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• W2020661012 abstract "The Arg123–Tyr166central and Ala190–Gln243 carboxyl-terminal pairs of helices of apoA-I were substituted with the pair of helices of apoA-II, resulting in the apoA-I(Δ(Arg123–Tyr166),∇A-II(Ser12–Ala75)) and apoA-I(Δ(Ala190–Gln243),∇A-II(Ser12–Gln77)) chimeras, respectively. The structures of these chimeras in aqueous solution and in reconstituted high density lipoproteins (rHDL) and the lecithin:cholesterol acyltransferase (LCAT) activation properties of the rHDL were studied. Recombinant human apoA-I and the chimeras were expressed in Escherichia coli and purified from the periplasmic space. Binding of the apolipoproteins with palmitoyloleoylphosphatidylcholine was associated with a similar shift of Trp fluorescence maxima from 337 to 332 nm, from 339 to 334 nm, and from 337 to 333 nm, respectively. All rHDL had a Stokes radius of 4.8 nm and contained 2 apolipoprotein molecules/particle. Circular dichroism measurements revealed eight α-helices per apoA-I and per chimera molecule. The catalytic efficiencies of LCAT activation were 1.5 ± 0.33 (mean ± S.D.; n = 3), 0.054 ± 0.009 (p < 0.001 versusapoA-I), and 1.3 ± 0.32 (p = not significantversus apoA-I) nmol of cholesteryl ester/h/μm, respectively. The lower LCAT activity of the central domain chimera was due to a 27-fold reducedV max with unaltered K m. Binding of radiolabeled LCAT to rHDL of apoA-I and apoA-I(Δ(Arg123–Tyr166),∇A-II(Ser12–Ala75)) was very similar. In conclusion, although substitution of the Arg123–Tyr166 central or Ala190–Gln243 carboxyl-terminal pair of helices of apoA-I with the pair of helices of apoA-II yields chimeras with structure similar to that of native apoA-I, exchange of the central domain (but not the carboxyl-terminal domain) of apoA-I reduces the rate of LCAT activity that is independent of binding to rHDL. The Arg123–Tyr166central and Ala190–Gln243 carboxyl-terminal pairs of helices of apoA-I were substituted with the pair of helices of apoA-II, resulting in the apoA-I(Δ(Arg123–Tyr166),∇A-II(Ser12–Ala75)) and apoA-I(Δ(Ala190–Gln243),∇A-II(Ser12–Gln77)) chimeras, respectively. The structures of these chimeras in aqueous solution and in reconstituted high density lipoproteins (rHDL) and the lecithin:cholesterol acyltransferase (LCAT) activation properties of the rHDL were studied. Recombinant human apoA-I and the chimeras were expressed in Escherichia coli and purified from the periplasmic space. Binding of the apolipoproteins with palmitoyloleoylphosphatidylcholine was associated with a similar shift of Trp fluorescence maxima from 337 to 332 nm, from 339 to 334 nm, and from 337 to 333 nm, respectively. All rHDL had a Stokes radius of 4.8 nm and contained 2 apolipoprotein molecules/particle. Circular dichroism measurements revealed eight α-helices per apoA-I and per chimera molecule. The catalytic efficiencies of LCAT activation were 1.5 ± 0.33 (mean ± S.D.; n = 3), 0.054 ± 0.009 (p < 0.001 versusapoA-I), and 1.3 ± 0.32 (p = not significantversus apoA-I) nmol of cholesteryl ester/h/μm, respectively. The lower LCAT activity of the central domain chimera was due to a 27-fold reducedV max with unaltered K m. Binding of radiolabeled LCAT to rHDL of apoA-I and apoA-I(Δ(Arg123–Tyr166),∇A-II(Ser12–Ala75)) was very similar. In conclusion, although substitution of the Arg123–Tyr166 central or Ala190–Gln243 carboxyl-terminal pair of helices of apoA-I with the pair of helices of apoA-II yields chimeras with structure similar to that of native apoA-I, exchange of the central domain (but not the carboxyl-terminal domain) of apoA-I reduces the rate of LCAT activity that is independent of binding to rHDL. ApoA-I is synthesized as a prepropeptide, cotranslationally cleaved to pro-apoA-I, and, upon secretion, processed to mature 243-amino acid apoA-I (1Brewer Jr., H.B. Fairwell T. LaRue A. Ronan R. Houser A. Bronzert T.J. Biochem. Biophys. Res. Commun. 