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A high density lipoprotein from Piaractus mesopotamicus, pacu, (Osteichthyes, Characidae), is associated with paraoxonase activity
Biochimie 83 (2001) 945−951 © 2001 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. S0300908401013426/FLA
A high density lipoprotein from Piaractus mesopotamicus, pacu, (Osteichthyes, Characidae), is associated with paraoxonase activity Evelize Follya, Vera Lucia Cunha Bastosb, Marcelo V. Alvesb, Jayme Cunha Bastosb, Georgia C. Atellaa* a
Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Av. Bauhinia, 400, bloco H, 2o andar, sala 031, Ilha do Fundão, Rio de Janeiro, R.J., CEP. 21940-590, Brazil b Departamento de Bioquímica, Instituto Roberto Alcântara Gomes, Universidade do Estado do Rio de Janeiro, RJ, Brazil (Received 23 March 2001; accepted 24 September 2001) Abstract — We have characterized the serum lipoprotein profile and localized the serum paraoxonase activity of pacu, Piaractus mesopotamicus, a tropical fish species. The total lipoprotein profile of pacu serum obtained after KBr density ultracentrifugation shows the predominance of HDL (1.1267 g/mL). SDS-PAGE electrophoresis revealed a negligible amount of LDL. Pacu HDL was purified by gel filtration column on HPLC, and its molecular mass was estimated to be 246 kDa. Protein composition was 35%, and comprised four protein components with molecular masses of 45, 38, 25 and 12.5 kDa. Lipids represent 58% of total HDL, comprising 40% neutral lipids and 18% phospholipids by weight. The HDL contains 7% of carbohydrates, and mannose was the only sugar detected by paper chromatography in HDL hydrolysates. HDL-containing fractions showed the major paraoxonase activity. Purification of HDL resulted in a 23-fold enrichment of this activity. This is the first experimental evidence demonstrating the association of paraoxonase activity with a HDL in fish. © 2001 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. paraoxonase / HDL / LDL / fish lipoprotein / fish biochemistry
1. Introduction Blood plasma lipoprotein biochemistry has received increasing attention since it became evident that changes in their levels and dysfunction in the synthesis of their apoproteins are related to cardiovascular disease in humans [1–5]. Lipoproteins are classified according to the densities at which they are isolated such as high-density lipoproteins (HDL), intermediate-density lipoproteins (IDL), low-density lipoproteins (LDL), very low-density lipoproteins (VLDL), and those that are secreted by the intestine following a dietary fat load, the chylomicrons [6]. Considering the amount of information about mammals, knowledge about fish plasma lipoprotein is limited to a small number of species, most from the northern hemisphere [7–9]. It has been reported that HDL dominates the lipoprotein profile in plasma from Salmonidae and may constitute up to 36% of the total plasma protein [8–11]. Nevertheless, many aspects of HDL metabolism in *Correspondence and reprints. E-mail address: [email protected] (G.C. Atella). Abbreviations: HPLC, high performance liquid chromatography; KBr, potassium bromide; SDS-PAGE, polyacrylamide gel electrophoresis with sodium duodecyl sulphate; native-PAGE, polyacrylamide gel eletrophoresis without sodium duodecyl sulphate.
Salmonidae are not well understood, as well as the functions of their apoproteins. The isolation and biochemical characterization of such apoproteins from an increasing number of fish species should facilitate the comprehension of the role that lipoproteins play in fish lipid metabolism. The heterogeneity of lipid-associated proteins in vertebrate plasma HDL is remarkable. Serum paraoxonase (EC 3.1.8.1) is a calcium-dependent HDL-associated ester hydrolase named after its ability to catalyze the hydrolysis of paraoxon, an organophosphate toxicant, into p-nitrophenol and diethyl phosphate [12, 13]. Blatter et al. [14] have isolated a novel HDL-associated 45 kDa apolipoprotein from mouse serum that was identified as paraoxonase. In early work we have detected hepatic and serum paraoxonase activities in several neotropical American fish species. However, we did not determine to which protein it was associated in serum [15, 16]. Several studies have suggested that paraoxonase impairs the induction of inflammation in artery walls [17–19] by rapidly destroying biologically active mildly oxidized inflammatory lipids present in LDL [20–22]. Pacu, Piaractus mesopotamicus (Osteichthyes, Characidae), is a South-American fish of economic importance. This fish has been receiving special attention from Brazilian scientists who aim at improving its intensive culture. However, no information is available about their
946 lipoproteins. Moreover, it might be of importance to know whether any HDL-associated paraoxonase present in fish plasma could be related to some coronary arterial changes already observed in these animals during their sexual maturation [23, 24]. To address these questions, our laboratory has purified plasma lipoproteins from pacu. Here, we report the characterization of HDL from pacu plasma and we demonstrate that this fish lipoprotein contains a combined paraoxonase activity. 2. Materials and methods 2.1. Fish Specimens of immature pacu, Piaractus mesopotamicus, were kindly donated by the Centro Nacional de Pesquisa em Peixes Tropicais (CEPTA) of the Instituto Brasileiro do Meio Ambiente e Recursos Naturais (IBAMA). Pacus measured around 18 cm and on average weighed approximately 220 g. The animals were kept in 500 L aerated tanks equipped with biologic filter containing dechlorinated water filtered through activated charcoal. Fish were acclimated for at least 25 days before being used.
