ApoA-I secretion from HepG2 cells: evidence for the secretion of both lipid-poor apoA-I and intracellularly assembled nascent HDL
2002; Elsevier BV; Volume: 43; Issue: 1 Linguagem: Inglês
10.1016/s0022-2275(20)30184-x
ISSN1539-7262
AutoresJeffrey W. Chisholm, Ellen R. Burleson, Gregory S. Shelness, John S. Parks,
Tópico(s)Lipid metabolism and disorders
ResumoThe goal of this study was to determine whether apolipoprotein A-I (apoA-I) is lipidated before secretion by HepG2 cells. ApoA-I was extracted from microsomes after radiolabeling with [35S]Met/Cys. After ultracentrifugal flotation, d < 1.25 g/ml and d > 1.25 g/ml fractions were immunoprecipitated and analyzed by SDS-PAGE. Under steady state radiolabeling conditions, 20% of extracted microsomal apoA-I floated at d < 1.25 g/ml. Pulse-chase experiments demonstrated that the percentage of microsomal apoA-I associated with lipid peaked between 2 and 8 min postsynthesis. Density gradient ultracentrifugation, and nondenaturing gradient gel electrophoresis of HepG2 cell medium, indicated that 50% of secretory apoA-I existed as small HDL particles with a diameter of ~7.5 nm. These and additional data suggested that ~20% of newly secreted apoA-I is lipidated intracellularly and another 30% is secreted in lipid-free or lipid-poor form but acquires sufficient lipid to become small HDL within 1 h of secretion, with little further maturation over the time course of the incubation (2 h). These results indicate that a process exists for the presecretory intracellular assembly of apoA-I with lipid in HepG2 cells and that apoA-I is secreted in both lipid-poor and lipidated forms. —Chisholm, J. W., E. R. Burleson, G. S. Shelness, and J. S. Parks. ApoA-I secretion from HepG2 cells: evidence for the secretion of both lipid-poor apoA-I and intracellularly assembled nascent HDL. J. Lipid Res. 2002. 43: 36–44. The goal of this study was to determine whether apolipoprotein A-I (apoA-I) is lipidated before secretion by HepG2 cells. ApoA-I was extracted from microsomes after radiolabeling with [35S]Met/Cys. After ultracentrifugal flotation, d < 1.25 g/ml and d > 1.25 g/ml fractions were immunoprecipitated and analyzed by SDS-PAGE. Under steady state radiolabeling conditions, 20% of extracted microsomal apoA-I floated at d < 1.25 g/ml. Pulse-chase experiments demonstrated that the percentage of microsomal apoA-I associated with lipid peaked between 2 and 8 min postsynthesis. Density gradient ultracentrifugation, and nondenaturing gradient gel electrophoresis of HepG2 cell medium, indicated that 50% of secretory apoA-I existed as small HDL particles with a diameter of ~7.5 nm. These and additional data suggested that ~20% of newly secreted apoA-I is lipidated intracellularly and another 30% is secreted in lipid-free or lipid-poor form but acquires sufficient lipid to become small HDL within 1 h of secretion, with little further maturation over the time course of the incubation (2 h). These results indicate that a process exists for the presecretory intracellular assembly of apoA-I with lipid in HepG2 cells and that apoA-I is secreted in both lipid-poor and lipidated forms. —Chisholm, J. W., E. R. Burleson, G. S. Shelness, and J. S. Parks. ApoA-I secretion from HepG2 cells: evidence for the secretion of both lipid-poor apoA-I and intracellularly assembled nascent HDL. J. Lipid Res. 2002. 43: 36–44. Apolipoprotein A-I (apoA-I) is a 28-kDa amphipathic plasma protein secreted by both the liver and intestine, and is the primary protein component of HDL (1Brewer Jr., H.B. Fairwell T. LaRue A. Ronan R. Houser A. Bronzert T.J. The amino acid sequence of human APOA-I, an apolipoprotein isolated from high density lipoproteins.Biochem. Biophys. Res. Commun. 