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• W2141016353 abstract "Apolipoprotein A-I (apoA-I) Nichinan, a naturally occurring variant with ΔE235 in the C terminus, is associated with low plasma HDL levels. Here, we investigated the tertiary structure, lipid-binding properties, and ability to induce cellular cholesterol efflux of apoA-I Nichinan and its C-terminal peptide. Thermal and chemical denaturation experiments demonstrated that the ΔE235 mutation decreased the protein stability compared with wild type (WT). ApoA-I Nichinan exhibited capabilities to bind to or solubilize lipid vesicles that are intermediate to that of WT and a L230P/L233P/Y236P variant in which the C-terminal α-helix folding is completely disrupted and forms relatively larger and unstable discoidal complexes, indicating that perturbation of the C-terminal α-helical structure by the ΔE235 mutation leads to reduced lipid binding. Supporting this, apoA-I 209-241/ΔE235 peptide showed significantly decreased ability to form α-helix both in the lipid-free and lipid-bound states, and reduced efficiency to solubilize vesicles. In addition, both apoA-I Nichinan and its C-terminal peptide exhibited reduced activity in ABCA1-mediated cellular cholesterol efflux. Thus, the disruption of the ability of the C-terminal region to form α-helix caused by the E235 deletion appears to be the important determinant of impaired lipid binding and cholesterol efflux ability and, consequently, the low plasma HDL levels of apoA-I Nichinan probands. Apolipoprotein A-I (apoA-I) Nichinan, a naturally occurring variant with ΔE235 in the C terminus, is associated with low plasma HDL levels. Here, we investigated the tertiary structure, lipid-binding properties, and ability to induce cellular cholesterol efflux of apoA-I Nichinan and its C-terminal peptide. Thermal and chemical denaturation experiments demonstrated that the ΔE235 mutation decreased the protein stability compared with wild type (WT). ApoA-I Nichinan exhibited capabilities to bind to or solubilize lipid vesicles that are intermediate to that of WT and a L230P/L233P/Y236P variant in which the C-terminal α-helix folding is completely disrupted and forms relatively larger and unstable discoidal complexes, indicating that perturbation of the C-terminal α-helical structure by the ΔE235 mutation leads to reduced lipid binding. Supporting this, apoA-I 209-241/ΔE235 peptide showed significantly decreased ability to form α-helix both in the lipid-free and lipid-bound states, and reduced efficiency to solubilize vesicles. In addition, both apoA-I Nichinan and its C-terminal peptide exhibited reduced activity in ABCA1-mediated cellular cholesterol efflux. Thus, the disruption of the ability of the C-terminal region to form α-helix caused by the E235 deletion appears to be the important determinant of impaired lipid binding and cholesterol efflux ability and, consequently, the low plasma HDL levels of apoA-I Nichinan probands. Apolipoprotein A-I (apoA-I) is the major protein of plasma HDL and functions as a critical mediator in reverse cholesterol transport, a process by which excess cholesterol in peripheral cells is transferred via HDL to the liver for catabolism (1Yokoyama S. Assembly of high density lipoprotein by the ABCA1/apolipoprotein pathway.Curr. Opin. Lipidol. 2005; 16: 269-279Crossref PubMed Scopus (54) Google Scholar, 2Krimbou L. Marcil M. Genest J. New insights into the biogenesis of human high-density lipoproteins.Curr. Opin. Lipidol. 