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• W2165906160 abstract "Abstract In this study, we investigated the role of positively and negatively charged amino acids within the 89-99 region of apolipoprotein A-I (apoA-I), which are highly conserved in mammals, on plasma lipid homeostasis and the biogenesis of HDL. We previously showed that deletion of the 89-99 region of apoA-I increased plasma cholesterol and phospholipids, but it did not affect plasma triglycerides. Functional studies using adenovirus-mediated gene transfer of two apoA-I mutants in apoA-I-deficient mice showed that apoA-I[D89A/E91A/E92A] increased plasma cholesterol and caused severe hypertriglyceridemia. HDL levels were reduced, and approximately 40% of the apoA-I was distributed in VLDL/IDL. The HDL consisted of mostly spherical and a few discoidal particles and contained preβ1 and α4-HDL subpopulations. The lipid, lipoprotein, and HDL profiles generated by the apoA-I[K94A/K96A] mutant were similar to those of wild-type (WT) apoA-I. Coexpression of apoA-I[D89A/E91A/E92A] and human lipoprotein lipase abolished hypertriglyceridemia, restored in part the α1,2,3,4 HDL subpopulations, and redistributed apoA-I in the HDL2/HDL3 regions, but it did not prevent the formation of discoidal HDL particles. Physicochemical studies showed that the apoA-I[D89A/E91A/E92A] mutant had reduced α-helical content and effective enthalpy of thermal denaturation, increased exposure of hydrophobic surfaces, and increased affinity for triglyceride-rich emulsions. We conclude that residues D89, E91, and E92 of apoA-I are important for plasma cholesterol and triglyceride homeostasis as well as for the maturation of HDL. Abstract In this study, we investigated the role of positively and negatively charged amino acids within the 89-99 region of apolipoprotein A-I (apoA-I), which are highly conserved in mammals, on plasma lipid homeostasis and the biogenesis of HDL. We previously showed that deletion of the 89-99 region of apoA-I increased plasma cholesterol and phospholipids, but it did not affect plasma triglycerides. Functional studies using adenovirus-mediated gene transfer of two apoA-I mutants in apoA-I-deficient mice showed that apoA-I[D89A/E91A/E92A] increased plasma cholesterol and caused severe hypertriglyceridemia. HDL levels were reduced, and approximately 40% of the apoA-I was distributed in VLDL/IDL. The HDL consisted of mostly spherical and a few discoidal particles and contained preβ1 and α4-HDL subpopulations. The lipid, lipoprotein, and HDL profiles generated by the apoA-I[K94A/K96A] mutant were similar to those of wild-type (WT) apoA-I. Coexpression of apoA-I[D89A/E91A/E92A] and human lipoprotein lipase abolished hypertriglyceridemia, restored in part the α1,2,3,4 HDL subpopulations, and redistributed apoA-I in the HDL2/HDL3 regions, but it did not prevent the formation of discoidal HDL particles. Physicochemical studies showed that the apoA-I[D89A/E91A/E92A] mutant had reduced α-helical content and effective enthalpy of thermal denaturation, increased exposure of hydrophobic surfaces, and increased affinity for triglyceride-rich emulsions. We conclude that residues D89, E91, and E92 of apoA-I are important for plasma cholesterol and triglyceride homeostasis as well as for the maturation of HDL. Apolipoprotein A-I (apoA-I) is the major protein component of HDL, and it plays an essential role in the biogenesis, maturation, and the functions of HDL (1Chroni A. Liu T. Gorshkova I. Kan H.Y. Uehara Y. von Eckardstein A. Zannis V.I. The central helices of apoA-I can promote ATP-binding cassette transporter A1 (ABCA1)-mediated lipid efflux. Amino acid residues 220-231 of the wild-type apoA-I are required for