1978; 80: 623-630Google Scholar, 2Law S.W. Brewer Jr., H.B. Nucleotide sequence and the encoded amino acids of human apolipoprotein A-I mRNA.Proc. Natl. Acad. Sci. USA. 1984; 81: 66-70Google Scholar). HDL functions in the transport of cholesterol from extrahepatic tissues back to the liver through the reverse cholesterol transport pathway (3Glomset J.A. The plasma lecithin:cholesterol acyltransferase reaction.J. Lipid Res. 1968; 9: 155-167Google Scholar, 4Fielding C.J. Fielding P.E. Molecular physiology of reverse cholesterol transport.J. Lipid Res. 1995; 36: 211-228Google Scholar, 5Barter P.J. Rye K.A. Molecular mechanisms of reverse cholesterol transport.Curr. Opin. Lipidol. 1996; 7: 82-87Google Scholar, 6Hill S.A. Mcqueen J. Reverse cholesterol transport—a review of the process and its clinical implications.Clin. Biochem. 1997; 30: 517-525Google Scholar). Increased plasma HDL levels may reflect increased plasma reverse cholesterol transport conferring resistance to cholesterol deposition in extrahepatic tissues. This concept is consistent with studies demonstrating that plasma HDL levels are inversely correlated with the risk of developing atherosclerosis (7Miller N.E. High-density lipoprotein: a major risk factor for coronary atherosclerosis.Baillieres Clin. Endocrinol. Metab. 1987; 1: 603-622Google Scholar, 8Castelli W.P. Garrison R.J. Wilson P.W. Abbott R.D. Kalousdian S. Kannel W.B. Incidence of coronary heart disease and lipoprotein cholesterol levels. The Framingham Study.J. Am Med. Assoc. 1986; 256: 2835-2838Google Scholar, 9Schaefer E.J. Lamon-Fava S. Ordovas J.M. Cohn S.D. Schaefer M.M. Castelli W.P. Wilson P.W. Factors associated with low and elevated plasma high density lipoprotein cholesterol and apolipoprotein A-I levels in the Framingham Offspring Study.J. Lipid Res. 1994; 35: 871-882Google Scholar). Although considerable research has been directed toward understanding HDL metabolism, it currently remains unclear how HDL are assembled. Two possibilities have been proposed to explain the assembly of HDL particles: intracellular lipidation of apoA-I and extracellular lipidation after apoA-I secretion from either the liver or intestine. In the intestine, apoA-I appears to be secreted on chylomicron particles and, after lipolysis of core triacylglycerol, apoA-I and redundant surface lipid are either moved onto existing HDL and/or create new nascent HDL (10Tall A.R. Small D.M. Body cholesterol removal: role of plasma high-density lipoproteins.Adv. Lipid Res. 1980; 17: 1-51Google Scholar). In contrast, mammalian liver apoA-I is not secreted on triglyceride-rich apoB-containing lipoproteins and the extent of lipidation of hepatic apoA-I is unknown. ApoA-I has been shown to undergo extracellular assembly with lipid when incubated with cells in culture (11Forte T.M. Bielicki J.K. Goth-Goldstein R. Selmek J. McCall M.R. Recruitment of cell phospholipids and cholesterol by apolipoproteins A-II and A-I: formation of nascent apolipoprotein-specific HDL that differ in size, phospholipid composition, and reactivity with LCAT.J. Lipid Res. 1995; 36: 148-157Google Scholar, 12Forte T.M. Goth-Goldstein R. Nordhausen R.W. McCall M.R. Apolipoprotein A-I-cell membrane interaction: extracellular assembly of heterogeneous nascent HDL particles.J. Lipid Res. 1993; 34: 317-324Google Scholar). This appears to occur through apoA-I interaction with the cell surface (13Oram J.F. Yokoyama S. Apolipoprotein-mediated removal of cellular cholesterol and phospholipids.J. Lipid Res. 1996; 37: 2473-2491Google Scholar, 14Rothblat G.H. de la Llera-Moya M. Atger V. Kellner-Weibel G. Williams D.L. Phillips M.C. Cell cholesterol efflux: integration of old and new observations provides new insights.J. Lipid Res. 1999; 40: 781-796Google Scholar, 15Gillotte K.L. Davidson W.S. Lund-Katz