2006; 17: 258-267Crossref PubMed Scopus (45) Google Scholar, 3Tang C. Oram J.F. The cell cholesterol exporter ABCA1 as a protector from cardiovascular disease and diabetes.Biochim. Biophys. Acta. 2009; 1791: 563-572Crossref PubMed Scopus (99) Google Scholar). It is generally thought that the first step in the reverse cholesterol transport pathway involves the efflux of cellular lipids to lipid-poor apoA-I, which is mediated by its interactions with ABCA1 (1Yokoyama S. Assembly of high density lipoprotein by the ABCA1/apolipoprotein pathway.Curr. Opin. Lipidol. 2005; 16: 269-279Crossref PubMed Scopus (54) Google Scholar, 2Krimbou L. Marcil M. Genest J. New insights into the biogenesis of human high-density lipoproteins.Curr. Opin. Lipidol. 2006; 17: 258-267Crossref PubMed Scopus (45) Google Scholar, 3Tang C. Oram J.F. The cell cholesterol exporter ABCA1 as a protector from cardiovascular disease and diabetes.Biochim. Biophys. Acta. 2009; 1791: 563-572Crossref PubMed Scopus (99) Google Scholar). Thus, mutations in the genes of apoA-I (4Frank P.G. Marcel Y.L. Apolipoprotein A-I: structure-function relationships.J. Lipid Res. 2000; 41: 853-872Abstract Full Text Full Text PDF PubMed Google Scholar, 5Sorci-Thomas M.G. Thomas M.J. The effects of altered apolipoprotein A-I structure on plasma HDL concentration.Trends Cardiovasc. Med. 2002; 12: 121-128Crossref PubMed Scopus (168) Google Scholar) or ABCA1 (6Oram J.F. Tangier disease and ABCA1.Biochim. Biophys. Acta. 2000; 1529: 321-330Crossref PubMed Scopus (201) Google Scholar, 7Attie A.D. Kastelein J.P. Hayden M.R. Pivotal role of ABCA1 in reverse cholesterol transport influencing HDL levels and susceptibility to atherosclerosis.J. Lipid Res. 2001; 42: 1717-1726Abstract Full Text Full Text PDF PubMed Google Scholar) lead to inadequate lipid transport from cells to extracellular spaces, resulting in the failure of HDL biogenesis and, consequently, low plasma HDL levels. Human apoA-I is a 243-residue polypeptide that contains 11- and 22-residue repeats of amphipathic α-helices (8Segrest J.P. Jones M.K. De Loof H. Brouillette C.G. Venkatachalapathi Y.V. Anantharamaiah G.M. The amphipathic helix in the exchangeable apolipoproteins: a review of secondary structure and function.J. Lipid Res. 1992; 33: 141-166Abstract Full Text PDF PubMed Google Scholar). It has been demonstrated that the apoA-I molecule folds into two tertiary structure domains, comprising an N-terminal α-helix bundle spanning residues 1–187 and a separate less organized C-terminal region spanning the remainder of the molecule (9Davidson W.S. Hazlett T. Mantulin W.W. Jonas A. The role of apolipoprotein AI domains in lipid binding.Proc. Natl. Acad. Sci. USA. 1996; 93: 13605-13610Crossref PubMed Scopus (131) Google Scholar, 10Saito H. Lund-Katz S. Phillips M.C. Contributions of domain structure and lipid interaction to the functionality of exchangeable human apolipoproteins.Prog. Lipid Res. 2004; 43: 350-380Crossref PubMed Scopus (186) Google Scholar, 11Ajees A.A. Anantharamaiah G.M. Mishra V.K. Hussain M.M. Murthy H.M. Crystal structure of human apolipoprotein A-I: insights into its protective effect against cardiovascular diseases.Proc. Natl. Acad. Sci. USA. 2006; 103: 2126-2131Crossref PubMed Scopus (194) Google Scholar). The helical segments in the N-terminal domain are involved in the activation of LCAT (12Sorci-Thomas M. Kearns M.W. Lee J.P. Apolipoprotein A-I domains involved in lecithin-cholesterol acyltransferase activation. Structure:function relationships.J. Biol. Chem. 1993; 268: 21403-21409Abstract Full Text PDF PubMed Google Scholar, 13Wu Z. Wagner M.A. Zheng L. Parks J.S. Shy 3rd, J.M. Smith J.D. Gogonea V. Hazen S.L. The refined structure of nascent HDL reveals a key functional domain for particle maturation and dysfunction.Nat. Struct. Mol. Biol. 2007; 14: 861-868Crossref PubMed Scopus (179) Google Scholar), whereas the C-terminal helix is involved in the strong lipid-binding properties of this protein (14Fang Y. Gursky O. Atkinson D. Lipid-binding studies of human apolipoprotein A-I and its terminally truncated mutants.Biochemistry. 