Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Effects of phospholipase A2 and its products on structural stability of human LDL: relevance to formation of LDL-derived lipid droplets

2011; Elsevier BV; Volume: 52; Issue: 3 Linguagem: Inglês

10.1194/jlr.m012567

ISSN

1539-7262

Autores

Shobini Jayaraman, Donald Gantz, Olga Gursky,

Tópico(s)

Diabetes, Cardiovascular Risks, and Lipoproteins

Resumo

Hydrolysis and oxidation of LDL stimulate LDL entrapment in the arterial wall and promote inflammation and atherosclerosis via various mechanisms including lipoprotein fusion and lipid droplet formation. To determine the effects of FFA on these transitions, we hydrolyzed LDL by phospholipase A2 (PLA2), removed FFA by albumin, and analyzed structural stability of the modified lipoproteins. Earlier, we showed that heating induces LDL remodeling, rupture, and coalescence into lipid droplets resembling those found in atherosclerotic lesions. Here, we report how FFA affect these transitions. Circular dichroism showed that mild LDL lipolysis induces partial β-sheet unfolding in apolipoprotein B. Electron microscopy, turbidity, and differential scanning calorimetry showed that mild lipolysis promotes LDL coalescence into lipid droplets. FFA removal by albumin restores LDL stability but not the protein conformation. Consequently, FFA enhance LDL coalescence into lipid droplets. Similar effects of FFA were observed in minimally oxidized LDL, in LDL enriched with exogenous FFA, and in HDL and VLDL. Our results imply that FFA promote lipoprotein coalescence into lipid droplets and explain why LDL oxidation enhances such coalescence in vivo but hampers it in vitro. Such lipid droplet formation potentially contributes to the pro-atherogenic effects of FFA. Hydrolysis and oxidation of LDL stimulate LDL entrapment in the arterial wall and promote inflammation and atherosclerosis via various mechanisms including lipoprotein fusion and lipid droplet formation. To determine the effects of FFA on these transitions, we hydrolyzed LDL by phospholipase A2 (PLA2), removed FFA by albumin, and analyzed structural stability of the modified lipoproteins. Earlier, we showed that heating induces LDL remodeling, rupture, and coalescence into lipid droplets resembling those found in atherosclerotic lesions. Here, we report how FFA affect these transitions. Circular dichroism showed that mild LDL lipolysis induces partial β-sheet unfolding in apolipoprotein B. Electron microscopy, turbidity, and differential scanning calorimetry showed that mild lipolysis promotes LDL coalescence into lipid droplets. FFA removal by albumin restores LDL stability but not the protein conformation. Consequently, FFA enhance LDL coalescence into lipid droplets. Similar effects of FFA were observed in minimally oxidized LDL, in LDL enriched with exogenous FFA, and in HDL and VLDL. Our results imply that FFA promote lipoprotein coalescence into lipid droplets and explain why LDL oxidation enhances such coalescence in vivo but hampers it in vitro. Such lipid droplet formation potentially contributes to the pro-atherogenic effects of FFA. In atherosclerosis, LDL-derived lipids are deposited in the subendothelium of the arterial wall. According to the "response to retention" hypothesis, atherogenesis is initiated upon LDL retention by the arterial proteoglycans and LDL modification by the resident hydrolases and oxidative agents (1Williams K.J. Tabas I. The response-to-retention hypothesis of early atherogenesis.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 551-561Crossref PubMed Google Scholar, 2Camejo G. Hurt-Camejo E. Wiklund O. Bondjers G. Association of apo B lipoproteins with arterial proteoglycans: pathological significance and molecular basis.Atherosclerosis. 