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• W1964558380 abstract "In the present study, the lipoprotein association of apoA-I, an apoA-I (ΔAla190-Gln243) deletion mutant and an apoA-I (Asp1-Leu189)/apoA-II (Ser12-Gln77) chimera were compared. At equilibrium, 80% of the 125I-labeled apolipoproteins associated with lipoproteins in rabbit or human plasma but with very different distribution profiles. High density lipoprotein (HDL)2,3-associated fractions were 0.60 for apoA-I, 0.30 for the chimera, and 0.15 for the deletion mutant, and corresponding very high density lipoprotein-associated fractions were 0.20, 0.50, and 0.65. Clearance curves after intravenous bolus injection of 125I-labeled apolipoproteins (3 µg/kg) in normolipemic rabbits could be adequately fitted with a sum of three exponential terms, yielding overall plasma clearance rates of 0.028 ± 0.0012 ml·min−1 for apoA-I (mean ± S.E.; n = 6), 0.10 ± 0.008 ml·min−1 for the chimera (p < 0.001 versus apoA-I) and 0.38 ± 0.022 ml·min−1 for the deletion mutant (p < 0.001 versus apoA-I and versus the chimera). Fractions that were initially cleared with a t1/2 of 3 min, most probably representing free apolipoproteins, were 0.30 ± 0.04, 0.50 ± 0.06 (p = 0.02 versus apoA-I), and 0.64 ± 0.07 (p = 0.002 versus apoA-I), respectively. At 20 min after the bolus, the fractions of injected material associated with HDL2,3 were 0.55 ± 0.06, 0.25 ± 0.03 (p = 0.001 versus apoA-I), and 0.09 ± 0.01 (p < 0.001 versus apoA-I and versus the chimera), respectively, whereas the fractions associated with very high density lipoprotein were 0.15 ± 0.006, 0.25 ± 0.03 (p = 0.008 versus apoA-I), and 0.27 ± 0.03 (p = 0.003 versus apoA-I), respectively. The ability of the different apolipoproteins to bind to HDL3 particles and displace apoA-I in vitro were compared. The molar ratios at which 50% of 125I-labeled apoA-I was displaced from the surface of HDL3 particles were 1:1 for apoA-I, 3:1 for the chimera and 12:1 for the deletion mutant, indicating 3- and 12-fold reductions of the affinities for HDL3 of the chimera and the deletion mutant, respectively. These data suggest that the carboxyl-terminal pair of helices of apoA-I are involved in the initial rapid binding of apoA-I to the lipid surface of HDL. Although the lipid affinity of apoA-II is higher than that of apoA-I, substitution of the carboxyl-terminal helices of apoA-I with those of apoA-II only partially restores its lipoprotein association. Thus, this substitution may affect cooperative interactions with the middle amphipathic helices of apoA-I that are critical for its specific distribution over the different HDL species. In the present study, the lipoprotein association of apoA-I, an apoA-I (ΔAla190-Gln243) deletion mutant and an apoA-I (Asp1-Leu189)/apoA-II (Ser12-Gln77) chimera were compared. At equilibrium, 80% of the 125I-labeled apolipoproteins associated with lipoproteins in rabbit or human plasma but with very different distribution profiles. High density lipoprotein (HDL)2,3-associated fractions were 0.60 for apoA-I, 0.30 for the chimera, and 0.15 for the deletion mutant, and corresponding very high density lipoprotein-associated fractions were 0.20, 0.50, and 0.65. Clearance curves after intravenous bolus injection of 125I-labeled apolipoproteins (3 µg/kg) in normolipemic rabbits could be adequately fitted with a sum of three exponential terms, yielding overall plasma clearance rates of 0.028 ± 0.0012 ml·min−1 for apoA-I (mean ± S.E.; n = 6), 0.10 ± 0.008 ml·min−1 for the chimera (p < 0.001 versus apoA-I) and 0.38 ± 0.022 ml·min−1 for the deletion mutant (p < 0.001 versus apoA-I and versus the chimera). Fractions that were initially cleared with a t1/2 of 3 min, most probably representing free apolipoproteins, were 0.30 ± 0.04, 0.50 ± 0.06 (p = 0.02 versus apoA-I), and 0.64 ± 0.07 (p = 0.002 versus apoA-I), respectively. At 20 min after the bolus, the fractions of injected material associated with HDL2,3 were 0.55 ± 0.06, 0.25 ± 0.03 (p = 0.001 versus apoA-I), and 0.09 ± 0.01 (p < 0.001 versus apoA-I and versus the chimera), respectively, whereas the fractions associated with very high density lipoprotein were 0.15 ± 0.006, 0.25 ± 0.03 (p = 0.008 versus