Identification of a Domain That Mediates Association of Platelet-activating Factor Acetylhydrolase with High Density Lipoprotein
2008; Elsevier BV; Volume: 283; Issue: 25 Linguagem: Inglês
10.1074/jbc.m802394200
ISSN1083-351X
AutoresAlison A. Gardner, Ethan C. Reichert, Matthew K. Topham, Diana M. Stafforini,
Tópico(s)Blood Coagulation and Thrombosis Mechanisms
ResumoThe plasma form of platelet-activating factor (PAF) acetylhydrolase (PAF-AH), also known as lipoprotein-associated phospholipase A2 (Lp-PLA2) inactivates potent lipid messengers such as PAF and modified phospholipids generated in settings of oxidant stress. In humans, PAF-AH circulates in blood in fully active form and associates with high and low density lipoproteins (HDL and LDL). Several studies suggest that the location of PAF-AH affects both the catalytic efficiency and the function of the enzyme in vivo. The distribution of PAF-AH among lipoproteins varies widely among mammals. Here, we report that mouse and human PAF-AHs associate with human HDL particles of different density. We made use of this observation in the development of a binding assay to identify domains required for association of human PAF-AH with human HDL. Sequence comparisons among species combined with domain-swapping and site-directed mutagenesis studies led us to the identification of C-terminal residues necessary for the association of human PAF-AH with human HDL. Interestingly, the region identified is not conserved among PAF-AHs, suggesting that PAF-AH interacts with HDL particles in a manner that is unique to each species. These findings contribute to our understanding of the mechanisms responsible for association of human PAF-AH with HDL and may facilitate future studies aimed at precisely determining the function of PAF-AH in each lipoprotein particle. The plasma form of platelet-activating factor (PAF) acetylhydrolase (PAF-AH), also known as lipoprotein-associated phospholipase A2 (Lp-PLA2) inactivates potent lipid messengers such as PAF and modified phospholipids generated in settings of oxidant stress. In humans, PAF-AH circulates in blood in fully active form and associates with high and low density lipoproteins (HDL and LDL). Several studies suggest that the location of PAF-AH affects both the catalytic efficiency and the function of the enzyme in vivo. The distribution of PAF-AH among lipoproteins varies widely among mammals. Here, we report that mouse and human PAF-AHs associate with human HDL particles of different density. We made use of this observation in the development of a binding assay to identify domains required for association of human PAF-AH with human HDL. Sequence comparisons among species combined with domain-swapping and site-directed mutagenesis studies led us to the identification of C-terminal residues necessary for the association of human PAF-AH with human HDL. Interestingly, the region identified is not conserved among PAF-AHs, suggesting that PAF-AH interacts with HDL particles in a manner that is unique to each species. These findings contribute to our understanding of the mechanisms responsible for association of human PAF-AH with HDL and may facilitate future studies aimed at precisely determining the function of PAF-AH in each lipoprotein particle. The platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) 2The abbreviations used are: PAF, platelet-activating factor, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; HDL, high density lipoprotein; hHDL, human HDL; LCAT, lecithin cholesterol acyltransferase; LDL, low density lipoprotein; Lp-PLA2, lipoprotein-associated phospholipase A2; p-hHDL, Pefabloc-treated human HDL; p-mHDL, Pefabloc-treated mouse HDL; PAF-AH, PAF acetylhydrolase; PLTP, phospholipid transfer protein. acetylhydrolase (PAF-AH) activity expressed in mammalian plasma is a phospholipase A2 secreted by cells of the hematopoietic system, primarily macrophages (1Stafforini D.M. Elstad M.R. McIntyre T.M. Zimmerman G.A. Prescott S.M. J. Biol. Chem. 1990; 265: 9682-9687Abstract Full Text PDF PubMed Google Scholar). This enzyme catalyzes the hydrolysis of short and/or oxidized acyl groups present in biologically active lipids such as PAF, oxidatively fragmented glycerophospholipids, esterified F2-isoprostanes, and