lipid efflux in vitro and high density lipoprotein formation in vivo.J. Biol. Chem. 2003; 278: 6719-6730Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 2Laccotripe M. Makrides S.C. Jonas A. Zannis V.I. The carboxyl-terminal hydrophobic residues of apolipoprotein A-I affect its rate of phospholipid binding and its association with high density lipoprotein.J. Biol. Chem. 1997; 272: 17511-17522Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 3Liu T. Krieger M. Kan H.Y. Zannis V.I. The effects of mutations in helices 4 and 6 of apoA-I on scavenger receptor class B type I (SR-BI)-mediated cholesterol efflux suggest that formation of a productive complex between reconstituted high density lipoprotein and SR-BI is required for efficient lipid transport.J. Biol. Chem. 2002; 277: 21576-21584Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 4Zannis V.I. Chroni A. Kypreos K.E. Kan H.Y. Cesar T.B. Zanni E.E. Kardassis D. Probing the pathways of chylomicron and HDL metabolism using adenovirus-mediated gene transfer.Curr. Opin. Lipidol. 2004; 15: 151-166Crossref PubMed Scopus (61) Google Scholar). It is generally believed that the biogenesis of HDL occurs through a complex pathway that requires apoA-I, ATP-binding cassette transporter A1 (ABCA1), LCAT, and several other proteins (4Zannis V.I. Chroni A. Kypreos K.E. Kan H.Y. Cesar T.B. Zanni E.E. Kardassis D. Probing the pathways of chylomicron and HDL metabolism using adenovirus-mediated gene transfer.Curr. Opin. Lipidol. 2004; 15: 151-166Crossref PubMed Scopus (61) Google Scholar). Population studies have shown that genetic mutations in apoA-I, ABCA1, and LCAT occur with high frequency in subjects with low HDL levels (5Kiss R.S. Kavaslar N. Okuhira K. Freeman M.W. Walter S. Milne R.W. McPherson R. Marcel Y.L. Genetic etiology of isolated low HDL syndrome: incidence and heterogeneity of efflux defects.Arterioscler. Thromb. Vasc. Biol. 2007; 27: 1139-1145Crossref PubMed Scopus (53) Google Scholar, 6Cohen J.C. Kiss R.S. Pertsemlidis A. Marcel Y.L. McPherson R. Hobbs H.H. Multiple rare alleles contribute to low plasma levels of HDL cholesterol.Science. 2004; 305: 869-872Crossref PubMed Scopus (904) Google Scholar, 7Frikke-Schmidt R. Nordestgaard B.G. Jensen G.B. Tybjaerg-Hansen A. Genetic variation in ABC transporter A1 contributes to HDL cholesterol in the general population.J. Clin. Invest. 2004; 114: 1343-1353Crossref PubMed Scopus (233) Google Scholar). In previous studies, systematic mutagenesis and gene transfer of human apoA-I mutants in apoA-I deficient (apoA-I−/−) mice disrupted specific steps along the pathway of biogenesis of HDL or induced other lipid and lipoprotein abnormalities (8Zannis V.I. Zanni E.E. Papapanagiotou A. Kardassis D. Chroni A. ApoA-I functions and synthesis of HDL: insights from mouse models of human HDL metabolism.High-Density Lipoproteins. From Basic Biology to Clinical Aspects. Wiley-VCH, Weinheim, 2006: 237-265Google Scholar). The phenotypes generated by apoA-I gene transfer in mice were discrete and included inhibition of the biogenesis of HDL (1Chroni A. Liu T. Gorshkova I. Kan H.Y. Uehara Y. von Eckardstein A. Zannis V.I. The central helices of apoA-I can promote ATP-binding cassette transporter A1 (ABCA1)-mediated lipid efflux. Amino acid residues 220-231 of the wild-type apoA-I are required for lipid efflux in vitro and high density lipoprotein formation in vivo.J. Biol. Chem. 2003; 278: 6719-6730Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar); generation of unstable intermediates (9Koukos G. Chroni A. Duka A. Kardassis D. Zannis V.I. Naturally occurring and bioengineered apoA-I mutations that inhibit the conversion of discoidal to spherical HDL: the abnormal HDL phenotypes can be corrected by treatment with LCAT.Biochem. J. 2007; 406: 167-174Crossref PubMed Scopus (27) Google Scholar, 10Koukos G. Chroni A. Duka A. Kardassis D. Zannis V.I. LCAT can rescue the abnormal phenotype produced by the natural ApoA-I mutations (Leu141Arg)Pisa and (Leu159Arg)FIN.Biochemistry. 