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• W2000312682 abstract "Recombinant adenoviruses with cDNAs for human apolipoprotein A-I (wild type (wt) apoA-I) and three mutants, referred to as Δ4-5A-I, Δ5-6A-I, and Δ6-7A-I, that have deletions removing regions coding for amino acids 100–143, 122–165, and 144–186, respectively, were created to study structure/function relationships of apoA-I in vivo. All mutants were expressed at lower concentrations than wt apoA-I in plasma of fasting apoA-I-deficient mice. The Δ5-6A-I mutant was found primarily in the lipid-poor high density lipoprotein (HDL) pool and at lower concentrations than Δ4-5A-I and Δ6-7A-I that formed more buoyant HDL2/3particles. At an elevated adenovirus dose and earlier blood sampling from fed mice, both Δ5-6A-I and Δ6-7A-I increased HDL-free cholesterol and phospholipid but not cholesteryl ester. In contrast, wt apoA-I and Δ4-5A-I produced significant increases in HDL cholesteryl ester. Further analysis showed that Δ6-7A-I and native apoA-I could bind similar amounts of phospholipid and cholesterol that were reduced slightly for Δ5-6A-I and greatly for Δ4-5A-I. We conclude from these findings that amino acids (aa) 100–143, specifically helix 4 (aa 100–121), contributes to the maturation of HDL through a role in lipid binding and that the downstream sequence (aa 144–186) centered around helix 6 (aa 144–165) is responsible for the activation of lecithin-cholesterol acyltransferase. Recombinant adenoviruses with cDNAs for human apolipoprotein A-I (wild type (wt) apoA-I) and three mutants, referred to as Δ4-5A-I, Δ5-6A-I, and Δ6-7A-I, that have deletions removing regions coding for amino acids 100–143, 122–165, and 144–186, respectively, were created to study structure/function relationships of apoA-I in vivo. All mutants were expressed at lower concentrations than wt apoA-I in plasma of fasting apoA-I-deficient mice. The Δ5-6A-I mutant was found primarily in the lipid-poor high density lipoprotein (HDL) pool and at lower concentrations than Δ4-5A-I and Δ6-7A-I that formed more buoyant HDL2/3particles. At an elevated adenovirus dose and earlier blood sampling from fed mice, both Δ5-6A-I and Δ6-7A-I increased HDL-free cholesterol and phospholipid but not cholesteryl ester. In contrast, wt apoA-I and Δ4-5A-I produced significant increases in HDL cholesteryl ester. Further analysis showed that Δ6-7A-I and native apoA-I could bind similar amounts of phospholipid and cholesterol that were reduced slightly for Δ5-6A-I and greatly for Δ4-5A-I. We conclude from these findings that amino acids (aa) 100–143, specifically helix 4 (aa 100–121), contributes to the maturation of HDL through a role in lipid binding and that the downstream sequence (aa 144–186) centered around helix 6 (aa 144–165) is responsible for the activation of lecithin-cholesterol acyltransferase. high density lipoproteins apolipoprotein A-I lecithin-cholesterol acyltransferase cholesteryl esters native human apoA-I amino acids apoA-I containing a deletion of aa 100–143 apoA-I containing a deletion of aa 122–165 apoA-I containing a deletion of aa 144–186 recombinant adenovirus(es) phosphate-buffered saline plaque-forming units adenovirus carrying the firefly luciferase cDNA free cholesterol phospholipid apolipoprotein E fractional cholesterol esterification rate reconstituted lipoproteins containing two apoA-I molecules per particle very low density lipoproteins wild type polyacrylamide gel electrophoresis fast protein liquid chromatography Epidemiological studies have shown that the levels of high density lipoprotein cholesterol (HDL-C)1 and the major protein of HDL, apolipoprotein A-I (apoA-I), are inversely correlated with the incidence of coronary artery disease (1.Miller N.E. Am. Heart J. 1987; 113: 589-597Crossref PubMed Scopus (404) Google Scholar, 2.Miller N.E. Thelle D.S. Forde O.H. Mjos O.D. Lancet. 