apoA-I), and 0.27 ± 0.03 (p = 0.003 versus apoA-I), respectively. The ability of the different apolipoproteins to bind to HDL3 particles and displace apoA-I in vitro were compared. The molar ratios at which 50% of 125I-labeled apoA-I was displaced from the surface of HDL3 particles were 1:1 for apoA-I, 3:1 for the chimera and 12:1 for the deletion mutant, indicating 3- and 12-fold reductions of the affinities for HDL3 of the chimera and the deletion mutant, respectively. These data suggest that the carboxyl-terminal pair of helices of apoA-I are involved in the initial rapid binding of apoA-I to the lipid surface of HDL. Although the lipid affinity of apoA-II is higher than that of apoA-I, substitution of the carboxyl-terminal helices of apoA-I with those of apoA-II only partially restores its lipoprotein association. Thus, this substitution may affect cooperative interactions with the middle amphipathic helices of apoA-I that are critical for its specific distribution over the different HDL species. Low plasma levels of high density lipoproteins (HDL) 1The abbreviations used are: HDLhigh density lipoprotein(s); apoA-I: apolipoprotein A-I; apoA-II: apolipoprotein A-II; apoA-I (ΔAla190-Gln243), apoA-I mutant with deletion of the Ala190-Gln243 segment; apoA-I (Asp1-Leu189)/apoA-II (Ser12-Gln77), chimera containing the Asp1-Leu189 segment of apoA-I linked to the Ser12-Gln77 segment of apoA-IIVHDLvery high density lipoprotein(s). and of their major protein component, apolipoprotein A-I (apoA-I), correlate with an increased risk for coronary heart disease (1Gordon D.J. Rifkind B.M. N. Engl. J. Med. 1989; 321: 1311-1316Google Scholar), and family and twin studies have suggested that decreased HDL levels are partially hereditary (2Christian J.C. Carmelli D. Castelli W.P. Fabsitz R. Grim C.E. Meaney F.J. Norton Jr., J.A. Reed T. Williams C.J. Wood P.D. Arteriosclerosis. 1990; 10: 1020-1025Google Scholar, 3De Backer G. Hulstaert F. De Munck K. Rosseneu M. Van Parijs L. Dramaix M. Am. Heart. J. 1986; 112: 478-484Google Scholar, 4Hunt S.C. Hasstedt S.J. Kuida H. Stults B.M. Hopkins P.N. Williams R.R. Am. J. Epidemiol. 1989; 129: 625-638Google Scholar, 5Pometta D. Micheli H. Suenram A. Jornot C. Atherosclerosis. 1979; 34: 419-429Google Scholar). In addition, HDL and their apolipoproteins increase the net efflux of cellular unesterified cholesterol (6Daniels R.J. Guertler L.S. Parker T.S. Steinberg D. J. Biol. Chem. 1981; 256: 4978-4983Google Scholar, 7Fielding C.J. Fielding P.E. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 3911-3914Google Scholar, 8Oram J.F. Albers J.J. Cheung M.C. Bierman E.L. J. Biol. Chem. 1981; 256: 8348-8356Google Scholar) and remove excess cholesterol from peripheral (nonhepatic) cells (9Glomset J.A. J. Lipid Res. 1968; 9: 155-167Google Scholar), which may explain the inverse correlation between risk of coronary heart disease and HDL levels (10Miller N.E. La Ville A. Crook D. Nature. 1985; 314: 109-111Google Scholar). high density lipoprotein(s); apoA-I: apolipoprotein A-I; apoA-II: apolipoprotein A-II; apoA-I (ΔAla190-Gln243), apoA-I mutant with deletion of the Ala190-Gln243 segment; apoA-I (Asp1-Leu189)/apoA-II (Ser12-Gln77), chimera containing the Asp1-Leu189 segment of apoA-I linked to the Ser12-Gln77 segment of apoA-II very high density lipoprotein(s). ApoA-I, the major protein component of HDL, is an important determinant of the concentration of HDL in plasma (11Assman G. Brewer Jr., H.B. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 989-993Google Scholar). ApoA-I binds and transports plasma lipids, serves as a cofactor for the enzyme lecithin:cholesterol acyltransferase, and increases cholesterol efflux from peripheral tissues (12Fielding C.J. Shore V.G. Fielding P.E. Biochem. Biophys. Res. Commun. 