FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2073276199> ?p ?o ?g. }

• W2073276199 endingPage "25151" @default.
• W2073276199 startingPage "25145" @default.
• W2073276199 abstract "To understand how the lipid composition of high density lipoprotein mediates the efflux of cellular cholesterol, we have characterized the effects of variations in the lipid composition of well defined model sonicated apolipoprotein A-I (apoA-I)-containing lipoprotein (LpA-I) particle on cholesterol efflux from cultured human skin fibroblasts. LpA-I particles with varying content of phosphatidylcholine (POPC), phosphatidylinositol, sphingomyelin, cholesterol ester, and triolein were prepared by co-sonication. Association of as little as 5 mol of phosphatidylcholine with apoA-I is sufficient to transform lipid-free apoA-I into a distinct lipoprotein-like particle that is a significantly better acceptor of cellular cholesterol. Increasing the ratio of POPC/apoA-I from 5/1 to 35.5/1 in the sonicated LpA-I is associated with a significant increase in the release of cellular cholesterol. At low POPC/apoA-I ratios, native gradient gel electrophoresis of the LpA-I shows these lipoproteins to be small complexes (around 5-6 nm), with only 1 molecule of apoA-I (Lp1A-I). At a POPC/apoA-I ratio above 11/1, LpA-I form well defined complexes that contain 2 molecules of apoA-I (Lp2A-I) and range in size from 7.6 to 7.7 nm. Inclusion of sphingomyelin into an Lp1A-I further stimulates cholesterol efflux significantly. In contrast, inclusion of either sphingomyelin or phosphatidylinositol into a sonicated Lp2A-I has no effect on cholesterol efflux. Incorporation of cholesterol ester and/or triolein into an Lp2A-I particle is associated with a small reduction in cholesterol efflux to these lipoproteins. Therefore, cholesterol efflux from human fibroblasts is directly proportional to the amount and type of phospholipid in a sonicated LpA-I particle. Changes in the conformation and charge of apoA-I that result from changes in the lipid composition of a sonicated LpA-I particle appear to directly affect the ability of the lipoprotein to bind and retain cholesterol molecules. These data therefore suggest that the adsorption/desorption of cholesterol molecules to/from a sonicated LpA-I complex may be less sensitive to interfacial lipid-lipid interactions, but may depend on a conformation-dependent ability of apoA-I to bind cholesterol. To understand how the lipid composition of high density lipoprotein mediates the efflux of cellular cholesterol, we have characterized the effects of variations in the lipid composition of well defined model sonicated apolipoprotein A-I (apoA-I)-containing lipoprotein (LpA-I) particle on cholesterol efflux from cultured human skin fibroblasts. LpA-I particles with varying content of phosphatidylcholine (POPC), phosphatidylinositol, sphingomyelin, cholesterol ester, and triolein were prepared by co-sonication. Association of as little as 5 mol of phosphatidylcholine with apoA-I is sufficient to transform lipid-free apoA-I into a distinct lipoprotein-like particle that is a significantly better acceptor of cellular cholesterol. Increasing the ratio of POPC/apoA-I from 5/1 to 35.5/1 in the sonicated LpA-I is associated with a significant increase in the release of cellular cholesterol. At low POPC/apoA-I ratios, native gradient gel electrophoresis of the LpA-I shows these lipoproteins to be small complexes (around 5-6 nm), with only 1 molecule of apoA-I (Lp1A-I). At a POPC/apoA-I ratio above 11/1, LpA-I form well defined complexes that contain 2 molecules of apoA-I (Lp2A-I) and range in size from 7.6 to 7.7 nm. Inclusion of sphingomyelin into an Lp1A-I further stimulates cholesterol efflux significantly. In contrast, inclusion of either sphingomyelin or phosphatidylinositol into a sonicated Lp2A-I has no effect on cholesterol efflux. Incorporation of cholesterol ester and/or triolein into an Lp2A-I particle is associated with a small reduction in cholesterol efflux to these lipoproteins. Therefore, cholesterol efflux from human fibroblasts is directly proportional to the amount and type of phospholipid in a sonicated LpA-I particle. Changes in the conformation and charge of apoA-I that result from changes in the lipid composition of a sonicated LpA-I