1977; 1: 965-968Abstract PubMed Scopus (975) Google Scholar). ApoA-I circulates as a 243-amino acid protein (3.Brewer Jr., H.B. Fairwell T. LaRue A. Ronan R. Houser A. Bronzert T.J. Biochem. Biophys. Res. Commun. 1978; 80: 623-630Crossref PubMed Scopus (223) Google Scholar) and plays an important role in determining the concentrations of plasma HDL-C. This is accomplished through the ability of apoA-I to promote the removal of cholesterol and phospholipids from cells and subsequently activate the enzyme lecithin-cholesterol acyltransferase (LCAT) (4.Fielding C.J. Shore V.G. Fielding P.E. Biochem. Biophys. Res. Commun. 1972; 46: 1493-1498Crossref PubMed Scopus (523) Google Scholar, 5.Soutar A.K. Garner C.W. Baker H.N. Sparrow J.T. Jackson R.L. Gotto A.M. Smith L.C. Biochemistry. 1975; 14: 3057-3064Crossref PubMed Scopus (264) Google Scholar) that catalyzes the formation of cholesteryl esters (CE) on HDL. Additional interactions of apoA-I with the cell surface scavenger receptor class B, type I (6.Rigotti A. Trigatti B. Babitt J. Penman M. Xu S.H. Krieger M. Curr. Opin. Lipidol. 1997; 8: 181-188Crossref PubMed Scopus (177) Google Scholar), and possibly with cholesteryl ester transfer protein may also influence circulating HDL-C levels. Domains of apoA-I required for lipid association and the activation of LCAT have been the focus of many in vitro investigations. The initial binding of apoA-I to lipids is thought to occur via the extreme N-terminal (aa 44–64) and C-terminal (aa 220–243) amphipathic α-helices that may or may not require additional interactions with other regions of the protein (7.Palgunachari M.N. Mishra V.K. Lund-Katz S. Phillips M.C. Adeyeye S.O. Alluri S. Anantharamaiah G.M. Segrest J.P. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 328-338Crossref PubMed Scopus (204) Google Scholar, 8.Mishra V.K. Palgunachari M.N. Datta G. Phillips M.C. Lund-Katz S. Adeyeye S.O. Segrest J.P. Anantharamaiah G.M. Biochemistry. 1998; 37: 10313-10324Crossref PubMed Scopus (79) Google Scholar, 9.Holvoet P. Zhao Z.A. Vanloo B. Vos R. Deridder E. Dhoest A. Taveirne J. Brouwers E. Demarsin E. Engelborghs Y. Rosseneu M. Collen D. Brasseur R. Biochemistry. 1995; 34: 13334-13342Crossref PubMed Scopus (87) Google Scholar, 10.Laccotripe M. Makrides S.C. Jonas A. Zannis V.I. J. Biol. Chem. 1997; 272: 17511-17522Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). In the lipid-bound state the central helices (aa 100 to 186) have been proposed to contain a putative hinge domain (11.Brouillette C.G. Anantharamaiah G.M. Biochim. Biophys. Acta. 1995; 1256: 103-129Crossref PubMed Scopus (167) Google Scholar, 12.Calabresi L. Meng Q.-H. Castro G.R. Marcel Y.L. Biochemistry. 1993; 32: 6477-6484Crossref PubMed Scopus (70) Google Scholar) thought to enable apoA-I to adopt distinct conformations on HDL, as well as the major LCAT activating domain (13.Banka C.L. Bonnet D.J. Black A.S. Smith R.S. Curtiss L.K. J. Biol. Chem. 1991; 266: 23886-23892Abstract Full Text PDF PubMed Google Scholar, 14.Dhoest A. Zhao Z.A. De Geest B. Deridder E. Sillen A. Engelborghs Y. Collen D. Holvoet P. J. Biol. Chem. 1997; 272: 15967-15972Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 15.Lindholm E.M. Bielicki J.K. Curtiss L.K. Rubin E.M. Forte T.M. Biochemistry. 1998; 37: 4863-4868Crossref PubMed Scopus (32) Google Scholar, 16.Meng Q.H. Calabresi L. Fruchart J.C. Marcel Y.L. J. Biol. Chem. 1993; 268: 16966-16973Abstract Full Text PDF PubMed Google Scholar, 17.Sorci-Thomas M.G. Curtiss L. Parks J.S. Thomas M.J. Kearns M.W. Landrum M. J. Biol. Chem. 1998; 273: 11776-11782Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 18.Frank P.G. N′Guyen D. Franklin V. Neville T. Desforges M. Rassart E. Sparks D.L. Marcel Y.L. Biochemistry. 