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• W1887901770 abstract "Five mutants of apolipoprotein A-I (apoA-I), apoA-I(Δ63–73), apoA-I(Δ140–150), apoA-I(63–[email protected]–150), apoA-I(R149V), and apoA-I(P143A) were compared with human plasma apoA-I for their ability to promote cholesterol and phospholipid efflux from HepG2 cells. A significantly lower capacity to promote cholesterol and phospholipid efflux was observed with lipid-free apoA-I(Δ63–73), while mutations apoA-I(Δ140–150) and apoA-I(P143A) affected phospholipid efflux only. When added as apoA-I/palmitoyloleoyl phosphatidylcholine (POPC) complex, mutations apoA-I(63–[email protected]–150) and apoA-I(Δ140–150) affected cholesterol efflux. None of the mutations affected α-helicity of the lipid-free mutants or their self-association. Five natural mutations of apoA-I, apoA-I(A95D), apoA-I (Y100H), apoA-I(E110K), apoA-I(V156E), and apoA-I (H162Q) were studied for their ability to bind lipids and promote cholesterol efflux. None of the mutations affected lipid-binding properties, cholesterol efflux, or α-helicity of lipid-free mutants. Two mutations affected self-association of apoA-I: apoA-I(A95D) was more prone to self-association, while apoA-I(E100H) did not self-associate.The following conclusions could be made from the combined data: i) regions 210–243 and 63–100 are the lipid-binding sites of apoA-I and are also required for the efflux of lipids to lipid-free apoA-I, suggesting that initial lipidation of apoA-I is rate limiting in efflux; ii) in addition to the lipid-binding regions, the central region is important for cholesterol efflux to lipidated apoA-I, suggesting its possible involvement in interaction with cells. Five mutants of apolipoprotein A-I (apoA-I), apoA-I(Δ63–73), apoA-I(Δ140–150), apoA-I(63–[email protected]–150), apoA-I(R149V), and apoA-I(P143A) were compared with human plasma apoA-I for their ability to promote cholesterol and phospholipid efflux from HepG2 cells. A significantly lower capacity to promote cholesterol and phospholipid efflux was observed with lipid-free apoA-I(Δ63–73), while mutations apoA-I(Δ140–150) and apoA-I(P143A) affected phospholipid efflux only. When added as apoA-I/palmitoyloleoyl phosphatidylcholine (POPC) complex, mutations apoA-I(63–[email protected]–150) and apoA-I(Δ140–150) affected cholesterol efflux. None of the mutations affected α-helicity of the lipid-free mutants or their self-association. Five natural mutations of apoA-I, apoA-I(A95D), apoA-I (Y100H), apoA-I(E110K), apoA-I(V156E), and apoA-I (H162Q) were studied for their ability to bind lipids and promote cholesterol efflux. None of the mutations affected lipid-binding properties, cholesterol efflux, or α-helicity of lipid-free mutants. Two mutations affected self-association of apoA-I: apoA-I(A95D) was more prone to self-association, while apoA-I(E100H) did not self-associate. The following conclusions could be made from the combined data: i) regions 210–243 and 63–100 are the lipid-binding sites of apoA-I and are also required for the efflux of lipids to lipid-free apoA-I, suggesting that initial lipidation of apoA-I is rate limiting in efflux; ii) in addition to the lipid-binding regions, the central region is important for cholesterol efflux to lipidated apoA-I, suggesting its possible involvement in interaction with cells. Apolipoprotein A-I (apoA-I) is the principal apolipoprotein of HDL and a key element in the reverse cholesterol transport pathway. ApoA-I is a single polypeptide of 243 amino acids and its characteristic feature is that when bound to lipids it is organized into a series of 22-mer or 11-mer amphipathic α-helices (1Segrest 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). These helices are positioned either perpendicularly to the surface of the lipid disk according to the picket fence model (2Phillips J.C. Wriggers W. Li Z. Jonas A. Schulten K. Predicting the structure of apolipoprotein A-I in reconstituted high-density lipoprotein