1978; 80: 623-630Crossref PubMed Scopus (222) Google Scholar). It is folded into amphipathic α-helices with hydrophilic and hydrophobic surfaces (2Segrest J.P. Jackson R.L. Morrisett J.D. Gotto Jr., A.M. FEBS Lett. 1974; 38: 247-258Crossref PubMed Scopus (485) Google Scholar, 3Segrest J.P. De Loof H. Dohlman J.G. Brouillette C.G. Anantharamaiah G.M. Proteins. 1990; 8 (; Correction (1991) Proteins9, 79): 103-117Crossref PubMed Scopus (594) Google Scholar, 4Segrest J.P. Jones M.K. De Loof H. Brouillette C.G. Venkatachalapathi Y.V. Anantharamaiah G.M. J. Lipid Res. 1992; 33: 141-166Abstract Full Text PDF PubMed Google Scholar), as demonstrated with complexes of phospholipids with apoA-I or model peptides (5Kroon D.J. Kupferberg J.P. Kaiser E. Kezdy F.J. J. Am. Chem. Soc. 1978; 100: 5975-5977Crossref Scopus (21) Google Scholar, 6Fukushima D. Yokoyama S. Kroon D.J. Kezdy F.J. Kaiser E.T. J. Biol. Chem. 1980; 255: 10651-10657Abstract Full Text PDF PubMed Google Scholar, 7Sparrow J.T. Gotto Jr., A.M. Ann. N. Y. Acad. Sci. 1980; 348: 187-211Crossref PubMed Scopus (66) Google Scholar, 8Anantharamaiah G.M. Jones J.L. Brouillette C.G. Schmidt C.F. Chung B.H. Hughes T.A. Bhown A.S. Segrest J.P. J. Biol. Chem. 1985; 260: 10248-10255Abstract Full Text PDF PubMed Google Scholar, 9Nakagawa S.H. Lau H.S.H. Kezdy F.J. Kaiser E.T. J. Am. Chem. Soc. 1985; 107: 7087-7092Crossref Scopus (62) Google Scholar, 10Srinivas R.V. Venkatachalapathi Y.U. Rui Z. Owens R.J. Gupta K.B. Srinivas S.K. Anantharamaiah G.M. Segrest J.P. Compans R.W. J. Cell. Biochem. 1991; 45: 224-237Crossref PubMed Scopus (55) Google Scholar, 11Vanloo B. Morrison J. Fidge N. Lorent G. Lins L. Brasseur R. Ruysschaert J.M. Baert J. Rosseneu M. J. Lipid Res. 1991; 32: 1253-1264Abstract Full Text PDF PubMed Google Scholar). ApoA-I, when associated with phospholipids in discoidal complexes (12Brasseur R. De Meutter J. Vanloo B. Goormaghtigh E. Ruysschaert J.M. Rosseneu M. Biochim. Biophys. Acta. 1990; 1043: 245-252Crossref PubMed Scopus (88) Google Scholar, 13Wald J.H. Goormaghtigh E. De Meutter J. Ruysschaert J.M. Jonas A. J. Biol. Chem. 1990; 265: 20044-20050Abstract Full Text PDF PubMed Google Scholar, 14Brasseur R. J. Biol. Chem. 1991; 266: 16120-16127Abstract Full Text PDF PubMed Google Scholar), contains eight putative amphipathic α-helices oriented around the edge of the discs, parallel to the acyl chains of the phospholipids, with their hydrophobic surface toward the lipid core and their hydrophilic surface toward the aqueous phase. The first amino-terminal domain (residues 44–63) has the lowest helical structure probability, whereas the second α-helix (residues 69–85) is not involved in a pair (12Brasseur R. De Meutter J. Vanloo B. Goormaghtigh E. Ruysschaert J.M. Rosseneu M. Biochim. Biophys. Acta. 1990; 1043: 245-252Crossref PubMed Scopus (88) Google Scholar, 13Wald J.H. Goormaghtigh E. De Meutter J. Ruysschaert J.M. Jonas A. J. Biol. Chem. 1990; 265: 20044-20050Abstract Full Text PDF PubMed Google Scholar, 14Brasseur R. J. Biol. Chem. 1991; 266: 16120-16127Abstract Full Text PDF PubMed Google Scholar). The six carboxyl-terminal α-helical structures most likely form pairs of antiparallel α-helices stabilized by protein-protein interactions. A minimum length of 17–20 amino acids (five to six helical turns) appears to be required for effective phospholipid binding and LCAT 1The abbreviations used are: LCAT, lecithin:cholesterol acyltransferase; rHDL, reconstituted high density lipoprotein(s); apoA-I(Δ(Arg123–Tyr166),∇A-II(Ser12–Ala75)), chimera with the Arg123–Tyr166 segment of apoA-I substituted with the Ser12–Ala75segment of apoA-II; apoA-I(Δ(Ala190–Gln243),∇A-II(Ser12–Gln77)), chimera with the Ala190–Gln243 segment of apoA-I substituted with the Ser12–Gln77segment of apoA-II; POPC, palmitoyloleoylphosphatidylcholine; PAGE, polyacrylamide gel electrophoresis. 