S. Rothblat G.H. Phillips M.C. Removal of cellular cholesterol by pre-β-HDL involves plasma membrane microsolubilization.J. Lipid Res. 1998; 39: 1918-1928Google Scholar, 16Gillotte K.L. Zaiou M. Lund-Katz S. Anantharamaiah G.M. Holvoet P. Dhoest A. Palgunachari M.N. Segrest J.P. Weisgraber K.H. Rothblat G.H. Phillips M.C. Apolipoprotein-mediated plasma membrane microsolubilization. Role of lipid affinity and membrane penetration in the efflux of cellular cholesterol and phospholipid.J. Biol. Chem. 1999; 274: 2021-2028Google Scholar) and the ATP-binding cassette A1 (ABCA1) transporter (17Brooks-Wilson A. Marcil M. Clee S.M. Zhang L.-H. Roomp K. Van Dam M. Yu L. Brewer C. Collins J.A. Molhuizen H.O.F. Loubser O. Ouelette B.F.F. Fichter K. Ashbourne-Excoffon K.J.D. Senson C.W. Scherer S. Mott S. Denis M. Martindale D. Frohlich J. Morgan K. Koop B. Pimstone S. Kastelein J.J.P. Hayden M.R. Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency.Nat. Genet. 1999; 22: 336-345Google Scholar, 18Rust S. Rosier M. Funke H. Real J. Amoura Z. Piette J.C. Deleuze J.F. Brewer H.B. Duverger N. Denefle P. Assmann G. Tangier disease is caused by mutations in the gene encoding ATP-binding cassette transporter 1.Nat.Genet. 1999; 22: 352-355Google Scholar, 19Bodzioch M. Orso E. Klucken J. Langmann T. Bottcher A. Diederich W. Drobnik W. Barlage S. Buchler C. Porsch-Ozcurumez M. Kaminski W.E. Hahmann H.W. Oette K. Rothe G. Aslanidis C. Lackner K.J. Schmitz G. The gene encoding ATP-binding cassette transporter 1 is mutated in Tangier disease.Nat. Genet. 1999; 22: 347-351Google Scholar, 20Lawn R.M. Wade D.P. Garvin M.R. Wang X. Schwartz K. Porter J.G. Seilhamer J.J. Vaughan A.M. Oram J.F. The Tangier disease gene product ABC1 controls the cellular apolipoprotein-mediated lipid removal pathway.J. Clin. Invest. 1999; 104: R25-R31Google Scholar). Gillotte et al. (15Gillotte K.L. Davidson W.S. Lund-Katz S. Rothblat G.H. Phillips M.C. Removal of cellular cholesterol by pre-β-HDL involves plasma membrane microsolubilization.J. Lipid Res. 1998; 39: 1918-1928Google Scholar) have demonstrated in fibroblasts that the extracellular lipidation of apoA-I through cell surface interactions is rapid, but of limited capacity, suggesting perhaps that lipid availability or some other factor, such as ABCA1 expression (21Bortnick A.E. Rothblat G.H. Stoudt G. Hoppe K.L. Royer L.J. McNeish J. Francone O.L. The correlation of ATP-binding cassette 1 mRNA levels with cholesterol efflux from various cell lines.J. Biol. Chem. 2000; 275: 28634-28640Google Scholar), is important in determining the extracellular assembly of apoA-I with lipid. Several studies using chicken (22Banerjee D. Redman C.M. Biosynthesis of high density lipoprotein by chicken liver: nature of nascent intracellular high density lipoprotein.J. Cell Biol. 1983; 96: 651-660Google Scholar, 23Banerjee D. Redman C.M. Biosynthesis of high density lipoprotein by chicken liver: conjugation of nascent lipids with apoprotein A1.J. Cell Biol. 1984; 99: 1917-1926Google Scholar) and rat hepatocytes (24Howell K.E. Palade G.E. Heterogeneity of lipoprotein particles in hepatic Golgi fractions.J. Cell Biol. 1982; 92: 833-845Google Scholar) have provided evidence for the secretion of intracellularly lipidated apoA-I [reviewed in ref. (25Dixon J.L. Ginsberg H.N. Hepatic synthesis of lipoproteins and apolipoproteins.Semin. Liver Dis. 1992; 12: 364-372Google Scholar)]. Howell and Palade (24Howell K.E. Palade G.E. Heterogeneity of lipoprotein particles in hepatic Golgi fractions.J. Cell Biol. 1982; 92: 833-845Google Scholar), using the HDL density fraction of rat liver Golgi extracts, found both HDL-sized particles and apoA-I, raising the possibility that apoA-I was assembled with lipid inside the cell and secreted as