disks.Biophys. J. 1997; 73: 2337-2346Abstract Full Text PDF PubMed Scopus (117) Google Scholar) or parallel to the surface according to the belt (3Segrest J.P. Jones M.K. Klon A.E. Sheldahl C.J. Hellinger M. De Loof H. Harvey S.C. A detailed molecular belt model for apolipoprotein A-I in discoidal high density lipoprotein.J. Biol. Chem. 1999; 274: 31755-31758Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar) and hairpin (4Tricerri M.A. Behling Agree A.K. Sanchez S.A. Bronski J. Jonas A. Arrangement of apolipoprotein A-I in reconstituted high-density lipoprotein disks: an alternative model based on fluorescence resonance energy transfer experiments.Biochemistry. 2001; 40: 5065-5074Crossref PubMed Scopus (84) Google Scholar) models. It follows from all three models that the correct secondary structure, i.e., correct length, amphipathicity, and orientation of the helices is essential for enabling apoA-I to maintain correct structure of HDL and to carry out its functions. That brings about the question of whether a specific amino acid sequence for apoA-I is required for carrying out its functions and how important this requirement might be in relation to that of secondary structure. Mutagenized apoA-I, synthetic peptides, and monoclonal antibodies have been used to probe the structure-function relationship of apoA-I [for review see (5Frank 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)]. These studies suggest that the central α-helical region (residues 137–186) is involved in LCAT activation, cellular cholesterol efflux, and interaction with a cell surface binding site (6Minnich A. Collet X. Roghani A. Cladaras C. Hamilton R.L. Fielding C.J. Zannis V.I. Site-directed mutagenesis and structure-function analysis of the human apolipoprotein A-I.J. Biol. Chem. 1992; 267: 16553-16560Abstract Full Text PDF PubMed Google Scholar, 7Sorci-Thomas M.G. Curtiss L. Parks J.S. Thomas M.J. Kearns M.W. Landrum M. The hydrophobic face orientation of apolipoprotein A-I amphipathic helix domain 143–164 regulates lecithin:cholesterol acyltransferase activation.J. Biol. Chem. 1998; 273: 11776-11782Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 8Sorci-Thomas M.G. Curtiss L. Parks J.S. Thomas M.J. Kearns M.W. Alteration in apolipoprotein A-I 22-mer repeat order results in a decrease in lecithin:cholesterol acyltransferase reactivity.J. Biol. Chem. 1997; 272: 7278-7284Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 9Sviridov D. Hoang A. Sawyer W. Fidge N. Identification of a sequence of apolipoprotein A-I associated with activation of lecithin:cholesterol acyltransferase.J. Biol. Chem. 2000; 275: 19707-19712Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 10Sviridov D. Pyle L. Fidge N. Identification of a sequence of apolipoprotein A-I associated with the efflux of intracellular cholesterol to human serum and apolipoprotein A-I containing particles.Biochemistry. 1996; 35: 189-196Crossref PubMed Scopus (57) Google Scholar, 11Sviridov D. Pyle L. Fidge N. Efflux of cellular cholesterol and phospholipid to apolipoprotein A-I mutants.J. Biol. Chem. 1996; 271: 33277-33283Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The C-terminus (residues 193–243) of apoA-I appears to play a role in protein–lipid interaction, cholesterol efflux from the plasma membrane, and in vivo HDL catabolism (11Sviridov D. Pyle L. Fidge N. Efflux of cellular cholesterol and phospholipid to apolipoprotein A-I mutants.J. Biol. Chem. 1996; 271: 33277-33283Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 12Palgunachari 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 (203) Google Scholar, 13Holvoet P. Zhao Z. Vanloo B. Vos R. Deridder E. Dhoest A. Taveirne J. Brouwers P. Demarsin E. Engelborghs Y. Rosseneu M. Collen D. Brasseur R. Phospholipid binding and lecithin-cholesterol acyltransferase activation properties of apolipoprotein A-I mutants.Biochemistry. 