1The abbreviations used are: LCAT, lecithin:cholesterol acyltransferase; rHDL, reconstituted high density lipoprotein(s); apoA-I(Δ(Arg123–Tyr166),∇A-II(Ser12–Ala75)), chimera with the Arg123–Tyr166 segment of apoA-I substituted with the Ser12–Ala75segment of apoA-II; apoA-I(Δ(Ala190–Gln243),∇A-II(Ser12–Gln77)), chimera with the Ala190–Gln243 segment of apoA-I substituted with the Ser12–Gln77segment of apoA-II; POPC, palmitoyloleoylphosphatidylcholine; PAGE, polyacrylamide gel electrophoresis. activation (12Brasseur R. De Meutter J. Vanloo B. Goormaghtigh E. Ruysschaert J.M. Rosseneu M. Biochim. Biophys. Acta. 1990; 1043: 245-252Crossref PubMed Scopus (88) Google Scholar, 13Wald J.H. Goormaghtigh E. De Meutter J. Ruysschaert J.M. Jonas A. J. Biol. Chem. 1990; 265: 20044-20050Abstract Full Text PDF PubMed Google Scholar, 14Brasseur R. J. Biol. Chem. 1991; 266: 16120-16127Abstract Full Text PDF PubMed Google Scholar).The structures in apoA-I involved in phospholipid binding and/or LCAT activation remain largely unidentified. Reported differences in LCAT activity of apoA-I and deletion mutants may result from altered folding and/or organization of these molecules in rHDL rather than from deletion of a functional domain (15Jonas A. Wald J.H. Toohill K.L.H. Krul E.S. Kezdy K.E. J. Biol. Chem. 1990; 265: 22123-22129Abstract Full Text PDF PubMed Google Scholar, 16Holvoet P. Zhao Z. 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). Therefore, in this study, the apoA-I(Δ(Arg123–Tyr166),∇A-II(Ser12–Ala75)) and apoA-I(Δ(Ala190–Gln243),∇A-II(Ser12–Gln77)) chimeras were produced, in which the Arg123–Tyr166 central or Ala190–Gln243 carboxyl-terminal pair of α-helices of apoA-I was deleted (Δ) and substituted (∇) with the pair of α-helices of apoA-II. The average structural properties in solution and in reconstituted high density lipoprotein particles of the two chimeras were found to be unaltered, but the central domain chimera had a markedly reduced LCAT activity.RESULTSFig. 1 is a schematic representation of the predicted amphipathic helical regions in apoA-I, apoA-II, apoA-I(Δ(Arg123–Tyr166),∇A-II(Ser12–Ala75)), and apoA-I(Δ(Ala190–Gln243),∇A-II(Ser12–Gln77)). The predicted number of amphipathic helices, according to Brasseuret al. (12Brasseur R. De Meutter J. Vanloo B. Goormaghtigh E. Ruysschaert J.M. Rosseneu M. Biochim. Biophys. Acta. 1990; 1043: 245-252Crossref PubMed Scopus (88) Google Scholar), was eight for apoA-I and for the two chimeras. Wild-type apoA-I and the apoA-I/apoA-II chimeras were expressed in the periplasmic space of E. coli cells and purified to homogeneity as evidenced by their migration as single bands on 10–15% SDS-polyacrylamide gels (Fig. 2). The molecular masses of the apolipoproteins, calculated from a plot of the logarithm of the molecular masses of the standard proteins versus the migration distance, were 28.3 kDa for apoA-I and 30.4 and 29.8 kDa for the respective chimeras and thus were in agreement with those calculated on the basis of the respective amino acid compositions. Wild-type apoA-I migrated in the same position as plasma apoA-I. The identity of each band was confirmed by immunoblot analysis using polyclonal rabbit anti-human apoA-I antibodies (data not shown).The α-helical contents of the apolipoproteins in aqueous solution, as determined by circular dichroism scanning, were 48% for apoA-I and 43 and 41% for the respective chimeras. The recovery of apolipoproteins in discoidal