Folly et al. 2.4. Polyacrylamide gel electrophoresis Polyacrylamide slab gels (15 × 15 × 0.1 cm) were run under denaturing conditions with SDS [26] or under non-denaturing conditions [27] at a constant current of 100 V. The gels were stained with Coomassie Brilliant Blue G or Sudan Black B. 2.5. Lipid analysis Lipids associated with pacu HDL were extracted using the method described by Bligh and Dryer [28]. Extracted lipids were analyzed by one-dimensional thin-layer chromatography for neutral lipids [29], or by two-dimensional thin-layer chromatography for phospholipids [30]. The thin-layer plates (TLC) were stained with iodine vapor and charred by dipping the plate in a solution of 10% cupric sulfate (w/v) in 8% phosphoric acid (v/v) after the chromatography [31]. 2.6. Paraoxonase activity assay
Blood was withdrawn with a syringe by puncture of the dorsal aorta, and transferred to glass centrifuge tubes kept at 25 °C until total clotting. After being refrigerated for 30 min the tubes were centrifuged at 3 000 rpm for 10 min and the serum used in the experiments.
Paraoxonase activity was carried out by measuring p-nitrophenol (p-NP) released from serum paraoxon hydrolysis as follows: 50 µL of serum, 0.1 M Tris-HCl buffer solution, pH 8.5, 50 µL of a 3 M NaCl and 12 mM CaCl2 solution in a final volume of 0.3 mL, at 30 ºC. Absorbance in each tube, at alkaline pH, was registered at 400 nm using a Shimadzu UV-160A spectrophotometer in 1 cm optical path cuvettes. A p-nitrophenol standard curve (1.5 to 75 nmol concentration range) was used for calculating the amount of p-nitrophenol released. Further conditions were as described in Cunha Bastos et al. [15].
2.3. HDL purification
2.7. Sugar analysis
Pacu serum samples of 12 mL were mixed with KBr to produce a mixture with a density of 1.3 g/mL. The density was checked using a refractometer. Over this mixture a volume of 0.9% NaCl was carefully added using a peristaltic pump into a 40 mL centrifuge tube. The tube was then centrifuged for 4 h at 45 000 rpm in a Beckman 50VTi vertical rotor. After centrifugation, two distinct bands were visualized in the gradient tubes; one gray band of low density next to the top and a large yellow band (HDL) in the middle. The content of each tube was fractionated from top to bottom using a peristaltic pump. HDL-containing fractions were collected and applied on a Superose 6 HR HPLC gel filtration column, equilibrated with 0.01 M sodium phosphate, 0.15 M NaCl, pH 7.4 (PBS), at a flow rate of 0.5 mL/min. In order to obtain a highly purified HDL fraction, the peak obtained from the first chromatography was applied on a second run in the same column, as above. Protein concentration in each fraction was determined using a modified Lowry assay [25].
Total sugar was determined using the DuBois et al. assay [32]. Fractions containing HDL purified from HPLC were heated with 2 M trifluoracetic acid at 100 ºC for 2 h. The hydrolysate was then centrifuged in a clinical centrifuge for 10 min, the supernatant evaporated and analyzed through descending paper chromatography in isobutyric acid-ammonium hydroxide-water (3:2:1, v/v) using Whatman No. 1T paper. Subsequently, the chromatograms were dried and stained with silver nitrate [33].