2003; 42: 13260-13268Crossref PubMed Scopus (50) Google Scholar, 15Saito H. Dhanasekaran P. Nguyen D. Holvoet P. Lund-Katz S. Phillips M.C. Domain structure and lipid interaction in human apolipoproteins A-I and E, a general model.J. Biol. Chem. 2003; 278: 23227-23232Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 16Tanaka M. Koyama M. Dhanasekaran P. Nguyen D. Nickel M. Lund-Katz S. Saito H. Phillips M.C. Influence of tertiary structure domain properties on the functionality of apolipoprotein A-I.Biochemistry. 2008; 47: 2172-2180Crossref PubMed Scopus (42) Google Scholar). The interaction between the N- and C-terminal helical regions appears to contribute to the overall conformational stability of the apoA-I molecule in solution (17Fang Y. Gursky O. Atkinson D. Structural studies of N- and C-terminally truncated human apolipoprotein A-I.Biochemistry. 2003; 42: 6881-6890Crossref PubMed Scopus (38) Google Scholar, 18Silva R.A. Hilliard G.M. Fang J. Macha S. Davidson W.S. A three-dimensional molecular model of lipid-free apolipoprotein A-I determined by cross-linking/mass spectrometry and sequence threading.Biochemistry. 2005; 44: 2759-2769Crossref PubMed Scopus (95) Google Scholar, 19Tanaka M. Dhanasekaran P. Nguyen D. Ohta S. Lund-Katz S. Phillips M.C. Saito H. Contributions of the N- and C-terminal helical segments to the lipid-free structure and lipid interaction of apolipoprotein A-I.Biochemistry. 2006; 45: 10351-10358Crossref PubMed Scopus (68) Google Scholar, 20Koyama M. Tanaka M. Dhanasekaran P. Lund-Katz S. Phillips M.C. Saito H. Interaction between the N- and C-terminal domains modulates the stability and lipid binding of apolipoprotein A-I.Biochemistry. 2009; 48: 2529-2537Crossref PubMed Scopus (38) Google Scholar). In humans, over 40 natural mutations in apoA-I have been reported to date, of which roughly half can reduce plasma HDL levels and increase the incidence of cardiovascular disease (4Frank P.G. Marcel Y.L. Apolipoprotein A-I: structure-function relationships.J. Lipid Res. 2000; 41: 853-872Abstract Full Text Full Text PDF PubMed Google Scholar, 5Sorci-Thomas M.G. Thomas M.J. The effects of altered apolipoprotein A-I structure on plasma HDL concentration.Trends Cardiovasc. Med. 2002; 12: 121-128Crossref PubMed Scopus (168) Google Scholar). The large majority of mutations associated with low plasma HDL levels are clustered in the N-terminal helix bundle domain, whereas very few mutations are in the C-terminal domain (5Sorci-Thomas M.G. Thomas M.J. The effects of altered apolipoprotein A-I structure on plasma HDL concentration.Trends Cardiovasc. Med. 2002; 12: 121-128Crossref PubMed Scopus (168) Google Scholar). ApoA-I Nichinan, which contains the deletion of residue E235 in the C-terminal helical region, is known to be associated with low plasma HDL cholesterol and apoA-I levels (21Han H. Sasaki J. Matsunaga A. Hakamata H. Huang W. Ageta M. Taguchi T. Koga T. Kugi M. Horiuchi S. et al.A novel mutant, ApoA-I nichinan (Glu235→0), is associated with low HDL cholesterol levels and decreased cholesterol efflux from cells.