You are on page 1of 6

The FASEB Journal Research Communication

Paraoxonase 1 protects against protein N-homocysteinylation in humans


Joanna Pera-Kaja n*, and Hieronim Jakubowski*,,,1
*Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry New JerseyNew Jersey Medical School, International Center for Public Health, Newark, New Jersey, USA; Department of Biochemistry and Biotechnology, University of Life Sciences, Poznan , Poland; and Institute of Bioorganic Chemistry, Poznan , Poland Genetic or nutritional disorders in homocysteine (Hcy) or folate metabolism elevate plasma Hcy-thiolactone and lead to vascular and/or brain pathologies. Hcy-thiolactone has the ability to form isopeptide bonds with protein lysine residues, which generates N-Hcy-protein with autoimmunogenic and prothrombotic properties. Paraoxonase (PON1), carried on high-density lipoproteins (HDLs) in the blood, hydrolyzes Hcy-thiolactone and protects against the accumulation of N-Hcy-protein in vitro. To determine its role in vivo, we studied how natural variation in Hcy-thiolactonase activity of PON1 affects plasma NHcy-protein levels in cystathionine -synthase-decient patients (n28). We found that plasma N-Hcy-protein was negatively correlated with serum Hcy-thiolactonase activity (r0.43, P0.01), i.e., the higher the Hcythiolactonase activity, the lower N-Hcy protein levels. This relation was faithfully replicated in vitro in experiments with radiolabeled Hcy-thiolactone. We also found that enzymatic activities of the PON1 protein measured with articial substrates correlated less strongly (r0.36, P0.025 for paraoxonase activity) or did not correlate at all (phenylacetate hydrolase and TBLase activities) with plasma N-Hcy protein. These ndings provide evidence that the Hcy-thiolactonase activity of PON1 is a determinant of plasma N-Hcyprotein levels and that Hcy-thiolactonase/PON1 protects proteins against N-homocysteinylation in vivo, a novel mechanism likely to contribute to atheroprotective roles of HDL in humans.Pera-Kaja n, J., Jakubowski, H. Paraoxonase 1 protects against protein N-homocysteinylation in humans. FASEB J. 24, 931936 (2010). www.fasebj.org
ABSTRACT

Key Words: cystathionine -synthase deciency enzymatic activities of high-density lipoprotein homocysteine-thiolactonase protein modication by homocysteine-thiolactone Homocysteine (Hcy), A SULFUR-CONTAINING nonprotein amino acid, is an intermediate metabolite that forms from the essential dietary protein amino acid methionine (Met). Levels of Hcy are regulated by remethylation to Met, catalyzed by Met synthase and betaine-Hcy methyltransferase, and transsulfuration to cysteine, the rst step of which is catalyzed by cystathionine -syn0892-6638/10/0024-0931 FASEB

thase (CBS) (1). Hcy is also metabolized to the thioester Hcy-thiolactone in an error-editing reaction in protein biosynthesis when Hcy is selected in place of Met by methionyl-tRNA synthetase (2). Hcy-thiolactone forms isopeptide bonds with protein lysine residues (35), which impairs or alters the proteins function (6 8). Severe hyperhomocysteinemia observed in CBS deciency is associated with brain and cardiovascular pathologies and leads to premature death due to vascular complications (1). Treatment with vitamin B6 in combination with folate or betaine lowers plasma Hcy and improves vascular outcomes in patients with CBS deciency, suggesting that Hcy or its metabolite plays a causal role in atherothrombosis. For example, untreated patients with CBS deciency suffer 1 vascular event/25 patient-yr (1), while treated patients suffer only 1 vascular event/263 patient-yr (relative risk 0.091, P0.001) (9). How hyperhomocysteinemia leads to specic clinical manifestations is not entirely clear. Because of parallel changes in the concentrations of Hcy metabolites under most clinical and experimental circumstances, it has not been possible to assign the observed toxicity to a specic metabolite. Hcy itself, S-adenosyl-Hcy, Hcythiolactone, and Hcy-thiolactone-modied protein (NHcy-protein) are potential candidates, and possible mechanisms have been assigned to each (10 12). One hypothesis suggests that metabolic conversion to Hcythiolactone and N-Hcy-protein contributes to the pathophysiology of Hcy excess (3, 6) and is involved in atherothrombotic disease in humans (10, 1316). Consistent with this hypothesis, Hcy-thiolactone and N-Hcyprotein, including prothrombotic N-Hcy-brinogen, are elevated in patients with CBS deciency (17). Genetic or nutritional disorders in Hcy or folate metabolism increase N-Hcy-protein levels also in mice (18). Furthermore, immunohistochemical studies show that N-Hcy-protein accumulates within atherosclerotic
1 Correspondence: Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, International Center for Public Health, 225 Warren St. Newark, NJ 07101-1709, USA. E-mail: jakubows@umdnj.edu doi: 10.1096/fj.09-144410