2.2. Blood serum
3. Results Total lipoprotein profile of immature pacu serum obtained after KBr density gradient ultracentrifugation shows the predominance of HDL. A thin gray layer can be visualized at the top of the gradient, which was supposed to be VLDL. LDL was not visually distinguishable. The equilibrium density of HDL particle was determined to be 1.1267 g/mL (figure 1). KBr gradient fractions were then
A HDLP from pacu is associated with paraoxonase activity
947
Figure 1. KBr gradient density ultracentrifugation fractions of pacu serum. After KBr gradient density, protein content of fractions and paraoxonase activity were determined. The insert shows a typical KBr gradient density ultracentrifugation tube where it can be observed a large band which corresponds to HDL. The arrow indicates the position of the HDL band.
analyzed by SDS-PAGE, and we observed a very low amount of apo B-100, which indicated the presence of a low amount of the LDL (figure 2). KBr gradient fractions were assayed for paraoxonase activity and maximal paraoxon hydrolysis was associated with HDL-containing fractions (figure 1). Fractions enriched in HDL were pooled and applied to a Superose 6 HR 10/30 HPLC
Figure 2. Analysis of the KBr density gradient fractions by SDS-PAGE (5–20% polyacrylamide gradient). Lane 1, human VLDL; lane 2, human LDL; lane 3, top of the KBr density gradient; lane 17, bottom of the KBr density gradient; lane 18, molecular mass standards (phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, trypsin inhibitor and α-lactalbumin; values are indicated on the right); lane 19, human HDL. In lanes 7, 8, and 9 a very low amount of apo-B100 can be observed which indicates the presence of LDL.
column for HDL purification (figure 3A, B). After the second chromatography (figure 3B), the analysis of the major protein peak by native-PAGE demonstrated that HDL was free of any contaminant proteins (figure 3D). Analytical gel filtration chromatography showed that plasma HDL from pacu has a native molecular mass of 246 kDa (data not shown). SDS-PAGE analysis of purified
948
Folly et al.
Figure 3. Purification of pacu HDL by gel filtration on Superose 6HR HPLC. HDL containing fractions were applied on a Superose 6HR HPLC gel filtration column. Fractions of the HDL peak were analyzed by native-PAGE. The top of the peak with partially purified pacu HDL was applied to a second chromatography. Fractions were again analyzed by native-PAGE. A. Profile of first chromatography. B. Analysis of the fractions from the first gel filtration chromatography by native-PAGE (5–20% polyacrylamide gradient). C. Profile of second chromatography. D. Analysis of the fractions from the second gel filtration chromatography by native-PAGE. The arrow indicates pacu HDL.
HDL revealed that it is made up of four protein components, apoHDL-I (∼45 kDa), apoHDL-II (∼38 kDa), apoHDL-III (∼25 kDa, AI) and apoHDL-IV (∼12.5 kDa, AII) (figure 4). The apoHDL-III is the only glycosylated apoprotein as visualized by periodate-Schiff staining on SDS-PAGE (data not shown). Mannose is the only neutral sugar detected by paper chromatography in the HDL hydrolysate (data not shown). The chemical composition of the purified lipoprotein was 58% lipid, 7% sugar and 35% protein by weight (table IA). Neutral lipids and phospholipids were 40 and 18% of the total lipid content by weight, respectively (table IB). The lipid composition of pacu HDL was obtained by one-dimension and two-dimension-TLC. Following a decreasing order of abundance, phosphatidylethanolamine (PE), phosphatidylcholine (PC) and phosphatidylserine (PS) were identified (data not shown). The major neutral lipids found were cholesteryl ester and
triacylglycerol. Minor amounts of fatty acids, 1,3 and 1,2 diacylglycerols and monoacylglycerol were also observed (data not shown). Table II summarizes the purification of pacu HDL. Paraoxonase activity was assayed at various stages of purification and is also shown. The purification procedure resulted in a 23-fold enrichment of paraoxonase activity with a yield of about 21%. The final protein preparation can be stored at 0–4 °C in phosphate buffer containing 20% KBr for at least 2 months with no appreciable loss of paraoxonase activity. We observed a gradient of paraoxonase activity following an enrichment on HDL purification suggesting an association between both proteins. 4. Discussion In contrast to mammals, which mobilize carbohydrates, fish preferentially utilize lipids as their source of energy.