2007; 46: 10713-10721Crossref PubMed Scopus (26) Google Scholar); inhibition of the activation of LCAT (9Koukos G. Chroni A. Duka A. Kardassis D. Zannis V.I. Naturally occurring and bioengineered apoA-I mutations that inhibit the conversion of discoidal to spherical HDL: the abnormal HDL phenotypes can be corrected by treatment with LCAT.Biochem. J. 2007; 406: 167-174Crossref PubMed Scopus (27) Google Scholar, 10Koukos G. Chroni A. Duka A. Kardassis D. Zannis V.I. LCAT can rescue the abnormal phenotype produced by the natural ApoA-I mutations (Leu141Arg)Pisa and (Leu159Arg)FIN.Biochemistry. 2007; 46: 10713-10721Crossref PubMed Scopus (26) Google Scholar, 11Chroni A. Duka A. Kan H.Y. Liu T. Zannis V.I. Point mutations in apolipoprotein a-I mimic the phenotype observed in patients with classical lecithin:cholesterol acyltransferase deficiency.Biochemistry. 2005; 44: 14353-14366Crossref PubMed Scopus (31) Google Scholar); and increase in plasma cholesterol or increase in both plasma cholesterol and triglycerides (12Chroni A. Kan H.Y. Shkodrani A. Liu T. Zannis V.I. Deletions of helices 2 and 3 of human apoA-I are associated with severe dyslipidemia following adenovirus-mediated gene transfer in apoA-I-deficient mice.Biochemistry. 2005; 44: 4108-4117Crossref PubMed Scopus (29) Google Scholar, 13Chroni A. Kan H.Y. Kypreos K.E. Gorshkova I.N. Shkodrani A. Zannis V.I. Substitutions of glutamate 110 and 111 in the middle helix 4 of human apolipoprotein A-I (apoA-I) by alanine affect the structure and in vitro functions of apoA-I and induce severe hypertriglyceridemia in apoA-I-deficient mice.Biochemistry. 2004; 43: 10442-10457Crossref PubMed Scopus (50) Google Scholar). These studies also showed that deletion of the 89-99 region increased the levels and density distribution of plasma cholesterol and phospholipids and generated discoidal HDL particles but did not affect plasma triglyceride levels (12). In the present study, we investigated the importance of the conserved positively and negatively charged residues present in the 89-99 domain of apoA-I for cholesterol and triglyceride homeostasis and the biogenesis of HDL. In the lipid-bound form, charged residues within the 89-99 region are juxtaposed with the complementary amino acid of apoA-I dimer or hairpin-shaped monomer (14Wu Z. Gogonea V. Lee X. May R.P. Pipich V. Wagner M.A. Undurti A. Tallant T.C. Baleanu-Gogonea C. Charlton F. et al.The low resolution structure of ApoA1 in spherical high density lipoprotein revealed by small angle neutron scattering.J. Biol. Chem. 2011; 286: 12495-12508Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 15Silva R.A. Huang R. Morris J. Fang J. Gracheva E.O. Ren G. Kontush A. Jerome W.G. Rye K.A. Davidson W.S. Structure of apolipoprotein A-I in spherical high density lipoproteins of different sizes.Proc. Natl. Acad. Sci. USA. 2008; 105: 12176-12181Crossref PubMed Scopus (162) Google Scholar) and can form solvent-inaccessible salt bridges (16Bashtovyy D. Jones M.K. Anantharamaiah G.M. Segrest J.P. Sequence conservation of apolipoprotein A-I affords novel insights into HDL structure-function.J. Lipid Res. 2011; 52: 435-450Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). We have found that changes in the positively charged residues K94 and K96 caused structural changes but did not affect the lipid and lipoprotein levels and the biogenesis of HDL. In contrast, substitutions of the negatively