1972; 46: 1493-1498Google Scholar, 13Oram J.F. McKnight G.L. Hart C.B. Atherosclerosis Rev. 1990; 20: 103-107Google Scholar, 14Hara H. Yokoyama S. J. Biol. Chem. 1991; 266: 3080-3086Google Scholar). In addition, apoA-I is an important ligand in the binding of HDL to cell membranes (15Slotte J.P. Oram J.F. Bierman E.L. J. Biol. Chem. 1987; 262: 12904-12907Google Scholar, 16Morrison J.R. McPherson G.A. Fidge N.H. J. Biol. Chem. 1992; 267: 13205-13209Google Scholar). These characteristics contribute to the ability of HDL to induce reverse cholesterol transport and thus to the protective effect of HDL on cardiovascular disease. ApoA-I is synthesized as a prepropeptide, which is cotranslationally cleaved to pro-apoA-I, and then cleaved during secretion to form the mature 243-amino acid apoA-I protein (17Brewer Jr., H.B. Fairwell T. LaRue A. Ronan R. Houser A. Bronzert T.J. Biochem. Biophys. Res. Commun. 1978; 80: 623-630Google Scholar). The secondary structure of apoA-I contains amphipathic helices composed of hydrophilic and hydrophobic surfaces (18Segrest J.P. Jackson R.L. Morrisett J.D. Gotto Jr., A.M. FEBS Lett. 1974; 38: 247-258Google Scholar, 19Segrest J.P. De Loof H. Dohlman J.G. Brouillette C.G. Anantharamaiah G.M. Proteins. 1990; 8 (Correction (1991) Proteins979): 103-117Google Scholar, 20Segrest J.P. Jones M.K. De Loof H. Brouillette C.G. Venkatachalapathi Y.V. Anantharamaiah G.M. J. Lipid. Res. 1992; 33: 141-166Google Scholar). It has been demonstrated that the carboxyl-terminal domain of apoA-I plays an important role in lipid binding and in the interaction with cell membranes (16Morrison J.R. McPherson G.A. Fidge N.H. J. Biol. Chem. 1992; 267: 13205-13209Google Scholar, 21Morrison J. Fidge N.H. Tozuka M. J. Biol. Chem. 1991; 266: 18780-18785Google Scholar, 22Dalton M.B. Swaney J.B. J. Biol. Chem. 1993; 268: 19273-19283Google Scholar, 23Minnich A. Collet X. Roghani A. Cladaras C. Hamilton R.L. Fielding C.J. Zannis V.I. J. Biol. Chem. 1992; 267: 16553-16560Google Scholar, 24Ji Y. Jonas A. J. Biol. Chem. 1995; 270: 11290-11297Google Scholar, 25Holvoet 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: 13334-13342Google Scholar). Analysis of patients with reduced plasma concentration of HDL revealed that accelerated catabolism, possibly due to enzymatic degradation, of apoA-I is the most common cause of low HDL levels (26Rader D.J. Gregg R.E. Meng M.S. Schaefer J.R. Zech L.A. Benson M.D. Brewer Jr., H.B. J. Lipid Res. 1992; 33: 755-763Google Scholar, 27Deeb S.S. Cheung M.C. Peng R. Wolf A.C. Stern R. Albers J.J. Knopp R.H. J. Biol. Chem. 1991; 266: 13654-13660Google Scholar, 28Brinton E.A. Eisenberg S. Breslow J.L. J. Clin. Invest. 1991; 87: 536-544Google Scholar, 29Le N.A. Ginsberg H.N. Metabolism. 1988; 37: 614-617Google Scholar). Schmidt et al. (30Schmidt H.H.J. Remaley A.T. Stonik J.A. Ronan R. Wellmann A. Thomas F. Zech L.A. Brewer Jr., H.B. Hoeg J.M. J. Biol. Chem. 1995; 270: 5469-5475Google Scholar) demonstrated that deletion of the carboxyl-terminal domain of apoA-I results in decreased in vivo lipoprotein association, in an altered distribution pattern in HDL, and in an increased clearance rate. Recently, we have demonstrated (25Holvoet 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: 13334-13342Google Scholar) that deletion of the carboxyl-terminal domain of apoA-I decreases the rate but not the extent of in vitro phospholipid association and that this interaction results in the formation of larger discoidal particles with increased apoA-I/phospholipid ratios. The present study compares the in vitro and in vivo lipoprotein binding properties of wild-type human apoA-I, an apoA-I (ΔAla190-Gln243) carboxyl-terminal deletion mutant, and an apoA-I (Asp1-Leu189)/apoA-II (Ser12-Gln77) chimera, in which the carboxyl-terminal pair of helices of apoA-I have been substituted with the pair of helices of apoA-II. Oligonucleotides were obtained by custom synthesis (Pharmacia, Brussels, Belgium). DNA sequencing was performed on a Pharmacia ALF DNA sequencer. Chromatography materials were obtained from Pharmacia. All DNA manipulations were carried out essentially as described (25Holvoet 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: 13334-13342Google Scholar). cDNAs for expression of wild-type apoA-I and of apoA-I (ΔAla190-Gln243) in Escherichia coli were obtained as described previously (25Holvoet 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: 13334-13342Google Scholar). The DNA fragment for the Ser12-Gln77 segment of apoA-II was amplified by polymerase chain reaction in an automated DNA thermal cycler (Perkin-Elmer) using the 5′-deoxyoligonucleotide