particle appear to directly affect the ability of the lipoprotein to bind and retain cholesterol molecules. These data therefore suggest that the adsorption/desorption of cholesterol molecules to/from a sonicated LpA-I complex may be less sensitive to interfacial lipid-lipid interactions, but may depend on a conformation-dependent ability of apoA-I to bind cholesterol. HDL 1The abbreviations used are: HDLhigh density lipoproteinapoA-Iapolipoprotein A-ILpA-IapoA-I-containing lipoproteinLp2A-ILpA-I containing 2 apoA-I/particlePCphosphatidylcholinePOPC1-palmitoyl 2-oleoyl phosphatidylcholinePIphosphatidylinositolSMsphingomyelinUCfree cholesterolCEcholesteryl linoleateTGtrioleinDMEMDulbecco's modified Eagle's mediumNDGGEnon-denaturing gradient gel electrophoresis. are a highly heterogeneous class of lipoproteins of various origins formed during the catabolism of triglyceride-rich lipoproteins and the synthesis and secretion of nascent HDL particles by the liver or intestine (Banerjee and Redman, 4Banerjee D. Redman C.M. J. Cell Biol. 1983; 96: 651-660Crossref PubMed Scopus (30) Google Scholar; Eisenberg, 13Eisenberg S. J. Lipid Res. 1984; 25: 1017-1058Abstract Full Text PDF PubMed Google Scholar; McCall et al., 34McCall M.R. Forte T.M. Shore V.G. J. Lipid Res. 1988; 29: 1127-1137Abstract Full Text PDF PubMed Google Scholar, 35McCall M.R. Nichols A.V. Blanche P.J. Shore V.G. Forte T.M. J. Lipid Res. 1989; 30: 1579-1589Abstract Full Text PDF PubMed Google Scholar; Castle et al., 9Castle C.K. Pape M.E. Marotti K.R. Melchior G.W. J. Lipid Res. 1991; 32: 439-447Abstract Full Text PDF PubMed Google Scholar; Thrift et al., 53Thrift R.N. Forte T.M. Cahoon B.E. Shore V.G. J. Lipid Res. 1986; 27: 236-250Abstract Full Text PDF PubMed Google Scholar). Recent studies have suggested that HDL may also be generated by the stepwise lipidation of apoA-I by acquisition of lipids from other lipoproteins (Hussain et al., 23Hussain M.M. Zanni E.E. Kelly M. Zannis V.I. Biochim. Biophys. Acta. 1989; 1001: 90-101Crossref PubMed Scopus (21) Google Scholar) or from extrahepatic cells (Hara and Yokoyama, 20Hara H. Yokoyama S. J. Biol. Chem. 1991; 266: 3080-3086Abstract Full Text PDF PubMed Google Scholar, 21Hara H. Yokoyama S. Biochemistry. 1992; 31: 2040-2046Crossref PubMed Scopus (87) Google Scholar; Bielicki et al., 6Bielicki J.K. Johnson W.J. Glick J.M. Rothblat G.H. Biochim. Biophys. Acta Lipids Lipid Metab. 1991; 1085: 7-14Crossref PubMed Scopus (20) Google Scholar, 7Bielicki J.K. Johnson W.J. Weinberg R.B. Glick J.M. Rothblat G.H. J. Lipid Res. 1992; 33: 1699-1709Abstract Full Text PDF PubMed Google Scholar; Forte et al., 16Forte T.M. Goth-Goldstein R. Nordhausen R.W. McCall M.R. J. Lipid Res. 1993; 34: 317-324Abstract Full Text PDF PubMed Google Scholar). Several lines of evidence suggest that HDL may be assembled extracellularly. First, apoA-I, the predominant apolipoprotein of HDL, appears to be secreted by the liver and the intestine mainly in the lipid free form; however, only about 3% of this apolipoprotein is present in plasma as lipid-free form (Neary and Gowland, 38Neary R.H. Gowland E. Clin. Chem. 1987; 33: 1163-1169Crossref PubMed Scopus (44) Google Scholar). Additionally, lipid-free apoA-I is able to release both phospholipid and cholesterol from extrahepatic cells (Bielicki et al., 7Bielicki J.K. Johnson W.J. Weinberg R.B. Glick J.M. Rothblat G.H. J. Lipid Res. 1992; 33: 1699-1709Abstract Full Text PDF PubMed Google Scholar; Hara and Yokoyama, 20Hara H. Yokoyama S. J. Biol. Chem. 1991; 266: 3080-3086Abstract Full Text PDF PubMed Google Scholar). Finally, the incubation of lipid-free apoA-I with Chinese hamster ovary cells in serum-free medium generates LpA-I with a gradual size increment (Forte et al., 16Forte T.M. Goth-Goldstein R. Nordhausen R.W. McCall M.R. J. Lipid Res. 1993; 34: 317-324Abstract Full Text PDF PubMed Google Scholar). A subspecies of small particles (7.3 nm) composed of 94% apoA-I and 6% phospholipids has also been identified in these studies, which appears analogous both in composition and size to the human plasma pre-β-HDL reported earlier (Kunitake et al., 29Kunitake S.T. Lasala K.L. Kane J.P. J. Lipid Res. 1985; 26: 549-555Abstract Full Text PDF PubMed Google Scholar) and may be analogous to the particles reported to be highly active in cellular cholesterol efflux (Castro and Fielding, 10Castro G.R. Fielding C.J. Biochemistry. 