phospholipid hydroperoxides (2Stremler K.E. Stafforini D.M. Prescott S.M. Zimmerman G.A. McIntyre T.M. J. Biol. Chem. 1989; 264: 5331-5334Abstract Full Text PDF PubMed Google Scholar, 3Stremler K.E. Stafforini D.M. Prescott S.M. McIntyre T.M. J. Biol. Chem. 1991; 266: 11095-11103Abstract Full Text PDF PubMed Google Scholar, 4Stafforini D.M. Sheller J.R. Blackwell T.S. Sapirstein A. Yull F.E. McIntyre T.M. Bonventre J.V. Prescott S.M. Roberts 2nd., L.J. J. Biol. Chem. 2006; 281: 4616-4623Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 5Kriska T. Marathe G.K. Schmidt J.C. McIntyre T.M. Girotti A.W. J. Biol. Chem. 2007; 282: 100-108Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). We and others (6Stafforini D.M. McIntyre T.M. Zimmerman G.A. Prescott S.M. Crit. Rev. Clin. Lab. Sci. 2003; 40: 643-672Crossref PubMed Scopus (184) Google Scholar) have proposed that the most likely function of this enzymatic activity is to provide a safety mechanism to limit the levels of pro-inflammatory mediators, the accumulation of which can have undesirable consequences. In human plasma, two-thirds of the PAF-AH activity are found associated with LDL and one-third circulates as a complex with HDL. This distribution profile varies among species, possibly because of differences among PAF-AH orthologs, combined with a wide diversity of lipoprotein levels and composition. Our previous studies suggested that intrinsic properties of PAF-AH played important roles as determinants of the location of the enzyme in vivo (7Stafforini D.M. Tjoelker L.W. McCormick S.P. Vaitkus D. McIntyre T.M. Gray P.W. Young S.G. Prescott S.M. J. Biol. Chem. 1999; 274: 7018-7024Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). These findings led to the identification of PAF-AH domains essential for the human enzyme to associate with LDL (7Stafforini D.M. Tjoelker L.W. McCormick S.P. Vaitkus D. McIntyre T.M. Gray P.W. Young S.G. Prescott S.M. J. Biol. Chem. 1999; 274: 7018-7024Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). A number of clinical studies from various laboratories indicate that altered location of PAF-AH correlates with human diseases such as coronary artery disease (8Karabina S.A. Elisaf M. Bairaktari E. Tzallas C. Siamopoulos K.C. Tselepis A.D. Eur. J. Clin. Investig. 1997; 27: 595-602Crossref PubMed Scopus (66) Google Scholar), hypercholesterolemia (9Tsimihodimos V. Karabina S.A. Tambaki A.P. Bairaktari E. Miltiadous G. Goudevenos J.A. Cariolou M.A. Chapman M.J. Tselepis A.D. Elisaf M. J. Lipid Res. 2002; 43: 256-263Abstract Full Text Full Text PDF PubMed Google Scholar), paroxysmal atrial fibrillation (10Okamura K. Miura S. Zhang B. Uehara Y. Matsuo K. Kumagai K. Saku K. Circ. J. 2007; 71: 214-219Crossref PubMed Scopus (25) Google Scholar), and chronic kidney disease (11Papavasiliou E.C. Gouva C. Siamopoulos K.C. Tselepis A.D. Nephrol. Dial Transplant. 2006; 21: 1270-1277Crossref PubMed Scopus (24) Google Scholar). These observations suggest that the distribution of PAF-AH in lipoproteins may define its physio-pathological function in humans. We previously reported that PAF-AH can migrate among lipoproteins (12Stafforini D.M. McIntyre T.M. Carter M.E. Prescott S.M. J. Biol. Chem. 1987; 262: 4215-4222Abstract Full Text PDF PubMed Google Scholar), found that the location of the enzyme impacts its catalytic activity (13Stafforini D.M. Carter M.E. Zimmerman G.A. McIntyre T.M. Prescott S.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2393-2397Crossref PubMed Scopus (86) Google Scholar), and presented evidence supporting a model wherein HDL may act as a transport system to distribute PAF-AH among LDL particles (14Stafforini D.M. Zimmerman G.A. McIntyre T.M. Prescott S.M. Trans. Assoc. Am Physicians. 