the human serum paraoxonase activity polymorphism.Nature Genet. 1993; 3: 73-76Google Scholar). A Q→R substitution at position 192 is determinant for activity, the R allele coding for a protein that displays several-fold higher activity towards paraoxon hydrolysis than the Q allele (16Adkins S. Gan K.N. Mody M. La Du B.N. Molecular basis for the polymorphic forms of human serum paraoxonase/arylesterase: glutamine or arginine at position 191, for the respective A or B allozymes.Am. J. Hum. Genet. 1993; 52: 598-608Google Scholar, 17Nevin D.N. Zambon A. Furlong C.E. Richter R.J. Humbert R. Hokanson J.E. Brunzell J.D. Paraoxonase genotypes, lipoprotein lipase activity, and HDL.Arterioscler. Thromb. Vasc. Biol. 1996; 16: 1243-1249Google Scholar). An M→L substitution at position 55 has a lesser effect on activity, while a strong linkage disequilibrium exists between L-55 and A-192 (18Blatter Garin M.C. James R.W. Dussoix P. Blanché H. Passa P. Froguel P. Ruiz J. Paraoxonase polymorphism Met-Leu 54 is associated with modified serum concentrations of the enzyme. A possible link between the paraoxonase gene and increased cardiovascular risk in diabetes.J. Clin. Invest. 1997; 99: 62-66Google Scholar). Arylesterase activity borne by the same protein (PON ar.ase) is not affected by either polymorphism and can be considered an index of actual protein concentrations, independent of PON1 variability. If, indeed, protection by PON of LDL against oxidation is a meaningful phenomenon, then the R allele should confer increased cardiovascular protection compared to Q. Recent studies performed on the basis of this hypothesis have, on the contrary, revealed that the RR genotype was more prevalent among subjects with a history of cardiovascular disease than in controls (19Ruiz J. Blanché H. James R.W. Garin M-C.S. Vaisse C. Charpentier G. Cohen N. Morabia A. Passa P. Froguel P. Gln-Arg 192 polymorphism of paraoxonase and coronary heart disease in type 2 diabetes.Lancet. 1995; 346: 869-872Abstract Google Scholar, 20Serrato S. Marian A.J. A variant of human paraoxonase/arylesterase (HUMPONA) gene is a risk factor for coronary artery disease.J. Clin. Invest. 1995; 96: 3005-3008Google Scholar, 21Sanghera D.K. Saha N. Aston C.E. Kamboh M.I. Genetic polymorphism of paraoxonase and the risk of coronary heart disease.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1067-1073Google Scholar, 22Odawara M. Tachi Y. Yamashita K. Paraoxonase polymorphism (Gln 192-Arg) is associated with coronary heart disease in Japanese non-insulin-dependent diabetes mellitus.J. Clin. Endocrinol. Metab. 1997; 82: 2257-2260Google Scholar). These results were, however, quite controversial (23Antikainen M Murtomäki S. Syvänne M. Pahlman R. Tahvanainen E. Jauhianen M. Frick M.H. Ehnholm C. The Gln-Arg 191 polymorphism of the human paraoxonase gene (HUMPONA) is not associated with the risk of coronary artery disease in Finns.J. Clin. Invest. 1996; 98: 883-885Google Scholar, 24Herrmann S.M. Blanc H. Poirier O. Arveiler D. Luc G. Evans A. Marques-Vidal P. Bard J.M. Cambien F. The Gln/Arg polymorphism of human paraoxonase (PON 192) is not related to myocardial infarction in the ECTIM study.Atherosclerosis. 1996; 126: 299-303Google Scholar, 25Suehiro T. Y. M. K. H. N. K. Paraoxonase gene polymorphism in Japanese subjects with coronary heart J. 1996; Scholar) and have, that this polymorphism of PON1 has effect on the vascular of diabetic patients as by a A. A. S. P. S. F. P. of between carotid and paraoxonase gene polymorphism in non-insulin-dependent diabetes Scholar). a of whether PON is at in protection of LDL against whether PON activity as measured by paraoxon hydrolysis the of the and whether the effect not be that of other HDL The seems to be that PON is as purified of the protein capable of oxidized a of oxidized LDL (13Watson A.D. Berliner J.A. Hama S.Y. La Du B.N. Faull K.F. Fogelman A.M. Navab M. Protective effect of high density lipoprotein associated paraoxonase: inhibition of the biological activity of minimally oxidized low density lipoprotein.J. Clin. Invest. 