1988; 27: 25-29Crossref PubMed Scopus (564) Google Scholar). high density lipoprotein apolipoprotein A-I apoA-I-containing lipoprotein LpA-I containing 2 apoA-I/particle phosphatidylcholine 1-palmitoyl 2-oleoyl phosphatidylcholine phosphatidylinositol sphingomyelin free cholesterol cholesteryl linoleate triolein Dulbecco's modified Eagle's medium non-denaturing gradient gel electrophoresis. When lipid-free apoA-I is incubated with extrahepatic cells, the incorporation of phospholipids into apoA-I does not seem to parallel that of cholesterol as evidenced by the following. (i) The acquisition of phospholipid and cholesterol by human apoA-I from isolated microsomal membrane (Nunez and Swaney, 40Nunez J.F. Swaney J.B. J. Biol. Chem. 1984; 259: 9141-9148Abstract Full Text PDF PubMed Google Scholar) as well as from intact human fibroblasts or mouse microphages (Li et al., 31Li Q. Kamaba A. Yokoyama S. Biochemistry. 1993; 32: 4597-4603Crossref PubMed Scopus (57) Google Scholar; Li and Yokoyama, 30Li Q. Yokoyama S. J. Biol. Chem. 1995; 270: 26216-26223Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar; Beliecki et al., 1992; Yancey et al., 54Yancey P.G. Bielicki J.K. Johnson W.J. Lund-Katz S. Palgunachari M.N. Anantharamaiah G.M. Segrest J.P. Phillips M.C. Rothblat G.H. Biochemistry. 1995; 34: 7955-7965Crossref PubMed Scopus (192) Google Scholar) is non-stoichiometric; (ii) phospholipid efflux to lipid-free apoA-I precedes and facilitates the efflux of cholesterol (Yancey et al., 54Yancey P.G. Bielicki J.K. Johnson W.J. Lund-Katz S. Palgunachari M.N. Anantharamaiah G.M. Segrest J.P. Phillips M.C. Rothblat G.H. Biochemistry. 1995; 34: 7955-7965Crossref PubMed Scopus (192) Google Scholar); (iii) association of phospholipids with apoHDL greatly increases its ability to release cellular cholesterol compared to the delipidated apoHDL (Stein and Stein, 49Stein O. Stein Y. Biochim. Biophys. Acta. 1973; 326: 232-244Crossref PubMed Scopus (79) Google Scholar), while treatment of HDL with either phospholipase A2 or heparin-releasable rat hepatic lipase reduced cholesterol efflux (Johnson et al., 24Johnson W.J. Bamberger M.J. Latta R.A. Rapp P.E. Phillips M.C. Rothblat G.H. J. Biol. Chem. 1986; 261: 5766-5776Abstract Full Text PDF PubMed Google Scholar). Therefore, it appears that the formation of apoA-I-phospholipid complexes is an important preliminary step before apoA-I can significantly promote the efflux of cellular cholesterol. However, there is up to now no direct evidence for a relationship between the progressive lipidation of apoA-I and the ability of the corresponding complexes to release cellular cholesterol in short term incubations, and the corresponding changes in certain physical parameters assumed by the apoA-I-lipid complexes during their progressive lipidation. We have attempted to answer these questions by preparation of model complexes generated in vitro by co-sonication of phospholipid and apolipoprotein as described previously (Hirz and Scanu, 22Hirz R. Scanu A.M. Biochim. Biophys. Acta. 1970; 207: 364-367Crossref PubMed Scopus (43) Google Scholar; Sparks et al., 47Sparks D.L. Davidson S.W. Lund-Katz S. Phillips M.C. J. Biol. Chem. 1995; 270: 26910-26917Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). We first prepared a series of reconstituted sonicated LpA-I complexes with varying 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC)/apoA-I ratios in order to answer the above questions. In addition, two other series of POPC·apoA-I complexes have also been prepared to investigate the contribution of HDL lipid composition, specifically surface phospholipid components, sphingomyelin (SM) and phosphatidylinositol (PI), or core neutral lipids, cholesterol ester (CE) and triolein (TG), to cellular cholesterol efflux. These studies were carried out using reconstituted LpA-I designed to mimic the high affinity acceptor, pre-β1-LpA-I, which has been identified in whole plasma and lymph (Fielding and Fielding, 14Fielding C.J. Fielding P.E. J. Lipid Res. 1995; 36: 211-228Abstract Full Text PDF PubMed Google Scholar), and to investigate the effect of progressive incorporation of different lipids into apoA-I on its function in cellular cholesterol efflux. POPC, bovine brain SM, and bovine liver PI were purchased from Avanti Polar Lipids Inc. (Birmingham, AL). TG, CE, free cholesterol (UC), and essentially fatty acid-free bovine serum albumin were obtained from Sigma. 