Composition and lipid spatial distribution of HDL particles in subjects with low and high HDL-cholesterol
2010; Elsevier BV; Volume: 51; Issue: 8 Linguagem: Inglês
10.1194/jlr.m006494
ISSN1539-7262
AutoresLaxman Yetukuri, Sanni Söderlund, Artturi Koivuniemi, Tuulikki Seppänen‐Laakso, Perttu Niemelä, M. Hyvönen, Marja‐Riitta Taskinen, Ilpo Vattulainen, Matti Jauhiainen, Matej Orešič,
Tópico(s)Cholesterol and Lipid Metabolism
ResumoA low level of high density lipoprotein cholesterol (HDL-C) is a powerful risk factor for cardiovascular disease. However, despite the reported key role of apolipo-proteins, specifically, apoA-I, in HDL metabolism, lipid molecular composition of HDL particles in subjects with high and low HDL-C levels is currently unknown. Here lipidomics was used to study HDL derived from well-characterized high and low HDL-C subjects. Low HDL-C subjects had elevated triacylglycerols and diminished lysophosphatidylcholines and sphingomyelins. Using information about the lipid composition of HDL particles in these two groups, we reconstituted HDL particles in silico by performing large-scale molecular dynamics simulations. In addition to confirming the measured change in particle size, we found that the changes in lipid composition also induced specific spatial distributions of lipids within the HDL particles, including a higher amount of triacylglycerols at the surface of HDL particles in low HDL-C subjects. Our findings have important implications for understanding HDL metabolism and function. For the first time we demonstrate the power of combining molecular profiling of lipoproteins with dynamic modeling of lipoprotein structure. A low level of high density lipoprotein cholesterol (HDL-C) is a powerful risk factor for cardiovascular disease. However, despite the reported key role of apolipo-proteins, specifically, apoA-I, in HDL metabolism, lipid molecular composition of HDL particles in subjects with high and low HDL-C levels is currently unknown. Here lipidomics was used to study HDL derived from well-characterized high and low HDL-C subjects. Low HDL-C subjects had elevated triacylglycerols and diminished lysophosphatidylcholines and sphingomyelins. Using information about the lipid composition of HDL particles in these two groups, we reconstituted HDL particles in silico by performing large-scale molecular dynamics simulations. In addition to confirming the measured change in particle size, we found that the changes in lipid composition also induced specific spatial distributions of lipids within the HDL particles, including a higher amount of triacylglycerols at the surface of HDL particles in low HDL-C subjects. Our findings have important implications for understanding HDL metabolism and function. For the first time we demonstrate the power of combining molecular profiling of lipoproteins with dynamic modeling of lipoprotein structure. High-density lipoprotein (HDL) is one of the five major lipoproteins (chylomicrons, VLDL, IDL, LDL, and HDL). HDL is the smallest and densest of the lipoproteins because it contains the highest proportion of protein. A low level of HDL cholesterol (HDL-C) is a powerful risk factor for cardiovascular disease (1.Gordon T. Castelli W.P. Hjortland M.C. Kannel W.B. Dawber T.R. High density lipoprotein as a protective factor against coronary heart disease. The Framingham Study.Am. J. Med. 1977; 62: 707-714Abstract Full Text PDF PubMed Scopus (4037) Google Scholar, 2.Chirovsky D.R. Fedirko V. Cui Y. Sazonov V. Barter P. Prospective studies on the relationship between high-density lipoprotein cholesterol and cardiovascular risk: a systematic review.Eur. J. Cardiovasc. Prev. Rehabil. 2009; 16: 404-423Crossref PubMed Scopus (43) Google Scholar, 3.Cooney M.T. Dudina A. De Bacquer D. Wilhelmsen L. Sans S. Menotti A. De Backer G. Jousilahti P. Keil U. 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A recent study of HDL proteome revealed many important changes in the protein composition of HDL in cardiovascular patients without changes in serum HDL-C (6.Vaisar T. Pennathur S. Green P.S. Gharib S.A. Hoofnagle A.N. Cheung M.C. Byun J. Vuletic S. Kassim S. Singh P. et al.Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL.J. Clin. Invest. 2007; 117: 746-756Crossref PubMed Scopus (772) Google Scholar). Detailed characterization of lipoprotein fractions and changes in their molecular lipid profiles may help identify novel biomarkers in lipid metabolism (7.Wiesner P. Leidl K. Boettcher A. Schmitz G. Liebisch G. Lipid profiling of FPLC-separated lipoprotein fractions by electrospray ionization tandem mass spectrometry.J. Lipid Res. 2009; 50: 574-585Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). Main lipid constituents of HDL particles include glycerophospholipids, cholesteryl esters (ChoE), sphingomyelins (SM), and triacylglycerols (TG). Lysophosphatidylcholines (lysoPC) are known to be associated with proatherogenic conditions (8.Glass C.K. Witztum J.L. Atherosclerosis: the road ahead.Cell. 2001; 104: 503-516Abstract Full Text Full Text PDF PubMed Scopus (2576) Google Scholar). Enrichment of HDL phospholipids, such as phosphatidylcholines (PC) and SM, improves the net efflux of cholesterol from scavenger receptor-BI expressing cells (9.Yancey P.G. de la Llera-Moya M. Swarnakar S. Monzo P. Klein S.M. Connelly M.A. Johnson W.J. Williams D.L. Rothblat G.H. High density lipoprotein phospholipid composition is a major determinant of the bi-directional flux and net movement of cellular free cholesterol mediated by scavenger receptor BI.J. Biol. Chem. 2000; 275: 36596-36604Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar), and phospholipid composition may have a major impact in the process of reverse cholesterol transport (RCT) (10.Brewer Jr., H.B. Increasing HDL cholesterol levels.N. Engl. J. Med. 2004; 350: 1491-1494Crossref PubMed Scopus (127) Google Scholar). We also demonstrated using HDL derived either from low or high HDL-C subjects that cholesterol efflux from human THP-1 macrophages correlated with phospholipids, particle size, and particle mass of HDL (11.Nakanishi S. Vikstedt R. Soderlund S. Lee-Rueckert M. Hiukka A. Ehnholm C. Muilu M. Metso J. Naukkarinen J. Palotie L. et al.Serum, but not monocyte macrophage foam cells derived from low HDL-C subjects, displays reduced cholesterol efflux capacity.J. Lipid Res. 2009; 50: 183-192Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). This is consistent with earlier efflux studies demonstrating that the phospholipid content of HDL is an important determinant of cholesterol egress (12.Wang N. Lan D. Chen W. Matsuura F. Tall A.R. ATP-binding cassette transporters G1 and G4 mediate cellular cholesterol efflux to high-density lipoproteins.Proc. Natl. Acad. Sci. USA. 2004; 101: 9774-9779Crossref PubMed Scopus (870) Google Scholar, 13.Fournier N. Paul J-L. Atger V. Cogny A. Soni T. de la Llera-Moya M. Rothblat G. Moatti N. HDL phospholipid content and composition as a major factor determining cholesterol efflux capacity from Fu5AH cells to human serum.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 2685-2691Crossref PubMed Scopus (137) Google Scholar). Additionally, the activity of HDL in the first step of RCT is affected by the fatty acyl chain length of the phospholipids (14.Davidson W.S. Gillotte K.L. Lund-Katz S. Johnson W.J. Rothblat G.H. Phillips M.C. The effect of high density lipoprotein phospholipid acyl chain composition on the efflux of cellular free cholesterol.J. Biol. Chem. 1995; 270: 5882-5890Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Together, the information on lipid molecular composition of HDL may provide better insights into the mechanisms behind the anti-atherogenic role of HDL particles. Conventional lipoprotein analyses have relied on analyses of total protein, phospholipid, free cholesterol, ChoE, and TG content (15.Vance D.E. Vance, editors J.E. Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier, Hungary2008Google Scholar). However, molecular level concentrations may provide more precise markers of specific metabolic phenotypes than total lipid class concentrations (16.Kotronen A. Velagapudi V. Yetukuri L. Westerbacka J. Bergholm R. Ekroos K. Makkonen J. Taskinen M.R. Oresic M. Yki-Järvinen H. Serum saturated fatty acids containing triacylglycerols are better markers of insulin resistance than total serum triacylglycerol concentrations.Diabetologia. 