1998; 37: 13902-13909Crossref PubMed Scopus (35) Google Scholar, 19.Uboldi P. Spoladore M. Fantappiè S. Marcovina S. Catapano A.L. J. Lipid Res. 1996; 37: 2557-2568Abstract Full Text PDF PubMed Google Scholar). We have previously generated a series of mutants in which a pair of adjacent helices were deleted sequentially within the central domain of apoA-I (20.Bergeron J. Frank P.G. Emmanuel F. Latta M. Zhao Y.W. Sparks D.L. Rassart E. Denèfle P. Marcel Y.L. Biochim. Biophys. Acta Lipids Lipid Metab. 1997; 1344: 139-152Crossref PubMed Scopus (47) Google Scholar). These mutants denoted here as Δ4-5A-I, Δ5-6A-I, and Δ6-7A-I contain deletions in which amino acids 100–143, 122–165, and 144–186 have been removed, respectively. Our rationale in choosing these deletions was that removing a pair of helices would minimally modify the periodicity of the amphipathic α-helices and their putative interactions. Subsequent characterization of these mutantsin vitro confirmed that the amphipathic α-helices within residues 100–186 were involved in interactions with phospholipids and may contribute to the overall lipid binding capacity of the apoA-I in the formation of HDL (21.Frank P.G. Bergeron J. Emmanuel F. Lavigne J.P. Sparks D.L. Denèfle P. Rassart E. Marcel Y.L. Biochemistry. 1997; 36: 1798-1806Crossref PubMed Scopus (35) Google Scholar). Removal of helices 4 and 5 (aa 100–143) caused a slight decrease in LCAT activation in vitro,whereas deletion of either helices 5 and 6 (aa 122–165) or 6 and 7 (aa 144–186) almost abolished cholesterol esterification (18.Frank P.G. N′Guyen D. Franklin V. Neville T. Desforges M. Rassart E. Sparks D.L. Marcel Y.L. Biochemistry. 1998; 37: 13902-13909Crossref PubMed Scopus (35) Google Scholar). This finding is in agreement with previous work suggesting that helix 6 (aa 144–165) is necessary for optimum LCAT activation (17.Sorci-Thomas M.G. Curtiss L. Parks J.S. Thomas M.J. Kearns M.W. Landrum M. J. Biol. Chem. 1998; 273: 11776-11782Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) although other domains of apoA-I may also be involved (9.Holvoet P. Zhao Z.A. Vanloo B. Vos R. Deridder E. Dhoest A. Taveirne J. Brouwers E. Demarsin E. Engelborghs Y. Rosseneu M. Collen D. Brasseur R. Biochemistry. 1995; 34: 13334-13342Crossref PubMed Scopus (87) Google Scholar, 22.Sorci-Thomas M. Kearns M.W. Lee J.P. J. Biol. Chem. 1993; 268: 21403-21409Abstract Full Text PDF PubMed Google Scholar, 23.Anantharamaiah G.M. Venkatachalapathi Y.V. Brouillette C.G. Segrest J.P. Arteriosclerosis. 1990; 10: 95-105Crossref PubMed Google Scholar). Despite the insights gained from these in vitro studies, the roles of apoA-I central domain amphipathic α-helices in HDL maturationin vivo are still not fully understood. In this study, we generated recombinant adenoviruses containing cDNAs for human wild-type apoA-I (wt apoA-I) and the three central domain deletion mutants for the purpose of structure/function analysis of apoA-I in vivo. Native apoA-I and the mutants were analyzed following injections of the recombinant adenoviruses into apoA-I-deficient mice. These studies demonstrate that specific amphipathic α-helices within the central domain of apoA-I contribute to the in vivo maturation of HDL through roles in either lipid binding or the activation of LCAT. The cDNAs for firefly luciferase (luc), wt apoA-I, Δ4-5A-I, Δ5-6A-I, and Δ6-7A-I were subcloned into the vector pCA13 under the control of the cytomegalovirus promoter (Microbix Biosystems Inc., Toronto, Ontario, Canada). Recombinant adenovirus (Ad5) constructs carrying these cDNAs were prepared following co-transfection of 293 cells (Microbix Biosystems Inc.) with