apolipoprotein-POPC-cholesterol complexes after gel filtration was 90% for wild-type apoA-I and the chimeras. The Stokes radius of all rHDL was 4.8 nm (Fig. 3 and TableI). Cross-linking of the apolipoprotein molecules within all the discoidal apolipoprotein-POPC-cholesterol particles revealed 2 apolipoprotein molecules/particle (Fig. 4 and Table I). The phospholipid surface was calculated from the circumference of the disc using the measured diameter minus 3 nm, i.e. 2 × radius of an α-helix, multiplied by a disc height of 3.8 nm. The number of phospholipid molecules was calculated from the calculated surface divided by 0.45 nm2, the surface area/condensed phospholipid molecule. The calculated apolipoprotein/phospholipid molar ratios were 1:90, 1:83, and 1:89, respectively, and were in agreement with the ratios calculated on the basis of the composition of the rHDL.Figure 3Determination of Stokes radii of rHDL by native polyacrylamide gel scanning. rHDL particles, isolated by gel filtration on a Superdex 200 HR column, were subjected to electrophoresis on 4–15% gradient polyacrylamide gels under nondenaturing conditions. A, apoA-I; B, apoA-I(Δ(Arg123–Tyr166),∇A-II(Ser12–Ala75));C, apoA-I(Δ(Ala190–Gln243),∇A-II(Ser12–Gln77));D, protein calibration mixture containing apoferritin (Stokes radius of 6.1 nm), catalase (5.2 nm), and lactate dehydrogenase (4.1 nm).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IProperties of discoidal apolipoprotein-POPC-cholesterol complexesPropertiesApolipoproteinApoA-I 1-aValues for plasma apoA-I and recombinant apoA-I were identical.ApoA-I(Δ(Arg123–Tyr166), ∇A-II(Ser12–Ala75))ApoA-I(Δ(Ala190–Gln243), ∇A-II(Ser12–Gln17))Stokes radius (nm)1-bSizes of rHDL particles were determined by PAGE.4.84.84.8ApoA-I molecules/disc (n)1-cThe number of apolipoprotein molecules/HDL particle was determined by SDS-PAGE after cross-linking with bis(sulfosuccinimidyl) suberate.222α-Helical content (%)1-dThe α-helical content was determined by CD scanning.747572Measured α-helices/apoA-I molecule (n)1-eThe number of α-helices/apolipoprotein molecule was calculated from the α-helical content and from the protein length assuming a length of 16 amino acids for the amphipathic helices and 6 amino acids for the adjacent β-strands (2, 12).888Estimated no. α-helices/apoA-I (n)1-fValues represent the estimated number according to the model of Brasseur et al. (12) (Fig. 1).888Fraction of phospholipid surface covered with α-helices1-gThe phospholipid surface was calculated from the circumference of the disc, using the measured diameter minus 3 nm, multiplied by a disc height of 3.8 nm. The area covered by an α-helix containing 16 amino acids was estimated to be 4 nm2.0.770.770.771-a Values for plasma apoA-I and recombinant apoA-I were identical.1-b Sizes of rHDL particles were determined by PAGE.1-c The number of apolipoprotein molecules/HDL particle was determined by SDS-PAGE after cross-linking with bis(sulfosuccinimidyl) suberate.1-d The α-helical content was determined by CD scanning.1-e The number of α-helices/apolipoprotein molecule was calculated from the α-helical content and from the protein length assuming a length of 16 amino acids for the amphipathic helices and 6 amino acids for the adjacent β-strands (2Segrest J.P. Jackson R.L. Morrisett J.D. Gotto Jr., A.M. FEBS Lett. 1974; 38: 247-258Crossref PubMed Scopus (485) Google Scholar, 12Brasseur R. De Meutter J. Vanloo B. Goormaghtigh E. Ruysschaert J.M. Rosseneu M. Biochim. Biophys. Acta. 1990; 1043: 245-252Crossref PubMed Scopus (88) Google Scholar).1-f Values represent the estimated number according to the model of Brasseur et al. (12Brasseur R. De Meutter J. Vanloo B. Goormaghtigh E. Ruysschaert J.M. Rosseneu M. Biochim. Biophys. Acta. 1990; 1043: 