1998; 139: 205-222Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 3Skålén K. Gustafsson M. Rydberg E.K. Hultén L.M. Wiklund O. Innerarity T.L. Borén J. Subendothelial retention of atherogenic lipoproteins in early atherosclerosis.Nature. 2002; 417: 750-754Crossref PubMed Scopus (716) Google Scholar). These modifications trigger a cascade of pro-inflammatory and pro-apoptotic responses that are caused, in part, by the toxic effects of the oxidized phospholipids and their hydrolytic products such as FFA and lyso-phosphatidylcholine (PC) (4Chisolm G.M. Steinberg D. The oxidative modification hypothesis of atherogenesis: an overview.Free Radic. Biol. Med. 2000; 28: 1815-1826Crossref PubMed Scopus (670) Google Scholar, 5de Winther M.P. Hofker M.H. Scavenging new insights into atherogenesis.J. Clin. Invest. 2000; 105: 1039-1041Crossref PubMed Scopus (56) Google Scholar). Hydrolytic and oxidative modifications can also induce LDL aggregation, fusion, and coalescence into lipid droplets, which further enhance LDL retention in the arterial wall (6Oörni K. Pentikäinen M.O. Ala-Korpela M. Kovanen P.T. Aggregation, fusion, and vesicle formation of modified low density lipoprotein particles: molecular mechanisms and effects on matrix interactions.J. Lipid Res. 2000; 41: 1703-1714Abstract Full Text Full Text PDF PubMed Google Scholar). LDL-derived small extracellular lipid droplets (30–400 nm) are prominent in early atherosclerotic lesions (7Guyton J.R. Klemp K.F. Development of the atherosclerotic core region. Chemical and ultrastructural analysis of microdissected atherosclerotic lesions from human aorta.Arterioscler. Thromb. 1994; 14: 1305-1314Crossref PubMed Scopus (120) Google Scholar) and are observed in the experimental models of atherosclerosis [(8De Spirito M. Brunelli R. Mei G. Bertani F.R. Ciasca G. Greco G. Papi M. Arcovito G. Ursini F. Parasassi T. Low density lipoprotein aged in plasma forms clusters resembling subendothelial droplets: aggregation via surface sites.Biophys. J. 2006; 90: 4239-4247Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) and references therein]. Most of the lipids found in fibrous atherosclerotic plaques are present in such droplets [reviewed in (9Guyton J.R. Phospholipid hydrolytic enzymes in a 'cesspool' of arterial intimal lipoproteins: a mechanism for atherogenic lipid accumulation.Arterioscler. Thromb. Vasc. Biol. 2001; 21: 884-886Crossref PubMed Scopus (24) Google Scholar)]. Moreover, fusion of modified LDL accelerates LDL uptake by arterial macrophages, eventually leading to foam cell formation and progression of atherosclerotic plaques containing large (400–6,000 nm) LDL-derived intracellular lipid droplets (7Guyton J.R. Klemp K.F. Development of the atherosclerotic core region. Chemical and ultrastructural analysis of microdissected atherosclerotic lesions from human aorta.Arterioscler. Thromb. 1994; 14: 1305-1314Crossref PubMed Scopus (120) Google Scholar). Hence, the atherogenic potential of LDL is linked to their propensity to fuse and coalesce into lipid droplets.Because nonmodified LDL do not fuse under physiologic conditions, modifications such as oxidation, lipolysis, and proteolysis are thought to be prerequisites for lipoprotein fusion [(1Williams K.J. Tabas I. The response-to-retention hypothesis of early atherogenesis.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 551-561Crossref PubMed Google Scholar, 2Camejo G. Hurt-Camejo E. Wiklund O. Bondjers G. Association of apo B lipoproteins with arterial proteoglycans: pathological significance and molecular basis.Atherosclerosis. 