2009; 52: 684-690Crossref PubMed Scopus (144) Google Scholar). Recent advances in mass spectrometry (MS)-based analytical platforms and bioinformatic approaches for managing large volumes of data have made it possible to study lipid species at the molecular level (17.Han X. Gross R.W. Shotgun lipidomics: electrospray ionization mass spectrometric analysis and quantitation of cellular lipidomes directly from crude extracts of biological samples.Mass Spectrom. Rev. 2005; 24: 367-412Crossref PubMed Scopus (890) Google Scholar, 18.Oresic M. Hänninen V.A. Vidal-Puig A. Lipidomics: a new window to biomedical frontiers.Trends Biotechnol. 2008; 26: 647-652Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). The lipidomics platform based on ultra performance liquid chromatography mass spectrometry (UPLC/MS) was recently utilized to characterize molecular lipids, including triacylglycerols, glycerophospholipids, sphingomyelins, cholesteryl esters, and ceramides in different lipoprotein fractions (16.Kotronen A. Velagapudi V. Yetukuri L. Westerbacka J. Bergholm R. Ekroos K. Makkonen J. Taskinen M.R. Oresic M. Yki-Järvinen H. Serum saturated fatty acids containing triacylglycerols are better markers of insulin resistance than total serum triacylglycerol concentrations.Diabetologia. 2009; 52: 684-690Crossref PubMed Scopus (144) Google Scholar). Lipid molecular composition of HDL particles in subjects with high and low HDL-cholesterol levels has not been studied systematically. Here we combine clinical cohort study and global lipidomics with molecular simulations of HDL particles. We apply UPLC/MS-based lipidomics to study HDL fractions from well-characterized high and low HDL-C subjects from a large Finnish population cohort, and we identify many specific changes in HDL lipidomes among subjects with high and low HDL-C. Using information about the lipid composition of HDL particles in these two groups, we reconstitute HDL particles in silico by performing large-scale molecular dynamics simulations. In addition to confirming the measured change in particle size, we show that HDL particles derived from high HDL-C subjects have a surprisingly different spatial distribution of triacylglycerols. The study comprised 47 subjects: 24 low HDL subjects and 23 high HDL subjects who were participants in the Health 2000 Health Examination Survey (19.Aromaa A. Koskinen, editors S. Health and functional capacity in Finland: baseline results of the Health 2000 Health Examination Survey. National Public Health Institute, Helsinki, Finland2004Google Scholar). The subjects represented the extreme ends of HDL-C levels (≤10th percentile and ≥90th percentile). HDL-C limits were as follows: low HDL-C men ≤ 1.03 mmol/l, low HDL-C women ≤ 1.23 mmol/l, high HDL-C men ≥ 1.79 mmol/l, and high HDL-C women ≥ 2.24 mmol/l. Subjects with diabetes, alcohol abuse, or malignancy were excluded. Alcohol abuse was defined as >160 g of alcohol per week for women and >310 g of alcohol per week for men. In addition, subjects using systemic estrogen, corticosteroid therapy, statins, or other drugs affecting HDL metabolism were excluded. Each study subject gave a written informed consent before participating in the study. The samples were collected in accordance with the Helsinki declaration, and the ethics committees of the participating centers approved the study design. Table 1 presents the characteristics of the low and high HDL-C subjects.TABLE 1Clinical and biochemical characteristics of the study subjectsLow HDL Subjects (Median IQR)High HDL Subjects (Median IQR)PaP-value from Mann-Whitney U test.N (men/women)bapoA-II measurements of three subjects in both low and high HDL-C groups are not available; hence, calculations are based on measurements from the remaining subjects.24 (12/12)23 (12/11)Age (years)53 (51–56)54 (50–60)0.564Body mass index (kg/m2)27.9 (24.3–31.5)22.8 (21.4–24.6)<0.001Systolic blood pressure (mmHg)131 (118–146)132 (121–147)0.647Diastolic blood pressure (mmHg)81 (75–89)81 (75–87)0.882Total cholesterol (mmol/l)5.15 (4.55–5.60)5.80 (5.30–6.10)<0.001HDL cholesterol (mmol/l)0.94 (0.86–1.10)2.52 (2.12–2.61)<0.001LDL cholesterol (mmol/l)3.30 (2.87–3.67)3.01 (2.68–3.55)0.225Insulin (mU/l)9.15 (7.05–10.93)6.00 (5.00–7.90)0.0015Triglycerides (mmol/l)1.80 (1.23–2.25)0.70 (0.60–0.90)<0.001apoA-I (mg/dl)134 (121–144)222 (206–234)<0.001apoA-II (mg/dl)28.0 (27.0–34.5)39.0 (35.0–45.0)<0.001apoB (mg/dl)124.5 (106.3–135.0)99.0 (84.0–109.0)<0.001PLTP activity (nmol/ml/h)4765 (4109–5549)5304 (4798–5810)0.058PLTP mass (µg/ml)7.5 (6.4–9.2)8.6 (6.8–9.5)0.328CETP activity (nmol/ml/h)28 (25–33)31 (27–38)0.250TNF-α (ng/l)6.5 (4.5–8.2)4.7 (4.1–5.8)0.038IL-6 (ng/l)1.6 (1.0–2.1)1.1 (0.8–2.0)0.163hs-CRP (mg/l)1.4 (0.8–2.7)0.7 (0.4–1.2)0.012HDL size (nm)9.0 (8.8–9.2)9.9 (9.7–10.2)<0.001Abbreviations: Apo, apolipoprotein; CETP, cholesteryl ester transfer protein; hs-CRP, high sensitivity C-reactive protein; IL, interleukin; IQR, interquartile range; PLTP, phospholipid transfer protein; TNF, tumor necrosis factor.a P-value from Mann-Whitney U test.b apoA-II measurements of three subjects in both low and high HDL-C groups are not available; hence, calculations are based on measurements from the remaining subjects. Open table in a new tab Abbreviations: Apo, apolipoprotein; CETP, cholesteryl ester transfer protein; hs-CRP, high sensitivity C-reactive protein; IL, interleukin; IQR, interquartile range; PLTP, phospholipid transfer protein; TNF, tumor necrosis factor. HDL for the lipidomic analysis was separated from plasma samples by ultracentrifugation (20.Taskinen M.R. Kuusi T. Helve E. Nikkila E.A. Yki-Jarvinen H. Insulin therapy induces antiatherogenic changes of serum lipoproteins in noninsulin-dependent diabetes.Arteriosclerosis. 1988; 8: 168-177Crossref PubMed Google Scholar). HDL subspecies distribution and HDL mean particle size were determined with native gradient gel electrophoresis (21.Blanche P.J. Gong E.L. Forte T.M. Nichols A.V. Characterization of human high-density lipoproteins by gradient gel electrophoresis.Biochim. Biophys. Acta. 1981; 665: 408-419Crossref PubMed Scopus (442) Google Scholar) with minor modifications as previously described (11.Nakanishi S. Vikstedt R. Soderlund S. Lee-Rueckert M. Hiukka A. Ehnholm C. Muilu M. Metso J. Naukkarinen J. Palotie L. et al.Serum, but not monocyte macrophage foam cells derived from low HDL-C subjects, displays reduced cholesterol efflux capacity.J. Lipid Res. 2009; 50: 183-192Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The molecular size intervals for HDL subspecies 2b, 2a, 3a, 3b, and 3c were used according to Blanche et al. (21.Blanche P.J. Gong E.L. Forte T.M. Nichols A.V. Characterization of human high-density lipoproteins by gradient gel electrophoresis.Biochim. Biophys. Acta. 1981; 665: 408-419Crossref PubMed Scopus (442) Google Scholar), and for each subspecies, the relative area under the densitometric scan were reported. HDL mean particle size was calculated by multiplying the mean size of each HDL subclass by its relative area under the densitometric scan (22.Perusse M. Pascot A. Despres J.P. Couillard C. Lamarche B. A new method for HDL particle sizing by polyacrylamide gradient gel electrophoresis using whole plasma.J. Lipid Res. 2001; 42: 1331-1334Abstract Full Text Full Text PDF PubMed Google Scholar). LDL peak particle size was measured with gradient gel electrophoresis as previously described in detail (23.Vakkilainen J. Jauhiainen M. Ylitalo K. Nuotio I.O. Viikari J.S. Ehnholm C. Taskinen M.R. LDL particle size in familial combined hyperlipidemia: effects of serum lipids, lipoprotein-modifying enzymes, and lipid transfer proteins.J. Lipid Res. 2002; 43: 598-603Abstract Full Text Full Text PDF PubMed Google Scholar). Venous blood samples were drawn after an overnight fast. Serum and EDTA plasma samples were stored at −70°C before analysis. Serum total cholesterol (TC), TG, and HDL-C were measured with Olympus AU400 clinical chemistry analyzer (Olympus, Hamburg, Germany) by fully enzymatic methods (Olympus kits OSR 6116 and 6133 for TC and TG, respectively, and Roche Diagnostics kit 3030024 for HDL-C) (Roche Diagnostics GmbH, Mannheim, Germany). LDL-C was calculated using the Friedewald formula (24.Friedewald W.T. Levy R.I. Fredrickson D.S. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge.Clin. Chem. 1972; 18: 499-502Crossref PubMed Scopus (62) Google Scholar). Concentrations of apoA-I and apolipoprotein B (apoB) were measured with Olympus AU400 analyzer by immunoturbidometric methods (kits 64265 and 67249 from Orion Diagnostica, Espoo, Finland). Serum apolipoprotein A-II (apoA-II) was measured with Cobas Mira analyzer (Hoffman-La Roche, Basel, Switzerland) immunoturbidometrically (Wako Chemicals GmbH, Neuss, Germany, and own polyclonal antibody produced in rabbits against purified human apoA-II). Serum apoE concentration was quantitated by ELISA (25.Siggins S. Jauhiainen M. Olkkonen V.M. Tenhunen J. Ehnholm C. PLTP secreted by HepG2 cells resembles the high-activity PLTP form in human plasma.J. Lipid Res. 2003; 44: 1698-1704Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Plasma glucose was measured by the glucose dehydrogenase method (Merck Diagnostica, Darmstadt, Germany). Plasma insulin was measured by radioimmunoassay (Pharmacia AB, Uppsala, Sweden). Phospholipid transfer protein (PLTP) activity was measured using the radiometric assay as previously described (26.Damen J. Regts J. Scherphof G. Transfer of [14C] phosphatidylcholine between liposomes and human plasma high density lipoprotein. Partial purification of a transfer-stimulating plasma factor using a rapid transfer assay.Biochim. Biophys. Acta. 1982; 712: 444-452Crossref PubMed Scopus (131) Google Scholar) with minor modifications (27.Jauhiainen M. Ehnholm C. Determination of human plasma phospholipid transfer protein mass and activity.Methods. 2005; 36: 97-101Crossref PubMed Scopus (51) Google Scholar). PLTP concentration was measured with ELISA (28.Siggins S. Karkkainen M. Tenhunen J. Metso J. Tahvanainen E. Olkkonen V.M. Jauhiainen M. Ehnholm C. Quantitation of the active and low-active forms of human plasma phospholipid transfer protein by ELISA.J. Lipid Res. 2004; 45: 387-395Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Cholesterol ester transfer protein (CETP) activity was measured with radiometric assay as described by Groener et al. (29.Groener J.E. Pelton R.W. Kostner G.M. Improved estimation of cholesteryl ester transfer/exchange activity in serum or plasma.Clin. Chem. 1986; 32: 283-286Crossref PubMed Scopus (151) Google Scholar). Paraoxonase activity was measured with spectrophotometry (30.Kleemola P. Freese R. Jauhiainen M. Pahlman R. Alfthan G. Mutanen M. Dietary determinants of serum paraoxonase activity in healthy humans.Atherosclerosis. 2002; 160: 425-432Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Nitrotyrosine concentration was measured with ELISA using kit HK 501 (HyCult Biotechnology, Uden, The Netherlands). The measurements of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and C-reactive protein (CRP) were done using the immuno-chemiluminometric assay (Immulite, DPC, Siemens Healthcare Diagnostics, USA). Blood pressure values are the mean values from three consecutive measurements carried out in 1–2 min intervals. Data on alcohol consumption was collected from questionnaires filled in by the study subjects. An internal standard mixture containing 10 lipid compounds was added to each sample (20 µl of total HDL fraction). Lipids were extracted with chloroform/methanol (2:1, v/v, 100 µl) solvent. The samples were centrifuged (10000 rpm, 3 min), 60 µl of the lower lipid extract was taken into an HPLC vial insert, and another standard mixture containing three labeled lipid compounds was added. The internal standards include PC(17:0/ 0:0), PC (17:0/17:0), PE(17:0/17:0), PG(17:0/17:0)[rac], Cer(d18:1/17:0), PS(17:0/17:0), PA(17:0/17:0) and D-erythro-Sphingosine-1-Phosphate (C17 Base) from Avanti Polar Lipids (Alabaster, AL) and MG(17:0/0:0/0:0)[rac], DG(17:0/17:0/0:0)[rac], and TG(17:0/17:0/17:0) from Larodan Fine Chemicals (Malmö, Sweden). The labeled standards include PC(16:0/0:0-D3), PC(16:0/16:0-D6), and TG(16:0/16:0/16:0-13C3 from Larodan Fine Chemicals. Lipid extracts (2 µl injections) were analyzed on a Waters