1995; 34: 13334-13342Crossref PubMed Scopus (87) Google Scholar, 14Laccotripe 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, 15Huang 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). The importance of individual amino acid residues for apoA-I function has also been examined by investigating naturally occurring apoA-I mutants. ApoA-I(L141R)Pisa, apoA-I(P143R)Giessen, apoA-I (V156E)Oita, apoA-I(L159R)Fin, apoA-I(R160L)Oslo, apoA-I (P165R), and apoA-I(ΔE235)Nichinan have been shown to affect either LCAT activation or cholesterol efflux (16Miccoli R. Zhu Y. Daum U. Wessling J. Huang Y. Navalesi R. Assmann G. von Eckardstein A. A natural apolipoprotein A-I variant, apoA-I (L141R)Pisa, interferes with the formation of alpha-high density lipoproteins (HDL) but not with the formation of pre beta 1-HDL and influences efflux of cholesterol into plasma.J. Lipid Res. 1997; 38: 1242-1253Abstract Full Text PDF PubMed Google Scholar, 17Utermann G. Haas J. Steinmetz A. Paetzold R. Rall Jr., S.C. Weisgraber K.H. Mahley R.W. Apolipoprotein A-IGiessen (Pro143—-Arg). A mutant that is defective in activating lecithin:cholesterol acyltransferase.Eur. J. Biochem. 1984; 144: 325-331Crossref PubMed Scopus (42) Google Scholar, 18Huang W. Sasaki J. Matsunaga A. Nanimatsu H. Moriyama K. Han H. Kugi M. Koga T. Yamaguchi K. Arakawa K. A novel homozygous missense mutation in the apo A-I gene with apo A-I deficiency.Arterioscler. Thromb. Vasc. Biol. 1998; 18: 389-396Crossref PubMed Scopus (45) Google Scholar, 19von Eckardstein A. Castro G. Wybranska I. Theret N. Duchateau P. Duverger N. Fruchart J.C. Ailhaud G. Assmann G. Interaction of reconstituted high density lipoprotein discs containing human apolipoprotein A-I (ApoA-I) variants with murine adipocytes and macrophages. Evidence for reduced cholesterol efflux promotion by apoA-I(Pro165→Arg).J. Biol. Chem. 1993; 268: 2616-2622Abstract Full Text PDF PubMed Google Scholar, 20Miettinen H.E. Jauhiainen M. Gylling H. Ehnholm S. Palomaki A. Miettinen T.A. Kontula K. Apolipoprotein A-IFIN (Leu159→Arg) mutation affects lecithin cholesterol acyltransferase activation and subclass distribution of HDL but not cholesterol efflux from fibroblasts.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 3021-3032Crossref PubMed Scopus (38) Google Scholar, 21Daum U. Leren T.P. Langer C. Chirazi A. Cullen P. Pritchard P.H. Assmann G. von Eckardstein A. Multiple dysfunctions of two apolipoprotein A-I variants, apoA-I(R160L)Oslo and apoA-I(P165R), that are associated with hypoalphalipoproteinemia in heterozygous carriers.J. Lipid Res. 1999; 40: 486-494Abstract Full Text Full Text PDF PubMed Google Scholar, 22Han H. Sasaki J. Matsunaga A. Hakamata H. Huang W. Ageta M. Taguchi T. Koga T. Kugi M. Horiuchi S. Arakawa K. 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). Most of these studies, however, did not address the issue of whether the effect of the mutation was due to its effect on the secondary structure of apoA-I or whether there is a requirement for a specific amino acid sequence for a particular function. In this paper, we analyze the effect of a number of apoA-I mutations, either naturally occurring or made by site-directed mutagenesis, on the ability of apoA-I to promote cholesterol and phospholipid efflux. We have previously demonstrated that monoclonal antibody directed against the central part of apoA-I (residues 140–150) inhibits the efflux of intracellular cholesterol to human plasma (10Sviridov D. Pyle L. Fidge N. Identification of a sequence of apolipoprotein A-I associated with the efflux of intracellular cholesterol to human serum and apolipoprotein A-I containing particles.Biochemistry. 