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1447-1455Crossref PubMed Scopus (27) Google Scholar, 22Huang W. Sasaki J. Matsunaga A. Han H. Li W. Koga T. Kugi M. Ando S. Arakawa K. A single amino acid deletion in the carboxy terminal of apolipoprotein A-I impairs lipid binding and cellular interaction.Arterioscler. Thromb. Vasc. Biol. 2000; 20: 210-216Crossref PubMed Scopus (28) Google Scholar). Interestingly, this ΔE235 mutant is associated with normal plasma LCAT activity but a decrease in the ratio of HDL2 to HDL3 particles. Because a number of studies have demonstrated the importance of the C-terminal region of apoA-I in the ABCA1-mediated lipid efflux from cells and HDL biogenesis (23Panagotopulos S.E. Witting S.R. Horace E.M. Hui D.Y. Maiorano J.N. Davidson W.S. The role of apolipoprotein A-I helix 10 in apolipoprotein-mediated cholesterol efflux via the ATP-binding cassette transporter ABCA1.J. Biol. Chem. 2002; 277: 39477-39484Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 24Chroni 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 (100) Google Scholar, 25Vedhachalam C. Liu L. Nickel M. Dhanasekaran P. Anantharamaiah G.M. Lund-Katz S. Rothblat G.H. Phillips M.C. Influence of ApoA-I structure on the ABCA1-mediated efflux of cellular lipids.J. Biol. Chem. 2004; 279: 49931-49939Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 26Chroni A. Koukos G. Duka A. Zannis V.I. The carboxy-terminal region of apoA-I is required for the ABCA1-dependent formation of alpha-HDL but not prebeta-HDL particles in vivo.Biochemistry. 2007; 46: 5697-5708Crossref PubMed Scopus (25) Google Scholar, 27Vedhachalam C. Ghering A.B. Davidson W.S. Lund-Katz S. Rothblat G.H. Phillips M.C. ABCA1-induced cell surface binding sites for ApoA-I.Arterioscler. Thromb. Vasc. Biol. 2007; 27: 1603-1609Crossref PubMed Scopus (117) Google Scholar, 28Vedhachalam C. Duong P.T. Nickel M. Nguyen D. Dhanasekaran P. Saito H. Rothblat G.H. Lund-Katz S. Phillips M.C. Mechanism of ATP-binding cassette transporter A1-mediated cellular lipid efflux to apolipoprotein A-I and formation of high density lipoprotein particles.J. Biol. Chem. 2007; 282: 25123-25130Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 29Hassan H.H. Denis M. Lee D.Y. Iatan I. Nyholt D. Ruel I. Krimbou L. Genest J. Identification of an ABCA1-dependent phospholipid-rich plasma membrane apolipoprotein A-I binding site for nascent HDL formation: implications for current models of HDL biogenesis.J. Lipid Res. 2007; 48: 2428-2442Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 29Hassan H.H. Denis M. Lee D.Y. Iatan I. Nyholt D. Ruel I. Krimbou L. Genest J. Identification of an ABCA1-dependent phospholipid-rich plasma membrane apolipoprotein A-I binding site for nascent HDL formation: implications for current models of HDL biogenesis.J. Lipid Res. 2007; 48: 2428-2442Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), it is possible that impaired lipid-binding ability caused by the deletion of E235 in apoA-I Nichinan could underlie the decreased plasma HDL concentrations seen in carriers of the mutation. However, the molecular basis for these effects, especially the structure-function relationship of apoA-I Nichinan, has not been established yet. In the present study, we investigated the effects of the E235 deletion in apoA-I and its C-terminal peptide on the structure, lipid-binding properties, and ability to induce ABCA1-mediated cellular cholesterol efflux. The results suggest that the disruption of the helix-forming ability of the C-terminal region caused by the E235 deletion is the critical determinant of impaired cholesterol efflux ability and, consequently, low plasma HDL levels in apoA-I Nichinan probands. The mutation in human apoA-I to delete residue E235 was made using the QuikChange site-directed mutagenesis kit (Stratagene). Human wild type (WT) apoA-I and engineered mutants were expressed and purified as described (15Saito H. Dhanasekaran P. Nguyen D. Holvoet P. Lund-Katz S. Phillips M.C. Domain structure and lipid interaction in human apolipoproteins A-I and E, a general model.J. Biol. Chem. 2003; 278: 23227-23232Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 16Tanaka M. Koyama M. Dhanasekaran P. Nguyen D. Nickel M. Lund-Katz S. Saito H. Phillips M.C. Influence of tertiary structure domain properties on the functionality of apolipoprotein A-I.Biochemistry. 