1998; 139: 205-222Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 3Skålén K. Gustafsson M. Rydberg E.K. Hultén L.M. Wiklund O. Innerarity T.L. Borén J. Subendothelial retention of atherogenic lipoproteins in early atherosclerosis.Nature. 2002; 417: 750-754Crossref PubMed Scopus (716) Google Scholar, 4Chisolm G.M. Steinberg D. The oxidative modification hypothesis of atherogenesis: an overview.Free Radic. Biol. Med. 2000; 28: 1815-1826Crossref PubMed Scopus (670) Google Scholar, 10Plihtari R. Hurt-Camejo E. Oörni K. Kovanen P.T. Proteolysis sensitizes LDL particles to phospholipolysis by secretory phospholipase A2 group V and secretory sphingomyelinase.J. Lipid Res. 2010; 51: 1801-1809Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) and references therein]. The effects of these modifications on LDL aggregation and fusion have been attributed to the packing defects in the particle surface (6Oörni K. Pentikäinen M.O. Ala-Korpela M. Kovanen P.T. Aggregation, fusion, and vesicle formation of modified low density lipoprotein particles: molecular mechanisms and effects on matrix interactions.J. Lipid Res. 2000; 41: 1703-1714Abstract Full Text Full Text PDF PubMed Google Scholar, 8De Spirito M. Brunelli R. Mei G. Bertani F.R. Ciasca G. Greco G. Papi M. Arcovito G. Ursini F. Parasassi T. Low density lipoprotein aged in plasma forms clusters resembling subendothelial droplets: aggregation via surface sites.Biophys. J. 2006; 90: 4239-4247Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), which may result from an imbalance between this surface and the apolar core (12Liu H. Scraba D.G. Ryan R.O. Prevention of phospholipase-C induced aggregation of low density lipoprotein by amphipathic apolipoproteins.FEBS Lett. 1993; 316: 27-33Crossref PubMed Scopus (78) Google Scholar). A similar imbalance leading to lipoprotein fusion and rupture can result from other perturbations such as heating, chemical denaturation, detergents, etc. [reviewed in (13Gursky O. Apolipoprotein structure and dynamics.Curr. Opin. Lipidol. 2005; 16: 287-294Crossref PubMed Scopus (40) Google Scholar)]. For example, heating leads to irreversible remodeling of LDL into smaller and larger particles; the former resemble small dense LDL and the latter are apparent products of LDL fusion (14Jayaraman S. Gantz D.L. Gursky O. Structural basis for thermal stability of human low-density lipoprotein.Biochemistry. 2005; 44: 3965-3971Crossref PubMed Scopus (33) Google Scholar). Further heating leads to irreversible rupture of these particles and release of their core lipids that coalesce into droplets; the size and morphology of these droplets resemble the extracellular lipid droplets found in atherosclerotic lesions (7Guyton J.R. Klemp K.F. Development of the atherosclerotic core region. Chemical and ultrastructural analysis of microdissected atherosclerotic lesions from human aorta.Arterioscler. Thromb. 1994; 14: 1305-1314Crossref PubMed Scopus (120) Google Scholar, 14Jayaraman S. Gantz D.L. Gursky O. Structural basis for thermal stability of human low-density lipoprotein.Biochemistry. 2005; 44: 3965-3971Crossref PubMed Scopus (33) Google Scholar). Hence, heating provides a useful tool to accelerate LDL remodeling and coalescence into lipid droplets and to monitor these transitions in real time.Surprisingly, in LDL isolated from human plasma, oxidation progressively inhibits heat-induced remodeling and rupture (15Jayaraman S. Gantz D.L. Gursky O. Effects of oxidation on the structure and stability of human low-density lipoprotein.Biochemistry. 2007; 46: 5790-5797Crossref PubMed Scopus (32) Google Scholar). Consequently, contrary to the accepted notion, oxidation per se inhibits rather than promotes LDL remodeling. This prompted us to postulate that fusion and coalescence of oxidized LDL in the arterial wall are facilitated by other factors, such as the enhanced binding of oxidized LDL to the arterial proteoglycans, the imbalance between the FFA generation by lipases and removal by albumin, etc. (15Jayaraman S. Gantz D.L. Gursky O. Effects of oxidation on the structure and stability of human low-density lipoprotein.Biochemistry. 