1-2n-[3H]Cholesterol, L-α[myo-inositol-2-3H]phosphatidylinositol ([3H]PI) and choline-[methyl-14C]sphingomyelin ([14C]SM) with specific activities of 52, 11, and 50 mCi/mmol, respectively, were obtained from Du Pont Canada Inc. (Mississauga, Canada). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, L-glutamine, and penicillin-streptomycin used for cell culture were purchased from Life Technologies, Inc. All other reagents were analytical grade. HDL was isolated by standard sequential ultracentrifugation from fresh plasma obtained from normolipidemic blood donors (Schumaker and Puppione, 42Schumaker V.N. Puppione D.L. Methods Enzymol. 1986; 128: 155-209Crossref PubMed Scopus (467) Google Scholar). The delipidation of HDL and purification of apoA-I was performed as described previously (Brewer et al., 8Brewer Jr., H.B. Ronan R. Meng M. Bishop C. Methods Enzymol. 1986; 128: 223-235Crossref PubMed Scopus (130) Google Scholar). A series of POPC·apoA-I complexes were prepared by a method described previously (Sparks et al., 48Sparks D.L. Anantharamaiah G.M. Segrest J.P. Phillips M.C. J. Biol. Chem. 1995; 270: 5151-5157Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Briefly, POPC in chloroform was dried under nitrogen in glass tubes. 900 μl of reconstitution buffer containing 10 mM Tris, 150 mM NaCl, 0.01% EDTA, 1 mM NaN3 (pH 8.0) was then added to the tubes and vortexed vigorously for 3 min to resuspend the POPC. The mixture was sonicated for 1 min at 100% duty cycle using a Branson 450 sonicator with a one-eighth-inch tapered microtip probe and an output control setting at 3 (manufacturer rated output of 40 watts), then incubated in a water bath at 37°C for 30 min, followed by sonication at 95% duty cycle for 5 min. All sonications were performed in 12 x 75-mm test tubes in a 15°C water bath and under nitrogen. ApoA-I at a concentration of 1.4 mg/ml was added to the tubes, and sonicated again for 4 x 1 min at 90% duty cycle. The resulting mixtures were passed through a 0.22-μm filter, and then reisolated by size exclusion chromatography on a Superose-6 column. For the preparation of sonicated spherical LpA-I with UC, SM, PI, CE, and/or TG, these components were mixed (at appropriate concentrations) with POPC and then processed as described for the POPC·apoA-I complexes. As control and for comparison with other model lipoproteins, reconstituted discoidal LpA-I were prepared in the presence of cholate as described previously by Sparks et al. (44Sparks D.L. Phillips M.C. Lund-Katz S. J. Biol. Chem. 1992; 267: 25830-25838Abstract Full Text PDF PubMed Google Scholar) from an initial mixture of POPC·apoA-I at a molar ratio of 40/1. Non-denaturing gradient gel electrophoresis (NDGGE) was carried out on precast gel (8-25%) using the Phast system (Pharmacia Biotech Inc.) to assess the homogeneity of LpA-I, and to estimate their Stoke's diameters calculated from a quadratic equation, derived from polynomial regression of the Stoke's diameters versus the migration distances of five standard proteins (high Mr standard, Pharmacia Biotech Inc.) (Nichols et al., 39Nichols A.V. Krauss R.M. Musliner T.A. Methods Enzymol. 1986; 128: 417-431Crossref PubMed Scopus (448) Google Scholar). The electrophoretic mobilities and surface potentials of LpA-I were determined by electrophoresis on 0.6% agarose gels (Beckman, Paragon Lipo kit) and calculated as described previously (Sparks and Phillips, 45Sparks D.L. Phillips M.C. J. Lipid Res. 1992; 33: 123-130Abstract Full Text PDF PubMed Google Scholar). The α-helix content of apoA-I in LpA-I was determined by circular dichroism (CD) spectroscopy at 222 nm (Sparks et al., 43Sparks D.L. Lund-Katz S. Phillips M.C. J. Biol. Chem. 1992; 267: 25839-25847Abstract Full Text PDF PubMed Google Scholar). ApoA-I number in each reconstituted LpA-I particle was estimated by cross-linking of apolipoproteins with dimethyl suberimidate (Swaney, 52Swaney J.B. Methods Enzymol. 1986; 128: 613-626Crossref PubMed Scopus (35) Google Scholar). The protein concentration was determined by the Lowry method (Lowry et al., 32Lowry O.H. Rosebrough M.