1992; 105: 44-63PubMed Google Scholar). These combined observations suggest that the lipoprotein environment regulates the function of PAF-AH, and they underscore the need to precisely characterize the nature of these interactions. Here, we report that individual species display unique lipoprotein distribution profiles and seem to utilize distinct PAF-AH domains to associate with the particles. In addition, we report the identification of key C-terminal residues required for association of human PAF-AH with human HDL. These studies are likely to facilitate the development of much needed in vivo model systems that faithfully recapitulate the unique lipoprotein distribution of human plasma PAF-AH and that can potentially be utilized to assess the role of the lipoprotein environment on the function of this enzyme. Materials—[3H-acetyl]PAF was purchased from Amersham Biosciences (Piscataway, NJ) and unlabeled PAF was from Avanti Polar Lipids (Alabaster, AL). Pfu was from Stratagene (La Jolla, CA), and dNTPs were purchased from Fermentas Inc. (Hanover, MD). Pefabloc was from Calbiochem. Secondary antibodies were purchased from BioSource International (Camarillo, CA). All other reagents were from Sigma. In some experiments we utilized recombinant human PAF-AH (Pafase®), which was a generous gift from ICOS Corporation (Bothell, WA). This protein was expressed as a truncation product starting at M-46 and was purified from bacterial sources. HDL particles were isolated as previously described (12Stafforini D.M. McIntyre T.M. Carter M.E. Prescott S.M. J. Biol. Chem. 1987; 262: 4215-4222Abstract Full Text PDF PubMed Google Scholar) and were treated with Pefabloc according to the instructions provided by the manufacturer. The Pefabloc-treated HDL (pHDL) was subjected to exhaustive dialysis against phosphate-buffered saline, at 4 °C. PAF-AH Activity Determinations—PAF-AH activity was determined by our previously described radiometric assay, using [3H-acetyl]PAF as the substrate (15Stafforini D.M. McIntyre T.M. Prescott S.M. Methods Enzymol. 1990; 187: 344-357Crossref PubMed Scopus (66) Google Scholar). We separated excess substrate from the product, [3H-acetate], by reverse phase column chromatography, as described (15Stafforini D.M. McIntyre T.M. Prescott S.M. Methods Enzymol. 1990; 187: 344-357Crossref PubMed Scopus (66) Google Scholar). Mutant Generation and Vectors—Site-directed mutagenesis was performed by a two-step amplification protocol using Pfu as the polymerase, as previously described (16MacRitchie A.N. Gardner A.A. Prescott S.M. Stafforini D.M. Faseb. J. 2007; 21: 1164-1176Crossref PubMed Scopus (33) Google Scholar). A FLAG tag was inserted at the N-terminal end to facilitate immunoblot detection and purification. The products were cloned into a pUC cloning vector under the control of the tryptophan promoter, as described (16MacRitchie A.N. Gardner A.A. Prescott S.M. Stafforini D.M. Faseb. J. 2007; 21: 1164-1176Crossref PubMed Scopus (33) Google Scholar). Plasmid DNA was purified using a plasmid miniprep purification kit (Qiagen Inc, Valencia, CA). Expression and Purification of Mutant and Chimeric Proteins—We generated various truncated forms of PAF-AH that started at Leu-41 and Ile-42 for mouse and human PAF-AH, respectively, and expressed the recombinant proteins in the Escherichia coli strain BL-21. Protein extracts were obtained as previously described (16MacRitchie A.N. Gardner A.A. Prescott S.M. Stafforini D.M. Faseb. J. 2007; 21: 1164-1176Crossref PubMed Scopus (33) Google Scholar). Where indicated, the supernatants were purified using anti-FLAG affinity beads, following the instructions provided by the manufacturer (Sigma). We determined enzymatic activity and protein content of the mutant preparations recovered after purification and assessed the level of expression by Western analyses using a monoclonal anti-FLAG (M2) antibody (Sigma), as described (3Stremler K.E. Stafforini D.M. Prescott S.M. McIntyre T.M. J. Biol. Chem. 1991; 266: 11095-11103Abstract Full Text PDF PubMed Google Scholar). Unless otherwise stated, the mutant proteins expressed significant levels of PAF-AH activity and mass, suggesting that the folding of the recombinant proteins was comparable to that of wild-type PAF-AH. HDL Binding Assay—To test binding to HDL we incubated a source of PAF-AH with pHDL (range: 4–29 mg) for 30–120 min at 37 °C, in a total volume of 500 μl. The amount of detergent was normalized and kept at a level that did not affect the integrity of HDL particles as judged by their ability to bind wild-type PAF-AH. The mixtures then were adjusted to a volume of 10 ml and a density of 1.3 g/ml with solid KBr. The solutions were layered with 0.9% NaCl and centrifuged at 50,000 rpm in a VTi 50 Beckman rotor, for 3 h at 4°C. The gradients were fractionated, and individual fractions were assayed for PAF-AH activity, as described (15Stafforini D.M. McIntyre T.M. Prescott S.M. Methods Enzymol. 