2007; 46: 5790-5797Crossref PubMed Scopus (32) Google Scholar). Here, we test the effects of PC hydrolysis by phospholipase A2 (PLA2) and removal of its products by albumin on heat-induced LDL remodeling, rupture, and lipid droplet formation.Enzymes from the PLA2 family hydrolyze PCs at the sn-2 position to generate lyso-PC and FFA. Several types of secretory PLA2 (16Hakala J.K. Oörni K. Pentikäinen M.O. Hurt-Camejo E. Kovanen P.T. Lipolysis of LDL by human secretory phospholipase A(2) induces particle fusion and enhances the retention of LDL to human aortic proteoglycans.Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1053-1058Crossref PubMed Scopus (111) Google Scholar, 17Oörni K. Kovanen P.T. Lipoprotein modification by secretory phospholipase A(2) enzymes contributes to the initiation and progression of atherosclerosis.Curr. Opin. Lipidol. 2009; 20: 421-427Crossref PubMed Scopus (33) Google Scholar) and the lipoprotein-associated PLA2 (Lp-PLA2) that preferentially hydrolyses oxidized PCs in LDL (18Davis B. Koster G. Douet L.J. Scigelova M. Woffendin G. Ward J.M. Smith A. Humphries J. Burnand K.G. Macphee C.H. Electrospray ionization mass spectrometry identifies substrates and products of lipoprotein-associated phospholipase A2 in oxidized human low density lipoprotein.J. Biol. Chem. 2008; 283: 6428-6437Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) provide biomarkers of inflammation and atherosclerosis [reviewed in (19Yedgar S. Cohen Y. Shoseyov D. Control of phospholipase A2 activities for the treatment of inflammatory conditions.Biochim. Biophys. Acta. 2006; 1761: 1373-1382Crossref PubMed Scopus (81) Google Scholar, 20Jönsson-Rylander A.C. Lundin S. Rosengren B. Pettersson C. Hurt-Camejo E. Role of secretory phospholipases in atherogenesis.Curr. Atheroscler. Rep. 2008; 10: 252-259Crossref PubMed Scopus (32) Google Scholar, 21Corson M.A. Emerging inflammatory markers for assessing coronary heart disease risk.Curr. Cardiol. Rep. 2009; 11: 452-459Crossref PubMed Scopus (19) Google Scholar, 22Rosenson R.S. Gelb M.H. Secretory phospholipase A2: a multifaceted family of proatherogenic enzymes.Curr. Cardiol. Rep. 2009; 11: 445-451Crossref PubMed Scopus (65) Google Scholar)]. Moreover, Lp-PLA2 has emerged as a causative agent of atherosclerosis and as a new therapeutic target (17Oörni K. Kovanen P.T. Lipoprotein modification by secretory phospholipase A(2) enzymes contributes to the initiation and progression of atherosclerosis.Curr. Opin. Lipidol. 2009; 20: 421-427Crossref PubMed Scopus (33) Google Scholar, 23Macphee C.H. Nelson J.J. Zalewski A. Lipoprotein-associated phospholipase A2 as a target of therapy.Curr. Opin. Lipidol. 2005; 16: 442-446Crossref PubMed Scopus (81) Google Scholar, 24Wilensky R.L. Shi Y. Mohler E.R. Hamamdzic D. Burgert M.E. Li J. Postle A. Fenning R.S. Bollinger J.G. Hoffman B.E. Inhibition of lipoprotein-associated phospholipase A2 reduces complex coronary atherosclerotic plaque development.Nat. Med. 2008; 14: 1059-1066Crossref PubMed Scopus (320) Google Scholar, 25Wilensky R.L. Macphee C.H. Lipoprotein-associated phospholipase A(2) and atherosclerosis.Curr. Opin. Lipidol. 2009; 20: 415-420Crossref PubMed Scopus (94) Google Scholar, 26Braun L.T. Davidson M.H. Lp-PLA2: a new target for statin therapy.Curr. Atheroscler. Rep. 2010; 12: 29-33Crossref PubMed Scopus (21) Google Scholar, 27White H. Editorial: why inhibition of lipoprotein-associated phospholipase A2 has the potential to improve patient outcomes.Curr. Opin. Cardiol. 