931

lesions in aortas of ApoE/ mice fed a normal chow diet, and the accumulation increases in the animals fed a hyperhomocysteinemic high-Met diet (19). In other animal models, treatment with Hcy-thiolactone causes pathophysiological changes reminiscent of those observed in human genetic hyperhomocysteinemia. For example, infusions with Hcy-thiolactone, or an Hcy-thiolactone-supplemented diet, produce atherosclerotic changes in baboons (20) or rats (21). Furthermore, treatment with Hcy-thiolactone causes developmental abnormalities in eyes of chick embryos, including optic lens dislocation (22), a diagnostic feature prevalent in CBS-decient human patients (1). Recent human clinical studies show that plasma N-Hcy-protein levels are associated with risk of coronary heart disease (23), whereas plasma Hcy-thiolactone levels are associated with the development and progression of diabetic macrovasculopathy (24). Because N-homocysteinylation impairs or alters protein function and leads to cell and tissue damage (10, 13, 16), it is likely that human beings evolved mechanisms that prevent excessive accumulation of N-Hcyprotein. One potential mechanism involves paroxonase 1 (PON1), a calcium-dependent enzyme carried on high-density lipoprotein (HDL) in the blood (25), known to protect against atherosclerosis in mice (26) and humans (27). Our previous work has shown that PON1 and HDL have the ability to hydrolyze Hcythiolactone (28) and protect against the accumulation of N-Hcy-protein in vitro (5, 29). The present work was undertaken to determine whether PON1 protects against protein N-homocysteinylation in vivo and to examine relationships between various enzymatic activities of PON1 and N-Hcy-protein levels in humans.