1996; 35: 189-196Crossref PubMed Scopus (57) Google Scholar). This finding was not confirmed, however, when we used truncated forms of apoA-I (11Sviridov D. Pyle L. Fidge N. Efflux of cellular cholesterol and phospholipid to apolipoprotein A-I mutants.J. Biol. Chem. 1996; 271: 33277-33283Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), presumably because the effects of truncation on the carboxyl-terminal half of apoA-I overshadowed the effect of removal of the targeted sequence. Therefore, we created apoA-I mutants more precisely targeting the region 140–150 of apoA-I; the mutations included those predictably affecting or not affecting the secondary structure of the region. Two mutations, Δ140–150 and P143A, were predicted to affect the secondary structure of the target region of apoA-I, while two others, R149V and 63–[email protected]–150, were not. The fifth mutation, Δ63–73, was designed as incurring changes to the secondary structure similar to the deletion of the target region, but located in a segment of apoA-I not thought to be involved in cholesterol efflux. It was recently demonstrated, however, that the region 63–73 might represent a second lipid-binding region of apoA-I (9Sviridov D. Hoang A. Sawyer W. Fidge N. Identification of a sequence of apolipoprotein A-I associated with activation of lecithin:cholesterol acyltransferase.J. Biol. Chem. 2000; 275: 19707-19712Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). In addition to mutations created by site-directed mutagenesis, we also investigated five natural mutations of apoA-I identified during screening of blood samples from a population survey. These natural mutations were also located in the central part of apoA-I between residues 95 and 162; the effect of these mutations on apoA-I structure and functions has not been previously described. Construction, expression, purification, and verification of the recombinant apoA-I mutants apoA-I(P143A), apoA-I(R149V), apopA-I (Δ63-73), apoA-I(Δ140–150), and apoA-I(63–[email protected]–150) are described in detail elsewhere (9Sviridov D. Hoang A. Sawyer W. Fidge N. Identification of a sequence of apolipoprotein A-I associated with activation of lecithin:cholesterol acyltransferase.J. Biol. Chem. 2000; 275: 19707-19712Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 23Sviridov D. Luong A. Pyle L. Fidge N. Effectivity of expression of mature forms of mutant human apolipoprotein A-I.Protein Expr. Purif. 1999; 17: 231-238Crossref PubMed Scopus (8) Google Scholar, 24Pyle L.E. Fidge N.H. Barton P.A. Luong A. Sviridov D. Production of mature human apolipoprotein A-I in a baculovirus-insect cell system: propeptide is not essential for intracellular processing but may assist rapid secretion.Anal. Biochem. 1997; 253: 253-258Crossref PubMed Scopus (19) Google Scholar). All apoA-I mutants were expressed in a baculovirus/insect cell expression system as described previously (24Pyle L.E. Fidge N.H. Barton P.A. Luong A. Sviridov D. Production of mature human apolipoprotein A-I in a baculovirus-insect cell system: propeptide is not essential for intracellular processing but may assist rapid secretion.Anal. Biochem. 1997; 253: 253-258Crossref PubMed Scopus (19) Google Scholar). Recombinant human apoA-Is containing natural mutations were expressed as a glutathione-S-transferase fusion protein in an Escherichia coli expression system as described previously (22Han H. Sasaki J. Matsunaga A. Hakamata H. Huang W. Ageta M. Taguchi T. Koga T. Kugi M. Horiuchi S. Arakawa K. 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, 25Huang W. Matsunaga A. Li W. Han H. Hoang A. Kugi M. Koga T. Sviridov D. Fidge N. Sasaki J. Recombinant proapoA-I(Lys107del) shows impaired lipid binding associated with reduced binding to plasma high density lipoprotein.Atherosclerosis. 2001; 159: 85-91Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Concentration of the proteins was measured according to Bradford (26Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 1976; 72: 248-256Crossref PubMed Scopus (215632) Google Scholar). Human plasma apoA-I was isolated and purified as described previously (27Morrison J.R. Fidge N.H. Grego B. Studies on the formation, separation, and characterization of cyanogen bromide fragments of human AI apolipoprotein.Anal. Biochem. 