245-252Crossref PubMed Scopus (88) Google Scholar) (Fig. 1).1-g The phospholipid surface was calculated from the circumference of the disc, using the measured diameter minus 3 nm, multiplied by a disc height of 3.8 nm. The area covered by an α-helix containing 16 amino acids was estimated to be 4 nm2. Open table in a new tab Figure 4Determination of the number of apoA-I molecules/rHDL particle by SDS-polyacrylamide gel scanning. rHDL particles, in which apoA-I molecules were cross-linked with bis(sulfosuccinimidyl) suberate, were subjected to electrophoresis on 10–15% gradient SDS-polyacrylamide gels. A, apoA-I;B, apoA-I(Δ(Arg123–Tyr166),∇A-II(Ser12–Ala75));C, apoA-I(Δ(Ala190–Gln243),∇A-II(Ser12–Gln77));D, protein calibration mixture containing phosphorylaseb (94 kDa), albumin (67 kDa), ovalbumin (43 kDa), and trypsin inhibitor (30 kDa).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The α-helical contents of the apoA-I proteins in discoidal apolipoprotein-POPC-cholesterol complexes were 74% for apoA-I and 75 and 72% for the respective chimeras. The number of α-helices, calculated from the α-helical content determined by circular dichroism scanning and from the protein length assuming a length of 22 amino acids/α-helical repeat (2Segrest J.P. Jackson R.L. Morrisett J.D. Gotto Jr., A.M. FEBS Lett. 1974; 38: 247-258Crossref PubMed Scopus (485) Google Scholar, 12Brasseur R. De Meutter J. Vanloo B. Goormaghtigh E. Ruysschaert J.M. Rosseneu M. Biochim. Biophys. Acta. 1990; 1043: 245-252Crossref PubMed Scopus (88) Google Scholar), was eight per apoA-I or per chimera molecule (Table I) and was thus in agreement with the predicted values (12Brasseur R. De Meutter J. Vanloo B. Goormaghtigh E. Ruysschaert J.M. Rosseneu M. Biochim. Biophys. Acta. 1990; 1043: 245-252Crossref PubMed Scopus (88) Google Scholar). The fraction of the phospholipid surface that was covered by the α-helix was calculated as the total phospholipid surface in nm2 divided by 4 nm2, the surface area of an α-helix, that contains 16 amino acids. The calculated fraction was 0.77 for apoA-I and the chimeras (Table I).Binding of apoA-I and the chimeras to POPC was associated with a shift of Trp fluorescence maxima to lower wavelengths from 337 to 332 nm for apoA-I (both for plasma apoA-I and recombinant apoA-I), from 339 to 334 nm for the central domain chimera, and from 337 to 333 nm for the carboxyl-terminal domain chimera (Table II), suggesting that lipid binding is associated with a translocation of Trp residues to a more apolar environment. The quenching parametersK SV and f a are summarized in Table II. For totally exposed Trp residues, in the absence of electrostatic or viscosity effects, K SV = 12m−1 and f a = 1, whereas for totally protected Trp residues, K SV = 0 andf a = 0. The quenchable fluorescence of all apolipoproteins in the respective rHDL represented, on average, 60% of the total fluorescence (Table II).Table IIFluorescence properties of apolipoproteins in rHDL particlesPropertiesApolipoproteinApoA-IApoA-I(Δ(Arg123–Tyr166), ∇A-II(Ser12–Ala75))ApoA-I(Δ(Ala190–Gln243), ∇A-II(Ser12–Gln77))Wavelength of maximum fluorescence (nm)332334333K SV(m−1) for quenching of Trp fluorescence with I−2.9 ± 0.73.4 ± 1.11.9 ± 0.06Quenchable fraction of Trp fluorescence (f a)0.61 ± 0.080.60 ± 0.050.58 ± 0.05Data represent mean ± S.D. for three independent measurements. Data for apoA-I and the chimeras were not significantly different. Values for plasma apoA-I and recombinant apoA-I were identical. Open table in a new tab Recombinant LCAT was obtained in serum-free conditioned medium of transfected 293 cells. The homogeneity of the recombinant LCAT preparation is illustrated in Fig. 5. LCAT activation by the discoidal apolipoprotein-POPC-cholesterol