charged residues D89, E91, and E92 by alanines altered the conformation of apoA-I, increased its affinity for VLDL/IDL, as well as for triglyceride-rich emulsions, affected the maturation of HDL, and caused severe hypertriglyceridemia. These findings combined with previous studies suggest that subtle changes in critical amino acids located in different domains of apoA-I may have severe effects not only on the biogenesis of HDL but also on plasma cholesterol and tri­glyceride homeostasis. Materials not mentioned in the experimental procedures have been obtained from sources described previously (3Liu T. Krieger M. Kan H.Y. Zannis V.I. The effects of mutations in helices 4 and 6 of apoA-I on scavenger receptor class B type I (SR-BI)-mediated cholesterol efflux suggest that formation of a productive complex between reconstituted high density lipoprotein and SR-BI is required for efficient lipid transport.J. Biol. Chem. 2002; 277: 21576-21584Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 13Chroni A. Kan H.Y. Kypreos K.E. Gorshkova I.N. Shkodrani A. Zannis V.I. Substitutions of glutamate 110 and 111 in the middle helix 4 of human apolipoprotein A-I (apoA-I) by alanine affect the structure and in vitro functions of apoA-I and induce severe hypertriglyceridemia in apoA-I-deficient mice.Biochemistry. 2004; 43: 10442-10457Crossref PubMed Scopus (50) Google Scholar). The apoA-I gene lacking the BglII restriction site, which is present at nucleotide position 181 of the genomic sequence relative to the ATG codon of the gene, was cloned into the pCDNA3.1 vector to generate the pCDNA3.1-apoA-I(ΔBglII) plasmid as described (10Koukos G. Chroni A. Duka A. Kardassis D. Zannis V.I. LCAT can rescue the abnormal phenotype produced by the natural ApoA-I mutations (Leu141Arg)Pisa and (Leu159Arg)FIN.Biochemistry. 2007; 46: 10713-10721Crossref PubMed Scopus (26) Google Scholar). This plasmid was used as a template to introduce the apoA-I mutations apoA-I[D89A/E91A/E92A] and apoA-I[K94A/K96A] using the QuickChange® XL mutagenesis kit (Stratagene, Santa Clara, CA) and the mutagenic primers shown in supplementary Table I. The recombinant adenoviruses were packaged in 911 cells, amplified in human embryonic kidney 293 (HEK 293) cells, purified, and titrated as described (10Koukos G. Chroni A. Duka A. Kardassis D. Zannis V.I. LCAT can rescue the abnormal phenotype produced by the natural ApoA-I mutations (Leu141Arg)Pisa and (Leu159Arg)FIN.Biochemistry. 2007; 46: 10713-10721Crossref PubMed Scopus (26) Google Scholar). The adenovirus expressing human lipoprotein lipase (hLPL) was a gift of Dr. Alex Vezeridis (17Vezeridis A.M. Drosatos K. Zannis V.I. Molecular etiology of a dominant form of type III hyperlipoproteinemia caused by R142C substitution in apoE4.J. Lipid Res. 2011; 52: 45-56Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). To assess the secretion of WT and mutant apoA-I forms, SW1783 human astrocytoma (HTB-13) cells grown to 80% confluence in Leibovitz's L-15 medium containing 2% heat-inactivated horse serum in 6-well plates were infected with adenoviruses expressing WT and mutant apoA-I forms at a multiplicity of infection of 10. At 24 h postinfection, the cells were washed twice with PBS and incubated in serum-free medium for 2 h. Following an additional wash with PBS, fresh serum-free medium was added, and 24 h later, it was collected and analyzed by SDS-PAGE for apoA-I expression. WT and the mutant apoA-I[D89A/E91A/E92A] were obtained from the culture media of HTB-13 cells grown in roller bottles following infection with adenoviruses expressing the corresponding proteins. For protein production, the culture medium was collected, concentrated 5-fold, dialyzed against 