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) and free cholesterol, total cholesterol, and phospholipid concentrations were measured using commercial enzymatic test kits (Boehringer Mannheim GmbH, Mannheim, Germany). The PI and SM contents of LpA-I were determined by inclusion in representative preparations of [3H]PI or [14C]SM. Normal human skin fibroblasts were purchased from Clonetics Inc. at the 9th passage, and maintained in a standard condition and used for efflux studies between the 16th and 22th passages. The conditions for seeding and labeling of the cells and for the study of cellular cholesterol efflux were described previously (Zhao and Marcel, 56Zhao Y. Marcel Y.L. Biochemistry. 1996; 35: 7174-7180Crossref PubMed Scopus (98) Google Scholar). Co-sonication of POPC and apoA-I results in well defined POPC·apoA-I complexes, wherein the POPC content can be changed by varying the POPC/apoA-I ratio in the initial mixture. POPC·apoA-I complexes are separated from the lipid-free apoA-I and from the non-incorporated lipid by size exclusion chromatography. As shown in Fig. 1, when the initial molar ratio of POPC/apoA-I is below 20/1, the purified POPC·apoA-I complexes appear as well defined particles on gradient acrylamide gels with sizes at about 5-6 nm. In contrast, large homogeneous LpA-I can be generated with initial POPC/apoA-I molar ratios at and above 20/1. These sonicated LpA-I particles prepared from the initial molar ratios of 20/1, 30/1, and 60/1 have estimated Stoke's diameters on NDGGE of 7.6, 7.6, and 7.7 nm, respectively. The slow migration of the lipid-free apoA-I is an artifact, which is a function of its concentration-dependent aggregation. Chemical cross-linking of the POPC· apoA-I complexes prepared from an initial POPC/apoA-I ratio below 20/1 indicates that only 1 molecule of apoA-I/complex is present, while those formed at 20/1 and above contain 2 molecules of apoA-I/particle. The composition of these sonicated POPC·apoA-I complexes re-isolated by gel filtration is summarized in Table I. The co-sonication of POPC and apoA-I allows the production of POPC·apoA-I complexes with as little as 2.4 mol of POPC/molecule of apoA-I. The lipid/protein ratio of these lipid-poor LpA-I complexes is analogous to that of the pre-β-HDL found in normolipidemic human plasma and the size of the complexes generated at the low ratios of POPC/apoA-I is analogous to that of pre-β-LpA-I (Kunitake et al., 29Kunitake S.T. Lasala K.L. Kane J.P. J. Lipid Res. 1985; 26: 549-555Abstract Full Text PDF PubMed Google Scholar; Castro and Fielding, 10Castro G.R. Fielding C.J. Biochemistry. 1988; 27: 25-29Crossref PubMed Scopus (564) Google Scholar; Francone and Fielding, 17Francone O.L. Fielding C.J. Eur. Heart J. 1990; 11: 218-224Crossref PubMed Google Scholar).Table I.Characterization of reconstituted LpA-I with varying ratios of POPC/apoA-ILpA-IPOPC/apoA-I Molar RatioApoA-IcEstimated by protein cross-linking with dimethyl suberimidate and subsequent SDS-polyacrylamide gel electrophoresis.POPCdTotal phospholipid content of each reconstituted LpA-I particle.SizeeHydrodynamic diameters of LpA-I determined from NDGGE (±0.5 nm S.D.). Particles for which size is not given are either heterogeneous or had a size outside of the standard range.α-HelixfDetermined from molar ellipticities at 222 nm in spectra (±4% S.D.).Surface PotentialgCalculated from the electrophoretic migration of LpA-I on agarose gel (±0.2 mV S.D.).(Initial)aThe POPC/apoA-I molar ratios of the initial mixtures for the preparation of LpA-I.(Final)bThe POPC/apoA-I molar ratios of reconstituted LpA-I after re-isolation (S.D. < 5%).mol/LpA-Inm%-mVApoA-IApoA-I0 :1478.3S15 :12.4 :112.44810.0S210 :15.0 :115.04910.0S320 :111.1 :1222.27.6519.6S430 :116.2 :1232.47.6539.4S560 :135.5 :1271.07.7599.2D1hA discoidal Lp2A-I prepared by cholate dialysis was included for comparison with results presented elsewhere.40 :138.7 :1279.09.3557.8a The POPC/apoA-I molar ratios of the initial mixtures for the preparation of LpA-I.b The POPC/apoA-I molar ratios of reconstituted LpA-I after re-isolation (S.D. < 5%).c Estimated by protein cross-linking with dimethyl suberimidate and subsequent SDS-polyacrylamide gel electrophoresis.d Total phospholipid content of each reconstituted LpA-I particle.e