Protein N-homocysteinylation in vitro D,L-[35S]Hcy-thiolactone was prepared from L-[35S]methionine (GE Healthcare, Piscataway, NJ, USA), as described previously (31). N-homocysteinylation of human serum protein in vitro was carried as follows: 5 l of human serum was incubated with 2.5 M D,L-[35S]Hcy-thiolactone at 37C for 3 h; during this time, 90% of the originally present D,L[35S]Hcy-thiolactone was incorporated into protein or hydrolyzed to [35S]Hcy (6, 29). The incorporation into protein was assayed by treatment with 5 mM dithiothreitol (DTT) and precipitation with 5% trichloroacetic acid; N-[35S]Hcy-protein was determined as DTT-resistant trichloroacetic acidprecipitable material. Enzymatic assays Serum Hcy-thiolactonase (HTase) activity assay monitors the formation of [35S]Hcy from [35S]Hcy-thiolactone, as described previously (28, 32). Briey, 5 l, 2.5 M, or 1 mM D,L-[35S]Hcy-thiolactone was dried in 0.5-ml microtubes on a Labconco concentrator (Labconco, Kansas City, MO, USA). Each tube received 5 l serum; after mixing, tubes were incubated at 37C for 30 min. The reaction was stopped by adding 15 l 5 mM disodium-EDTA (pH 8.0) containing 1 mM DTT. [35S]Hcy was separated from [35S]Hcy-thiolactone by thin-layer chromatography and quantied using a Beckman LS 6500 liquid scintillation counter (Beckman Coulter, Fullerton, CA, USA). HTase assays were performed in duplicates with average measurements for each sample calculated. Average coefcient of variance was 9.1% on 28 replicates performed on two different days. Blank assays, carried out with serum samples in which PON1 was inactivated with 10 mM EDTA, were subtracted from the results. Units of HTase activity are expressed as percentage [35S]Hcy-thiolactone hydrolyzed to [35S]Hcy in 30 min. We note that when HTase activity is expressed as percentage [35S]Hcy-thiolactone hydrolyzed to [35S]Hcy, the relative values of Hcy-thiolactone hydrolyzed are concentration independent when [Hcy-thiolactone] Km (Km value for Hcy-thiolactone is 23 mM; ref. 28). This was conrmed by separate assays with 2.5 M or 1 mM D,L-[35S]Hcy-thiolactone, which yielded identical HTase activity values. Thus, the variations in HTase activity described in Figs. 1-4 reect the physiological HTase activity variations. Paraoxonase (POase), arylesterase (PhAcase), and -thiobutyrolactonase (TBLase) assays were performed at 25C in 100-fold diluted serum (5 l serum/500 l assay) in reaction mixtures containing 50 mM K-HEPES buffer (pH 7.4), 1 mM CaCl2. Each measurement was an average of 2 4 replicates. Coefcients of variance were 6.1 and 2.9% for POase and PhAcase assays, respectively. One unit of activity is dened as a change in absorbance of 0.0001/min. For POase activity assays, the generation of p-nitrophenol from 2 mM paraoxon was monitored at 412 nm (13,000 M1 cm1) for 2-min time periods, and reaction rates (A412/ min) were calculated. For PhAcase activity assays, the generation of phenol from 5 mM phenyl acetate (PhAc) was monitored at 270 nm (1300 M1 cm1) for 2 min, and rates (A270/min) were calculated from the rst 0.5 min of the reaction. For TBLase assays (33), 100-fold diluted serum was rst preincubated in 0.3 mM 5,5-dithio-bis-2-nitrobenzoic acid, 50 mM K-HEPES buffer (pH 7.4), and 1 mM CaCl2 for 20 min at room temperature, then 2.5 mM -thiobutyrolactone (TBL) was added; A412 was then monitored for 4 min, and rates were calculated for the last 2 min of the reaction.
N AND JAKUBOWSKI PERA-KAJA

MATERIALS AND METHODS


CBS-decient human subjects We studied 28 Dutch patients (14 to 74 yr old) with homocystinuria due to mutations in the CBS gene (18). CBS activity in broblasts derived from these patients was 2.5% of the control CBS activity from unaffected individuals. Patients were initially diagnosed at ages 2 to 54 on the basis of clinical manifestations of CBS deciency (ectopia lentis, Marfanoid appearance, osteoporosis, severe hyperhomocysteinemia, and hypermethioninemia). Five patients with CBS deciency survived a vascular event before diagnosis. All patients were on Hcy-lowering treatment (vitamin B6) following diagnosis. Blood samples were taken into Vacutainer EDTA tubes for the preparation of plasma, as described previously (19, 20). Serum for PON1 enzymatic assays was obtained from serum separation tubes after a 30-min clotting time at room temperature. All specimens were stored at 80C. Determination of in vivo N-Hcy-protein levels in human plasma Plasma N-Hcy-protein was assayed as described previously (30).
932 Vol. 24 March 2010

The FASEB Journal www.fasebj.org

TABLE 1. Levels of enzymatic activities with different substrates of PON1 in human serum
Mean PON1 activity (U) Patients with CBS deciency Unaffected control subjects

Substrate

Hcy-thiolactone Paraoxon Phenyl acetate -Thiobutyrolactone

15.2 6.3 147 85 14,660 3805 105 29

9.9 3.5;a 8.55b 144 93;a 125b

Data are expressed as means sd. aData from ref. 29. bData from ref. 32.