25 mM ammonium bicarbonate, and lyophilized. The lyophilized apoA-I was combined with β-oleoyl-γ-palmitoyl-L-α-phosphatidylcholine (POPC), cholesterol, and sodium cholate at the ratio 1 mg/9.5 mg/0.47 mg/4.5 mg. The proteoliposomes formed were fractionated by density gradient ultracentrifugation, and the fractions that contained pure apoA-I were collected and delipidated three times using 2:1 v/v chloroform:methanol (18Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipides from animal tissues.J. Biol. Chem. 1957; 226: 497-509.Abstract Full Text PDF PubMed Google Scholar). ABCA1-mediated cholesterol efflux measurements using HEK293-EBNA cells and LCAT activation assays were performed as described (13Chroni A. Kan H.Y. Kypreos K.E. Gorshkova I.N. Shkodrani A. Zannis V.I. Substitutions of glutamate 110 and 111 in the middle helix 4 of human apolipoprotein A-I (apoA-I) by alanine affect the structure and in vitro functions of apoA-I and induce severe hypertriglyceridemia in apoA-I-deficient mice.Biochemistry. 2004; 43: 10442-10457Crossref PubMed Scopus (50) Google Scholar, 19Fitzgerald M.L. Mendez A.J. Moore K.J. Andersson L.P. Panjeton H.A. Freeman M.W. ATP-binding cassette transporter A1 contains an NH2-terminal signal anchor sequence that translocates the protein's first hydrophilic domain to the exoplasmic space.J. Biol. Chem. 2001; 276: 15137-15145Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Male apoA-I−/− (ApoA1tm1Unc) C57BL/6J mice (20Williamson R. Lee D. Hagaman J. Maeda N. Marked reduction of high density lipoprotein cholesterol in mice genetically modified to lack apolipoprotein A-I.Proc. Natl. Acad. Sci. USA. 1992; 89: 7134-7138Crossref PubMed Scopus (190) Google Scholar) were purchased from Jackson Laboratories (Bar Harbor, ME). The mice were maintained on a 12-h light/dark cycle and standard rodent chow. All procedures performed on the mice were in accordance with National Institutes of Health and institutional guidelines. ApoA-I−/− mice, 6–8 weeks of age, were injected via the tail vein with 2 × 109 plaque forming units (pfu) of recombinant adenovirus per animal. The animals were euthanized four days postinjection following a 4 h fast. The concentration of total cholesterol, free cholesterol, phospholipids, and triglycerides of plasma drawn four days postinfection was determined using the Total Cholesterol E, Free Cholesterol C, and Phospholipids C reagents (Wako Chemicals USA, Inc., Richmond, VA) and INFINITY triglycerides reagent (ThermoScientific, Waltham, MA), respectively, according to the manufacturer's instructions. The concentration of cholesteryl esters was determined by subtracting the concentration of free cholesterol from the concentration of total cholesterol. Plasma apoA-I levels were determined by a turbidometric assay using AutoKit A-I (Wako Chemicals). For fast-protein liquid chromatography (FPLC) analysis of plasma, 17 μl plasma obtained from mice infected with adenovirus-expressing WT or mutant apoA-I forms were loaded onto a Sepharose 6 PC column (Amersham Biosciences, Piscataway, NJ) in a SMART micro FPLC system (Amersham Biosciences) and eluted with PBS. A total of 25 fractions of 50 μl volume each were collected for further analysis. The concentration of lipids in the FPLC fractions was determined as described above. The plasma HDL subpopulations were separated by two-dimensional electrophoresis. The proteins were then transferred to a nitrocellulose membrane and apoA-I was detected by immunoblotting, using the goat polyclonal anti-human apoA-I antibody AB740 (Chemicon International, Billerica, MA) (12Chroni A. Kan H.Y. Shkodrani A. Liu T. Zannis V.I. Deletions of helices 2 and 3 of human apoA-I are associated with severe dyslipidemia following adenovirus-mediated gene transfer in apoA-I-deficient mice.Biochemistry. 