A HDLP from pacu is associated with paraoxonase activity
949
Table I. A. Chemical composition of pacu HDL. Protein content was determined by modified Lowry assay. Total sugar content of pacu HDL was determined by DuBois et al. [32] assay. Lipids were extracted using the method described by Bligh and Dryer [28]. B. Lipids were extracted and analyzed by two TLC (oneand two-dimensional). Extracted lipids were analyzed by onedimensional TLC for neutral lipids, or by two-dimensional TLC for phospholipids. TLC plates were stained with iodine vapor and charred with a solution of cupric sulfate and phosphoric acid. PS, phosphatidylserine; PC, phosphatidylcholine; PE, phosphatidylethanolamine. Each value is the mean of four experiments. A. Component
Weight (%) 35 ± 8.4 7 ± 1.2 58 ± 9.1
Protein Sugar Lipids B. Lipids
(%) 40.0 ± 5.1 18.0 ± 2.2 9.3 ± 0.8 7.4 ± 1.1 1.3 ± 0.04
Neutral Phospholipid PE PC PS
This leads to an increasing utilization of fishes as a model for studying lipid metabolism and transport [9, 34, 35]. Plasma lipoproteins from fish have become the focus of significant studies more than fifteen years ago, although detailed reports on the functions of their apoproteins have not yet been produced [36]. In the present study we demonstrate the first experimental evidence concerning the association of paraoxonase activity with HDL in fishes. At present, we have no clear understanding of what role paraoxonase could carry out in pacu. Since we have found very low detectable LDL levels in pacu plasma, as well as low levels of paraoxonase activity, probably both proteins are associated with the protection of LDL against oxidation. Considering the large evolutionary divergence and the differences between human and fish environments, plasma HDL molecules of pacu were remarkably similar to
Figure 4. Analysis of the HDL apolipoproteins by SDS-PAGE (5–20% polyacrylamide gradient). Lane 1, HDL apolipoproteins; lane 2, rainbow standard with molecular masses indicated.
human HDL in chemical and physical properties. Pacu HDL hydrated density of 1.1267g/mL was similar to that of human HDL3. Apoprotein organization of pacu HDL consists of 4 apolipoprotein with molecular masses similar to those described in other fish species and to human HDL [37, 38]. It contains two major species with 25 kDa
Table II. Paraoxonase activity during pacu HDL purification. Fraction
Serum HDL fraction Superose 1 Superose 2
Volume (mL)
14.00 1.70 5.10 10.20
Protein total (mg)
518.28 19.43 14.38 4.59
Paraoxonase activity U/mg protein (× 103)
U total (× 103)
Yield (%)
Purification factor (fold)
0.17 0.58 1.43 3.95
87.02 11.34 20.56 18.13
100 13.04 23.63 20.83
1 3.48 8.41 23.48
950 (apoAI like) and 12.5 kDa (apoAII like) and two minor species (45 kDa and 38 kDa). The relative molecular mass of the native complex was determined by gel filtration as approximately 246 kDa. Several studies have determined that the molecular mass value for HDL from human and different fish species ranges from > 180 kDa to > 360 kDa by gel filtration or gel electrophoresis [9]. In Dicentrarchus labrax it was obtained a molecular mass of approximately 200 kDa by gel filtration [39]; ∼180 kDa by analytical ultracentrifugation in Oncorynchus gorbuscha [40] and 50 kDa by electron microscopy volume calculations in Oncorynchus mykiss [11]. Thereby, those results demonstrated the several differences in the native HDL molecular mass in fishes. As in many other fish species [9] the HDL of Piaractus mesopotamicus contained carbohydrate and a large percentage of lipid (table I). We have detected 7% of carbohydrate in pacu HDL which is considerable higher when compared with other fish species, as described for Oncorynchus gorbuscha (pink salmon) with 2.4%. Mannose was the only sugar detected in the acid hydrolysate of HDL (data not shown) [40]. The distribution of lipid classes of pacu HDL, including neutral lipids and phospholipids, is very similar to that reported on many other vertebrate species [41]. The most abundant phospholipids were phosphatidylethanolamine and phosphatidylcholine as in Salmonidae, pacific sardine and humans [40, 42]. The percentage of triacylglycerols and cholesteryl ester we have found in pacu plasma HDL is higher than the reported values on different vertebrates species [9, 41]. Altogether, the above results show that as it is known for other Osteichthyes, with the exception of the coelacanth [9, 42], HDL dominates the lipoprotein profile in pacu plasma. This suggests that, as in Salmonidae and in the Characidae pacu most plasma cholesterol and triacylglycerols are transported by HDL as a result of their abundance. After purification pacu HDL exhibited a paraoxonase activity. This is the first report of a HDLassociated paraoxonase activity in a non-mammalian vertebrate. This suggest that fish lipoproteins, and their enzymatic activities, could be used for understanding the biochemical and physiological mechanisms related to the evolution of cardiovascular diseases.
Acknowledgments We wish to express our gratitude to Laboratório de Tecido Conjuntivo from Departamento de Bioquímica Médica, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro for helping us with the sugar analysis; Heloisa S.L. Coelho and Lilian Soares da Cunha Gomes for excellent technical assistance; to Dr. Mario A.C. Silva-Neto, Dr. Adriana Costero, Daniel Marques Golodne and Dr. Mohammed Shahabuddin (National Institutes of Health) for a critical reading of the manuscript. This
Folly et al. work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ).