1990; 186: 145-152Crossref PubMed Scopus (44) Google Scholar). The reconstituted HDL (rHDL) was prepared by the sodium cholate dialysis method according to Jonas et al. (28Matz C.E. Jonas A. Micellar complexes of human apolipoprotein A-I with phosphatidylcholines and cholesterol prepared from cholate-lipid dispersions.J. Biol. Chem. 1982; 257: 4535-4540Abstract Full Text PDF PubMed Google Scholar, 29Jonas A. Kezdy K.E. Wald J.H. Defined apolipoprotein A-I conformations in reconstituted high density lipoprotein discs.J. Biol. Chem. 1989; 264: 4818-4824Abstract Full Text PDF PubMed Google Scholar) using palmitoyloleoyl phosphatidylcholine (POPC) (Sigma, Castle Hill, NSW, Australia), apoA-I, and sodium cholate (Sigma) in a molar ratio of 80:1:80. The characteristics of the particles were reported previously (9Sviridov D. Hoang A. Sawyer W. Fidge N. Identification of a sequence of apolipoprotein A-I associated with activation of lecithin:cholesterol acyltransferase.J. Biol. Chem. 2000; 275: 19707-19712Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 25Huang W. Matsunaga A. Li W. Han H. Hoang A. Kugi M. Koga T. Sviridov D. Fidge N. Sasaki J. Recombinant proapoA-I(Lys107del) shows impaired lipid binding associated with reduced binding to plasma high density lipoprotein.Atherosclerosis. 2001; 159: 85-91Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Efflux of plasma membrane and intracellular cholesterol as well as cellular phospholipid to apoA-I modified by site directed mutagenesis was tested in a previously described model utilizing HepG2 cells (10Sviridov D. Pyle L. Fidge N. Identification of a sequence of apolipoprotein A-I associated with the efflux of intracellular cholesterol to human serum and apolipoprotein A-I containing particles.Biochemistry. 1996; 35: 189-196Crossref PubMed Scopus (57) Google Scholar, 11Sviridov D. Pyle L. Fidge N. Efflux of cellular cholesterol and phospholipid to apolipoprotein A-I mutants.J. Biol. Chem. 1996; 271: 33277-33283Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Briefly, to label plasma membrane cholesterol and phospholipid, cells were incubated in serum-containing medium with [1α,2α(n)-3H]cholesterol (Amersham Pharmacia Biotech; specific radioactivity 1.81 TBq/mmol, final radioactivity 75 KBq/ml) and [methyl-14C]choline (Amersham Pharmacia Biotech; specific activity 2.1 GBq/mmol, final activity 0.2 MBq/ml) for 48 h in a CO2 incubator. The cells were then washed and cooled on ice. Leibovitz L-15 serum-free medium containing [1-14C]acetic acid sodium salt [(ICN; specific radioactivity 2.2 GBq/mmol, final radioactivity 18 MBq/ml) was added and cells were incubated for 3 h at 15°C to label newly synthesized (intracellular) cholesterol. Under these conditions, intracellular cholesterol trafficking is blocked while cholesterol biosynthesis proceeds (30Kaplan M.R. Simoni R.D. Transport of cholesterol from the endoplasmic reticulum to the plasma membrane.J. Cell Biol. 1985; 101: 446-453Crossref PubMed Scopus (170) Google Scholar, 31Urbani L. Simoni R.D. Cholesterol and vesicular stomatitis virus G protein take separate routes from the endoplasmic reticulum to the plasma membrane.J. Biol. Chem. 1990; 265: 1919-1923Abstract Full Text PDF PubMed Google Scholar). After labeling, cells were incubated for 3 h or indicated periods of time at 37°C with serum-free medium containing 1,000-fold excess of unlabeled sodium acetate, and lipid-free apoA-I at a final concentration of 1 μM, or rHDL at a final POPC concentration of 80 μM. Lipids were extracted from aliquots of media and cells, and cholesterol and phospholipid were isolated by TLC as described previously (32Sviridov D. Fidge N. Efflux of intracellular vs plasma membrane cholesterol in HepG2 cells: different availability and regulation by apolipoprotein A-I.J. Lipid Res. 1995; 36: 1887-1896Abstract Full Text PDF PubMed Google Scholar). Efflux of cellular cholesterol to apoA-I containing natural mutations was tested in a previously described model utilizing human skin fibroblasts (33Sviridov D. Pyle L.E. Jauhiainen M. Ehnholm C. Fidge N.H. Deletion of the pro-peptide of apolipoprotein