2008; 47: 2172-2180Crossref PubMed Scopus (42) Google Scholar). The apoA-I preparations were at least 95% pure as assessed by SDS-PAGE. The C-terminal apoA-I 209-241 and 209-241/ΔE235 peptides were synthesized using Fmoc chemistry as described (30Tanaka M. Tanaka T. Ohta S. Kawakami T. Konno H. Akaji K. Aimoto S. Saito H. Evaluation of lipid-binding properties of the N-terminal helical segments in human apolipoprotein A-I using fragment peptides.J. Pept. Sci. 2009; 15: 36-42Crossref PubMed Scopus (14) Google Scholar, 31Tanaka T. Tanaka M. Sugiura M. Kawakami T. Aimoto S. Saito H. Deletion of single amino acid E235 affects the structure and lipid interaction of human apolipoprotein A-I C-terminal peptides.Chem. Pharm. Bull. (Tokyo). 2009; 57: 499-503Crossref PubMed Scopus (2) Google Scholar). The N and C termini were capped with an acetyl group and an amide group, respectively. Peptide purity was verified by analytical HPLC (>97%) and mass spectrometry. In all experiments, apoA-I variants and peptides were freshly dialyzed from 6 M guanidine hydrochloride (GdnHCl) solution into the appropriate buffer before use. Near- and far-ultraviolet (UV) circular dichroism (CD) spectra were recorded with a Jasco J-810 or an Aviv 62DS spectropolarimeter. After dialysis from 6 M GdnHCl solution, the apoA-I protein or peptide solutions of 25–50 μg/ml in 10 mM sodium phosphate buffer (pH 7.4) were subjected to near-UV measurements by scanning from 185 to 260 nm in a 1–2 mm cuvette, and solutions of 0.3 mg/ml apoA-I concentration were used for near-UV measurements (270–320 nm) in a 1 cm cuvette. For the mixture with small unilamellar vesicles (SUVs), the apoA-I protein or peptide was incubated for 1 h prior to the measurement with egg phosphatidylcholine (PC) SUVs prepared by sonication as described before (32Saito H. Dhanasekaran P. Nguyen D. Deridder E. Holvoet P. Lund-Katz S. Phillips M.C. α-Helix formation is required for high affinity binding of human apolipoprotein A-I to lipids.J. Biol. Chem. 2004; 279: 20974-20981Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). The results were corrected by subtracting the baseline for an appropriate blank sample. The α-helix content was calculated from the molar ellipticity at 222 nm ([θ]222) using the equation: % α-helix = [(–[θ]222 + 3000)/(36000 + 3000)] × 100 (33Sparks D.L. Lund-Katz S. Phillips M.C. The charge and structural stability of apolipoprotein A-I in discoidal and spherical recombinant high density lipoprotein particles.J. Biol. Chem. 1992; 267: 25839-25847Abstract Full Text PDF PubMed Google Scholar). Thermal unfolding was monitored from the change in [θ]222 over the temperature range of 20–90°C, as described (34Acharya P. Segall M.L. Zaiou M. Morrow J. Weisgraber K.H. Phillips M.C. Lund-Katz S. Snow J. Comparison of the stabilities and unfolding pathways of human apolipoprotein E isoforms by differential scanning calorimetry and circular dichroism.Biochim. Biophys. Acta. 2002; 1584: 9-19Crossref PubMed Scopus (63) Google Scholar). Although there was some variability in the initial and final α-helix contents, the apoA-I variants exhibited reversible thermal unfolding. The van’t Hoff enthalpy, ΔHv, was calculated