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A high density lipoprotein from Piaractus mesopotamicus, pacu, (Osteichthyes, Characidae), is associated with paraoxonase activity Evelize Follya, Vera Lucia Cunha Bastosb, Marcelo V. Alvesb, Jayme Cunha Bastosb, Georgia C. Atellaa* a
Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Av. Bauhinia, 400, bloco H, 2o andar, sala 031, Ilha do Fundão, Rio de Janeiro, R.J., CEP. 21940-590, Brazil b Departamento de Bioquímica, Instituto Roberto Alcântara Gomes, Universidade do Estado do Rio de Janeiro, RJ, Brazil (Received 23 March 2001; accepted 24 September 2001) Abstract — We have characterized the serum lipoprotein profile and localized the serum paraoxonase activity of pacu, Piaractus mesopotamicus, a tropical fish species. The total lipoprotein profile of pacu serum obtained after KBr density ultracentrifugation shows the predominance of HDL (1.1267 g/mL). SDS-PAGE electrophoresis revealed a negligible amount of LDL. Pacu HDL was purified by gel filtration column on HPLC, and its molecular mass was estimated to be 246 kDa. Protein composition was 35%, and comprised four protein components with molecular masses of 45, 38, 25 and 12.5 kDa. Lipids represent 58% of total HDL, comprising 40% neutral lipids and 18% phospholipids by weight. The HDL contains 7% of carbohydrates, and mannose was the only sugar detected by paper chromatography in HDL hydrolysates. HDL-containing fractions showed the major paraoxonase activity. Purification of HDL resulted in a 23-fold enrichment of this activity. This is the first experimental evidence demonstrating the association of paraoxonase activity with a HDL in fish. © 2001 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved. paraoxonase / HDL / LDL / fish lipoprotein / fish biochemistry
1. Introduction Blood plasma lipoprotein biochemistry has received increasing attention since it became evident that changes in their levels and dysfunction in the synthesis of their apoproteins are related to cardiovascular disease in humans [1–5]. Lipoproteins are classified according to the densities at which they are isolated such as high-density lipoproteins (HDL), intermediate-density lipoproteins (IDL), low-density lipoproteins (LDL), very low-density lipoproteins (VLDL), and those that are secreted by the intestine following a dietary fat load, the chylomicrons [6]. Considering the amount of information about mammals, knowledge about fish plasma lipoprotein is limited to a small number of species, most from the northern hemisphere [7–9]. It has been reported that HDL dominates the lipoprotein profile in plasma from Salmonidae and may constitute up to 36% of the total plasma protein [8–11]. Nevertheless, many aspects of HDL metabolism in *Correspondence and reprints. E-mail address: [email protected] (G.C. Atella). Abbreviations: HPLC, high performance liquid chromatography; KBr, potassium bromide; SDS-PAGE, polyacrylamide gel electrophoresis with sodium duodecyl sulphate; native-PAGE, polyacrylamide gel eletrophoresis without sodium duodecyl sulphate.
Salmonidae are not well understood, as well as the functions of their apoproteins. The isolation and biochemical characterization of such apoproteins from an increasing number of fish species should facilitate the comprehension of the role that lipoproteins play in fish lipid metabolism. The heterogeneity of lipid-associated proteins in vertebrate plasma HDL is remarkable. Serum paraoxonase (EC 3.1.8.1) is a calcium-dependent HDL-associated ester hydrolase named after its ability to catalyze the hydrolysis of paraoxon, an organophosphate toxicant, into p-nitrophenol and diethyl phosphate [12, 13]. Blatter et al. [14] have isolated a novel HDL-associated 45 kDa apolipoprotein from mouse serum that was identified as paraoxonase. In early work we have detected hepatic and serum paraoxonase activities in several neotropical American fish species. However, we did not determine to which protein it was associated in serum [15, 16]. Several studies have suggested that paraoxonase impairs the induction of inflammation in artery walls [17–19] by rapidly destroying biologically active mildly oxidized inflammatory lipids present in LDL [20–22]. Pacu, Piaractus mesopotamicus (Osteichthyes, Characidae), is a South-American fish of economic importance. This fish has been receiving special attention from Brazilian scientists who aim at improving its intensive culture. However, no information is available about their
946 lipoproteins. Moreover, it might be of importance to know whether any HDL-associated paraoxonase present in fish plasma could be related to some coronary arterial changes already observed in these animals during their sexual maturation [23, 24]. To address these questions, our laboratory has purified plasma lipoproteins from pacu. Here, we report the characterization of HDL from pacu plasma and we demonstrate that this fish lipoprotein contains a combined paraoxonase activity. 2. Materials and methods 2.1. Fish Specimens of immature pacu, Piaractus mesopotamicus, were kindly donated by the Centro Nacional de Pesquisa em Peixes Tropicais (CEPTA) of the Instituto Brasileiro do Meio Ambiente e Recursos Naturais (IBAMA). Pacus measured around 18 cm and on average weighed approximately 220 g. The animals were kept in 500 L aerated tanks equipped with biologic filter containing dechlorinated water filtered through activated charcoal. Fish were acclimated for at least 25 days before being used.