1990; 187: 344-357Crossref PubMed Scopus (66) Google Scholar). Where indicated, we utilized "modified" KBr density gradients designed to improve resolution in the heavy density region. These gradients were identical to those described above except that they were generated by layering 20 ml of a 1.3 g/ml KBr solution containing pHDL and PAF-AH with 0.9% NaCl. The Association of Plasma PAF-AH with Lipoproteins and the Total Levels of Enzymatic Activity Vary among Species—Previous studies reported differences in the distribution of PAF-AH activity in human compared with mouse or rat plasma, but to our knowledge no comparative studies among species have been presented to date. We investigated the distribution of PAF-AH activity in freshly isolated plasma from six different species and found vast differences in the distribution pattern among lipoproteins (Fig. 1). PAF-AH associated with both LDL and HDL in plasma from bovine, canine, and human sources (Fig. 1, A–C). However, the distribution between lipoprotein particles varied among the three species; notably, humans were the only species in which most of the circulating PAF-AH associated with LDL. In rat plasma, PAF-AH associated exclusively with HDL particles, in agreement with a previous report (Fig. 1D) (17Pritchard P.H. Biochem. J. 1987; 246: 791-794Crossref PubMed Scopus (36) Google Scholar). Initial studies revealed that the densities of HDL particles with which PAF-AH associated were higher in guinea pig and mouse compared with other species (not shown), so it was necessary to adapt the standard fractionation protocol to improve resolution in the heavy density region. We adjusted the density of the gradients as described under "Experimental Procedures" and found almost complete association of guinea pig and mouse PAF-AH activities with HDL particles (Fig. 1, E and F), as previously reported (18Theilmeier G. De Geest B. Van Veldhoven P.P. Stengel D. Michiels C. Lox M. Landeloos M. Chapman M.J. Ninio E. Collen D. Himpens B. Holvoet P. Faseb. J. 2000; 14: 2032-2039Crossref PubMed Scopus (136) Google Scholar, 19Tsaoussis V. Vakirtzi-Lemonias C. J. Lipid Mediat. Cell Signal. 1994; 9: 317-331PubMed Google Scholar). Our studies also provided a quantitative comparison of the total amount of plasma PAF-AH activity expressed in six species tested. Interestingly, species in which PAF-AH associated with both HDL and LDL expressed lower total levels of activity compared with those in which the location of the enzyme was limited to HDL particles (Table 1).TABLE 1Expression levels of PAF-AH activity in freshly isolated plasma samples from various speciesPlasma sourcePAF-AH activitynmol/min/mlnmol/min/mgBovine47.7 ± 0.60.33 ± 0.01Human73.2 ± 6.81.23 ± 0.23Dog61.4 ± 2.02.32 ± 0.15Rat183.1 ± 3.13.89 ± 0.13Guinea pig234.1 ± 9.510.78 ± 0.88Mouse630.0 ± 5.215.72 ± 0.26 Open table in a new tab Development and Characterization of a PAF-AH/HDL Binding Assay—To characterize the nature of the interaction between PAF-AH and HDL particles, we developed a binding assay that consisted of incubating either the purified recombinant enzyme, or solubilized extracts expressing various mutant and chimeric PAF-AH constructs, with Pefabloc-treated human HDL particles that lacked enzymatic activity (p-hHDL). To assess the extent of binding to p-hHDL, we subjected the mixtures to ultracentrifugation, fractionation, and activity determinations. We found that under the conditions described under "Experimental Procedures" the human wild-type enzyme, supplied in either purified form or as a solubilized bacterial extract, associated with particles of density identical to that of natural PAF-AH-containing human HDL particles (compare Fig. 1C and supplemental Fig. S1A). Optimal binding was observed after incubation for 1 h at 37°C (supplemental Fig. S1B). To ensure that the amount of p-hHDL supplemented to each assay was not the factor limiting the extent of binding, we varied the amount of p-hHDL using a fixed level of PAF-AH and found comparable results when p-hHDL ranged between 9 and 29 mg (supplemental