from the slope of the line fitted by linear regression to the equation, ln KD = − (ΔHv/R) 1/T + constant, where KD is the equilibrium constant describing the unfolding of apoA-I at each temperature, R is the gas constant, and T is temperature. The kinetics of thermal unfolding of apoA-I in discoidal complexes with 1-palmitoyl-2-oleoyl PC (POPC) prepared using the cholate dispersion method (35Thuahnai S.T. Lund-Katz S. Williams D.L. Phillips M.C. Scavenger receptor class B, type I-mediated uptake of various lipids into cells. Influence of the nature of the donor particle interaction with the receptor.J. Biol. Chem. 2001; 276: 43801-43808Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) was monitored by temperature-jump analysis from 25 to 80–90°C (36Gursky O. Gantz Ranjana D.L. Complex of human apolipoprotein C-1 with phospholipid: thermodynamic or kinetic stability?.Biochemistry. 2002; 41: 7373-7384Crossref PubMed Scopus (56) Google Scholar). For monitoring chemical denaturation, lipid-free proteins at a concentration of 25–50 µg/ml were incubated overnight at 4°C with GdnHCl or urea at various concentrations. When complexed with POPC, apoA-I was completely denatured by incubating for 72 h (37Reijngoud D.J. Phillips M.C. Mechanism of dissociation of human apolipoprotein A-I from complexes with dimyristoylphosphatidylcholine as studied by guanidine hydrochloride denaturation.Biochemistry. 1982; 21: 2969-2976Crossref PubMed Scopus (68) Google Scholar). KD at a given denaturant concentration was calculated from the change in either [θ]222 or wavelength of maximum fluorescence (WMF) of intrinsic Trp residues (15Saito H. Dhanasekaran P. Nguyen D. Holvoet P. Lund-Katz S. Phillips M.C. Domain structure and lipid interaction in human apolipoproteins A-I and E, a general model.J. Biol. Chem. 2003; 278: 23227-23232Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). The Trp emission fluorescence spectrum was recorded from 300 to 420 nm using an excitation wavelength of 295 nm with a Hitachi F-7000 fluorescence spectrophotometer in Tris buffer (10 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.02% NaN3, pH 7.4). The Gibbs free energy of denaturation in the absence of denaturant, ΔGD°, the midpoint of denaturation, D1/2, and m value, which reflects the cooperativity of denaturation in the transition region, were determined by the linear equation, ΔGD = ΔGD° − m[denaturant], where ΔGD = − RT ln KD (19Tanaka M. Dhanasekaran P. Nguyen D. Ohta S. Lund-Katz S. Phillips M.C. Saito H. Contributions of the N- and C-terminal helical segments to the lipid-free structure and lipid interaction of apolipoprotein A-I.Biochemistry. 2006; 45: 10351-10358Crossref PubMed Scopus (68) Google Scholar, 33Sparks D.L. Lund-Katz S. Phillips M.C. The charge and structural stability of apolipoprotein A-I in discoidal and spherical recombinant high density lipoprotein particles.J. Biol. Chem. 1992; 267: 25839-25847Abstract Full Text PDF PubMed Google Scholar). 8-Anilino-1-naphthalenesulfonic acid (ANS) fluorescence measurements were carried out with a Hitachi F-4500 fluorescence spectrophotometer. The extent of ANS binding to hydrophobic sites on the apoA-I variants was determined by measuring ANS fluorescence spectra recorded from 400 to 600 nm at an excitation wavelength of 395 nm, in the absence or presence of 50 μg/ml protein and an excess of ANS (250 μM) (15Saito H. Dhanasekaran P. Nguyen D. Holvoet P. Lund-Katz S. Phillips M.C. Domain structure and lipid interaction in human apolipoproteins A-I and E, a general model.J. Biol. Chem. 2003; 278: 23227-23232Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Heats of apoA-I