nascent HDL. In contrast, Hamilton, Moorehouse, and Havel (26Hamilton R.L. Moorehouse A. Havel R.J. Isolation and properties of nascent lipoproteins from highly purified rat hepatocytic Golgi fractions.J. Lipid Res. 1991; 32: 529-543Google Scholar) were unable to visualize discoidal particles that resembled nascent HDL using highly purified rat Golgi fractions and concluded that intracellular HDL does not exist. The reason for these opposing results remains unclear. Banerjee and Redman (22Banerjee D. Redman C.M. Biosynthesis of high density lipoprotein by chicken liver: nature of nascent intracellular high density lipoprotein.J. Cell Biol. 1983; 96: 651-660Google Scholar, 23Banerjee D. Redman C.M. Biosynthesis of high density lipoprotein by chicken liver: conjugation of nascent lipids with apoprotein A1.J. Cell Biol. 1984; 99: 1917-1926Google Scholar) have performed more detailed studies of the intracellular lipidation of apoA-I, using chicken hepatocytes. They characterized a heterogeneous population of phospholipid-rich apoA-I-containing nascent HDL (22Banerjee D. Redman C.M. Biosynthesis of high density lipoprotein by chicken liver: nature of nascent intracellular high density lipoprotein.J. Cell Biol. 1983; 96: 651-660Google Scholar) that, on the basis of lipid radiolabeling studies, was assembled in the Golgi apparatus (23Banerjee D. Redman C.M. Biosynthesis of high density lipoprotein by chicken liver: conjugation of nascent lipids with apoprotein A1.J. Cell Biol. 1984; 99: 1917-1926Google Scholar). However, the relevance of chicken hepatocytes to the study of mammalian apoA-I secretion and lipidation remains unclear, as chicken apoA-I, unlike mammalian apoA-I, has been shown to have a broad tissue distribution and is often found in circulation associated with apoB-containing lipoprotein particles. Because chickens lack apoE, it has been suggested that chicken apoA-I may function more like mammalian apoE (27Rajavashisth T.B. Dawson P.A. Williams D.L. Shackleford J.E. Lebherz H. Lusis A.J. Structure, evolution, and regulation of chicken apolipoprotein A-I.J. Biol. Chem. 1987; 262: 7058-7065Google Scholar). Clearly, strong evidence demonstrating the intracellular assembly of mammalian apoA-I with lipid is lacking. The following studies, using HepG2 cells, were designed to investigate whether and to what extent mammalian apoA-I is associated with lipid inside the cell. The results of these studies suggest that apoA-I is secreted by the liver in both lipid-poor form and as nascent HDL. HepG2 cells were maintained in MEM (Mediatech, Ormond Beach, FL) that was supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 U/ml)-streptomycin (100 μg/ml), and 2× vitamins (Mediatech). For all experiments, cells were split into 150-mm dishes and experiments were initiated once the cells were near confluence. Cell monolayers were then washed three times with PBS (Mediatech) and used as indicated in the figure legends. For steady state radiolabeling of intracellular apoA-I, washed cell monolayers were incubated for 1–2 h in 5 ml of Met/Cys-free MEM (ICN, Costa Mesa, CA) that was supplemented with [35S]Met/Cys (0.1 mCi/ml) (EasyTag Express; New England Nuclear, Boston, MA). For pulse-chase experiments, intracellular Met and Cys pools were depleted by incubation in serum-free, Met/Cys-free medium for 20–30 min. The medium was then removed and 5 ml of radiolabeling medium containing radiolabel at 0.1 mCi/ml was added for a pulse of 2–10 min. After the pulse period, 5 ml of serum-free medium containing 1 mM Cys and 2.5 mM Met (chase medium) was added to the radiolabeling medium. In some experiments cycloheximide (Sigma, St. Louis, MO), a protein synthesis inhibitor, was included at a final concentration of 0.1 mM. The medium was then