Hydrodynamic diameters of LpA-I determined from NDGGE (±0.5 nm S.D.). Particles for which size is not given are either heterogeneous or had a size outside of the standard range.f Determined from molar ellipticities at 222 nm in spectra (±4% S.D.).g Calculated from the electrophoretic migration of LpA-I on agarose gel (±0.2 mV S.D.).h A discoidal Lp2A-I prepared by cholate dialysis was included for comparison with results presented elsewhere. Open table in a new tab The molar range of POPC/apoA-I allowing the formation of sonicated LpA-I complexes varies from a few molecules to a maximum of about 36 mol, while 30-133 mol of POPC are needed to form the homogeneous discoidal LpA-I prepared in the presence of cholate (Bergeron et al., 5Bergeron J. Frank P.G. Scales D. Meng Q.-H. Castro G. Marcel Y.L. J. Biol. Chem. 1995; 270: 27429-27438Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar; Sparks et al., 44Sparks D.L. Phillips M.C. Lund-Katz S. J. Biol. Chem. 1992; 267: 25830-25838Abstract Full Text PDF PubMed Google Scholar, 43Sparks D.L. Lund-Katz S. Phillips M.C. J. Biol. Chem. 1992; 267: 25839-25847Abstract Full Text PDF PubMed Google Scholar, 48Sparks D.L. Anantharamaiah G.M. Segrest J.P. Phillips M.C. J. Biol. Chem. 1995; 270: 5151-5157Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Although the increase in the POPC/apoA-I ratios alters only slightly the sizes of the sonicated POPC·apoA-I complexes as estimated on NDGGE, the secondary structure of apoA-I does change with the progressive increase of POPC in the sonicated complexes, as demonstrated by the modification in α-helical contents and electrophoretic mobilities. This also provides indirect evidence of the association of the phospholipids with apoA-I. The α-helical content increases slightly with addition of 2.4 mol of POPC into apoA-I and continues to increase as more POPC associates with apoA-I (Table I). Upon electrophoresis, POPC·apoA-I complexes prepared by sonication migrate to intermediate positions between pre-β- and α-HDL, which tend to decrease with an increase of POPC/apoA-I ratio (Fig. 2A). The change in the surface charge of these complexes is summarized in Table I. Next to PC, SM and PI are the two major phospholipids associated with HDL. SM is also one of the major phospholipids of nascent LpA-I, including those described as high affinity acceptors for cellular cholesterol (Castro and Fielding, 10Castro G.R. Fielding C.J. Biochemistry. 1988; 27: 25-29Crossref PubMed Scopus (564) Google Scholar; Hara and Yokoyama, 20Hara H. Yokoyama S. J. Biol. Chem. 1991; 266: 3080-3086Abstract Full Text PDF PubMed Google Scholar; Fielding and Fielding, 14Fielding C.J. Fielding P.E. J. Lipid Res. 1995; 36: 211-228Abstract Full Text PDF PubMed Google Scholar). We have tested a large range of ratios of SM or PI/POPC in order to mimic the different phospholipid to apoA-I molar ratios found in typical plasma LpA-I. We have observed that SM competes effectively with POPC for association with apoA-I. Typically, starting from a molar ratio of 40:1:4:20 (POPC:apoA-I:UC:SM), we obtained purified Lp2A-I particles with ratios of 14:1:2.1:11.2, as shown in Table II. The presence of SM does not significantly change the particle size (Table II) or electrophoretic mobility of sonicated LpA-I complexes (Fig. 2B). As should be expected, the inclusion of PI in the sonicated complexes significantly increases the electrophoretic mobility (Fig. 2B), which can slightly affects the determination of particle size (Table II). We have also generated a species of SM-containing complexes with a low phospholipid/apoA-I ratio (S9, Table II), which are Lp1A-I particles with a size similar to its POPC/apoA-I counterpart (less than 6 nm, S3, Table I) and an electrophoretic mobility within the pre-β range (Fig. 2B).Table IICharacterization of sonicated LpA-I complexes with sphingomyelin, phosphatidylinositol, or HDL core neutral lipidsLpA-IComposition molar ratioApoA-ISizeSurface Potential(InitialFinal)mol/LpA-Inm-mVVariation in sphingomyelin and phosphatidylinositol (POPC/ApoA-I/UC/SM/PI)S660:1:4:0:029:1:1.8:0:027.79.6S740:1:4:20:014:1:2.1:11.2:027.89.1S850:1:4:0:1024:1:1.5:0:6.127.512.9S910:1:0:10:047:1:0:5.4:018.3Variation in neutral core lipids (POPC/ApoA-I/UC/CE/TG)S1060:1:3:6:046:1:1.2:2.9:027.89.3S1160:1:3:0:638:1:1.3:0:6.427.710.1S1260:1:3:6:643:1:1.2:2.6:6.527.6510.2 