RESULTS Variations in serum HTase activity vs. enzymatic activities with articial substrates In addition to its natural substrate Hcy-thiolactone, the PON1 protein hydrolyzes articial substrates, such as paraoxon (after which the PON1 protein was named), phenyl actetate, and -thiobutyrolactone (33). The assays with these articial substrates are used in clinical studies of a relation between PON1 and human disease. However, whether PON1 activity with an articial substrate reects the physiological activity with Hcy-thiolactone was unclear. To answer this question, enzymatic activities of the PON1 protein with different substrates were assayed in sera from patients with CBS deciency, and relations between these activities were examined. HTase activity in the studied population varied from 1.0 to 27.9 U, with a mean of 15.2 6.3 U, higher than in other populations (Table 1). POase activity in patients with CBS deciency varied from 36 to 305 U, with a mean of 147 81 U, similar to that in other populations. PhAcase activity varied from 2200 to 20,200 U, with a mean of 14,660 3805 U. TBLase activity varied from 71 to 179 U, with a mean of 105 29 U (Table 1). POase activity was signicantly correlated with the HTase activity (R20.80, P0.001) (Fig. 1A). Less strong but signicant correlation was also observed between TBLase and HTase activity (Fig. 1C). However, there was no signicant correlation between PhAcase and HTase activity (Fig. 1B).
Figure 2. In vitro accumulation of N-Hcy-protein is inversely dependent on HTase activity in human serum.

High HTase activity minimizes protein N-homocysteinylation in vitro Sera from patients with CBS deciency were incubated with [35S]Hcy-thiolactone at 37C for 3 h, and N-[35S]Hcy-protein formation was determined as described in Materials and Methods and plotted as a function of serum HTase activity. As shown in Fig. 2, there was a signicant negative correlation between the amount of N-[35S]Hcy-protein formed in vitro and the serum HTase activity, i.e., the higher the HTase activity, the lower the amount of N-[35S]Hcy-protein formed. High HTase activity protects against protein N-homocysteinylation in humans Plasma N-Hcy-protein, assayed as described in the Materials and Methods section, was plotted as a function of serum HTase activity. As shown in Fig. 3, there was a signicant negative correlation between the in vivo concentration of N-Hcy-protein and the serum HTase activity, i.e., the higher the HTase activity, the lower the concentration of N-Hcy-protein accumulated in vivo in the human plasma.

Figure 1. HTase activity is positively correlated with POase (A) and TBLase activities (C), but not with PhAcase activity (B) in serum from patients with CBS deciency.
PON1 AND PROTEIN N-HOMOCYSTEINYLATION 933

Figure 3. In vivo human plasma N-Hcy-protein levels are negatively correlated with serum HTase activity.

PhAcase and TBLase activities do not correlate with the concentration of N-Hcy-protein in humans To determine whether other activities of PON1 would predict levels of N-Hcy-protein, relationships between POase, PhAcase, or TBLase activity and N-Hcy-protein in humans were examined. There was a signicant negative correlation between the in vivo concentration of N-Hcy-protein and POase activity (Fig. 4A), albeit somewhat weaker than that with HTase activity. However, there was no signicant correlation between PhAcase or TBLase activity and the in vivo N-Hcy-protein levels (Fig. 4B, C).

DISCUSSION Biochemical mechanisms underlying physiological role of PON1 were largely unknown. The present study, taking advantage of our new assays for the quantication of N-linked Hcy content in proteins and for the determination of HTase activity, provides evidence that one of the physiological roles of PON1 is to protect against N-Hcy-protein accumulation in vivo, a novel mechanism likely to contribute to atheroprotective roles of HDL in humans. Specically, we show that plasma N-Hcy-protein levels in humans are negatively correlated