binding to SUV were measured with a MicroCal MCS isothermal titration calorimeter at 25°C (32Saito H. Dhanasekaran P. Nguyen D. Deridder E. Holvoet P. Lund-Katz S. Phillips M.C. α-Helix formation is required for high affinity binding of human apolipoprotein A-I to lipids.J. Biol. Chem. 2004; 279: 20974-20981Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). To ensure that the injected protein bound completely to the SUV surface, the PC to protein molar ratio was kept over 10,000. Heat of dilution determined by injecting apoA-I solution into buffer was subtracted from the heat for the corresponding apoA-I-SUV binding experiments. The decay rate constants for the heats of binding were obtained from fitting the titration curves to a one-phase exponential decay model (15Saito H. Dhanasekaran P. Nguyen D. Holvoet P. Lund-Katz S. Phillips M.C. Domain structure and lipid interaction in human apolipoproteins A-I and E, a general model.J. Biol. Chem. 2003; 278: 23227-23232Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). The kinetics of solubilization of dimyristoyl PC (DMPC) vesicles by the apoA-I variants or peptides were measured by monitoring the time-dependent decrease in turbidity at 24.6°C. For the apoA-I proteins, DMPC large unilamellar vesicles (LUVs) extruded through a 200-nm filter at a concentration of 0.25 mg/ml were mixed with apoA-I samples (0.02–0.2 mg/ml), and incubated for 15 min to monitor the light scattering intensity at 325 nm with a Shimadzu UV-2450 spectrophotometer (20Koyama M. Tanaka M. Dhanasekaran P. Lund-Katz S. Phillips M.C. Saito H. Interaction between the N- and C-terminal domains modulates the stability and lipid binding of apolipoprotein A-I.Biochemistry. 2009; 48: 2529-2537Crossref PubMed Scopus (38) Google Scholar, 38Sakamoto T. Tanaka M. Vedhachalam C. Nickel M. Nguyen D. Dhanasekaran P. Phillips M.C. Lund-Katz S. Saito H. Contributions of the carboxyl-terminal helical segment to the self-association and lipoprotein preferences of human apolipoprotein E3 and E4 isoforms.Biochemistry. 2008; 47: 2968-2977Crossref PubMed Scopus (39) Google Scholar). For the apoA-I peptides, much lower concentrations of DMPC multilamellar vesicles (MLVs) and peptides (typically 34 μg/ml DMPC and 2–20 μg/ml peptides) were used to avoid the aggregation of DMPC/peptide complexes during experiments. The decrease in turbidity was monitored by the right-angle light scattering intensity using a Hitachi F-4500 spectrophotometer with both excitation and emission wavelengths set at 600 nm (31Tanaka T. Tanaka M. Sugiura M. Kawakami T. Aimoto S. Saito H. Deletion of single amino acid E235 affects the structure and lipid interaction of human apolipoprotein A-I C-terminal peptides.Chem. Pharm. Bull. (Tokyo). 2009; 57: 499-503Crossref PubMed Scopus (2) Google Scholar, 39Mishra V.K. Palgunachari M.N. Datta G. Phillips M.C. Lund-Katz S. Adeyeye S.O. Segrest J.P. Anantharamaiah G.M. Studies of synthetic peptides of human apolipoprotein A-I containing tandem amphipathic α-helixes.Biochemistry. 1998; 37: 10313-10324Crossref PubMed Scopus (78) Google Scholar). The relative affinities of apoA-I peptides for the lipid-water interface were determined with a surface balance technique, as described previously (40Palgunachari M.N. Mishra V.K. Lund-Katz S. Phillips M.C. Adeyeye S.O. Alluri S. Anantharamaiah G.M. Segrest J.P. Only the two end helixes of eight tandem amphipathic helical domains of human apo A-I have significant lipid affinity. Implications for HDL assembly.Arterioscler. Thromb. Vasc. Biol. 1996; 16: 328-338Crossref PubMed Scopus (202) Google Scholar, 41Gillotte 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. et al.