Fig. S1C). Additional studies showed that p-hHDL levels could be further reduced to 4 mg without affecting the extent of binding (not shown). We also found that a fixed amount of p-hHDL (9 mg) supported binding of PAF-AH over a relatively wide range of enzyme concentrations (supplemental Fig. S1D). These combined results identified experimental conditions that resulted in optimal binding of PAF-AH to exogenous human HDL. In addition, the studies demonstrated that solubilized bacterial extracts behaved in a manner equivalent to that of purified enzyme sources. Subjecting various types of PAF-AH-containing samples to the binding assay in the absence of exogenous p-hHDL resulted in complete loss of enzymatic activity (not shown). Mouse and Human PAF-AHs Associate with Human HDL Particles of Different Density—Our next goal was to investigate whether association of PAF-AH with HDL was defined by the enzyme, the lipoprotein particles, or both. To address this issue, we compared the ability of the mouse and human enzymes to associate with heterologous p-HDL particles. Because the density of mouse PAF-AH-containing HDL particles was higher than that of human particles (Fig. 1), we adjusted the density of the gradients to optimize separation within the HDL region, as described above for Fig. 1, E and F. This enabled us to clearly identify a peak of PAF-AH-containing HDL particles in mouse serum (fractions 5–9, Fig. 2A). The association of endogenous PAF-AH with lipoproteins was not affected by this technical adjustment as judged by the similar behavior of the human plasma enzyme in the two types of gradients (compare Figs. 1C and 2B). We next found that mouse and human PAF-AHs associated with Pefabloc-treated murine HDL (p-mHDL) particles of the same density (i.e. fractions 5–9, Fig. 2C). In contrast, we observed that human PAF-AH associated with lighter p-hHDL particles compared with mouse PAF-AH (Fig. 2D). These results suggested that the mechanisms that govern association of PAF-AH with HDL vary among species and include contributions from both the enzyme and the lipoprotein particles. In addition, these results provided the basis for the next series of experiments. The C-terminal End of Human PAF-AH Mediates Binding to Human HDL—We next focused our studies on the identification of domain(s) responsible for the association of human PAF-AH with p-hHDL. The observation that the mouse and human enzymes associated with p-hHDL particles of different density (Fig. 2D) provided us with a tool to search for discrete protein domains that contributed to the interaction. Our strategy consisted of replacing regions within human PAF-AH with corresponding sequences derived from the mouse ortholog, and then testing binding of the chimeric constructs to p-hHDL (Fig. 3A, Ref. 16MacRitchie A.N. Gardner A.A. Prescott S.M. Stafforini D.M. Faseb. J. 2007; 21: 1164-1176Crossref PubMed Scopus (33) Google Scholar). We found that replacement of residues 339–441 in the human protein with the corresponding murine sequences (construct V) resulted in a chimeric protein whose behavior differed from that of wild-type PAF-AH (compare Fig. 3, B and G). In contrast, the remaining chimeric constructs (constructs I-IV) displayed binding to p-hHDL similar to that of the wild-type human enzyme (Fig. 3, C–F). To further investigate the role of individual PAF-AH domains in binding to p-hHDL, we utilized a complementary approach. We generated a second set of chimeric constructs in which we increased the contribution of sequences derived from human PAF-AH, as we proportionately decreased representation of the mouse protein (Fig. 4A). We next tested the ability of these constructs to associate with p-hHDL and found evidence confirming a requirement for the human C terminus in binding to p-hHDL (Fig. 4, B–E). These studies supported and refined our previous findings as they revealed that human PAF-AH associated with p-hHDL through a domain comprised of amino acids 340 and 415.FIGURE 4The C-terminal end of human PAF-AH confers the mouse ortholog the ability to associate with human HDL. A, schematic representation of mouse/human chimeric constructs generated and tested for binding to human HDL. B, comparison of human and mouse PAF-AH association with human HDL. The data presented in Fig. 3B are reproduced for comparison with panels C–E. C–E, binding of chimeric constructs VI-VIII to human HDL. Solubilized extracts expressing the chimeric constructs indicated (5 nmol/min) were incubated with p-hHDL (14.2 mg) and subjected to ultracentrifugation. All the studies depicted in this figure were conducted using standard KBr density gradients.