with serum HTase activity, and that this in vivo relationship can be faithfully reproduced in vitro. That these ndings reect a physiologically relevant function of PON1 is further supported by our observations that activities of PON1 with articial substrates (e.g., phenyl acetate, -thiobutyrolactone) are not correlated with the in vivo N-Hcy-protein levels. In addition to promoting reverse cholesterol transport, HDL also promotes anti-inammatory and antioxidant effects. The HDL-associated PON1 protein contributes to the anti-inammatory and antioxidant activities of the lipoprotein (34), thereby preventing atherosclerosis in animal models (26) and humans (27). However, what mechanisms are responsible for PON1-mediated systemic antioxidant effects is not clear. A possibility that PON1 protects against atherosclerosis due to its ability to detoxify oxidized lipids, often alluded to in the literature (27, 35), has no biochemical basis and thus is unlikely. The POase or PhAcase activities measured in clinical studies cannot be mechanistically related to a putative antioxidative function of PON1. Paraoxon and phenylacetate are articial substrates convenient for monitoring hydrolytic activity of PON1, but to the best of our knowledge, there is no assay for monitoring a putative redox activity of PON1. Although it has been suggested that PON1 inhibits oxidation of LDL in vitro (36), more recent studies demonstrate that the PON1 protein has no intrinsic ability to protect against lipid oxidation or inactivate harmful oxidation products (3739). Hcy-thiolactone is the only known naturally occurring (40, 41) substrate of PON1 (4, 28). Hcythiolactone, generated by methionyl-tRNA synthetase in an error-editing reaction during protein synthesis to remove the nonprotein amino acid Hcy (2), has the ability to chemically modify proteins (3, 6). Protein modication by Hcy-thiolactone, similar to other pathologically relevant protein modications, such as the modication by glucose or products of lipid oxidation, impairs or alters the proteins structure and function (6), including the redox function (7, 8), and has been implicated in the vascular and neurological pathology of human genetic hyperhomocysteinemia (16, 17). Our present nding that the HTase activity of PON1 minimizes protein damage by

Figure 4. In vivo human plasma N-Hcy-protein levels are negatively correlated with serum POase (A), but not with serum PhAcase (B) and TBLase (C) activities.
934 Vol. 24 March 2010 The FASEB Journal www.fasebj.org N AND JAKUBOWSKI PERA-KAJA

N-homocysteinylation in CBS-decient human patients suggests that PON1 is likely to play a protective role against vascular and neurological pathology in hyperhomocysteinemic patients. Consistent with this suggestion is our previous nding that the HTase activity of PON1 is a predictor of coronary heart disease in the general population (42). The conclusion that PON1 protects proteins against N homocysteinylation by hydrolyzing Hcy-thiolactone in vivo is supported by our observation that the relationship between the in vivo N-Hcy-protein levels and HTase activity (Fig. 3) has been reproduced in vitro using radiolabeled Hcy-thiolactone (Fig. 2). The present work also demonstrates that insights into N-Hcy-protein metabolism obtained in previous ex vivo studies with cultured human cells and serum are valid for the human body. For example, supplementation with physiological levels of HDL inhibits the accumulation of Hcy-thiolactone and N-Hcyprotein in cultures of human vascular endothelial cells (5). When normal human or animal sera are incubated with Hcy-thiolactone, the extent of N-Hcyprotein formation is lower in sera from high HTase activity donors than in sera from low-activity donors; the extent of the in vitro N-Hcy-protein formation is the lowest in serum from rabbits, which have 510fold higher levels of serum HTase/PON1 activity than an average human serum does (29). The present work shows similar effects of the natural variation in serum HTase activity on N-Hcy-protein levels in vivo in the human body. In addition to its natural substrate Hcy-thiolactone, PON1 has the ability to hydrolyze articial substrates. Measurements of enzymatic activities of PON1 with paraoxon, phenyl acetate, or -thiobutyrolactone are used in clinical studies examining relationships between PON1 and cardiovascular disease (27, 33, 34). Whether these activities reect a physiologically relevant functional activity was unknown. Our present ndings demonstrate that of the three activities measured with articial substrates only POase, but not PhAcase or TBLase, reects physiologically relevant HTase activity of PON1. For example, we found that plasma N-Hcy-protein correlates signicantly not only with HTase (Fig. 3) but also with the POase activity (Fig. 4A). Furthermore, POase and HTase activities are strongly correlated in the present study population (Fig. 1A) and in other populations (29, 32). In contrast, the correlation between TBLase and HTase is much weaker (Fig. 1C), and there is no correlation between PhAcase and HTase activities (Fig. 1B). Taken together, these ndings indicate that POase is a good surrogate for the physiological HTase activity of PON1 (32), whereas PhAcase and TBLase do not reect the physiological activity. In conclusion, by showing that the HTase activity of PON1 is a determinant of plasma N-Hcy-protein levels and that HTase/PON1 protects against accumulation of the proatherogenic N-Hcy-protein in vivo, our ndings provide evidence for a novel mechanism
PON1 AND PROTEIN N-HOMOCYSTEINYLATION