the plasmid pJM17 and the appropriate pCA13 plasmid. LipofectAMINE (Life Technologies, Inc.) was used as the transfection reagent. After an 8-h incubation with the transfection mixture in Opti-MEM (Life Technologies, Inc.), the 293 cells were incubated overnight with Eagle's minimum essential medium containing 10% fetal bovine serum and then overlaid with 0.65% SeaPlaque agar (FMC BioProducts, Rockland, ME) in Eagle's minimum essential medium containing fetal bovine serum (5%), penicillin (100 units/ml), and streptomycin sulfate (100 μg/ml). Plaques were picked (1–2 weeks later) from the agar and resuspended in sterile phosphate-buffered saline (PBS, 137 mm NaCl, 8.2 mmNa2HPO4, 1.5 mmKH2PO4, 2.7 mm KCl) containing CaCl2 (0.68 mm), MgCl2 (0.50 mm) (PBS2+), and glycerol (10%). These plaques were used to infect subsequent confluent monolayers of 293 cells in order to amplify the recombinant adenoviruses. Polymerase chain reaction of DNA prepared from SDS- (0.5%) and Pronase (0.05%)-lysed 293 cells infected with the appropriate Ad5 construct was performed to detect the apoA-I cDNAs or luciferase cDNA (luc). All adenoviruses were subjected to several rounds of amplification prior to a final purification on two successive CsCl gradients. The purified virus stocks were dialyzed extensively against PBS2+ containing 10% glycerol and aliquoted through sterile 0.22-μm filters (Millipore Corporation, Bedford, MA), and plaque-forming units (pfu) per ml were determined by incubating fresh monolayers of 293 cells (60-mm dishes) with the appropriate dilution (usually 10−9) of adenovirus stock. ApoA-I-deficient (Apoa1 tm1Unc) C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME) were maintained on a 12-h light/12-h dark schedule on a normal chow diet (Charles River Rodent Animal Diet, catalog number 5075–18% protein, 4.5% fat). All experiments performed were in accordance with protocols approved by the University of Ottawa Animal Care Committee. All mice used, unless otherwise indicated, were 3–10-month-old females. Lipid measurements were performed on the plasma of fasted (9–11 h) and non-fasted mice (fed state). Briefly, blood was collected into EDTA(K3) microcapillary tubes (Sarstedt Inc., St-Leonard, Québec, Canada) following clipping of the tail vein, and plasma was subsequently isolated by centrifugation (5 min at 4000 × g). Total (TC) and free (FC) cholesterol levels were measured with enzymatic kits (Roche Molecular Biochemicals) as was phospholipid (PL) (Wako Chemicals, Neuss, Germany) using freshly isolated plasma samples. Cholesteryl ester levels were determined by subtracting FC from TC. These values were used for pre-adenovirus injection lipid levels. Mice were allowed to recover at least 2 days prior to the injection of the recombinant adenoviruses. All adenoviruses were injected into the tail vein at a dosage of either 2 × 109 or 1 × 1010 pfu. To monitor the efficiency of each tail vein injection, 2 × 107 pfu of luc.Ad5 were included in the injections of the Ad5 carrying the apoA-I cDNAs. Mice were either fasted prior to blood collection 96 h post-adenovirus injection or alternatively blood was taken from fed mice (on the chow diet) 40 h postinjection. Mice were anesthetized, and blood was collected into EDTA(K3) vacutainer tubes following a laparotomy and subsequent clipping of the descending aorta. The liver was removed, weighed (typically 1–1.3 g wet weight), and homogenized on ice in luciferase lysis buffer (1 ml). Luciferase activity was measured in a Lumat LB9507 luminometer (EG 38 G Berthold) following addition of 10 μl of luciferase assay reagent (Promega Corp., Madison, WI). Plasma was isolated by centrifugation at 3000 × g for 13 min, and TC, FC, CE, and PL levels were determined as described earlier. Plasma (1.5 μl) isolated from fasted mice prior to injection or following injection of luc.Ad5, Δ4-5A-I.Ad5, or Δ5-6A-I.Ad5 (2 × 109 pfu of each) was subjected to 12% SDS-PAGE. Following transfer to nitrocellulose, the membrane was probed with a polyclonal anti-mouse apolipoprotein E (apoE) antibody (BIODESIGN International, Kennebunk, ME) and visualized by chemiluminescence (West Pico SuperSignal substrate, Pierce) after incubation with horseradish peroxidase-conjugated anti-rabbit IgG (Amersham Pharmacia Biotech). The relative intensities of the apoE signals were determined with software from Bio-Rad (Quantity One, version 4.10). Plasma (500 μl) isolated following the different Ad5 injections (96 h) was loaded on two Superdex 200 columns (analytical grade, Amersham Pharmacia Biotech) connected in series with a total bed volume of approximately 400 ml and void volume of 100 ml. The columns were standardized with a mixture of high and low molecular weight markers of known Stokes' diameters (Amersham Pharmacia Biotech). Plasma was passed down the columns at a flow rate of 0.1 ml per min, and 5-ml fractions were collected. The VLDL and low density lipoprotein component of the plasma appeared in the void volume (fractions 9–12) on these columns. Large size HDL2 particles were localized in fractions 15–19 (12.1 to 10.2 nm), larger HDL3particles in fractions 20–23 (9.7 to 8.2 nm), and smaller HDL fractions containing albumin were in fractions 24–28. Total cholesterol was determined for the various fractions. Briefly, samples were concentrated (5-fold by lyophilization) and resuspended in PBS, and cholesterol levels were quantified by standard enzymatic kits as described. Aliquots (100 μl) from each fraction were analyzed for apoA-I by slot blot (Bio-Rad Bio-Dot SF unit) analysis. The nitrocellulose was probed with biotinylated monoclonal antibodies directed against apoA-I (a combination of 4H1 (against the extreme N terminus) and 5F6 (against the central region)). The antibodies were biotinylated with Sulfo-NHS-Biotin (Pierce). The presence of the apoA-I proteins in each of the FPLC Superdex 200 fractions was detected by chemiluminescence (Pierce SuperSignal substrate) following treatment with streptavidin-conjugated horseradish peroxidase (Amersham Pharmacia Biotech). No background signal could be detected as indicated by analysis of plasma fractions isolated following luc.Ad5 injections. Lipid levels (TC, FC, CE, and PL) in the HDL2/HDL3 FPLC fractions were measured as described earlier. The concentrations of the different apoA-I variants in the plasma of apoA-I-deficient mice were determined by a solid-phase radioimmunoassay as described previously (12.Calabresi L. Meng Q.-H. Castro G.R. Marcel Y.L. Biochemistry. 1993; 32: 6477-6484Crossref PubMed Scopus (70) Google Scholar, 24.Calabresi L. Banfi C. Sirtori C.R. Franceschini G. Biochim. Biophys. Acta Lipids Lipid Metab. 1992; 1124: 195-198Crossref PubMed Scopus (15) Google Scholar). ApoA-I-deficient mouse plasma was included with human apoA-I in generation of the standard curve to account for any cross-reactivity between the monoclonal antibodies and potential antigens in the plasma. No cross-reaction was detected. Plasma samples isolated following the Ad5 injections were subjected to discontinuous gradient density ultracentrifugation. Briefly, each sample (0.5 to 1.0 ml) was brought to a final volume of 3 ml (1 mm EDTA) with the addition of potassium bromide (1 g of KBr) and sucrose (50 mg). The discontinuous gradient consisted of plasma (3 ml on bottom) layered successively with ρ = 1.21 g/ml KBr (2 ml), ρ = 1.08 g/ml KBr (3 ml), and ρ = 1.00 g/ml (3 ml top). The tubes were spun for 18 h at 35,000 rpm on a Beckman L8–70M ultracentrifuge at 8 °C, and fractions (1 ml) were