Q-Tof Premier mass spectrometer combined with an Acquity Ultra Performance LC™ (UPLC™) (Waters Inc., Milford, MA). The column was an Acquity UPLC BEH C18 10 × 50 mm with 1.7 µm particles and the gradient solvent system included water (1% 1M NH4Ac, 0.1% HCOOH) and LC/MS grade (Rathburn) acetonitrile/isopropanol (5:2, 1% 1M NH4Ac, 0.1% HCOOH). The total run time, including a 5 min reequilibration step, was 18 min. The flow rate was 0.200 ml/min. The data were collected at mass range of m/z 300–1200 with a scan duration of 0.2 s in ESI+ mode. Statistical analyses were performed using a freely available R language (http://www.r-project.org/). False discovery rate (FDR) q-values were computed using statistical methods from R package "qvalue." Correlation analysis was performed using "gplots" library from R package. Supervised model was built for clustering and discrimination using partial least squares discriminant analysis (PLS/DA) (31.Geladi P. Kowalski B.R. Partial least-squares regression: a tutorial.Anal. Chim. Acta. 1986; 185: 1-17Crossref Scopus (5503) Google Scholar, 32.Barker M. Rayens W. Partial least squares for discrimination.J. Chemometr. 2003; 17: 166-173Crossref Scopus (1925) Google Scholar). The PLS/DA model attempts to get the latent variables by maximizing the covariance between measured data (X) (e.g., lipid profile data) and response variables of interest (Y) (e.g., high HDL-C and low HDL-C groups). The model was built by scaling X data to unit variance and zero mean, and Y data to zero mean. The random subsets cross validation method (33.Wise B.M. Gallagher N.B. Bro R. Shaver J.M. Windig W. Koch J.S. PLS Toolbox 3.5 for use with Matlab. Eigenvector Research, Inc, Manson, WA2005Google Scholar) and Q2 scores were used to optimize the models. The variable importance in the projection (VIP) values (34.Wold S. Esbensen K. Geladi P. Principal component analysis.Chemom. Intell. Lab. Syst. 1987; 2: 37-52Crossref Scopus (7499) Google Scholar) were computed to identify most important lipid species contributing to separation of low and high HDL-C groups in the PLS/DA model. PLS/DA model was built using Matlab, version 7.0 (Mathworks, Natick, MA) and PLS Toolbox, version 4.0, of the Matlab package (Eigenvector Research, Wenatchee, WA). In the case of apoA-I, we first built an all-atom model for apoA-I based on the previous data, and then we coarse grained the structure. The relative conformation of apoA-Is was similar to the belt-like structure of Borhani et al. who produced the solution X-ray structure of truncated apoA-I (residues 44-243) (35.Borhani D.W. Rogers D.P. Engler J.A. Brouillette C.G. Crystal structure of truncated human apolipoprotein A-I suggests a lipid-bound conformation.Proc. Natl. Acad. Sci. USA. 1997; 94: 12291-12296Crossref PubMed Scopus (408) Google Scholar). We also followed the approach of Segrest et al. who arranged atomistic apoA-Is around the lipid moiety in belt-like fashion (36.Segrest J.P. Jones M.K. Klon A.E. Sheldahl C.J. Hellinger M. De Loof H. Harvey S.C. A detailed molecular belt model for apolipoprotein A-I in discoidal high density lipoprotein.J. Biol. Chem. 1999; 274: 31755-31758Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar) so that the hydrophobic sides of amphiphilic α-helices were pointing toward the lipid moiety. This structure was further used to build a model for the full-length apoA-I by adding the previously absent N-terminal part (residues 1-43) of the apoA-I to our model. The conformation of this segment was determined to be α-helical as suggested by the previous NMR data (37.Okon M. Frank P. Marcel Y. Cushley R. Heteronuclear NMR studies of human serum apolipoprotein A-I. Part I. Secondary structure in lipid-mimetic solution.FEBS Lett. 2002; 517: 139-143Crossref PubMed Scopus (32) Google Scholar). The hydrophobic side of this helix was also orientated toward the lipid moiety. We directly coarse grained the atomistic structure and placed the apoA-Is around the previously simulated lipid droplet. The secondary structure of apoA-I was enforced almost completely to the α-helical conformation; only the N- and C-terminal ends were modeled