Open table in a new tab Four subspecies of sonicated LpA-I containing CE and/or TG were made as indicated in Table II. Three moles of UC, 6 mol of CE, and/or 6 mol of TG were added to POPC·apoA-I mixtures with an initial molar ratio of 60/1, a ratio that we have shown to generate homogeneous LpA-I particles. Compared to the LpA-I particles containing only POPC and apoA-I, the presence of UC, CE, and/or TG did not affect the homogeneity of these sonicated LpA-I particles as demonstrated by NDGGE. The presence of UC slightly reduced the level of POPC that could be incorporated into apoA-I; however, at the molar ratios used here, the presence of TG and especially that of CE appeared to increase POPC incorporation into LpA-I. TG and CE alone could be very efficiently incorporated into LpA-I, with TG having an incorporation efficiency close to 100%. In addition, the presence of CE did not modify either the size of LpA-I particles (Table II), or their electrophoretic mobility on agarose gel (Fig. 2B). The presence of TG alone or TG and CE together did not alter the size of these particles (Table II); however, their electrophoretic mobility was slightly increased with both CE and TG (Fig. 2B). In contrast to Lp2A-I prepared in the presence of cholate (discoidal LpA-I), where variations in POPC content have no effect on the ability of these lipoproteins to accept cholesterol, 2Y. Zhao, D. L. Sparks, and Y. L. Marcel, unpublished data. an increase in the POPC content of sonicated POPC·apoA-I complexes significantly enhanced their ability to promote the efflux of cellular cholesterol (Fig. 3). Within the first 90 min of incubation, the efflux followed the biphasic pattern usually observed with fibroblasts (Zhao and Marcel, 56Zhao Y. Marcel Y.L. Biochemistry. 1996; 35: 7174-7180Crossref PubMed Scopus (98) Google Scholar). The stimulating effect of the phospholipid content of the LpA-I complexes could also be observed in both phases. The second phase of cellular cholesterol efflux to these sonicated LpA-I complexes was linear and positively related to the phospholipid levels incorporated into LpA-I. No saturation was observed up to 90 min of incubation. Cellular cholesterol efflux is very sensitive to the association of phospholipid with apoA-I, as demonstrated by the significant increase in efflux observed with as few as 2.4 mol of POPC/mol of apoA-I compared to lipid free apoA-I (results not shown). The efflux of cholesterol from fibroblasts to S2 or S4 was concentration-dependent up to 100 μg of apoA-I/ml, where both particles were close to saturation (Fig. 4). Again, the LpA-I complex containing more POPC showed higher ability to accept cell-derived cholesterol at all concentrations tested. The calculated Vmax values are 16.9 and 12.6 μg/ml, respectively, for S2 and S4. At all concentrations tested, lipid free apoA-I was not efficient in promoting cholesterol efflux compared to the lipidated apoA-I.Fig. 4Effect of POPC content on concentration-dependent cellular cholesterol efflux to POPC·apoA-I complexes. Human skin fibroblasts were seeded and labeled as described in Fig. 3. For the efflux study, the washed cells were incubated with DMEM containing increasing protein concentration of sonicated lipid-free apoA-I, or POPC·apoA-I complexes at the final POPC/apoA-I molar ratio of 5/1 or 16.2/1 (S2 and S4, respectively). Aliquots of medium were taken at 90 min of incubation for radioactivity determination. Efflux is expressed as medium radioactivity/μg of cell protein (n = 4).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To compare the abilities of sonicated LpA-I and discoidal LpA-I (prepared by cholate dispersion) to accept cellular cholesterol from cultured human skin fibroblasts, discoidal Lp2" @default.
• W2073276199 created "2016-06-24" @default.
• W2073276199 creator A5049874756 @default.
• W2073276199 creator A5055362268 @default.
• W2073276199 creator A5073121857 @default.
• W2073276199 date "1996-10-01" @default.
• W2073276199 modified "2023-10-18" @default.
• W2073276199 title "Specific Phospholipid Association with Apolipoprotein A-I Stimulates Cholesterol Efflux from Human Fibroblasts" @default.
• W2073276199 cites W121664585 @default.
• W2073276199 cites W1449253847 @default.