complexes obeyed Michaelis-Menten kinetics, as shown by linear Lineweaver-Burk plots of the inverse of the initial activation rate (1/V 0) versus the inverse of the cholesterol concentrations (1/[C]). The apparent kinetic parameters V max and K m and the V max/K m ratios for the different apolipoprotein-POPC-cholesterol complexes are summarized in Table III. Exchange of the Arg123–Tyr166 paired helix of apoA-I with the pair of helices of apoA-II reduced the LCAT activity of apoA-I 27-fold due to a reduction of V max and not ofK m. In contrast, exchange of the Ala190–Gln243 carboxyl-terminal domain helices of apoA-I with the pair of helices of apoA-II had no effect on the LCAT activity of apoA-I.Figure 5SDS-PAGE of recombinant LCAT on 10–15% gradient gels. Lane 1, protein calibration mixture consisting of phosphorylase b (94 kDa), albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), and trypsin inhibitor (20 kDa); lane 2, recombinant LCAT.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6 illustrates the binding of radiolabeled LCAT to rHDL of apoA-I and the apoA-I(Δ(Arg123–Tyr166),∇A-II(Ser12–Ala75)) chimera. Fifty % of maximal binding was obtained with 34 μg/ml apoA-I and 27 μg/ml apoA-I(Δ(Arg123–Tyr166), ∇A-II(Ser12–Ala75)).Figure 6Binding of radiolabeled LCAT to discoidal apolipoprotein-POPC-cholesterol complexes. •, apoA-I; ▪, apoA-I (Δ(Arg123–Tyr166),∇A-II(Ser12–Ala75)).View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONReported differences in LCAT activity of apoA-I variants may be due to defective interaction with phospholipids, structural changes in rHDL, and/or deletion of functional domains. To further investigate the role of the central and carboxyl-terminal domains of apoA-I in LCAT activation, the apoA-I(Δ(Arg123–Tyr166),∇A-II(Ser12–Ala75)) and apoA-I(Δ(Ala190–Gln243),∇A-II(Ser12–Gln77)) chimeras were produced. The extent of in vitro phospholipid binding of these chimeras was similar to that of apoA-I, as demonstrated by comparable disc formation after mixing the apolipoproteins and phospholipids at equal weight ratios. This was evidenced by a shift of the maximum Trp fluorescence to a shorter wavelength and by a decreased accessibility of the Trp residues to I−. The sizes of rHDL reconstituted with apoA-I and the chimeras were identical: the respective rHDL contained 2 apolipoprotein molecules/particle, and circular dichroism scanning revealed eight α-helices per intact apoA-I molecule and per chimera molecule, in agreement with the predicted numbers according to Brasseur et al. (12Brasseur R. De Meutter J. Vanloo B. Goormaghtigh E. Ruysschaert J.M. Rosseneu M. Biochim. Biophys. Acta. 1990; 1043: 245-252Crossref PubMed Scopus (88) Google Scholar). The calculated apolipoprotein/phospholipid molar ratios of the different rHDL particles were very similar. Thus, substitution of the Ala123–Tyr166 central or Ala190–Gln243 carboxyl-terminal domain helices of apoA-I with the pair of helices of apoA-II did not affect the size and the composition of rHDL, and the conformation and helical distribution in the different apolipoproteins in these particles were very similar. Substitution of the carboxyl-terminal domain of apoA-I with the helices of apoA-II did not reduce LCAT activity, but substitution of the central domain resulted in a 27-fold reduction of LCAT activity, suggesting that the Ala123–Tyr166 segment is critical for LCAT activation. Binding experiments revealed that the reduced LCAT activity of the apoA-I(Δ(Arg123–Tyr166),∇A-II(Ser12–Ala75)) chimera was not due to reduced binding of LCAT to rHDL.Based on data obtained with synthetic peptides, it was concluded that the LCAT-activating domain of apoA-I resides in a 22-mer tandem repeat located between residues 66 and 121 (35Anantharamaiah G.M. Venkatachalapathi Y.V. Brouillette C.G. Segrest J.P. Arteriosclerosis. 