removed and replaced with 5 ml of fresh medium containing 1 mM Cys, 2.5 mM Met, and in some experiments 0.1 mM cycloheximide. All chase periods were stopped by placing the culture dishes on ice. For pulse-only samples, radiolabeling was stopped by adding ice-cold chase medium and placing the culture dishes on ice. For experiments in which lipid-poor apoA-I was added, the source of apoA-I was either delipidated (28Scanu A.M. Edelstein C. Solubility in aqueous solutions of ethanol of the small molecular weight peptides of the serum very low density and high density lipoproteins: relevance to the recovery problem during delipidation of serum lipoproteins.Anal. Biochem. 1971; 44: 576-588Google Scholar) [35S]Met/Cys-radiolabeled HepG2 cell medium (6 h of labeling in serum-free medium) or delipidated (28Scanu A.M. Edelstein C. Solubility in aqueous solutions of ethanol of the small molecular weight peptides of the serum very low density and high density lipoproteins: relevance to the recovery problem during delipidation of serum lipoproteins.Anal. Biochem. 1971; 44: 576-588Google Scholar) 125I-radiolabeled (29McFarlane A.A. Efficient trace labeling of proteins with iodine.Nature. 1958; 182: 53-57Google Scholar, 30Bilheimer D.W. Eisenberg S. Levy R.I. The metabolism of very low density lipoprotein proteins. I. Preliminary in vitro and in vivo observations.Biochim. Biophys. Acta. 1972; 260: 212-221Google Scholar) human plasma apoA-I (31Miller K.R. Parks J.S. Influence of vesicle surface composition on the interfacial binding of lecithin:cholesterol acyltransferase and apolipoprotein A-I.J. Lipid Res. 1997; 38: 1094-1102Google Scholar, 32Parks J.S. Rudel L.L. Isolation and characterization of high density lipoprotein apoproteins in the non-human primate (vervet).J. Biol. Chem. 1979; 254: 6716-6723Google Scholar) that had been denatured by chemical (16Gillotte K.L. Zaiou M. Lund-Katz S. Anantharamaiah G.M. Holvoet P. Dhoest A. Palgunachari M.N. Segrest J.P. Weisgraber K.H. Rothblat G.H. Phillips M.C. Apolipoprotein-mediated plasma membrane microsolubilization. Role of lipid affinity and membrane penetration in the efflux of cellular cholesterol and phospholipid.J. Biol. Chem. 1999; 274: 2021-2028Google Scholar) (6 M guanidine-HCl denaturation followed by refolding) or thermal (33Tall A.R. Shipley G.G. Small D.M. Conformational and thermodynamic properties of apo A-1 of human plasma high density lipoproteins.J. Biol. Chem. 1976; 251: 3749-3755Google Scholar) (60 °C for 30 min) methods. Lipid-free human plasma apoA-I had less than one molecule of phospholipid per molecule of apoA-I as determined by phosphorus assay (34Fiske C.H. SubbaRow Y. Colorimetric determination of phosphorus.J. Biol. Chem. 1925; 66: 357-400Google Scholar) of 1 mg of purified apoA-I. Microsomes were prepared by a modification of the method described by Ingram and Shelness (35Ingram M.F. Shelness G.S. Folding of the aminoterminal domain of apolipoprotein B initiates microsomal triglyceride transfer protein-dependent lipid transfer to nascent very low density lipoprotein.J. Biol. Chem. 1997; 272: 10279-10286Google Scholar). The medium was removed from plates and the cells were washed with 5 ml of ice-cold PBS. The cells were recovered by scraping into a 15-ml centrifuge tube in a volume of 5 ml of ice-cold PBS. Cells were pelleted at 1,500 g for 10 min and the supernatant was removed by aspiration. The cells were gently resuspended in 1 ml of hypotonic buffer (10 mM HEPES, pH 7.4), containing protease inhibitors [PI; 2× complete EDTA-free PI cocktail (Roche, Nutley, NJ) supplemented with pepstatin (2 μg/ml; Sigma) and 0.1% EDTA]. Cells were pelleted again by low speed centrifugation at 1,500 g for 10 min and then resuspended in 1 ml of hypotonic buffer