2010; 25: 299-301Crossref PubMed Scopus (14) Google Scholar). Lp-PLA2 (28Gaubatz J.W. Gillard B.K. Massey J.B. Hoogeveen R.C. Huang M. Lloyd E.E. Raya J.L. Yang C.Y. Pownall H.J. Dynamics of dense electronegative low density lipoproteins and their preferential association with lipoprotein phospholipase A(2).J. Lipid Res. 2007; 48: 348-357Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) and phospholipase C (29Bancells C. Villegas S. Blanco F.J. Benitez S. Gallego I. Beloki L. Perez-Cuellar M. Ordonez-Llanos J. Sanchez-Quesada J.L. Aggregated electronegative low-density lipoprotein in human plasma shows high tendency to phospholipolysis and particle fusion.J. Biol. Chem. 2010; 285: 32425-32435Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) are reportedly enriched in the small dense LDL and/or in the electronegative LDL, which may promote fusion of these LDL and contribute to their enhanced pro-atherogenic properties (29Bancells C. Villegas S. Blanco F.J. Benitez S. Gallego I. Beloki L. Perez-Cuellar M. Ordonez-Llanos J. Sanchez-Quesada J.L. Aggregated electronegative low-density lipoprotein in human plasma shows high tendency to phospholipolysis and particle fusion.J. Biol. Chem. 2010; 285: 32425-32435Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar).We hypothesize that the pro-atherogenic properties of PLA2 result in part from the direct effects of its products on LDL fusion and rupture. This hypothesis is based on the effects of PLA2 and its products, lyso-PC and FFA (which promote positive and negative bilayer curvature, respectively), on specific steps in lipid bilayer fusion (30Brown W.J. Chambers K. Doody A. Phospholipase A2 (PLA2) enzymes in membrane trafficking: mediators of membrane shape and function.Traffic. 2003; 4: 214-221Crossref PubMed Scopus (235) Google Scholar, 31Chernomordik L.V. Kozlov M.M. Mechanics of membrane fusion.Nat. Struct. Mol. Biol. 2008; 15: 675-683Crossref PubMed Scopus (705) Google Scholar). In addition, PLA2 enzymes can lyse various membranes, including cell membranes in erythrocytes as well as the membranes in various bacteria and viruses (32Desbois A.P. Smith V.J. Antibacterial free fatty acids: activities, mechanisms of action and biotechnological potential.Appl. Microbiol. Biotechnol. 2010; 85: 1629-1642Crossref PubMed Scopus (835) Google Scholar), which is important for the immune response. We speculate that the ability of PLA2 to promote membrane fusion and lysis may extend to lipoprotein fusion and rupture. This is suggested by studies from Hakala et al. (16Hakala J.K. Oörni K. Pentikäinen M.O. Hurt-Camejo E. Kovanen P.T. Lipolysis of LDL by human secretory phospholipase A(2) induces particle fusion and enhances the retention of LDL to human aortic proteoglycans.Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1053-1058Crossref PubMed Scopus (111) Google Scholar) showing that LDL hydrolysis by PLA2 in the presence of arterial proteoglycans causes LDL fusion; in those studies, FFA were removed from LDL by using near-physiologic concentrations of albumin (20 mg/ml) in an essentially FFA-free state. Even though albumin is believed to remove most FFA produced upon lipolysis of plasma lipoproteins, excess FFA generated locally can partition into lipoproteins (33Chung B.H. Tallis G.A. Cho B.H. Segrest J.P. Henkin Y. Lipolysis-induced partitioning of free fatty acids to lipoproteins: effect on the biological properties of free fatty acids.J. Lipid Res. 1995; 36: 1956-1970Abstract Full Text PDF PubMed Google Scholar), particularly in the acidic environment of atherosclerotic lesions where albumin has impaired ability to remove FFA (34Lähdesmäki K. Plihtari R. Soininen P. Hurt-Camejo E. Ala-Korpela M. Oörni K. Kovanen P.T. Phospholipase A(2)-modified LDL particles retain the generated hydrolytic products and are more atherogenic at acidic pH.Atherosclerosis. 