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Structural Basis of Specific Interactions of Lp-PLA 2 with HDL Revealed By Structural basis of specifi c interactions of Lp-PLA2 with HDL revealed by hydrogen deuterium exchange mass spectrometry 1, † † 2, Jian Cao, * Yuan-Hao Hsu, * Sheng Li, Virgil L. Woods , Jr., and Edward A. Dennis * Departments of Chemistry and Biochemistry and Pharmacology* and Department of Medicine and Biomedical Sciences Graduate Program, † School of Medicine, University of California, San Diego , La Jolla, CA 92093-0601 Abstract Lipoprotein-associated phospholipase A2 (Lp- Human lipoprotein-associated phospholipase A2 (Lp- PLA2 ), specifi cally Group VIIA PLA2 , is a member of the PLA 2 ), also known as plasma platelet activating factor phospholipase A superfamily and is found mainly associ- 2 acetylhydrolase, belongs to the phospholipase A2 super- ated with LDL and HDL in human plasma. Lp-PLA2 is family (designated as Group VIIA PLA ) ( 1 ). Lp-PLA is considered as a risk factor, a potential biomarker, a target 2 2 found mainly associated with lipoproteins in human for therapy in the treatment of cardiovascular disease, and plasma, about 70% with LDL and another 30% with HDL evidence suggests that the level of Lp-PLA2 in plasma is associated with the risk of future cardiovascular and stroke ( 2 ). Lp-PLA2 was fi rst identifi ed as an enzyme that inacti- events. The differential location of the enzyme in LDL/ vates platelet activating factor (PAF) by hydrolyzing the HDL lipoproteins has been suggested to affect Lp-PLA2 acetyl group at the sn -2 position to produce lyso-PAF and function and/or its physiological role and an abnormal dis- acetate ( 3 ). Later, it was found that Lp-PLA2 has compara- tribution of the enzyme may correlate with diseases. Al- ble activity toward phosphatidycholines (PCs) containing though a mutagenesis study suggested that a surface helix short acyl chains at the sn -2 position, oxidized PCs (oxPCs) (residues 362–369) mediates the association between Lp- and F2 -isoprostanes esterifi ed phospholipids, which are PLA2 and HDL, the molecular details and mechanism of as- sociation has remained unknown. We have now employed phospholipids with oxidized fatty acyl chains at the sn -2 hydrogen deuterium exchange mass spectrometry to char- position ( 4–6 ). Lp-PLA2 is secreted predominantly by mac- acterize the interaction between recombinant human Lp- rophages ( 7 ) and it has been shown that its expression is PLA2 and human HDL. We have found that specifi c residues signifi cantly increased during activation of macrophages 113–120, 192–204, and 360–368 likely mediate HDL binding. in atherosclerotic lesions ( 8 ). By inactivating PAF and hydro- In a previous study, we showed that residues 113–120 are lyzing oxPC in the oxidized LDL particles, Lp-PLA2 could important for Lp-PLA2 -liposome interactions. We now fi nd act as an anti-atherogenic enzyme ( 9 ). However, a large that residues 192–204 show a decreased deuteration level number of studies have suggested a pro-atherosclerotic when Lp-PLA is exposed to apoA-I, but not apoA-II, the 2 role ( 10–12 ). This is because Lp-PLA generates the pro- most abundant apoproteins in HDL, and additionally, resi- 2 dues 360–368 are only affected by HDL. The results sug- infl ammatory and pro-apoptotic lipid mediators lyso-PC and gest that apoA-I and phospholipid membranes play crucial oxidized nonesterifi ed fatty acids, which play an important roles in Lp-PLA2 localization to HDL. —Cao, J., Y.-H. Hsu, role in the development of atherosclerotic necrotic cores. S. Li, V. L. Woods, Jr., and E. A. Dennis. Structural basis Increased Lp-PLA2 mass or activity in plasma is believed of specifi c interactions of Lp-PLA2 with HDL revealed by to be associated with an increased risk of various cardiovas- hydrogen deuterium exchange mass spectrometry. J. Lipid cular diseases ( 13 ). Therefore, Lp-PLA2 is considered as a Res . 2013. 54: 127–133. biomarker for cardiovascular diseases and it has been approved as a diagonostic for stroke and coronary disease Supplementary key words lipoprotein-associated phospholipase A2 • high density lipoprotein • apolipoproteins • enzymology Abbreviations: DXMS, deuterium exchange mass spectrometry; Lp-PLA2 , lipoprotein-associated phospholipase A2 ; PAF, platelet activating This study was supported by the National Institutes of Health grant RO1- factor; PC, phosphatidycholine; oxPC, oxidized phosphatidycholine . GM20501 (E.A.D.). Its contents are solely the responsibility of the authors and 1 do not necessarily represent the offi cial views of the National Institutes of Current address: Department of Chemistry, Tunghai University, Health. Taichung, Taiwan. 