• W2073276199 cites W1484994078 @default.
• W2073276199 cites W1486434785 @default.
• W2073276199 cites W1487993287 @default.
• W2073276199 cites W1547690943 @default.
• W2073276199 cites W1552121147 @default.
• W2073276199 cites W1565539489 @default.
• W2073276199 cites W1569934993 @default.
• W2073276199 cites W1595123479 @default.
• W2073276199 cites W1604508313 @default.
• W2073276199 cites W1622026323 @default.
• W2073276199 cites W1775749144 @default.
• W2073276199 cites W1821419670 @default.
• W2073276199 cites W1878775584 @default.
• W2073276199 cites W1918185966 @default.
• W2073276199 cites W1947043095 @default.
• W2073276199 cites W1964151652 @default.
• W2073276199 cites W1964969550 @default.
• W2073276199 cites W1970374641 @default.
• W2073276199 cites W1974176003 @default.
• W2073276199 cites W1984500723 @default.
• W2073276199 cites W1992607538 @default.
• W2073276199 cites W2012859837 @default.
• W2073276199 cites W2019700131 @default.
• W2073276199 cites W2029209271 @default.
• W2073276199 cites W2029812497 @default.
• W2073276199 cites W2032350462 @default.
• W2073276199 cites W2034601582 @default.
• W2073276199 cites W2041395627 @default.
• W2073276199 cites W2046688695 @default.
• W2073276199 cites W2048965220 @default.
• W2073276199 cites W2049746692 @default.
• W2073276199 cites W2062417507 @default.
• W2073276199 cites W2063246145 @default.
• W2073276199 cites W2068646590 @default.
• W2073276199 cites W2078674094 @default.
• W2073276199 cites W2080488210 @default.
• W2073276199 cites W2083392018 @default.
• W2073276199 cites W2091162454 @default.
• W2073276199 cites W2109944550 @default.
• W2073276199 cites W2121990983 @default.
• W2073276199 cites W2129068391 @default.
• W2073276199 cites W2139266365 @default.
• W2073276199 cites W2163655701 @default.
• W2073276199 cites W2181399443 @default.
• W2073276199 cites W2181747163 @default.
• W2073276199 cites W2184241686 @default.
• W2073276199 cites W2270085378 @default.
• W2073276199 cites W2338151611 @default.
• W2073276199 cites W2347058726 @default.
• W2073276199 cites W2402922726 @default.
• W2073276199 cites W2416316246 @default.
• W2073276199 cites W80696700 @default.
• W2073276199 cites W964282702 @default.
• W2073276199 doi "https://doi.org/10.1074/jbc.271.41.25145" @default.
• W2073276199 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/8810270" @default.
• W2073276199 hasPublicationYear "1996" @default.
• W2073276199 type Work @default.
• W2073276199 sameAs 2073276199 @default.
• W2073276199 citedByCount "38" @default.
• W2073276199 countsByYear W20732761992015 @default.
• W2073276199 countsByYear W20732761992016 @default.
• W2073276199 crossrefType "journal-article" @default.
• W2073276199 hasAuthorship W2073276199A5049874756 @default.
• W2073276199 hasAuthorship W2073276199A5055362268 @default.
• W2073276199 hasAuthorship W2073276199A5073121857 @default.
• W2073276199 hasBestOaLocation W20732761991 @default.
• W2073276199 hasConcept C126322002 @default.
• W2073276199 hasConcept C185592680 @default.
• W2073276199 hasConcept C200082930 @default.
• W2073276199 hasConcept C2778163477 @default.
• W2073276199 hasConcept C2778918659 @default.
• W2073276199 hasConcept C2779134260 @default.
• W2073276199 hasConcept C41625074 @default.
• W2073276199 hasConcept C55493867 @default.
• W2073276199 hasConcept C57089818 @default.
• W2073276199 hasConcept C62746215 @default.
• W2073276199 hasConcept C71924100 @default.
• W2073276199 hasConcept C86803240 @default.
• W2073276199 hasConceptScore W2073276199C126322002 @default.
• W2073276199 hasConceptScore W2073276199C185592680 @default.
• W2073276199 hasConceptScore W2073276199C200082930 @default.
• W2073276199 hasConceptScore W2073276199C2778163477 @default.
• W2073276199 hasConceptScore W2073276199C2778918659 @default.
• W2073276199 hasConceptScore W2073276199C2779134260 @default.
• W2073276199 hasConceptScore W2073276199C41625074 @default.
• W2073276199 hasConceptScore W2073276199C55493867 @default.
• W2073276199 hasConceptScore W2073276199C57089818 @default.
• W2073276199 hasConceptScore W2073276199C62746215 @default.

• next