2009; 207: 352-359Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Here, we test the effects of FFA retained in LDL on the heat-induced lipoprotein fusion and coalescence into lipid droplets. To do so, we compare the effects of PC hydrolysis in native and in oxidized LDL in the presence and in the absence of albumin. The results imply a potentially important role of FFA in the in vivo formation of lipoprotein-derived lipid droplets.MATERIALS AND METHODSIsolation of lipoproteinsHuman lipoproteins from five healthy volunteers were used. Plasma was donated at a blood bank in compliance with the Institutional Review Board protocols and with written consent obtained from the volunteers. Single-donor lipoproteins were isolated from fresh EDTA-treated plasma by KBr density gradient ultracentrifugation in the density range 0.94–1.006 g/ml for VLDL, 1.019–1.063 g/ml for LDL, and 1.063–1.21 g/ml for HDL (35Schumaker V.N. Puppione D.L. Sequential flotation ultracentrifugation.Methods Enzymol. 1986; 128: 155-170Crossref PubMed Scopus (467) Google Scholar). Lipoproteins from each class migrated as a single band on the agarose gel and on the nondenaturing gel. Lipoprotein stock solutions were dialyzed against buffer A (10 mM Na phosphate buffer, 0.25 mM EDTA, 0.02% NaN3, pH 7.5), degassed, and stored in the dark at 4°C. The stock solutions were used within 2 weeks during which no protein degradation was detected by SDS PAGE and no changes in the net charge were observed on the agarose gel. Protein concentration was determined by a modified Lowry assay.Preparation and characterization of lipoproteins hydrolyzed by PLA2Lipoprotein solutions (3 mg/ml protein concentration) were dialyzed against buffer B (10 mM Tris, pH 7.5) and were incubated with porcine pancreatic PLA2 (Sigma) in buffer B containing 2 mM CaCl2 for 12 h at 37°C following established protocols (36Hakala J.K. Oörni K. Ala-Korpela M. Kovanen P.T. Lipolytic modification of LDL by phospholipase A2 induces particle aggregation in the absence and fusion in the presence of heparin.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1276-1283Crossref PubMed Scopus (46) Google Scholar). To obtain lipoproteins hydrolyzed to stage 1, 2, or 3, we used 0.05, 0.5, or 5 μg of PLA2, respectively. The reaction was stopped by adding EDTA to a final concentration of 15 mM. To remove FFA from the lipoproteins, 20 mg/ml of essentially fatty acid-free human serum albumin (HSA; Sigma) was included in some incubation mixtures (16Hakala J.K. Oörni K. Pentikäinen M.O. Hurt-Camejo E. Kovanen P.T. Lipolysis of LDL by human secretory phospholipase A(2) induces particle fusion and enhances the retention of LDL to human aortic proteoglycans.Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1053-1058Crossref PubMed Scopus (111) Google Scholar); this albumin concentration corresponds to the average value found in the interstitial fluid of the arterial intima (37Smith E.B. Transport, interactions and retention of plasma proteins in the intima: the barrier function of the internal elastic lamina.Eur. Heart J. 1990; 11: 72-81Crossref PubMed Google Scholar). The lipoproteins were reisolated by ultracentrifugation. The complete removal of albumin from the lipoproteins with which it was coincubated was confirmed by SDS PAGE (see Fig. 1B). Lipoproteins from the same plasma pool were subjected to identical incubation and reisolation procedures but without PLA2 to assess the effects of spontaneous hydrolysis at 37°C, i. e., hydrolysis in the absence of exogenous PLA2; such hydrolysis