A-I reduces protein expression, but stimulates effective conversion of preβ-HDL to α-HDL.J. Lipid Res. 2000; 41: 1872-1882Abstract Full Text Full Text PDF PubMed Google Scholar). It has been previously shown that properties of cholesterol efflux from fibroblasts and HepG2 cells are similar (34Sviridov D. Fidge N. Pathway of cholesterol efflux from human hepatoma cells.Biochim. Biophys. Acta. 1995; 1256: 210-230Crossref PubMed Scopus (30) Google Scholar). Fibroblasts, like HepG2 cells, contain ATP binding cassette transporter A1 (ABCA1) required for the efflux to lipid-free apoA-I (35Bortnick 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: 28634-28640Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Loading with cholesterol can increase cholesterol efflux to fibroblasts, but both loaded and non-loaded fibroblasts exert features of ABCA1-dependent specific efflux to lipid-free apoA-I (36Gillotte-Taylor K. Nickel M. Johnson W.J. Francone O.L. Holvoet P. Lund-Katz S. Rothblat G.H. Phillips M.C. Effects of enrichment of fibroblasts with unesterified cholesterol on the efflux of cellular lipids to apolipoprotein A-I.J. Biol. Chem. 2002; 277: 11811-11820Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Non-cholesterol-loaded fibroblasts have been used in this study; the efflux in the presence of lipid-free apoA-I was twice of that in its absence. Fibroblasts were labeled by incubating with [3H]cholesterol (Amersham Pharmacia Biotech; specific radioactivity 1.81 TBq/mmol, final radioactivity 75 KBq/ml) for 48 h at 37°C in the presence of 10% fetal calf serum and then incubated with DMEM containing different cholesterol acceptors for 3 h at 37°C. The final concentration of apoA-I was 1 μM. Lipids from cells and medium were extracted by incubation with hexane-isopropanol (3:2 v/v) and counted. Solubilization of dimyristoyl phosphatidylcholine (DMPC) by apoA-I was studied as described previously (9Sviridov D. Hoang A. Sawyer W. Fidge N. Identification of a sequence of apolipoprotein A-I associated with activation of lecithin:cholesterol acyltransferase.J. Biol. Chem. 2000; 275: 19707-19712Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 33Sviridov D. Pyle L.E. Jauhiainen M. Ehnholm C. Fidge N.H. Deletion of the pro-peptide of apolipoprotein A-I reduces protein expression, but stimulates effective conversion of preβ-HDL to α-HDL.J. Lipid Res. 2000; 41: 1872-1882Abstract Full Text Full Text PDF PubMed Google Scholar). Briefly, dry DMPC (Sigma) was sonicated in Tris buffer (pH 8.0) to form multilamellar liposomes. Apolipoproteins (final concentration 0.1 mg/ml) were pre-incubated for 10 min at 24°C and the reaction initiated by adding DMPC liposomes (final DMPC concentration 0.5 mg/ml). The reduction of absorption at 325 nm (which reflects the reduction in light scattering) was monitored for 1 h at 2 min intervals at 24°C to assess formation of apoA-I/DMPC complexes. The α-helical content of apoA-I was determined by measuring circular dichroism spectra at protein concentration 0.1 mg/ml in 20 mM phosphate buffer (pH 7.4) at 25°C. Circular dichroism spectra were measured on a JASCO 810 spectropolarimeter. Data were collected from 185 nm to 250 nm at 0.5 nm intervals. The percentage of α-helical content of apoA-I was calculated by the equation of Chen et al. (37Chen Y. Yang J.T. Martinez H.M. Determination of the secondary structures of proteins by circular dichroism and optical rotatory dispertion.Biochemistry. 1972; 11: 4120-4141Crossref PubMed Scopus (1904) Google Scholar). Self-association of apoA-I was studied as described previously (38Pyle L.E. Sawyer W.H. Fujiwara Y. Mitchell A. Fidge N.H. Structural and functional properties of full-length and truncated human proapolipoprotein AI expressed in Escherichia coli.Biochemistry. 