2005; 44: 4108-4117Crossref PubMed Scopus (29) Google Scholar). An aliquot of 300 μl of plasma obtained from adenovirus-infected mice was diluted with saline to a total volume of 0.5 ml and fractionated by density gradient ultracentrifugation. Following ultracentrifugation, 0.5 ml fractions were collected and analyzed by SDS-PAGE as previously described (12Chroni A. Kan H.Y. Shkodrani A. Liu T. Zannis V.I. Deletions of helices 2 and 3 of human apoA-I are associated with severe dyslipidemia following adenovirus-mediated gene transfer in apoA-I-deficient mice.Biochemistry. 2005; 44: 4108-4117Crossref PubMed Scopus (29) Google Scholar). Fractions 6-7 obtained by the density ultracentrifugation, which float in the HDL region, were analyzed by electron microscopy (EM) using a Philips CM-120 electron microscope. Total hepatic RNA was isolated by the Trizol® method (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. RNA samples were adjusted to 0.1 μg/μl, and cDNA was produced using the high-capacity reverse transcriptase cDNA kit (Applied Biosystems, Foster City, CA). Apo A-I mRNA was quantified using Applied Biosystems Gene Array TaqMan® primers for apoA-I cDNA (Cat# Hs00985000_g1) and 18s rRNA (Cat# 4319413E) with the TaqMan® Gene expression PCR Master Mix (Cat# 4370048), using the Applied Biosystems 7300 Real-Time PCR System (21Zhu L.J. Altmann S.W. mRNA and 18S-RNA coapplication-reverse transcription for quantitative gene expression analysis.Anal. Biochem. 2005; 345: 102-109Crossref PubMed Scopus (111) Google Scholar). Several days prior to the physicochemical analyses, the lyophilized proteins were dissolved in 4 M guanidine hydrochloride (GndHCl) and then refolded by subsequent dialysis against 2.5 M and 1.25 M GndHCl solutions in PBS, followed by extensive dialysis against the appropriate buffer [10 mM sodium phosphate and 0.02% NaN3 (pH 7.4) for circular dichroism (CD) experiments, PBS for fluorescence and dimyristoyl-L-α-phosphatidylcholine (DMPC) clearance measurements, or Tris for emulsion-binding experiments]. Far-UV CD spectra were recorded at 25°C on an AVIV 62DS or AVIV 215 spectropolarimeter (AVIV Associates, Inc., Lakewood, NJ) equipped with a thermoelectric temperature control at protein concentrations 20-55 μg/ml as described previously (22Gorshkova I.N. Liadaki K. Gursky O. Atkinson D. Zannis V.I. Probing the lipid-free structure and stability of apolipoprotein A-I by mutation.Biochemistry. 2000; 39: 15910-15919Crossref PubMed Scopus (45) Google Scholar, 23Gorshkova I.N. Liu T. Zannis V.I. Atkinson D. Lipid-free structure and stability of apolipoprotein A-I: probing the central region by mutation.Biochemistry. 2002; 41: 10529-10539Crossref PubMed Scopus (39) Google Scholar). For each protein, spectra were recorded at several protein concentrations, then normalized to the protein concentration and expressed as mean residue ellipticity [Θ], which was calculated by the equation: [Θ] = Θ × MRW / (10 × l × c), where Θ is the measured ellipticity, MRW is the mean residue weight (about 115), l is the cell path length (cm), and c is the protein concentration (g/ml). The α-helix content was estimated from the mean residue ellipticity at 222 nm, [Θ222] (24Chen Y.H. Yang J.T. Martinez H.M. Determination of the secondary structures of proteins by circular dichroism and optical rotatory dispersion.Biochemistry. 1972; 11: 4120-4131Crossref PubMed Scopus (1912) Google Scholar). Thermal unfolding of apoA-I in solution was monitored by changes in ellipticity at 222 nm. The thermodynamic parameters of the transitions, melting temperature (Tm) and van't Hoff enthalpy (ΔHv), were determined from the van't Hoff analysis of the melting curves as described previously (22Gorshkova I.N. Liadaki K. Gursky O. Atkinson D. Zannis V.I. Probing the lipid-free structure and stability of apolipoprotein A-I by mutation.Biochemistry. 