containing PI and incubated on ice for 15 min. The cells were then transferred to a 2-ml Dounce homogenizer and homogenized by 25 strokes, using the tight pestle. The homogenate was adjusted to 0.25 M sucrose and centrifuged for 10 min at 1,000 g. The supernatant was removed and centrifuged for an additional 10 min before being transferred to a thick-walled polycarbonate ultracentrifuge tube. Samples were centrifuged in a TL-100 centrifuge at 70,000 rpm for 15 min at 4°C, using a TLA 100.3 rotor (Beckman-Coulter, Fullerton, CA). After ultracentrifugation, the supernatant was aspirated and discarded and the microsomal pellet was used for carbonate extraction experiments. Microsomes were washed with 1 ml of ice-cold membrane buffer [10 mM HEPES (pH 7.4), 250 mM sucrose, 10 mM NaCl, 10 mM KCl, and PI], and the microsomes were resuspended in 300 μl of membrane buffer and transferred to a Dounce homogenizer. Thirty-three microliters of 1 M sodium carbonate, pH 11.5, was added and the microsomes were resuspended by 12 strokes of the Dounce homogenizer and placed in a thick-walled polycarbonate centrifuge tube. The Dounce homogenizer was washed with 1.33 ml of 0.1 M sodium carbonate, pH 11.5, and the wash was added to the resuspended microsomes. After a 1-h incubation on ice, the extracted membranes were separated from the microsomal lumenal contents by ultracentrifugation in a TL-100 centrifuge, using a TLA-100.3 rotor, for 25 min at 80,000 rpm at 4°C. The supernatant was removed and adjusted to 0.5% phenol red, 0.2 M NaCl, 0.05 M Tris, and 0.25% fatty acid-free BSA (Sigma). The final pH was adjusted to pH 7.4 with 20% HCl. Media or microsomal carbonate extracts were adjusted to d = 1.25 g/ml with solid KBr, transferred to quick-seal tubes, and floated by centrifugation in a TL-100 (TLA-100.3 rotor, 18 h, 80,000 rpm, 15°C). Samples floated by ultracentrifugation were recovered by tube cutting into ~1.0-ml tops and ~2.5-ml bottoms. In control experiments using either delipidated 35S- or 125I-radiolabeled apoA-I, 11.9 ± 1.1% (n = 6) of the apoA-I was found in the top fraction. This was the background for the assay, because a second ultracentrifugation spin of the bottom fraction resulted in a similar recovery of 12% in the top fraction. Because our data demonstrated that lipid-free apoA-I had less than one molecule of phospholipid per molecule of apoA-I and the recovery of lipid-free apoA-I in the top fraction was constant in all experiments, we subtracted this background from the percentage of apoA-I floated in the d < 1.25 g/ml top fraction. In some experiments, samples were adjusted to d = 1.21 g/ml and ultracentrifuged in a density gradient using an SW-41 rotor (Beckman-Coulter) for 44 h at 40,000 rpm and 15°C (36Chapman M.J. Goldstein S. Lagrange D. Laplaud P.M. A density gradient ultracentrifugal procedure for the isolation of the major lipoprotein classes from human serum.J. Lipid Res. 1981; 22: 339-358Google Scholar). In one experiment the density gradient was modified (7-ml sample at d = 1.25 g/ml, 2-ml sample at d = 1.20 g/ml, and 3-ml sample at d = 1.05 g/ml) to increase the separation of particles in the density range of HDL. Gradients were recovered in 1-ml fractions, using a manual digital pipette (Oxford) or an automated gradient fractionator (Auto Densi-Flow; Labconco, Kansas City, MO). The density of each fraction was obtained by weighing known volumes of the fractions recovered from blank gradients. Immunoprecipitations were performed on samples after buffer exchanging the samples three times into TBS (20 mM Tris-HCl, 150 mM NaCl, pH 7.4), using a Centricon-10 centrifugal concentrator (Millipore, Bedford, MA), or in the case of density gradient fractions after diluting 4-fold with deionized H2O. Samples were