as random coils. This approach has been used previously in the work of Catte et al. with truncated apoA-I (38.Catte A. Patterson J.C. Bashtovyy D. Jones M.K. Gu F. Li L. Rampioni A. Sengupta D. Vuorela T. Niemelä P. et al.Structure of spheroidal HDL particles revealed by combined atomistic and coarse-grained simulations.Biophys. J. 2008; 94: 2306-2319Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Thus, the flexibility of α-helical segments arises from the three-body angle and four-body dihedral potentials of backbone beads that were produced by Monticelli et al. to extend the MARTINI force field to proteins (39.Monticelli L. Kandasamy S.K. Periole X. Larson R.G. Tieleman D.P. Marrink S-J. The MARTINI coarse-grained force field: extension to proteins.J. Chem. Theory Comput. 2008; 4: 819-834Crossref PubMed Scopus (1693) Google Scholar). The radius of the ring formed by apoA-Is was ∼12.5 nm, and the average distance between apoA-Is was 2.5–3.0 nm. Next, to produce starting structures for the low HDL-C, normal HDL-C, and high HDL-C systems, the lipid composition of the previously simulated lipid droplet was tuned according to Table 2. All lipid molecules, except those that were added to the starting system, were inside the ring formed by apoA-Is. Next all three systems were energy minimized by the steepest descent algorithm, and 10 ns vacuum simulations were carried out to get lipids to the one unified phase, which meant that the added lipids diffused to the main lipid particle. Systems were solvated, and 18 water beads were changed to Na+ beads to neutralize the charges in the systems. Production simulations lasted for 2 μs, which corresponds to 8 μs of effective time as the MARTINI model speeds up the dynamics by an approximate factor of four (39.Monticelli L. Kandasamy S.K. Periole X. Larson R.G. Tieleman D.P. Marrink S-J. The MARTINI coarse-grained force field: extension to proteins.J. Chem. Theory Comput. 2008; 4: 819-834Crossref PubMed Scopus (1693) Google Scholar). System sizes were approximately 30,000–35,000 water beads and 4,300–5,000 lipid and protein beads in total. The box size in each simulation was 17 × 17 × 17 nm.TABLE 2Lipid compositions and concentrations used in simulationsLipid CompositionsaNumber of molecules per HDL particle. Determined by LipidomicsapoA-ISMPCFChoChoETGlysoPCNormal HDL-C21810950901910Low HDL-C2131092581245High HDL-C22310975991415Average ConcentrationsbUnits are μmol/l [lipid] / mg/dl [apoA-I]. of Lipid Classes Determined by LipidomicsSMHDL-CChoETGlysoPCLow HDL-C0.057 ± 0.0037.545 ± 0.2000.105 ± 0.0090.436 ± 0.0240.014 ± 0.007High HDL-C0.082 ± 0.00410.928 ± 0.2540.132 ± 0.0200.244 ± 0.0360.022 ± 0.002Abbreviations: Apo, apolipoprotein; ChoE, cholesteryl ester; FCho, free cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; lysoPC, lysophosphatidylcholine; SM, sphingomyelin; TG, triacylglycerol.a Number of molecules per HDL particle.b Units are μmol/l [lipid] / mg/dl [apoA-I]. Open table in a new tab Abbreviations: Apo, apolipoprotein; ChoE, cholesteryl ester; FCho, free cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; lysoPC, lysophosphatidylcholine; SM, sphingomyelin; TG, triacylglycerol. Simulations were performed by the GROMACS simulation package (v. 4.0) (40.Hess B. Kutzner C. van der Spoel D. Lindahl E. GROMACS 4: algorithms for highly efficient, load-balanced, and scalable molecular simulation.J. Chem. Theory Comput. 2008; 4: 435-447Crossref PubMed Scopus (11893) Google Scholar). The standard MARTINI lipid force field was used for PC (PC(16:0/18:1)), lysoPC (PC(16:0/0:0)), FCho, and SM (SM(d18:1/16:0)) molecules (41.Marrink S.J.H. Risselada J. Yefimov S.D. Tieleman P. de Vries A.H. The MARTINI force field: coarse grained model for biomolecular simulations.J. Phys. Chem. B. 2007; 111: 7812-7824Crossref PubMed Scopus (3583) Google Scholar). Protein part was modeled using the protein extension of the MARTINI description (39.Monticelli L. Kandasamy S.K. Periole X. Larson R.G. Tieleman D.P. Marrink S-J. The MARTINI coarse-grained force field: extension to proteins.J. Chem. Theory Compu
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