Folly et al. 2.4. Polyacrylamide gel electrophoresis Polyacrylamide slab gels (15 × 15 × 0.1 cm) were run under denaturing conditions with SDS [26] or under non-denaturing conditions [27] at a constant current of 100 V. The gels were stained with Coomassie Brilliant Blue G or Sudan Black B. 2.5. Lipid analysis Lipids associated with pacu HDL were extracted using the method described by Bligh and Dryer [28]. Extracted lipids were analyzed by one-dimensional thin-layer chromatography for neutral lipids [29], or by two-dimensional thin-layer chromatography for phospholipids [30]. The thin-layer plates (TLC) were stained with iodine vapor and charred by dipping the plate in a solution of 10% cupric sulfate (w/v) in 8% phosphoric acid (v/v) after the chromatography [31]. 2.6. Paraoxonase activity assay
Blood was withdrawn with a syringe by puncture of the dorsal aorta, and transferred to glass centrifuge tubes kept at 25 °C until total clotting. After being refrigerated for 30 min the tubes were centrifuged at 3 000 rpm for 10 min and the serum used in the experiments.
Paraoxonase activity was carried out by measuring p-nitrophenol (p-NP) released from serum paraoxon hydrolysis as follows: 50 µL of serum, 0.1 M Tris-HCl buffer solution, pH 8.5, 50 µL of a 3 M NaCl and 12 mM CaCl2 solution in a final volume of 0.3 mL, at 30 ºC. Absorbance in each tube, at alkaline pH, was registered at 400 nm using a Shimadzu UV-160A spectrophotometer in 1 cm optical path cuvettes. A p-nitrophenol standard curve (1.5 to 75 nmol concentration range) was used for calculating the amount of p-nitrophenol released. Further conditions were as described in Cunha Bastos et al. [15].
2.3. HDL purification
2.7. Sugar analysis
Pacu serum samples of 12 mL were mixed with KBr to produce a mixture with a density of 1.3 g/mL. The density was checked using a refractometer. Over this mixture a volume of 0.9% NaCl was carefully added using a peristaltic pump into a 40 mL centrifuge tube. The tube was then centrifuged for 4 h at 45 000 rpm in a Beckman 50VTi vertical rotor. After centrifugation, two distinct bands were visualized in the gradient tubes; one gray band of low density next to the top and a large yellow band (HDL) in the middle. The content of each tube was fractionated from top to bottom using a peristaltic pump. HDL-containing fractions were collected and applied on a Superose 6 HR HPLC gel filtration column, equilibrated with 0.01 M sodium phosphate, 0.15 M NaCl, pH 7.4 (PBS), at a flow rate of 0.5 mL/min. In order to obtain a highly purified HDL fraction, the peak obtained from the first chromatography was applied on a second run in the same column, as above. Protein concentration in each fraction was determined using a modified Lowry assay [25].
Total sugar was determined using the DuBois et al. assay [32]. Fractions containing HDL purified from HPLC were heated with 2 M trifluoracetic acid at 100 ºC for 2 h. The hydrolysate was then centrifuged in a clinical centrifuge for 10 min, the supernatant evaporated and analyzed through descending paper chromatography in isobutyric acid-ammonium hydroxide-water (3:2:1, v/v) using Whatman No. 1T paper. Subsequently, the chromatograms were dried and stained with silver nitrate [33].