collected from top to bottom. The densities of the fractions were determined by comparing the readings measured with a refractometer (Fisher) to values obtained for solutions with known densities. Aliquots of each of the fractions were dialyzed against PBS (0.025 μm filter disks, Millipore Corp.), placed in SDS sample buffer, and analyzed by 12% SDS-PAGE. The gels were either stained for protein (GELCODE Blue Stain, Pierce) or transferred to nitrocellulose for Western blot analysis probing for either the human apoA-I proteins or mouse apoE. The concentrations of the apoA-I proteins (wild-type and mutant) in the different lipoprotein fractions from very low density lipoproteins (VLDL) to lipid-poor HDL species were measured by radioimmunoassay as described. The discontinuous gradient density fractions were subjected to non-denaturing gradient gel electrophoresis followed by Western blot analysis to size the HDL species formed by wt apoA-I or the central domain deletion mutants. The samples were dialyzed against PBS before application to the gels. The gradient gels were run for 2200 V-h and included high molecular weight markers (Amersham Pharmacia Biotech) that were biotinylated with Sulfo-NHS-Biotin (Pierce). The gels were transferred at 4 °C for 4 h with one change of transfer buffer. The nitrocellulose membranes were probed with biotinylated anti-apoA-I antibodies 4H1 and 5F6 followed by treatment with streptavidin-horseradish peroxidase and detection with the Pierce West Pico SuperSignal substrate as described. HDL fractions were analyzed for their morphology by negative staining electron microscopy as described previously (25.Forte T.M. Nordhausen R.W. Methods Enzymol. 1986; 128: 442-457Crossref PubMed Scopus (183) Google Scholar). The HDL were concentrated to 100–200 μg/ml as determined by the Markwell Lowry method (26.Markwell M.A. Haas S.M. Bieber L.L. Tolbert N.E. Anal. Biochem. 1978; 87: 206-210Crossref PubMed Scopus (5346) Google Scholar) and mixed 1:1 (v/v) with 2% sodium phosphotungstate, pH 7.4 (previously filtered through 0.22-μm membrane), before applying (10 μl) to carbon-coated Formvar grids (200 mesh). After 30 s on the grid, the samples were dried and analyzed in a Hitachi H-7100 electron microscope at × 50,000 or × 70,000 magnification. Cholesterol esterification rates of plasma samples from apoA-I-deficient mice expressing wt apoA-I or Δ4-5A-I were analyzed as described (27.Dobiasova M. Stribrna J. Sparks D.L. Pritchard P.H. Frohlich J.J. Arteriosclerosis. 1991; 11: 64-70Crossref Scopus (81) Google Scholar) with the following modifications. The apoA-I-deficient plasma samples (30 μl) containing no apoA-I (luc.Ad5-injected mice), wt apoA-I (10 μg), or Δ4-5A-I (10 μg) were diluted in PBS (final volume 250 μl) and incubated with [3H]cholesterol on filter disks (5 μCi of [3H]FC) at 4 °C overnight. Two aliquots (50 μl each) were removed and used as the time 0 point, and 2 aliquots were placed at 37 °C for 30 min. Ethanol (1 ml) was added (2 h room temperature incubation) to the time 0 and 30-min samples. The supernatant collected following centrifugation (10 min at 10,000 × g) was incubated with hexane carrier (3 ml containing 50 μg/ml CE and 100 μg/ml FC) and then evaporated to dryness under N2. The samples were solubilized in chloroform (200 μl), spotted (50 μl) on thin layer chromatography (TLC) plates (Silica Glass Fiber-Gelman Science, Ann Arbor, MI), run in a hydrophobic solvent system (89% hexane, 10% diethyl ether, and 1% acetic acid), and FC and CE were visualized with iodine staining. Bands corresponding to FC and CE were cut, and radioactivity was determined. The fractional cholesterol esterification rate is expressed as the percentage of FC converted to CE per h. We first established