likely to contribute to atheroprotective roles of HDL in humans.


This work was supported in part by grants from the American Heart Association (0855919D) and the Ministry of Science and Higher Education, Warsaw, Poland (NN 401 230634, N401 065 32/1504, POIG.01.03.01-00-097/08).

REFERENCES
1. Mudd, S. H., Levy, H. L., and Krauss, J. P. (2001) Disorders of transsulfuration. In The Metabolic and Molecular Bases of Inherited Disease, Vol. 2, (Scriver, C. R., Beaudet, A. L., Sly, W. S., Valle, D., Childs, B., Kinzler, K. W., and Vogelstein, B., eds) pp. 2007 2056, Mc Graw-Hill, New York Jakubowski, H., and Goldman, E. (1993) Synthesis of homocysteine thiolactone by methionyl-tRNA synthetase in cultured mammalian cells. FEBS Lett. 317, 237240 Jakubowski, H. (1997) Metabolism of homocysteine thiolactone in human cell cultures. Possible mechanism for pathological consequences of elevated homocysteine levels. J. Biol. Chem. 272, 19351942 Jakubowski, H. (2000) Homocysteine thiolactone: metabolic origin and protein homocysteinylation in humans. J. Nutr. 130, 377S381S Jakubowski, H., Zhang, L., Bardeguez, A., and Aviv, A. (2000) Homocysteine thiolactone and protein homocysteinylation in human endothelial cells: implications for atherosclerosis. Circ. Res. 87, 4551 Jakubowski, H. (1999) Protein homocysteinylation: possible mechanism underlying pathological consequences of elevated homocysteine levels. FASEB J. 13, 22772283 Glowacki, R., and Jakubowski, H. (2004) Cross-talk between Cys34 and lysine residues in human serum albumin revealed by N-homocysteinylation. J. Biol. Chem. 279, 10864 10871 Pera-Kaja n, J., Marczak, L., Kajan, L., Skowronek, P., Twardowski, T., and Jakubowski, H. (2007) Modication by homocysteine thiolactone affects redox status of cytochrome c. Biochemistry 46, 6225 6231 Yap, S., Boers, G. H., Wilcken, B., Wilcken, D. E., Brenton, D. P., Lee, P. J., Walter, J. H., Howard, P. M., and Naughten, E. R. (2001) Vascular outcome in patients with homocystinuria due to cystathionine -synthase deciency-treated chronically: a multicenter observational study. Arterioscler. Thromb. Vasc. Biol. 21, 2080 2085 Jakubowski, H. (2007) The molecular basis of homocysteine thiolactone-mediated vascular disease. Clin. Chem. Lab. Med. 45, 1704 1716 Jakubowski, H. (2001) Protein N-homocysteinylation: implications for atherosclerosis. Biomed. Pharmacother. 55, 443 447 Pera-Kaja n, J., Twardowski, T., and Jakubowski, H. (2007) Mechanisms of homocysteine toxicity in humans. Amino Acids 32, 561572 Jakubowski, H. (2004) Molecular basis of homocysteine toxicity in humans. Cell. Mol. Life Sci. 61, 470 487 Jakubowski, H. (2005) Anti-N-homocysteinylated protein autoantibodies and cardiovascular disease. Clin. Chem. Lab. Med. 43, 10111014 Jakubowski, H. (2006) Pathophysiological consequences of homocysteine excess. J. Nutr. 136, 1741S1749S Jakubowski, H. (2008) The pathophysiological hypothesis of homocysteine thiolactone-mediated vascular disease. J. Physiol. Pharmacol. 59(Suppl. 9), 155167 Jakubowski, H., Boers, G. H., and Strauss, K. A. (2008) Mutations in cystathionine -synthase or methylenetetrahydrofolate reductase gene increase N-homocysteinylated protein levels in humans. FASEB J. 22, 4071 4076 Jakubowski, H., Pera-Kaja n, J., Finnell, R. H., Cabrera, R. M., Wang, H., Gupta, S., Kruger, W. D., Kraus, J. P., and Shih, D. M. (2009) Genetic or nutritional disorders in homocysteine or folate metabolism increase protein N-homocysteinylation in mice. FASEB J. 23, 17211727