2 To whom correspondence should be addressed. Manuscript received 5 July 2012 and in revised form 26 September 2012. e-mail: [email protected] Published, JLR Papers in Press, October 22, 2012 The online version of this article (available at http://www.jlr.org) DOI 10.1194/jlr.M030221 contains supplementary data in the form of fi ve fi gures. Copyright © 2013 by the American Society for Biochemistry and Molecular Biology, Inc. This article is available online at http://www.jlr.org Journal of Lipid Research Volume 54, 2013 127 risk by the US Food and Drug Administration ( 14 ). In EXPERIMENTAL METHODS addition to serving as a biomarker, Lp-PLA2 is also consid- ered as a target for the treatment of cardiovascular disease. Materials A GSK inhibitor, Darapladid or SB-40848, has been shown D2 O (99.6%) was obtained from Cambridge Isotope Laborato- 3 to be a good selective inhibitor and has been successfully ries. [ H-acteyl] PAF was purchased from Perkin Elmer. Unlabeled applied in a diabetic and hypercholesterolemic swine PAF and 1,2-dimyristoyl- sn -glycero-3-phosphocholine were from Avanti Polar Lipids (Alabaster, AL). Human human HDL, human model to reduce the development of advanced coronary apoA-I human apoA-II, and BSA were purchased from Sigma- atherosclerosis ( 15 ). Lp-PLA2 is found predominantly Aldrich. All other reagents were analytical reagent grade or better. bound to lipoproteins in human plasma and a small amount may also be bound to other microparticles ( 16 ). Preparation of recombinant protein and activity assay The location of the enzyme on LDL or HDL may alter the The recombinant human Lp-PLA2 protein including residues protein’s catalytic behavior. The enzyme associated with 47-441 with an additional alanine residue at the N terminus was LDL appears to be more active than the same enzyme prepared as described previously ( 25 ). Purifi ed Lp-PLA2 was associated with HDL ( 17 ). In vitro assays using low PAF concentrated to 2.5 mg/ml in protein buffer (20 mM Tris, 150 mM concentrations that mimic physiological levels showed that NaCl, 10% glycerol, pH 7.5, 1 mM DTT) with an Amicon Ultra-15 (Millipore) with the protein concentration determined by bicin- Lp-PLA2 associated with HDL particles is inactive ( 2 ). LDL- associated Lp-PLA plays a pro-infl ammatory and pro- choninic acid protein assay. The enzyme activity was determined 2 by a radiometric assay described elsewhere ( 30 ). The assay was per- atherogenic role because it can hydrolyze oxPC to generate formed in 100 mM HEPES buffer (pH = 7.5, 1 mM EGTA, 1 mM pro-atherogenic mediators, lyso-PC, and oxidized fatty DTT, 2 mM CHAPS) by using 0.1 mM [ 3 H-acteyl] PAF as substrate acids. Additionally, Lp-PLA2 shows preferential association in a fi nal volume of 500 µl. The reaction was initiated by adding with dense LDL and the very dense lipoprotein subfrac- 1 ng purifi ed Lp-PLA2 and then was incubated at 37°C for tion in human plasma, which contains the most athero- 20 min. The reaction was quenched using 500 µl 10 M acetic acid. 3 genic LDL particles ( 18 ). Although only about 30% of The released H-acetate was separated by C18 reversed-phase car- tridges (Phenomenex) and quantitated by scintillation counting. Lp-PLA2 in normal human plasma associates with HDL, this ratio varies greatly among different animal models and growing evidence supports an anti-atherogenic role of Preparation of samples for DXMS experiments D2 O buffer consisted of 12 mM Tris, 50 mM NaCl in 99% D2 O, HDL associated Lp-PLA2 ( 19, 20 ). The ratio of Lp-PLA2 associated with HDL or LDL to the total plasma enzyme pDread 7.5, as previously described ( 31–35 ). Exchange experi- ments were initiated by mixing 20 µ L of Lp-PLA2 (2.5 mg/ml) may serve as a useful biomarker of atherogenicity in patients in protein buffer, or Lp-PLA preincubated with human HDL, with dyslipidemias ( 21 ). Together, the distribution and 2 apoA-I, apoA-II, or BSA with 60 µ L of D2 O buffer to a fi nal con- location of Lp-PLA2 in LDL/HDL lipoproteins may regu- centration of 50% D2 O. The mixtures were incubated at 0°C for late Lp-PLA2 catalytic activity, function, and/or its physio- 10, 30, 100, 300, 1,000, 3,000, or 10,000 s and then the exchange logical roles. Therefore, a precise characterization of the reaction was quenched by adding 120 µl of ice-cold quench solu- (M guanidine hydrochloride 0.8 ف tion (0.96% formic acid, 0 interaction between Lp-PLA2 and lipoproteins is necessary. Previously, residues 115, 116, and 205 were suggested to resulting in samples with fi nal concentrations of 0.58% formic ف contribute to the association of Lp-PLA with LDL ( 22 ). acid and 0 0.5 M guanidine hydrochloride, pH 2.5. The samples 2 were then immediately frozen on dry ice and stored at Ϫ 80°C. The C terminus of apoB-100 could be important for LDL binding to Lp-PLA2 ( 22 ). Although a domain on Lp-PLA2 Proteolysis: LC/MS analysis of samples containing residues 367–370 has been proposed to mediate All steps were performed at 0°C as described previously ( 25 ).
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