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Identification of Specific Residues Involved in PAF-AH/HDL Interaction—Our next goal was to more precisely map the minimal domain necessary to confer binding to p-hHDL. To accomplish this, we cloned the guinea pig and rat PAF-AH cDNAs, expressed and purified the recombinant proteins, and then investigated whether they associated with p-hHDL. We found that the behavior of these proteins differed from that of human PAF-AH and resembled the binding pattern displayed by the murine ortholog (compare supplemental Fig. S2 and Fig. 4B). Next, we aligned the sequences comprised by amino acids 340–415 from the human, mouse, rat, and guinea pig orthologs and searched for residues in the human sequence that were absent in all the rodent orthologs (Fig. 5A). This led to the identification of Arg-347, His-367, Lys-370, Asn-378, Ala-379, Ser-384, and Ile-409 as candidates for further testing (Fig. 5A). We generated human R347K and N378R mutants and found that these mutants displayed normal binding to p-hHDL (Fig. 5, B and C). Next, we replaced the mouse string NKLT comprising residues 366–369 with HMLK, corresponding to amino acids 367–370 in the human protein. Interestingly, the resulting chimeric construct (mHMLK) associated with p-hHDL in a manner similar to that of the human wild-type protein (Fig. 5D). As expected, mHMLK constructs that mimicked the human protein at residues 384 and 409 behaved in a manner comparable to that of the mHMLK mutant (not shown). The role of Ala-379 will be discussed below. These results suggested a key role for the HMLK domain in the interaction of PAF-AH with p-hHDL. To characterize contributions from individual residues within the HMLK domain, we conducted additional experiments (Fig. 6A). We found that mutation of residues Met-368 and Leu-369 prevented binding to p-hHDL (Fig. 6, C and D). In addition, individual replacement of His-367 and Lys-370 with the corresponding mouse residues affected binding to a lesser extent (Fig. 6, B and E). These combined results further establish participation of the HMLK domain in the association of human PAF-AH with p-hHDL. Our results provide a possible explanation to account for the failure of rodent PAF-AH orthologs to associate with p-hHDL.FIGURE 6Individual contribution of residues within the HMLK domain to association with human HDL. A, amino acid alignment of human, mouse, rat, and guinea pig PAF-AHs in the C-terminal region comprised by amino acids 367–370. B–E, binding of human PAF-AH mutants H367N, M368K, L369A, and K370T to human HDL. Solubilized extracts expressing the proteins indicated (5 nmol/min) were incubated with p-hHDL (range: 12.1–4.1 mg) and subjected to ultracentrifugation. All the studies depicted in this figure were conducted using standard KBr density gradients.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Naturally Occurring Polymorphic Forms of PAF-AH Display Normal Binding to Human HDL—The human plasma PAF-AH gene displays several polymorphisms and three of them (R92H, I198T, and A379V) result in amino acid changes that have been described in the Caucasian population (20Karasawa K. Biochim. Biophys. Acta. 2006; 1761: 1359-1372Crossref PubMed Scopus (93) Google Scholar, 21Bell R. Collier D.A. Rice S.Q. Roberts G.W. MacPhee C.H. Kerwin R.W. Price J. Gloger I.S. Biochem. Biophys. Res. Commun. 1997; 241: 630-635Crossref PubMed Scopus (31) Google Scholar, 22Stafforini D.M. Pharmacogenomics. 