2. 3.

4. 5.

6. 7. 8.

9.

10. 11. 12. 13. 14. 15. 16. 17.

18.

935

19.

20. 21. 22.

23.

24.

25. 26.

27.

28. 29. 30. 31.

Pera-Kaja n, J., Stanger, O., Luczak, M., Ziolkowska, A., Malendowicz, L. K., Twardowski, T., Lhotak, S., Austin, R. C., and Jakubowski, H. (2008) Immunohistochemical detection of Nhomocysteinylated proteins in humans and mice. Biomed. Pharmacother. 62, 473 479 Harker, L. A., Slichter, S. J., Scott, C. R., and Ross, R. (1974) Homocystinemia. Vascular injury and arterial thrombosis. New Engl. J. Med. 291, 537543 Endo, N., Nishiyama, K., Otsuka, A., Kanouchi, H., Taga, M., and Oka, T. (2006) Antioxidant activity of vitamin B6 delays homocysteine-induced atherosclerosis in rats. Br. J. Nutr. 95, 1088 1093 Maestro de las Casas, C., Epeldegui, M., Tudela, C., VarelaMoreiras, G., and Perez-Miguelsanz, J. (2003) High exogenous homocysteine modies eye development in early chick embryos. Birth Defects Res. A Clin. Mol. Teratol. 67, 35 40 Yang, X., Gao, Y., Zhou, J., Zhen, Y., Yang, Y., Wang, J., Song, L., Liu, Y., Xu, H., Chen, Z., and Hui, R. (2006) Plasma homocysteine thiolactone adducts associated with risk of coronary heart disease. Clin. Chim. Acta 364, 230 234 Gu, W., Lu, J., Yang, G., Dou, J., Mu, Y., Meng, J., and Pan, C. (2008) Plasma homocysteine thiolactone associated with risk of macrovasculopathy in Chinese patients with type 2 diabetes mellitus. Adv. Ther. 25, 914 924 Camps, J., Marsillach, J., and Joven, J. (2009) The paraoxonases: role in human diseases and methodological difculties in measurement. Crit. Rev. Clin. Lab. Sci. 46, 83106 Shih, D. M., Gu, L., Xia, Y. R., Navab, M., Li, W. F., Hama, S., Castellani, L. W., Furlong, C. E., Costa, L. G., Fogelman, A. M., and Lusis, A. J. (1998) Mice lacking serum paraoxonase are susceptible to organophosphate toxicity and atherosclerosis. Nature 394, 284 287 Bhattacharyya, T., Nicholls, S. J., Topol, E. J., Zhang, R. L., Yang, X., Schmitt, D., Fu, X. M., Shao, M. Y., Brennan, D. M., Ellis, S. G., Brennan, M. L., Allayee, H., Lusis, A. J., and Hazen, S. L. (2008) Relationship of paraoxonase 1 (PON1) gene polymorphisms and functional activity with systemic oxidative stress and cardiovascular risk. JAMA 299, 12651276 Jakubowski, H. (2000) Calcium-dependent human serum homocysteine thiolactone hydrolase. A protective mechanism against protein N-homocysteinylation. J. Biol. Chem. 275, 39573962 Jakubowski, H., Ambrosius, W. T., and Pratt, J. H. (2001) Genetic determinants of homocysteine thiolactonase activity in humans: implications for atherosclerosis. FEBS Lett. 491, 3539 Jakubowski, H. (2008) New method for the determination of protein N-linked homocysteine. Anal. Biochem. 380, 257261 Jakubowski, H. (2007) Facile syntheses of [35S]homocysteinethiolactone, [35S]homocystine, [35S]homocysteine, and [S-nitroso-35S]homocysteine. Anal. Biochem. 370, 124 126