1990; 10: 95-105Crossref PubMed Google Scholar). This was further supported by the finding that monoclonal antibodies directed against an epitope that spanned residues 95–121 inhibited the LCAT activation with apoA-I (36Banka 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). Binding of antibodies to an epitope in the amino-terminal domain of apoA-I may, however, induce conformational changes in the central domain of apoA-I that may be responsible for the reduction of LCAT activity (37Meng Q.-H. Calabresi L. Fruchart J.-C. Marcel Y.L. J. Biol. Chem. 1993; 268: 16966-16973Abstract Full Text PDF PubMed Google Scholar). Deletion of the Leu44–Leu126amino-terminal domain of apoA-I indeed did not reduce its LCAT activity (16Holvoet P. Zhao Z. 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), suggesting that the amino-terminal domain of apoA-I is not critical for LCAT activation.Using deletion mutants of apoA-I, Minnich et al. (38Minnich A. Collet X. Roghani A. Cladaras C. Hamilton R.L. Fielding C.J. Zannis V.I. J. Biol. Chem. 1992; 267: 16553-16560Abstract Full Text PDF PubMed Google Scholar) found that deletion of the Met148–Gly186 segment resulted in decreased LCAT activity, whereas Sorci-Thomas et al. (39Sorci-Thomas M. Kearns M.W. Lee J.P. J. Biol. Chem. 1993; 268: 21403-21409Abstract Full Text PDF PubMed Google Scholar) found that deletion of the Pro143–Ala164 segment reduced LCAT activity. In previous studies, the conformation of deletion mutants in their respective rHDL was not investigated. However, it has been demonstrated that the decreased LCAT activity of deletion mutants of apoA-I may be due to differences in folding (decreased α-helical content) and/or organization of the apolipoproteins in rHDL (3 or 4 molecules/particle as compared with 2 for intact apoA-I) rather than to the removal of specific domains for LCAT activity (16Holvoet P. Zhao Z. 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). Indeed, it has been demonstrated that rHDL discs containing wild-type apoA-I may have discrete sizes, compositions (with 2, 3, or 4 protein molecules/particle), and apoA-I conformations (with six, seven, or eight α-helices/apoA-I molecule in contact with lipid) and that differences in the apoA-I structure in these particles correlate with their ability to activate LCAT (40Jonas A. Kézdy K.E. Wald J.H. J. Biol. Chem. 1989; 264: 4818-4824Abstract Full Text PDF PubMed Google Scholar).Chimeras in which α-helical segments of apoA-I are substituted with helical segments of apoA-II, which does not activate LCAT, in such a way that the average secondary structure of the apolipoprotein molecule as well as the organization of the apolipoprotein molecules in rHDL are not affected may be preferable reagents to address the function of a particular structural domain in LCAT activation. Indeed, rHDL containing apoA-II have a very low LCAT activity (41Vanloo B. Taveirne J. Baert J. Loren G. Lins L. Ruysschaert J.M. Rosseneu M. Biochim. Biophys. Acta. 1992; 1128: 258-266Crossref PubMed Scopus (25) Google Scholar), and the addition of apoA-II together with apoA-I to liposomes reduces LCAT activity by 70% (42Chen C.H. Albers J.J. Eur. J. Biochem. 