that wt apoA-I reached physiological levels (170 ± 40 mg/dl) in the plasma of fasted apoA-I-deficient mice 96 h post-Ad5 injection at a dose of 2 × 109 pfu. Under the same conditions, all apoA-I mutants were expressed at significantly lower levels. The highest expression was observed for Δ4-5A-I, followed by Δ6-7A-I, and lowest for Δ5-6A-I. No impairment in secretion rates of the apoA-I central domain deletion mutants were detected following infection of COS-7 cells in culture (>95% of all apoA-I forms were secreted in the media as measured by Western blot analysis 3 days postinfection, data not shown). This suggests that all apoA-I mutants are folded normally and secreted equally. When we increased the Ad5 doses and analyzed plasma in the fed state at earlier times postinjection (within 40 h), the levels of the apoA-I mutants obtained were similar to or greater than those found for wt apoA-I (Table I, 40-h fed column). The time course of expression showed that all mutants had early peak levels that decreased markedly by day 6 postinjection, whereas wt apoA-I reached a peak concentration 10 days postinjection and remained circulating for over 30 days (Fig.1).Table IThe protein concentrations of native human apoA-I and the three mutants in the plasma of apoA-I-deficient miceAd5 constructApoA-I levels96-H fasted (n)40-H fed (n)mg/dlLuciferase0.32 ± 0.22 (2)NDcND, not determined.wt apoA-I172 ± 43 (4)aAdenovirus dose = 2 × 109 pfu.178 ± 130 (3)aAdenovirus dose = 2 × 109 pfu.Δ4-5A-I27 ± 6 (4)aAdenovirus dose = 2 × 109 pfu.643 ± 206 (3)bAdenovirus dose = 1 × 1010 pfu.Δ5-6A-I3.6 ± 2.3 (7)aAdenovirus dose = 2 × 109 pfu.405 ± 230 (3)bAdenovirus dose = 1 × 1010 pfu.Δ6-7A-I9.3 ± 4.2 (3)aAdenovirus dose = 2 × 109 pfu.198 ± 90 (4)bAdenovirus dose = 1 × 1010 pfu.Each Ad5 construct was administered via the tail vein of apoA-I-deficient mice at either one of the two doses as described under “Experimental Procedures.” The number (n) of mice used for each mutant and either treatment is indicated. There are significant increases in the plasma protein concentrations of the apoA-I mutants when a higher Ad5 dose is combined with earlier sampling (40 h postinjection) and maintenance of the fed state.a Adenovirus dose = 2 × 109 pfu.b Adenovirus dose = 1 × 1010 pfu.c ND, not determined. Open table in a new tab Each Ad5 construct was administered via the tail vein of apoA-I-deficient mice at either one of the two doses as described under “Experimental Procedures.” The number (n) of mice used for each mutant and either treatment is indicated. There are significant increases in the plasma protein concentrations of the apoA-I mutants when a higher Ad5 dose is combined with earlier sampling (40 h postinjection) and maintenance of the fed state. Expression of the apoA-I mutants in plasma taken from fasted apoA-I-deficient mice (96 h postinjection) was not accompanied by any increase in plasma lipid levels but rather by moderate decreases (Table II). In contrast, wt apoA-I produced significant increases in plasma cholesterol and phospholipids. This increase in plasma cholesterol was due exclusively to an increase in the HDL-CE concentration. The control luc.Ad5-injected mice had slightly decreased lipid levels compared with non-injected mice, but this did not reach statistical significance. Therefore, the decrease in plasma lipid levels following expression of the apoA-I mutants, and in particular Δ5-6A-I (Table II), was significant (p < 0.05) and not explained by an effect of the adenovirus vector alone.Table IIFasted plasma lipid levels and the ratio of esterified to total cholesterol in apoA-I-deficient mice prior to or following injections of either luciferase or the various apoA-I recombinant adenovirus constructsAd5 injectionaAll adenovirus