results from the hydrolytic activity of apolipoprotein (apo)B (38Parthasarathy S. Barnett J. Phospholipase A2 activity of low density lipoprotein: evidence for an intrinsic phospholipase A2 activity of apoprotein B-100.Proc. Natl. Acad. Sci. USA. 1990; 87: 9741-9745Crossref PubMed Scopus (138) Google Scholar, 39Reisfeld N. Lichtenberg D. Dagan A. Yedgar S. Apolipoprotein B exhibits phospholipase A1 and phospholipase A2 activities.FEBS Lett. 1993; 315: 267-270Crossref PubMed Scopus (21) Google Scholar) and the LDL-associated Lp-PLA2. Such spontaneously hydrolyzed LDL (marked S) as well as those hydrolyzed to stages 1–3 by PLA2 (marked by the stage number) were dialyzed against buffer A for further studies.LDL enrichment with exogenous oleic acidSodium oleate (>99% purity, from Sigma) was used. Native LDL (2 mg/ml protein) and a freshly prepared emulsion of oleic acid (8 mM) in 10 mM Na phosphate buffer, 250 mM EDTA, were coincubated at 37°C for 4 h or 12 h. Unbound oleic acid was removed by gel filtration using Superose 6 10/300 GL column by elution in buffer A at a flow rate of 0.5 ml/min. The final concentrations of oleic acid incorporated into LDL, which were determined by quantitative TLC analysis, were 1.5 mM after 4 h and 3 mM after 12 h of incubation, a significant enrichment as compared with unmodified LDL (0.065–0.01 mM).Oxidation of LDLLDL minimally oxidized by Cu2+ (moxLDL) were obtained following established protocols [(15Jayaraman S. Gantz D.L. Gursky O. Effects of oxidation on the structure and stability of human low-density lipoprotein.Biochemistry. 2007; 46: 5790-5797Crossref PubMed Scopus (32) Google Scholar) and references therein]. Briefly, LDL solution (0.1 mg/ml protein) was incubated with 5 µM CuSO4 at 37°C in buffer B for 1 h. The reaction was quenched by adding EDTA to a final concentration of 250 mM, followed by cooling to 4°C and dialysis against buffer A. LDL oxidation under these conditions corresponds to the end of the lag phase during which the core antioxidants are consumed (as monitored by visible absorption spectra of core carotenoids, such as those shown in supplementary Fig. II A) and the beginning of the propagation phase during which conjugated dienes are produced [as monitored by absorbance at 234 nm (15Jayaraman S. Gantz D.L. Gursky O. Effects of oxidation on the structure and stability of human low-density lipoprotein.Biochemistry. 2007; 46: 5790-5797Crossref PubMed Scopus (32) Google Scholar, 40Esterbauer H. Gebicki J. Puhl H. Jürgens G. The role of lipid peroxidation and antioxidants in oxidative modification of LDL.Free Radic. Biol. Med. 1992; 13: 341-390Crossref PubMed Scopus (2132) Google Scholar)]. MoxLDL prepared by this method showed no protein fragmentation by SDS PAGE, no significant changes in the apoB conformation by circular dichroism (CD) spectroscopy (15Jayaraman S. Gantz D.L. Gursky O. Effects of oxidation on the structure and stability of human low-density lipoprotein.Biochemistry. 2007; 46: 5790-5797Crossref PubMed Scopus (32) Google Scholar), and no changes in lipid composition by TLC (see Fig. 8C). Hydrolysis of moxLDL by PLA2 to stage 1 (1·moxLDL) was done as described for native LDL in the absence or in the presence of 20 mg/ml FFA-free albumin to produce minimally oxidized and hydrolyzed FFA-free LDL (1·moxLDL·HSA). The LDL modified by these methods were reisolated by density gradient centrifugation, dialyzed against buffer A, and used for stability studies.Fig. 8Combined effects of mild oxidation and hydrolysis on LDL stability assessed by heating in CD experiments. Minimally oxidized LDL (mox), prepared as described in Methods, were hydrolyzed by PLA2 to stage 1 (1·mox) and treated with albumin to remove FFA (1·mox·HSA). The LDL (2 mg/ml in buffer A, placed in 1 mm path length cell) that were modified by these methods or were native (0) were heated at a rate of 11°C/h. The melting data were recorded at 280 nm by CD (A) and turbidity (dynode voltage) (B). TLC shows formation of FFA and lyso-PC upon hydrolysis of moxLDL to stage 1 (C). Quantitative lipid analysis based on the TLC data (D). Error bars indicate the standard error of the mean for five independent measurements.