2000; 39: 15910-15919Crossref PubMed Scopus (45) Google Scholar, 23Gorshkova I.N. Liu T. Zannis V.I. Atkinson D. Lipid-free structure and stability of apolipoprotein A-I: probing the central region by mutation.Biochemistry. 2002; 41: 10529-10539Crossref PubMed Scopus (39) Google Scholar). Fluorescence emission spectra of 8-anilino-1-naphthalene-sulfonate (ANS) were recorded in PBS buffer at ANS concentration of 125 μM in the presence of 25 μg/ml WT apoA-I, mutant apoA-I forms, or carbonic anhydrase, or in the absence of any protein using a FluoroMax-2 fluorescence spectrometer (25). The wavelength of maximum fluorescence (WMF) and the intensity of fluorescence emission at WMF were determined from each spectra after subtraction of the buffer baseline as described (25Gorshkova I.N. Kypreos K.E. Gantz D.L. Zannis V.I. Atkinson D. Biophysical properties of apolipoprotein E4 variants: implications in molecular mechanisms of correction of hypertriglyceridemia.Biochemistry. 2008; 47: 12644-12654Crossref PubMed Scopus (13) Google Scholar). The solubilization of DMPC multilamellar vesicles by apoA-I was monitored by the decrease in absorbance at 325 nm following the administration of apoA-I to a suspension of DMPC in PBS as described previously (25Gorshkova I.N. Kypreos K.E. Gantz D.L. Zannis V.I. Atkinson D. Biophysical properties of apolipoprotein E4 variants: implications in molecular mechanisms of correction of hypertriglyceridemia.Biochemistry. 2008; 47: 12644-12654Crossref PubMed Scopus (13) Google Scholar). Triglyceride-rich emulsions were prepared as described (26Derksen A. Small D.M. Interaction of ApoA-1 and ApoE-3 with triglyceride-phospholipid emulsions containing increasing cholesterol concentrations. Model of triglyceride-rich nascent and remnant lipoproteins.Biochemistry. 1989; 28: 900-906Crossref PubMed Scopus (35) Google Scholar, 27Gorshkova I.N. Atkinson D. Enhanced binding of apolipoprotein A-I variants associated with hypertriglyceridemia to triglyceride-rich particles.Biochemistry. 2011; 50: 2040-2047Crossref PubMed Scopus (7) Google Scholar). The particles, which were analyzed for phospholipid and triglyceride content as well as by EM to determine their morphology and size, were used for apoA-I binding assays within two days. For these assays, 120 µg of WT or mutant apoA-I (freshly dialyzed) were incubated for 1 h at 27°C with increasing amounts of emulsion in 1.8 ml of Tris buffer to give a phosphatidylcholine to protein molar ratio ranging from 180 to 710. The apoA-I bound to the emulsions was recovered by ultracentrifugation and quantitated as described (27Gorshkova I.N. Atkinson D. Enhanced binding of apolipoprotein A-I variants associated with hypertriglyceridemia to triglyceride-rich particles.Biochemistry. 2011; 50: 2040-2047Crossref PubMed Scopus (7) Google Scholar). Experiments were performed three or four times using emulsions from three different preparations. To assess the expression and secretion of the two mutant proteins relative to WT apoA-I, we infected HTB-13 cells with recombinant adenoviruses expressing the WT or the mutant apoA-I genes using a multiplicity of infection of 10. Analysis of the medium 24 h postinfection showed that the WT and the two mutant forms of apoA-I were secreted at comparable levels in the medium (supplementary Fig. IA). Plasma lipids, apoA-I, and hepatic apoA-I mRNA levels were determined four days postinfection of apoA-I−/−mice with adenoviruses expressing the WT apoA-I