1986; 155: 589-594Crossref PubMed Scopus (12) Google Scholar). Thus, substitution of sequences in apoA-I that are critical for the interaction with LCAT with sequences derived from apoA-II would result in decreased LCAT activity. Substitution of the carboxyl-terminal domain of apoA-I with helices of apoA-II was found not to affect LCAT activity, but substitution of the central domain resulted in a 25-fold reduction of LCAT activity, suggesting that the central domain (but not the carboxyl-terminal domain) is essential for LCAT activation.The differences in LCAT activity were due to differences in apparentV max values, which reflect the activated enzyme concentration, and not to differences in apparent K m values, which reflect the affinity of LCAT for rHDL (43Bolin D.J. Jonas A. J. Biol. Chem. 1994; 269: 7429-7434Abstract Full Text PDF PubMed Google Scholar). SimilarK m values are indeed in agreement with similar binding of LCAT to rHDL of apoA-I and the central domain chimera. Thus, the Ala123–Tyr166 segment of apoA-I appears to contain structures that are required for optimal phospholipid and cholesterol presentation to LCAT (44Sparks D.L. Anantharamaiah G.M. Segrest J.P. Phillips M.C. J. Biol. Chem. 1995; 270: 5151-5157Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) that cannot be mimicked by the apoA-II segment. It is possible that substitution of the central domain affects the conformation of a hinged domain that is crucial for LCAT activation because antibodies that interfere with the mobility of a hinged domain in the central part of apoA-I inhibit LCAT activation (37Meng Q.-H. Calabresi L. Fruchart J.-C. Marcel Y.L. J. Biol. Chem. 1993; 268: 16966-16973Abstract Full Text PDF PubMed Google Scholar). Previous data obtained with deletion mutants supported the existence of such a hinged domain overlapping either the Asn102–Lys140 or Ala124–His162 segment of apoA-I (16Holvoet P. Zhao Z. 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). Because conformational changes elsewhere in the molecule could not be excluded, these data were, however, not conclusive. The present study strongly suggests that this hinged domain most likely overlaps the Ala124–His162 domain of apoA-I.In conclusion, substitution of the central or carboxyl-terminal pair of helices of apoA-I with the helices of apoA-II does not affect its average structure in rHDL. Substitution of the central domain (but not the carboxyl-terminal domain) results in a significant reduction of the rate of LCAT activation, although the binding of LCAT to rHDL is not reduced. ApoA-I is s" @default.
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• W2020661012 cites W1503830583 @default.
• W2020661012 cites W1520043991 @default.
• W2020661012 cites W1524712279 @default.
• W2020661012 cites W1536020222 @default.
• W2020661012 cites W1561342323 @default.
• W2020661012 cites W1569934993 @default.
• W2020661012 cites W1573404455 @default.
• W2020661012 cites W1574745553 @default.
• W2020661012 cites W1576149482 @default.
• W2020661012 cites W1596092489 @default.
• W2020661012 cites W1606479506 @default.
• W2020661012 cites W165264657 @default.
• W2020661012 cites W1964558380 @default.
• W2020661012 cites W1966583531 @default.
• W2020661012 cites W1966686403 @default.
• W2020661012 cites W1970142406 @default.
• W2020661012 cites W1985488373 @default.
• W2020661012 cites W1994618293 @default.
• W2020661012 cites W1995725157 @default.
• W2020661012 cites W2028622989 @default.
• W2020661012 cites W2033299688 @default.
• W2020661012 cites W2049746692 @default.
• W2020661012 cites W2050125414 @default.
• W2020661012 cites W2059699637 @default.
• W2020661012 cites W2061704447 @default.
• W2020661012 cites W2062228103 @default.
• W2020661012 cites W2076530860 @default.
• W2020661012 cites W2081253788 @default.
• W2020661012 cites W2093004132 @default.
• W2020661012 cites W2100837269 @default.
• W2020661012 cites W2101108802 @default.
• W2020661012 cites W2114471659 @default.
• W2020661012 cites W2115125237 @default.
• W2020661012 cites W2129228470 @default.
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• W2020661012 cites W4293247451 @default.
• W2020661012 cites W80696700 @default.
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