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Gel electrophoresisSDS PAGE was performed using a 4–20% gradient system. The gels were run at 150 V for 2 h and stained with Denville Blue protein stain. Agarose gels were performed using a TITAN lipoprotein gel electrophoresis system. LDL samples containing 4 µg protein were loaded on the precast gels that were run using barbital-sodium barbital buffer at 60 V for 40 min and at 125 V for 7 min. The gels were dried at 70°C for 20 min, stained with 0.1% w/v Fat Red 7B stain in 95% methanol, destained in 75% methanol, and dried at 70°C.Lipid analysisNative and hydrolyzed LDL were analyzed by TLC for total lipid composition and by GC for FFA composition. The total amount of FFA was within the range reported for normal human plasma LDL (41Skipski V.P. Barclay M. Barclay R.K. Fetzer V.A. Good J.J. Archibald F.M. Lipid composition of human serum lipoproteins.Biochem. J. 1967; 104: 340-352Crossref PubMed Scopus (187) Google Scholar). The lipids were extracted by the Folch method (42Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipides from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar) with 2:1 chloroform:methanol and were dried under nitrogen. For TLC, known amounts of dry lipids were analyzed using hexane:ether:acetic acid (70:30:1) to separate apolar lipids, or chloroform:methanol:water:acetic acid (65:25:4:1) to separate polar lipids.For quantitative analysis of LDL lipids by TLC, Image J software (National Institutes of Health, Bethesda, MD) was used to calculate the band areas corresponding to PC, FFA, and lyso-PC. A calibration plot of the peak area versus the amount of sample was obtained from the analysis of the charred spots corresponding to known amounts of lipids; the plot was linear with R = 0.99. This plot was used to determine the relative amount of PC, FFA, and lyso-PC for each LDL sample. To minimize the errors resulting from variations in the length of the TLC run and in the charring conditions, the standards were spotted in each plate. The relative fraction of each lipid was plotted as a mean of five independent experiments with the standard error of mean reported for each fraction.For GC, fatty acid methyl esters of neutral, polar, and apolar lipids were prepared as described (43Morrison W.R. Smith L.M. Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride-methanol.J. Lipid Res. 1964; 5: 600-608Abstract Full Text PDF PubMed Google Scholar). Briefly, the extracted dry lipids were dissolved in 0.3 ml dry benzene, 0.35 ml dry methanol, and 0.35 ml dry boron trifluoride in methanol in a capped vial. The vials were kept at 100°C for 30 min. After cooling, 1.5 ml water was added to stop the reaction, 5 ml of hexane was added, and the mixture was vortexed for 30 s. The aqueous and organic phases were separated by centrifugation. The top hexane layer was removed and dried under N2. The dry lipids were resuspended in 100 µL ultrapure hexane and were injected into GC. The fatty acids were analyzed on a fused silica capillary column, 30 m × 0.25 mm (Supelco) installed in Shimadzu GC-14A gas chromatograph equipped with a hydrogen flame ionization detector. Hydrogen was the carrier gas at 3 psi. The injections were made at 100°C; after 30 s, the oven temperat

Referência(s)