2001; 2: 163-175Crossref PubMed Scopus (18) Google Scholar). The results depicted in Fig. 4 did not allow us to rule out contributions from these residues to binding to p-hHDL. First, Arg-92 is conserved between the human and mouse orthologs; second, there is a conservative substitution at position 198 of the mouse ortholog (Val-197); third, a valine replaces Ala-379 in the mouse ortholog. To investigate whether the presence of these polymorphisms affected association of human PAF-AH with p-hHDL, we expressed human constructs R92H, I198T, and A379V and then tested the ability of extracts expressing the recombinant proteins to bind to the lipoprotein. We found (Fig. 7) that the three naturally occurring PAF-AH polymorphic forms displayed normal binding to p-hHDL. In addition, introduction of a histidine or a threonine residue at positions 91 and 197, respectively, of the mouse HMLK mutant (see Fig. 5D) did not prevent association with p-hHDL (not shown). These combined data firmly establish that the polymorphic forms of PAF-AH R92H, I198T, and A379V retain the ability to associate with HDL. In recent years, PAF-AH has become known as Lp-PLA2 because of the fact that the activity circulates in plasma as a complex with LDL and HDL (12Stafforini D.M. McIntyre T.M. Carter M.E. Prescott S.M. J. Biol. Chem. 1987; 262: 4215-4222Abstract Full Text PDF PubMed Google Scholar, 23Tselepis A.D. Dentan C. Karabina S.A. Chapman M.J. Ninio E. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1764-1773Crossref PubMed Scopus (192) Google Scholar). Elevated expression of PAF-AH activity and/or protein has been reported in patients with coronary artery disease by a number of groups, and is thought to be an independent predictor of disease severity in humans (24Sabatine M.S. Morrow D.A. O'Donoghue M. Jablonksi K.A. Rice M.M. Solomon S. Rosenberg Y. Domanski M.J. Hsia J. Arterioscler. Thromb. Vasc. Biol. 2007; 27: 2463-2469Crossref PubMed Scopus (113) Google Scholar, 25Packard C.J. O'Reilly D.S. Caslake M.J. McMahon A.D. Ford I. Cooney J. Macphee C.H. Suckling K.E. Krishna M. Wilkinson F.E. Rumley A. Lowe G.D. N. Engl. J. Med. 2000; 343: 1148-1155Crossref PubMed Scopus (769) Google Scholar, 26Zalewski A. Nelson J.J. Hegg L. Macphee C. Clin. Chem. 2006; 52: 1645-1650Crossref PubMed Scopus (60) Google Scholar, 27Koenig W. Khuseyinova N. Lowel H. Trischler G. Meisinger C. Circulation. 2004; 110: 1903-1908Crossref PubMed Scopus (283) Google Scholar, 28Ballantyne C.M. Hoogeveen R.C. Bang H. Coresh J. Folsom A.R. Heiss G. Sharrett A.R. Circulation. 2004; 109: 837-842Crossref PubMed Scopus (560) Google Scholar, 29Oei H.H. van der Meer I.M. Hofman A. Koudstaal P.J. Stijnen T. Breteler M.M. Witteman J.C. Circulation. 2005; 111: 570-575Crossref PubMed Scopus (394) Google Scholar, 30Brilakis E.S. McConnell J.P. Lennon R.J. Elesber A.A. Meyer J.G. Berger P.B. Eur. Heart J. 2005; 26: 137-144Crossref PubMed Scopus (203) Google Scholar, 31Corsetti J.P. Rainwater D.L. Moss A.J. Zareba W. Sparks C.E. Clin. Chem. 2006; 52: 1331-1338Crossref PubMed Scopus (76) Google Scholar, 32Iribarren C. Gross M.D. Darbinian J.A. Jacobs Jr., D.R. Sidney S. Loria C.M. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 216-221Crossref PubMed Scopus (104) Google Scholar, 33Khuseyinova N. Koenig W. Mol. Diagn. Ther. 2007; 11: 203-217Crossref PubMed Scopus (18) Google Scholar). In addition, PAF-AH protein has been detected in atherosclerotic plaques of humans (34Kolodgie F.D. Burke A.P. Skorija K.S. Ladich E. Kutys R. Makuria A.T. Virmani R. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 2523-2529Crossref PubMed Scopus (303) Google Scholar, 35Papaspyridonos M. Smith A. Burnand K.G. Taylor P. Padayachee S. Suckling K.E. James C.H. Greaves D.R. Patel L. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 1837-1844Crossref PubMed Scopus (142) Google Scholar) and in experimental animals (36Hakkinen T. Luoma J.S. Hiltunen M.O. Macphee C.H. Milliner K.J. Patel L. Rice S.Q. Tew D.G. Karkola K. Yla-Herttuala S. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2909-2917Crossref PubMed Scopus (230) Google Scholar). These correlative findings led to the proposition that PAF-AH actively contributes to the pathogenesis of vascular disease and that inhibiting its enzymatic activity could be beneficial for the treatment of atherosclerosis and related disorders (37Macphee C.H. Nelson J. Zalewski A. Curr. Opin. Pharmacol. 2006; 6: 154-161Crossref PubMed Scopus (57) Google Scholar, 38Macphee C.H. Nelson J.J. Zalewski A. Curr. Opin. Lipidol. 2005; 16: 442-446Crossref PubMed Scopus (81) Google Scholar, 39Zalewski A. Macphee C. N
Referência(s)