32.

33.

34.

35.

36. 37.

38.

39. 40. 41.

42.

Lacinski, M., Skorupski, W., Cieslinski, A., Sokolowska, J., Trzeciak, W. H., and Jakubowski, H. (2004) Determinants of homocysteine-thiolactonase activity of the paraoxonase-1 (PON1) protein in humans. Cell. Mol. Biol. 50, 885 893 Kosaka, T., Yamaguchi, M., Motomura, T., and Mizuno, K. (2005) Investigation of the relationship between atherosclerosis and paraoxonase or homocysteine thiolactonase activity in patients with type 2 diabetes mellitus using a commercially available assay. Clin. Chim. Acta 359, 156 162 Jarvik, G. P., Rozek, L. S., Brophy, V. H., Hatsukami, T. S., Richter, R. J., Schellenberg, G. D., and Furlong, C. E. (2000) Paraoxonase (PON1) phenotype is a better predictor of vascular disease than is PON1(192) or PON1(55) genotype. Arterioscler. Thromb. Vasc. Biol. 20, 24412447 Mackness, M. I., Durrington, P. N., and Mackness, B. (2004) The role of paraoxonase 1 activity in cardiovascular disease: potential for therapeutic intervention. Am. J. Cardiovasc. Drugs 4, 211217 Mackness, M. I., Arrol, S., and Durrington, P. N. (1991) Paraoxonase prevents accumulation of lipoperoxides in lowdensity lipoprotein. FEBS Lett. 286, 152154 Marathe, G. K., Zimmerman, G. A., and McIntyre, T. M. (2003) Platelet-activating factor acetylhydrolase, and not paraoxonase-1, is the oxidized phospholipid hydrolase of high density lipoprotein particles. J. Biol. Chem. 278, 39373947 Teiber, J. F., Draganov, D. I., and La Du, B. N. (2004) Puried human serum PON1 does not protect LDL against oxidation in the in vitro assays initiated with copper or AAPH. J. Lipid Res. 45, 2260 2268 Connelly, P. W., Draganov, D., and Maguire, G. F. (2005) Paraoxonase-1 does not reduce or modify oxidation of phospholipids by peroxynitrite. Free Radic. Biol. Med. 38, 164 174 Chwatko, G., and Jakubowski, H. (2005) The determination of homocysteine-thiolactone in human plasma. Anal. Biochem. 337, 271277 Chwatko, G., Boers, G. H., Strauss, K. A., Shih, D. M., and Jakubowski, H. (2007) Mutations in methylenetetrahydrofolate reductase or cystathionine beta-synthase gene, or a high-methionine diet, increase homocysteine thiolactone levels in humans and mice. FASEB J. 21, 17071713 Domagaa, T. B., acinski, M., Trzeciak, W. H., Mackness, B., Mackness, M. I., and Jakubowski, H. (2006) The correlation of homocysteine-thiolactonase activity of the paraoxonase (PON1) protein with coronary heart disease status. Cell. Mol. Biol. 52, 39 Received for publication August 20, 2009. Accepted for publication October 8, 2009.

936

Vol. 24

March 2010

The FASEB Journal www.fasebj.org

N AND JAKUBOWSKI PERA-KAJA