1995; 96: 2882-2891Google Scholar) and of HDL against oxidation M. M. La Du B.N. Paraoxonase inhibits high-density lipoprotein oxidation and its Clin. Invest. Scholar). however, equally efficient despite in PON it is not that an difference the enzyme is associated with HDL or it is on A recent has a more protective effect of HDL bearing the low PON Q type protein during oxidative incubation of LDL M.I. Arrol S. B. Durrington P.N. of paraoxonase and of high-density lipoproteins in low-density lipoprotein against lipid 1997; Scholar, B. M.I. Arrol S. W. Durrington P.N. of the human serum paraoxonase 55 and 192 genetic on the protection by high density lipoprotein against low density lipoprotein oxidative Scholar). has as of a protective for the Q despite its 192 displayed protective properties towards oxidation of LDL incubation during which of was in the of the in this of by HDL, of of the The relative to the actual of PON1 genetic polymorphism on oxidative mechanisms have to this of to cardiovascular to low HDL more than to high type 2 to be affected by protection against We have HDL from type 2 diabetic patients for the 192 polymorphism and protective on both and of PON by subjects for this from a of patients on the basis of PON1 QQ and The was by was obtained from The patients the of a controlled type 2 and significant between QQ and RR for of the and in of and for and with of in of the ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± in a an in for HDL and of enzyme activity, on EDTA for LDL and from PON1 was by the substitution corresponding to position 192 an (15Humbert R. Adler D.A. Disteche C.M. Hassett C. Omiecinski C.J. Furlong C.E. The molecular basis of the human serum paraoxonase activity polymorphism.Nature Genet. 1993; 3: 73-76Google Scholar). A the the was by at for at for and at for products with by and by Q a and allele R and Paraoxon hydrolysis (PON p.ase) and arylesterase (PON ar.ase) activities measured to Gan K.N. A. La Du B.N. of human serum paraoxonase/arylesterase: for esterase both Metab. 1991; Scholar), paraoxon or as in the presence of in for PON and for PON as for PON paraoxon per min) and for PON per LDL was by from on LDL against with EDTA and in at to each EDTA was by against at The same LDL oxidation in HDL was from the serum of patients by and against at LDL was with HDL from patients for at in of LDL without copper or HDL, and of LDL with copper for the same (9Mackness M.I. Arrol S. Durrington P.N. Paraoxonase prevents accumulation of lipoperoxide in low-density lipoprotein.FEBS. 1991; 286: 152-154Google Scholar). Lipid peroxide concentrations measured at the the M. H. M. M. G. A assay for lipid in serum lipoproteins a Lipid Res. 1989; Scholar) on of the Oxidation was by of of EDTA and and incubation at in the was measured at and of lipid was a of Protection was as in lipid peroxide production with to oxidation of LDL in the of HDL electrophoretic mobility of LDL was by on and with The was measured by to and in mobility relative to of in the of LDL oxidation to HDL and to PON1 genotype by for at compared by PON was 6-fold higher < in the serum of patients with the RR genotype PON is of concentrations (15Humbert R. Adler D.A. Disteche C.M. Hassett C. Omiecinski C.J. Furlong C.E. The molecular basis of the human serum paraoxonase activity polymorphism.Nature Genet. 1993; 3: 73-76Google Scholar, S. Gan K.N. Mody M. La Du B.N. Molecular basis for the polymorphic forms of human serum paraoxonase/arylesterase: glutamine or arginine at position 191, for the respective A or B allozymes.Am. J. Hum. Genet. 1993; 52: 598-608Google Scholar) displayed a difference which was, significant < the of PON to PON of the activity of was higher in patients with the RR of HDL, PON activity per HDL protein was higher for the RR than QQ genotype ± vs. ± P < of for both induced peroxidation of LDL was to the same by HDL from either genotype (n = of whether by the accumulation of or the in electrophoretic mobility of LDL < for by for concentrations of 2 in the presence of HDL, protection was of at which accumulation of was by 60.6 ± 20.0 and 63.9 ± in the QQ and RR of incubation respective to ± and ± An was in protection against increased mobility which ± 21.8 and 61.4 ± in the QQ and RR at and ± and ± at PON was to the same in both