and the two apoA-I mutants. It was found that at comparable levels of mRNA expression the apoA-I[D89A/E91A/E92A] mutant caused dyslipidemia characterized by severe hypertriglyceridemia, increased plasma cholesterol and phospholipids, and decreased cholesteryl ester (CE) to total cholesterol (TC) ratio. The lipid parameters in mice expressing the apoA-I[K94A/K96A] mutant were comparable to those of mice expressing WT apoA-I with the exception of TC/apoA-I ratio, which was reduced for this mutant (Table 1).TABLE 1Plasma Lipids, ApoA-I, and Hepatic mRNA Levels of ApoA-I−/− Mice Expressing WT and Mutant Forms of ApoA-I Obtained Four Days PostinfectionProtein ExpressedTCTC/apoA-IFree CholesterolCE/TCPLTriglycerideRelative apoA-I mRNAPlasma apoA-Img/dlmg/dlmg/dlmg/dl%mg/dlGFP28 ± 7—12 ± 30.58 ± 0.0433 ± 2234 ± 3——WT apoA-I268 ± 550.95 ± 0.1675 ± 340.72 ± 0.06296 ± 9670 ± 11100 ± 32283 ± 84apoA-I [D89A/E91A/E92A]497 ± 1392.52 ± 0.84347 ± 1470.36 ± 0.31603 ± 4062106 ± 1629101 ± 24235 ± 106apoA-I [K94A/K96A]108 ± 230.53 ± 0.0446 ± 210.58 ± 0.15190 ± 1466 ± 956 ± 20220 ± 51apoA-I [D89A/E91A/E92A] + hLPL122 ± 561 ± 0.256 ± 200.44 ± 0.14255 ± 12849 ± 1641 ± 699 ± 18Values are means ± SD (n = 4-6). Open table in a new tab Values are means ± SD (n = 4-6). FPLC analysis of plasma from apoA-I−/− mice infected with the recombinant adenovirus expressing either WT apoA-I or the two apoA-I mutants showed that, in mice expressing WT apoA-I, cholesterol was distributed predominantly in the HDL2/HDL3 region. The distribution of cholesterol in the apoA-I[K94A/K96A] mutant was similar to that of WT apoA-I with an additional shoulder in the IDL and LDL regions. In contrast, in mice expressing the apoA-I[D89A/E91A/E92A] mutant, cholesterol was distributed predominantly in the VLDL/IDL/LDL region (Fig. 1A). All the triglycerides of the apoA-I[D89A/E91A/E92A] mutant were found in the VLDL region. The VLDL triglyceride peak of the WT apoA-I and apoA-I[K94A/K96A] mutant were negligible (Fig. 1B). Fractionation of plasma by density gradient ultracentrifugation and subsequent analysis of the resulting fractions by SDS-PAGE served two purposes: it gave important information of the distribution of apoA-I in different lipoprotein fractions, and it provided the HDL fractions that were used for EM analysis. It was found that the WT apoA-I and apoA-I[K94A/K96A] mutant are predominantly distributed in the HDL2 and HDL3 regions (Fig. 2A, B). In contrast, in the case of the apoA-I[D89A/E91A/E92A] mutant, approximately 40% of apoA-I was distributed in the VLDL/IDL/LDL fractions and the remaining in the HDL3 and, to a lesser extent, the HDL2 fractions (Fig. 2C). Analysis of the HDL fractions 6 and 7 obtained following density gradient ultracentrifugation by EM showed that the WT apoA-I and the apoA-I[K94A/K96A] mutant generated spherical particles (Fig. 2D, E) and the apoA-I[D89A/E91A/E92A] mutant generated mostly spherical and a few discoidal particles (Fig. 2F). Control experiments showed that HDL density fractions obtained from green fluorescent protein (GFP)-expressing apoA-I−/− mice, which cannot form HDL, contained very few spherical particles (supplementary Fig. II). Two-dimensional gel electrophoresis of plasma showed that WT apoA-I and the apoA-I[K94A/K96A] mutant formed normal α subpopulations with a small amount of preβ HDL particles (Fig. 2G, H). In contrast, the apoA-I[D89A/E91A/E92A] mutant formed predominantly preβ1 and α4 HDL particles at a ratio of approximately 2:1 (Fig. 2I). Lipoprotein lipase normalizes the lipid and lipoprotein abnormalities induced by the apoA-I[D89A/E91A/E92A] mutation. ApoA-I−/− mice were coinfected with 2" @default.
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