2.2. Blood serum
3. Results Total lipoprotein profile of immature pacu serum obtained after KBr density gradient ultracentrifugation shows the predominance of HDL. A thin gray layer can be visualized at the top of the gradient, which was supposed to be VLDL. LDL was not visually distinguishable. The equilibrium density of HDL particle was determined to be 1.1267 g/mL (figure 1). KBr gradient fractions were then
A HDLP from pacu is associated with paraoxonase activity
947
Figure 1. KBr gradient density ultracentrifugation fractions of pacu serum. After KBr gradient density, protein content of fractions and paraoxonase activity were determined. The insert shows a typical KBr gradient density ultracentrifugation tube where it can be observed a large band which corresponds to HDL. The arrow indicates the position of the HDL band.
analyzed by SDS-PAGE, and we observed a very low amount of apo B-100, which indicated the presence of a low amount of the LDL (figure 2). KBr gradient fractions were assayed for paraoxonase activity and maximal paraoxon hydrolysis was associated with HDL-containing fractions (figure 1). Fractions enriched in HDL were pooled and applied to a Superose 6 HR 10/30 HPLC
Figure 2. Analysis of the KBr density gradient fractions by SDS-PAGE (5–20% polyacrylamide gradient). Lane 1, human VLDL; lane 2, human LDL; lane 3, top of the KBr density gradient; lane 17, bottom of the KBr density gradient; lane 18, molecular mass standards (phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, trypsin inhibitor and α-lactalbumin; values are indicated on the right); lane 19, human HDL. In lanes 7, 8, and 9 a very low amount of apo-B100 can be observed which indicates the presence of LDL.
column for HDL purification (figure 3A, B). After the second chromatography (figure 3B), the analysis of the major protein peak by native-PAGE demonstrated that HDL was free of any contaminant proteins (figure 3D). Analytical gel filtration chromatography showed that plasma HDL from pacu has a native molecular mass of 246 kDa (data not shown). SDS-PAGE analysis of purified
948
Folly et al.
Figure 3. Purification of pacu HDL by gel filtration on Superose 6HR HPLC. HDL containing fractions were applied on a Superose 6HR HPLC gel filtration column. Fractions of the HDL peak were analyzed by native-PAGE. The top of the peak with partially purified pacu HDL was applied to a second chromatography. Fractions were again analyzed by native-PAGE. A. Profile of first chromatography. B. Analysis of the fractions from the first gel filtration chromatography by native-PAGE (5–20% polyacrylamide gradient). C. Profile of second chromatography. D. Analysis of the fractions from the second gel filtration chromatography by native-PAGE. The arrow indicates pacu HDL.
HDL revealed that it is made up of four protein components, apoHDL-I (∼45 kDa), apoHDL-II (∼38 kDa), apoHDL-III (∼25 kDa, AI) and apoHDL-IV (∼12.5 kDa, AII) (figure 4). The apoHDL-III is the only glycosylated apoprotein as visualized by periodate-Schiff staining on SDS-PAGE (data not shown). Mannose is the only neutral sugar detected by paper chromatography in the HDL hydrolysate (data not shown). The chemical composition of the purified lipoprotein was 58% lipid, 7% sugar and 35% protein by weight (table IA). Neutral lipids and phospholipids were 40 and 18% of the total lipid content by weight, respectively (table IB). The lipid composition of pacu HDL was obtained by one-dimension and two-dimension-TLC. Following a decreasing order of abundance, phosphatidylethanolamine (PE), phosphatidylcholine (PC) and phosphatidylserine (PS) were identified (data not shown). The major neutral lipids found were cholesteryl ester and
triacylglycerol. Minor amounts of fatty acids, 1,3 and 1,2 diacylglycerols and monoacylglycerol were also observed (data not shown). Table II summarizes the purification of pacu HDL. Paraoxonase activity was assayed at various stages of purification and is also shown. The purification procedure resulted in a 23-fold enrichment of paraoxonase activity with a yield of about 21%. The final protein preparation can be stored at 0–4 °C in phosphate buffer containing 20% KBr for at least 2 months with no appreciable loss of paraoxonase activity. We observed a gradient of paraoxonase activity following an enrichment on HDL purification suggesting an association between both proteins. 4. Discussion In contrast to mammals, which mobilize carbohydrates, fish preferentially utilize lipids as their source of energy.