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: 2021-2028Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Linear extrapolation of the initial surface pressure of egg PC monolayer in the absence of peptides (πi) versus the change in surface pressure in the presence of peptides (Δπi) curve to the point at which Δπi = 0 gave the monolayer exclusion pressure (πe). The πe value indicates the surface pressure at which the peptides are no longer able to penetrate into the egg PC monolayer. J774 murine macrophages were grown and maintained in RPMI 1640 supplemented with 10% FBS and 0.5% gentamycin. For efflux experiments, these cells were seeded in 12-well plates, grown to 80–90% confluence, and then labeled by incubating the cells for 24 h in RPMI medium supplemented with 1% FBS, 2 µg/ml CP-113,818 ACAT inhibitor, and 3 µCi/ml [3H]cholesterol (25Vedhachalam C. Liu L. Nickel M. Dhanasekaran P. Anantharamaiah G.M. Lund-Katz S. Rothblat G.H. Phillips M.C. Influence of ApoA-I structure on the ABCA1-mediated efflux of cellular lipids.J. Biol. Chem. 2004; 279: 49931-49939Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). After labeling, the cells were washed with MEM-Hepes and incubated with RPMI medium containing 0.2% BSA, 2 µg/ml CP-113,818 ACAT inhibitor, and 0.3 mM 8-(4-chlorophenylthio)-cAMP for 12 h, to upregulate the expression of ABCA1. Cells were then washed with MEM-Hepes and incubated with or without apoA-I proteins or peptides at the indicated concentrations for 4 h. To determine the free unesterified cholesterol efflux, aliquots were removed from the incubation medium at specific time points, filtered, and radioactivity was determined by liquid scintillation counting. The percent cholesterol efflux was calculated after subtracting the background cholesterol efflux (without apoA-I) as follows: (counts/min in medium at 4 h/cpm in cells at t = 0) × 100. For efflux experiments with baby hamster kidney (BHK) cells, BHK cells transfected with human ABCA1 (a generous gift from Dr. John Oram) were grown and maintained in DMEM containing 10% FBS and 0.5% gentamycin (42Vaughan A.M. Oram J.F. ABCA1 redistributes membrane cholesterol independent of apolipoprotein interactions.J. Lipid Res. 2003; 44: 1373-1380Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). These cells were seeded in 12-well plates and then labeled by incubating the cells for 24 h in DMEM medium supplemented with 2.5% FBS, 2 µg/ml CP-113,818 ACAT inhibitor, and 3 µCi/ml [3H]cholesterol. ABCA1 was induced by incubating the labeled cells for 18 h in DMEM containing 0.2% BSA and 10 nM mifepristone. Cells were then washed with MEM-Hepes and incubated with or without apoA-I proteins or peptides under the indicated conditions of concentration for 4 h and the percentage cholesterol efflux was calculated as mentioned above. The secondary structure and thermal unfolding of apoA-I Nichinan were analyzed by far-UV CD spectroscopy, in comparison with the apoA-I L230P/L233P/Y236P mutant in which the C-terminal α-helix is completely disrupted (19Tanaka M. Dhanasekaran P. Nguyen D. Ohta S. Lund-Katz S. Phillips M.C. Saito H. Contributions of the N- and C-terminal helical segments to the lipid-free structure and lipid interaction of apolipoprotein A-I.Biochemistry. 2006; 45: 10351-10358Crossref PubMed Scopus (68) Google Scholar, 25Vedhachalam C. Liu L. Nickel M. Dhanasekaran P. Anantharamaiah G.M. Lund-Katz S. Rothblat G.H. Phillips M.C. Influence of ApoA-I structure on the ABCA1-mediated efflux of cellular lipids.J. Biol. Chem. 2004; 279: 49931-49939Abstract F" @default.
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