during the of incubation and and of its activity at the of the while incubation of HDL with to a of activity between PON in the and protection by HDL as by production of change in electrophoretic mobility relative to QQ RR of oxidative protection by HDL considered was in with PON in the = P = for inhibition of peroxide production and = and P = for change mobility and the in both it for the RR to the of activity in patients with the the QQ The of the was, however, for the in a same of protection in both that PON to be related to protection of LDL against oxidation, the enzyme should be more than the PON is and In to whether protection was related to actual to the effect of PON inhibition in of HDL for 10 at prior to the in of PON Protection of LDL against oxidation, however, remained unaffected this protection remained correlated with PON in the = P < for = P < for results obtained was by EDTA of by HDL of protective and during the of the as as of PON activity between and 2 and a between and whether not the protective at the of that of HDL from a with genotype for either 2 or in the presence of copper of LDL antioxidant PON was from to and 2 per HDL protein 2 and incubation with of HDL from patients to between genotypes, this was performed concentrations of of from subjects with and RR during a incubation in the presence of LDL and In protection of LDL was by HDL concentrations of 2 per HDL from the QQ and subjects both protection, but from the RR at In light of the in 2 of protection by from QQ or RR this should however, be as a of the RR The same was with PON which is of A difference was, however, protection was as a of PON protection to of activity in the for the QQ for and high for PON has to HDL against oxidation and to in of vascular The whether PON is at in the protection of LDL against oxidation by copper or whether the of HDL or other HDL is was HDL from that high PON activity for of HDL this serum was obtained from a rabbit and HDL to the same as in the of human of HDL protein from to in the incubation corresponding to PON from to protection was obtained between and of HDL protein for PON between and in the same of activities as protection with the human enzyme but with protein concentrations to that protection is closely associated with more than with HDL In summary, results show that of HDL to LDL from oxidation is independent of the 192 Q→R genotype of that HDL protective for paraoxon hydrolysis is but that protection correlated PON measured that rabbit HDL that have high PON with to protein protective in with PON and not with HDL PON1 variability for either a glutamine or an arginine in the 192 results in a low activity for the Q allele and a higher for the R allele with effect on arylesterase activity, different activities for the protein bearing PON of or the other the QQ PON activity has to as as in to actual protein (15Humbert R. Adler D.A. Disteche C.M. Hassett C. Omiecinski C.J. Furlong C.E. The molecular basis of the human serum paraoxonase activity polymorphism.Nature Genet. 1993; 3: 73-76Google Scholar), is between activities in the hydrolysis by PON of lipid is for the protective effect HDL towards LDL oxidation, then the of the high PON RR allele among diabetic subjects with to be has that a more of the protective by PON1 the higher activity of the enzyme K.N. A. La Du B.N. of human serum paraoxonase/arylesterase: for esterase both Metab. 1991; Scholar). LDL from a source with a high of not difference between HDL from subjects in the of enzyme or LDL and the for PON1 in its protective effect against oxidation of LDL (13Watson A.D. Berliner J.A. Hama S.Y. La Du B.N. Faull K.F. Fogelman A.M. Navab M. Protective effect of high density lipoprotein associated paraoxonase: inhibition of the biological activity of minimally oxidized low density lipoprotein.J. Clin. Invest. 