constructs were injected at a final titer of 2 × 109 p/fu. Plasma was sampled 96 h postinjection from a given number (n) of fasted (9–11-h) apoA-I-deficient mice. The lipid and CE/TC ratio values for wt apoA-I and mutants are shown. Low concentrations of Δ5–6A-I and Δ6–7A-I (see Table I) reduce the already low plasma lipid levels found in apoA-I-deficient mice, whereas wt apoA-I increases plasma CE and PL as well as the CE/TC ratio. (n)Plasma lipid levelsCE/TCTCFCCEPLmg/dlNone (8)53 ± 526 ± 527 ± 666 ± 120.51 ± 0.09Luciferase (3)44 ± 323 ± 221 ± 546 ± 60.47 ± 0.08wt apoA-I (4)115 ± 14bValues that are statistically significant atp < 0.05 (Student's t test) from noninjected apoA-I deficient mice are indicated.34 ± 1181 ± 26bValues that are statistically significant atp < 0.05 (Student's t test) from noninjected apoA-I deficient mice are indicated.181 ± 25bValues that are statistically significant atp < 0.05 (Student's t test) from noninjected apoA-I deficient mice are indicated.0.70 ± 0.06bValues that are statistically significant atp < 0.05 (Student's t test) from noninjected apoA-I deficient mice are indicated.Δ4-5A-I (3)41 ± 1424 ± 1217 ± 444 ± 100.43 ± 0.10Δ5-6A-I (4)27 ± 4bValues that are statistically significant atp < 0.05 (Student's t test) from noninjected apoA-I deficient mice are indicated.15 ± 3bValues that are statistically significant atp < 0.05 (Student's t test) from noninjected apoA-I deficient mice are indicated.12 ± 1bValues that are statistically significant atp < 0.05 (Student's t test) from noninjected apoA-I deficient mice are indicated.28 ± 5bValues that are statistically significant atp < 0.05 (Student's t test) from noninjected apoA-I deficient mice are indicated.0.44 ± 0.03Δ6-7A-I (3)47 ± 433 ± 514 ± 2bValues that are statistically significant atp < 0.05 (Student's t test) from noninjected apoA-I deficient mice are indicated.46 ± 30.29 ± 0.05bValues that are statistically significant atp < 0.05 (Student's t test) from noninjected apoA-I deficient mice are indicated.a All adenovirus constructs were injected at a final titer of 2 × 109 p/fu. Plasma was sampled 96 h postinjection from a given number (n) of fasted (9–11-h) apoA-I-deficient mice. The lipid and CE/TC ratio values for wt apoA-I and mutants are shown. Low concentrations of Δ5–6A-I and Δ6–7A-I (see Table I) reduce the already low plasma lipid levels found in apoA-I-deficient mice, whereas wt apoA-I increases plasma CE and PL as well as the CE/TC ratio.b Values that are statistically significant atp < 0.05 (Student's t test) from noninjected apoA-I deficient mice are indicated. Open table in a new tab We tested the possibility that the reduced concentration of lipids in plasma of apoA-I-deficient mice following expression of the apoA-I mutants was correlated with decreases in the concentrations of other apolipoproteins normally found in these mice. It was previously reported that apoA-I-deficient mice have increased HDL apoE levels comprising 25% of the total HDL protein compared with control C57BL/6 mice in which apoE on HDL was barely detectable (28.Plump A.S. Azrolan N. Odaka H. Wu L. Jiang X. Tall A. Eisenberg S. Breslow J.L. J. Lipid Res. 1997; 38: 1033-1047Abstract Full Text PDF PubMed Google Scholar). We observed that plasma apoE levels (primarily in the HDL pool) were significantly reduced following expression of either Δ4-5A-I (Fig.2 A, lanes 6 and 7) or Δ5-6A-I (Fig. 2 A, lanes 8" @default.
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• W2000312682 title "Distinct Central Amphipathic α-Helices in Apolipoprotein A-I Contribute to the in Vivo Maturation of High Density Lipoprotein by Either Activating Lecithin-Cholesterol Acyltransferase or Binding Lipids" @default.
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