1996; 35: 12046-12052Crossref PubMed Scopus (27) Google Scholar). In brief, proteins in PBS were mixed in solution with a 5.6 molar excess of dithiobis(succinimidyl propionate) (DSP) (Pierce, Rockford, IL) dissolved in DMSO. Samples were incubated for 30 min at room temperature and then quenched with Tris/HCl, (pH 7.3) (final concentration 50 mM), for 15 min and analyzed on 10% SDS-PAGE. All experiments were performed in quadruplicate (i.e., determination from four dishes) and reproduced two or three times. Background values for the cholesterol and phospholipid efflux (i.e., the amount of radioactivity released to the medium in the absence of an acceptor) were subtracted. Means ± SE are presented. Statistical significance of differences was determined by Student's two-tail t-test. Predicted hydrophobicity (Kyte-Doolittle), average charge, and amphipathicity (Eisenberg) of the regions of apoA-I were calculated using Protean software (DNASTAR Inc). Wheel diagrams and predicted orientations of α-helices were generated using Antheprot v. 4.0 (Microsoft). Labeled cells were incubated for 3 h with equimolar (1 μM) concentrations of human plasma apoA-I or each of the five apoA-I mutants, and transfers of plasma membrane and newly synthesized intracellular cholesterol, and of cellular phospholipid, from cells to acceptor were assessed. When apoA-I was added in lipid-free form, efflux of both plasma membrane and intracellular cholesterol to four out of five mutants [apoA-I(Δ140-150), apoA-I(P143A), apoA-I(R149V), and apoA-I(63–[email protected]–150)] was similar to the efflux observed with wild-type apoA-I (Figs. 1A, B). Efflux of plasma membrane cholesterol to the mutant apoA-I(Δ63-73) was 15 times less than to the wild-type apoA-I, and efflux of intracellular cholesterol to this mutant was no more than efflux to the medium alone (Figs. 1A, 1B). When phospholipid efflux to lipid-free apoA-I was assessed, three mutations, apoA-I(Δ140–150), apoA-I(P143A), and apoA-I(Δ63–73), showed ∼50% inhibition of phospholipid efflux, whereas the two other mutations, apoA-I(R149V) and apoA-I(63–[email protected]–150), did not affect efflux of phospholipid (Fig. 1C). The ability of lipid-free apoA-I to promote cholesterol efflux could be affected by mutations through a number of mechanisms. First, a mutation may affect α-helicity of the lipid-free form, which in turn may affect the initial microsolubilization of the microdomains of plasma membrane (39Gillotte 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: 2021-2028Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). No effect of the mutations on the secondary structure of lipid-free mutants was, however, found (Table 1). It should be mentioned that the effect of mutations on the secondary structure of lipid-free apoA-I might be different from their effect on that of lipid-bound apoA-I, where α-helicity is much higher. Second, a mutation may affect the ability of apoA-I to self-associate. Enhanced self-association may decrease the apparent concentration of a monomer apoA-I. No effect of mutations on self-association of lipid-free apoA-I was, however, found (Fig. 2A). The amount of monomer protein was 55–65% for the wild-type and all mutated apoA-I. This is consistent with our previous finding indicating that the carboxyl-terminal end of apoA-I is essential for its self-association (38Pyle L.E. Sawyer W.H. Fujiwara Y. Mitchell A. Fidge N.H. Structural and functional properties of full-length and truncated human proapolipoprotein AI expressed in Escherichia coli.Biochemistry. 1996; 35: 12046-12052Crossref PubMed Scopus (27) Google Scholar). Third, the ability of apoA-I to promote cholesterol efflux could be affected by its lipid-binding properties (11Sviridov D. Pyle L. Fidge N. Efflux of cellular cholesterol and phospholipid to apolipoprotein A-I mutants.J. Biol. Chem. 1996; 271: 33277-33283Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 39Gillotte K.L. Zaiou M. Lund" @default.
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• W1887901770 title "Structure-function studies of apoA-I variants:site-directed mutagenesis and natural mutations" @default.
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