1995; 96: 2882-2891Google Scholar, M. M. La Du B.N. Paraoxonase inhibits high-density lipoprotein oxidation and its Clin. Invest. Scholar). the effect of the was on oxidized both to to the same the this oxidized has to a of human artery wall PON widely B. M.I. Arrol S. W. Durrington P.N. of the human serum paraoxonase 55 and 192 genetic on the protection by high density lipoprotein against low density lipoprotein oxidative Scholar). that the protective effect of HDL on LDL oxidation was not related to the for hydrolysis of results this HDL from diabetic patients for both PON by a factor of displayed protective towards peroxidation of LDL from a Protection was both by accumulation of products in the incubation and by of electrophoretic mobility of the LDL of PON by or not the protective of show that the of protection with incubation is to copper of PON1 in of of wall cells as a for oxidative modification of Watson (13Watson A.D. Berliner J.A. Hama S.Y. La Du B.N. Faull K.F. Fogelman A.M. Navab M. Protective effect of high density lipoprotein associated paraoxonase: inhibition of the biological activity of minimally oxidized low density lipoprotein.J. Clin. Invest. 1995; 96: 2882-2891Google Scholar) have that inhibition of PON in HDL to the of by EDTA protective from to a between PON and hydrolase in the protective effect. the basis of peroxide accumulation or not of PON to protection to extent, the of hydrolase to protection in this Recent however, show that of PON to totally protection against oxidation both of LDL and HDL M. M. La Du B.N. Paraoxonase inhibits high-density lipoprotein oxidation and its Clin. Invest. Scholar). is not to the to the PON the also with of determinant for of HDL with copper prior to the of LDL in the in peroxidation of as as of PON without of HDL protection by HDL that PON not act by to it in of the recent that a associated with PON a role in the antioxidant properties of HDL, the that other play a determinant show human HDL, protection is in by of HDL on the of 2 independent of PON in to R. Y. S. of by of the reducing from human Lipid Res. Scholar) and both and HDL to by to B. R. Oxidation of high density Evidence for of lipid by of and Biol. Chem. Scholar). which is associated with PON in HDL Richter R.J. Furlong C.E. J.A. is associated with paraoxonase in human 1994; Scholar) seems to prevent lipoperoxide in artery wall cells in is increased in or to while coronary patients high of to PON M. S. Lenten B.J. C.J. Lusis A.J. Fogelman A.M. oxidized LDL an increased Clin. Invest. 1997; 99: Scholar). An interaction of with PON to of its antioxidant activity, but is at to factor protein also play an role (13Watson A.D. Berliner J.A. Hama S.Y. La Du B.N. Faull K.F. Fogelman A.M. Navab M. Protective effect of high density lipoprotein associated paraoxonase: inhibition of the biological activity of minimally oxidized low density lipoprotein.J. Clin. Invest. 1995; 96: 2882-2891Google Scholar). of the results obtained to of to against a effect of PON in of a We show that protection by HDL in which PON remained correlated with PON in the and that in rabbit protection is by a of the show in the of low concentrations, the is relative to PON for the QQ than RR In the of concentrations, however, a effect is that is not different between a to be these be in of The higher of the in the QQ genotype to a significant of protection HDL concentrations is the in diabetic patients with coronary lesions and this for the of the R allele in this group. have a protective effect of PON towards of in the vascular cell Navab M. Lenten B.J. S.Y. A.M. Fogelman A.M. Lusis A.J. of in high density lipoproteins to Clin. Invest. 1997; Scholar), more towards oxidation of HDL or of LDL M. M. La Du B.N. Paraoxonase inhibits high-density lipoprotein oxidation and its Clin. Invest. Scholar). of PON in HDL with a chemical M. M. La Du B.N. Paraoxonase inhibits high-density lipoprotein oxidation and its Clin. Invest. Scholar) of protective against of by these results suggest that the protein bearing both PON and PON activities also possesses a third thermostable property, independent of closely associated with PON and unaffected by PON genetic variability. is not as the on a in the part of the and should that the for protection is from that for paraoxon hydrolysis was the of a from was by from and de and the of the of and de for efficient of the of this high density lipoprotein low density lipoprotein paraoxonase arylesterase hydrolysis activity of paraoxonase arylesterase activity of paraoxonase