A HDLP from pacu is associated with paraoxonase activity
949
Table I. A. Chemical composition of pacu HDL. Protein content was determined by modified Lowry assay. Total sugar content of pacu HDL was determined by DuBois et al. [32] assay. Lipids were extracted using the method described by Bligh and Dryer [28]. B. Lipids were extracted and analyzed by two TLC (oneand two-dimensional). Extracted lipids were analyzed by onedimensional TLC for neutral lipids, or by two-dimensional TLC for phospholipids. TLC plates were stained with iodine vapor and charred with a solution of cupric sulfate and phosphoric acid. PS, phosphatidylserine; PC, phosphatidylcholine; PE, phosphatidylethanolamine. Each value is the mean of four experiments. A. Component
Weight (%) 35 ± 8.4 7 ± 1.2 58 ± 9.1
Protein Sugar Lipids B. Lipids
(%) 40.0 ± 5.1 18.0 ± 2.2 9.3 ± 0.8 7.4 ± 1.1 1.3 ± 0.04
Neutral Phospholipid PE PC PS
This leads to an increasing utilization of fishes as a model for studying lipid metabolism and transport [9, 34, 35]. Plasma lipoproteins from fish have become the focus of significant studies more than fifteen years ago, although detailed reports on the functions of their apoproteins have not yet been produced [36]. In the present study we demonstrate the first experimental evidence concerning the association of paraoxonase activity with HDL in fishes. At present, we have no clear understanding of what role paraoxonase could carry out in pacu. Since we have found very low detectable LDL levels in pacu plasma, as well as low levels of paraoxonase activity, probably both proteins are associated with the protection of LDL against oxidation. Considering the large evolutionary divergence and the differences between human and fish environments, plasma HDL molecules of pacu were remarkably similar to
Figure 4. Analysis of the HDL apolipoproteins by SDS-PAGE (5–20% polyacrylamide gradient). Lane 1, HDL apolipoproteins; lane 2, rainbow standard with molecular masses indicated.
human HDL in chemical and physical properties. Pacu HDL hydrated density of 1.1267g/mL was similar to that of human HDL3. Apoprotein organization of pacu HDL consists of 4 apolipoprotein with molecular masses similar to those described in other fish species and to human HDL [37, 38]. It contains two major species with 25 kDa
Table II. Paraoxonase activity during pacu HDL purification. Fraction
Serum HDL fraction Superose 1 Superose 2
Volume (mL)
14.00 1.70 5.10 10.20
Protein total (mg)
518.28 19.43 14.38 4.59
Paraoxonase activity U/mg protein (× 103)
U total (× 103)
Yield (%)
Purification factor (fold)
0.17 0.58 1.43 3.95
87.02 11.34 20.56 18.13
100 13.04 23.63 20.83
1 3.48 8.41 23.48
950 (apoAI like) and 12.5 kDa (apoAII like) and two minor species (45 kDa and 38 kDa). The relative molecular mass of the native complex was determined by gel filtration as approximately 246 kDa. Several studies have determined that the molecular mass value for HDL from human and different fish species ranges from > 180 kDa to > 360 kDa by gel filtration or gel electrophoresis [9]. In Dicentrarchus labrax it was obtained a molecular mass of approximately 200 kDa by gel filtration [39]; ∼180 kDa by analytical ultracentrifugation in Oncorynchus gorbuscha [40] and 50 kDa by electron microscopy volume calculations in Oncorynchus mykiss [11]. Thereby, those results demonstrated the several differences in the native HDL molecular mass in fishes. As in many other fish species [9] the HDL of Piaractus mesopotamicus contained carbohydrate and a large percentage of lipid (table I). We have detected 7% of carbohydrate in pacu HDL which is considerable higher when compared with other fish species, as described for Oncorynchus gorbuscha (pink salmon) with 2.4%. Mannose was the only sugar detected in the acid hydrolysate of HDL (data not shown) [40]. The distribution of lipid classes of pacu HDL, including neutral lipids and phospholipids, is very similar to that reported on many other vertebrate species [41]. The most abundant phospholipids were phosphatidylethanolamine and phosphatidylcholine as in Salmonidae, pacific sardine and humans [40, 42]. The percentage of triacylglycerols and cholesteryl ester we have found in pacu plasma HDL is higher than the reported values on different vertebrates species [9, 41]. Altogether, the above results show that as it is known for other Osteichthyes, with the exception of the coelacanth [9, 42], HDL dominates the lipoprotein profile in pacu plasma. This suggests that, as in Salmonidae and in the Characidae pacu most plasma cholesterol and triacylglycerols are transported by HDL as a result of their abundance. After purification pacu HDL exhibited a paraoxonase activity. This is the first report of a HDLassociated paraoxonase activity in a non-mammalian vertebrate. This suggest that fish lipoproteins, and their enzymatic activities, could be used for understanding the biochemical and physiological mechanisms related to the evolution of cardiovascular diseases.
Acknowledgments We wish to express our gratitude to Laboratório de Tecido Conjuntivo from Departamento de Bioquímica Médica, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro for helping us with the sugar analysis; Heloisa S.L. Coelho and Lilian Soares da Cunha Gomes for excellent technical assistance; to Dr. Mario A.C. Silva-Neto, Dr. Adriana Costero, Daniel Marques Golodne and Dr. Mohammed Shahabuddin (National Institutes of Health) for a critical reading of the manuscript. This
Folly et al. work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ).
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