immunoprecipitated overnight at 4°C in TBS buffer containing 1% Triton X-100, 0.5% BSA, and 2× PI, using 5 μl of either polyclonal anti-human apoA-I (Roche) or monoclonal anti-human apoA-I antibody (37Curtiss L.K. Banka C.L. Selection of monoclonal antibodies for linear epitopes of an apolipoprotein yields antibodies with comparable affinity for lipid-free and lipid-associated apolipoprotein.J. Lipid Res. 1996; 37: 884-892Google Scholar). Antibody-antigen complexes were recovered by adding 50 μl of protein G-Sepharose (50%, v/v) (Amersham-Pharmacia Biotech, Piscataway, NJ) and incubating for 2 h at 4°C. The protein G-Sepharose was pelleted in a microcentrifuge and washed three times with TBS containing 1% Triton X-100. Pellets were then boiled for 10 min in SDS-PAGE sample buffer and fractionated by 15% SDS-PAGE. SDS-PAGE analysis was performed with Tris-glycine gels in a Bio-Rad (Hercules, CA) PROTEAN II system as previously described (38Irwin D. O'Looney P.A. Quinet E. Vahouny G.V. Application of SDS gradient polyacrylamide slab gel electrophoresis to analysis of apolipoprotein mass and radioactivity of rat lipoproteins.Atherosclerosis. 1984; 53: 163-172Google Scholar). Gels containing radiolabeled samples were dried and exposed to film at −70°C, using a Kodak (Rochester, NY) Transcreen-LE and either Biomax MS or MR film. Samples and high molecular weight standards (Amersham-Pharmacia Biotech) were electrophoresed (1,400 V ·h, 10°C), using a 4–30% nondenaturing gradient gel (39Rainwater D.L. Andres D.W. Ford A.L. Lowe F. Blanche P.J. Krauss R.M. Production of polyacrylamide gradient gels for the electrophoretic resolution of lipoproteins.J. Lipid Res. 1992; 33: 1876-1881Google Scholar). Samples were transferred from gels to nitrocellulose (0.2 μm for 7 h at 35 V, 4°C) and incubated sequentially with anti-human apoA-I (diluted 1:1,000; Roche) and anti-sheep IgG-horseradish peroxidase conjugate (diluted 1:20,000; Sigma). Bands were visualized by chemiluminescence (Pierce, Rockford, IL) and captured on film. In some experiments, the nitrocellulose blots were developed with anti-goat IgG conjugated with alkaline phosphatase (diluted 1:2,000; Vector, Burlingame, CA) and NBT/BCIP reagent (Promega, Madison, WI). Quantitation of sample bands after autoradiography was accomplished by scanning the film with a UMAX (Fremont, CA) Super-Vista S-12 scanner equipped with a transparency adapter. Images were imported into Scion Image (Scion, Frederick, MD) and densities were obtained from the grayscale image after calibrating the scanner with a density step table (Kodak). To validate the densitometry procedure, 125I-labeled apoA-I or 35S-labeled apoA-I bands were excised from the dried gels after autoradiography and counted either in a γ counter (125I) or in a scintillation counter (35S) after digestion with 30% hydrogen peroxide and addition of liquid scintillation cocktail. The correlation between densitometry and counting of the excised bands was r2 = 0.97 ± 0.02 (n = 4 separate experiments). Statistical differences were determined by ANOVA followed by a Bonferroni/Dunn post hoc comparison to identify significant differences between individual time points. Experiments were undertaken to examine the secretion and lipidation of apoA-I in HepG2 cells. Results shown in Fig. 1 demonstrate that after a 10-min pulse, apoA-I secretion was linear between 15 and 60 min. Immunoprecipitation of apoA-I from the top (d < 1.25 g/ml) and bottom (d > 1.25 g/ml) fractions of chase medium subjected to ultracentrifugation demonstrated that ~50% of the radiolabeled apoA-I was associated with sufficient lipid to float (lipidated apoA-I) by 120 min (Fig. 2). Because ~25% of secreted apoA-I was lipidated at the earliest time point sampled (Fig. 2; 15 min), we hypothesized that some of the lipidation of apoA-I occurred in HepG2 cells before secretion and that the difference in lipidation between the early time points and 120 min was due to an extracellular lipidation process.Fig. 2.Lipidation state of apoA-I secreted by HepG2 cells. HepG2 cells were pulse radiolabeled for 10 min and chased for the indicated times. Medium was ultracentrifuged at d = 1.25 g/ml and apoA-I in the top and bottom fractions was immunoprecipitated and analyzed by 15% SDS-PAGE and fluorography. The data are shown as the percentage of HepG2 cell medium apoA-I lipidated (i.e., percentage in d < 1.25 g/ml top fraction) during the chase as determined by densitometric analysis of the film. Different symbols indicate results from independent experiments. All data have been corrected for background in the separation method as described in Materials and Methods.View Large Image Figure ViewerDownload (PPT) To specifically assess the extent to which extracellular lipidation of apoA-I is responsible for HDL present in HepG2 cell medium, 1.5 μg of 35S-labeled apoA-I from delipidated HepG2 cell medium or 1 μg of lipid-free 125I-labeled human plasma apoA-I was incubated with HepG2 cells for the times indicated in Fig. 3. The quantity of lipid-free apoA-I added to medium was in the range of that secreted by HepG2 cells during a 1-h incubation, as determined by apoA-I enzyme-linked immunosorbent assay (40Koritnik D.L. Rudel L.L. Measurement of apolipoprotein A-I concentration in nonhuman primate serum by enzyme-linked immunosorbent assay (ELISA).J. Lipid Res. 1983; 24: 1639-1645Google Scholar) (0.4–1.5 μg of apoA-I per 150-mm dish per h). After adjusting for the small background of apoA-I that floated in the absence of cells (see Materials and Methods), ~20% of the exogenous apoA-I acquired sufficient lipid to float at d < 1.25 g/ml by 120 min (Fig. 3). The lipidation event appeared to become saturated by 30 min, indicating that lipid or some other factor might be limiting. By contrast, at 120 min ~50% of newly secreted apoA-Iwas lipidated (Fig. 2), indicating that the lipidation of endogenous, newly secreted apoA-I exceeded that of lipid-free apoA-I added to the medium exogenously (Fig. 3). The difference (~20–30% depending on the time point) in the relative lipidation of exogenous and endogenous apoA-I was attributed to the presence of an intracellular lipidation mechanism. To further explore the lipidation state of newly secreted apoA-I, we analyzed medium obtained from HepG2 cells after a 2-h incubation as well as lipid-free apoA-I by equilibrium density gradient ultracentrifugation and size fractionation on 4–30% nondenaturing gradient gels. Figure 4A shows a density gradient profile of lipid-free 125I-apoA-I. This preparation of apoA-I had less than one molecule of phospholipid per molecule of apoA-I, on the basis of chemical assay. The lipid-free apoA-I was distributed in the bottom four fractions of the density gradient (d > 1.223 g/ml), as expected. Figure 4B shows the results of nondenaturing gradient gel electrophoresis of HepG2 cell medium. In lane 1, HepG2 medium demonstrated two size ranges of immunoreactive apoA-I particles, one in the size range of albumin (7.1 nm in diameter) and another above the 7.1-nm marker. The latter size range corresponds to that of small HDL particles in plasma that contain two molecules of apoA-I per particle (41Colvin P.L. Moriguchi E. Barrett P.H. Parks J.S. Rudel L.L. Small HDL particles containing two apoA-I molecules are precursors in vivo to medium and large HDL particles containing three and four apoA-I molecules in nonhuman primates.J. Lipid Res. 1999; 40: 1782-1792Google Scholar). To characterize the nat
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