dATGGCGCCAGACTGTCTCAGTACTTCCAGAGGCGCCAGACTG primer, overlapping the apoA-I (Gly186-Leu189) and the apoA-II (Ser12-Gln16) segments, and the 3′-deoxyoligonucleotide dTAGGCGCCTCACTGGG TGGGTGGCAGGCTGTGTT reversed primer, overlapping the apoA-II (Thr72-Gln77) segment followed with a TGA stop codon and a NarI site. Thirty cycles were performed, consisting of 1 min of denaturation at 94°C, 2 min of annealing at 52°C, and 1.5 min of extension at 72°C. The polymerase chain reaction product was digested with NarI and ligated in the NarI-treated pMc-5-apoA-I transfection vector resulting in the pMC-5-apoA-I (Asp1-Leu189)/apoA-II (Ser12-Gln77) vector for the expression of apoA-I (Asp1-Leu189)/apoA-II (Ser12-Gln77) in E. coli. All cDNA constructs were confirmed by DNA sequencing, as described previously (25Holvoet 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: 13334-13342Google Scholar). Apolipoproteins were expressed in the periplasmic fractions of E. coli WK6 host cells as described (25Holvoet 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: 13334-13342Google Scholar). Standard apoA-I was isolated from normolipemic human plasma as described previously (17Brewer Jr., H.B. Fairwell T. LaRue A. Ronan R. Houser A. Bronzert T.J. Biochem. Biophys. Res. Commun. 1978; 80: 623-630Google Scholar). The purity of proteins was established by SDS-gel electrophoresis (31Laemmli U.K. Nature. 1970; 227: 680-685Google Scholar) and immunoblotting (32Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Google Scholar). Proteins were iodinated by the Bolton and Hunter method (33Bolton A.E. Hunter W.M. Biochim. Biophys. Acta. 1973; 329: 318-330Google Scholar). The pharmacokinetic properties of wild-type apoA-I, apoA-I (ΔAla190-Gln243), and apoA-I (Asp1-Leu189)/ApoA-II (Ser12-Gln77) in New Zealand White rabbits were determined by measurement of the residual radioactivity after bolus injection of 125I-labeled proteins (3 µg/kg) in blood samples that were taken at times 1, 2, 5, 10, 20, 30 min and at 1, 2, 3, 4, 5, 6, 7, 8, 24, 28, and 31 h. The results were plotted semilogarithmically, and the curves were fitted with a sum of three exponential terms C(t) = Ae−-αt+ Be−βt+ Ce−γt, by graphical curve peeling (34Gibaldi M. Perrier D. Pharmacokinetics. Marcel Dekker Inc., New York1983: 45Google Scholar). The coefficients A, B, and C were calculated from the intercepts on the ordinate, whereas the exponents α, β, and γ were calculated from the slopes. The following clearance parameters were calculated using standard formulas derived by Gibaldi and Perrier (34Gibaldi M. Perrier D. Pharmacokinetics. Marcel Dekker Inc., New York1983: 45Google Scholar): total volume of distribution VD = dose/C; extrapolated area under the curve (AUC)= A/α+ B/β+ C/γ, and plasma clearance rate Clp = dose/AUC. Statistical differences between these parameters were calculated using the Student t test. Continuous density gradient ultracentrifugation (35Cheung M.C. Segrest J.P. Albers J.J. Cone J.T. Brouillette C.G. Chung B.H. Kashyap M. Glasscock M.A. Anantharamaiah G.M. J. Lipid. Res. 1987; 28: 913-929Google Scholar) was performed using a table top T-100 ultracentrifuge (Analis, Namur, Belgium) in 5-ml tubes. Four hundred µl of rabbit plasma were analyzed by gel filtration on a Superose 6HR column equilibrated with 20 mM Tris-HCl buffer, pH 8.1, containing 0.15 M NaCl, 1 mM EDTA, and 0.02 mg/ml sodium azide in a fast protein liquid chromatography system (Waters Associates, Milford, MA). The levels of phospholipids and cholesterol were determined using standard enzymatic assays (Biomérieux, Marcy, France, and Boehringer Mannheim, Meylon, France, respectively), and the protein levels were determined according to Bradford (36Bradford M.M. Anal. Biochem. 1976; 72: 248-254Google Scholar). Human HDL3, containing 240 µg of apoA-I in 200 µl, was incubated with 30 µg of 125I-labeled apoA-I for 1 h at 37°C. Nonbound radiolabeled apoA-I was separated from the HDL3 fraction by filtration on a Centricon 100 filter (Amicon, Beverly, MA). Radiolabeled HDL3 was diluted 8-fold in 20 mM Tris-HCl buffer, and 50-µl aliquots, containing 0.80 µg of radiolabeled apoA-I, were mixed with 50 µl of solutions that contained 0.24, 0.48, 0.96, 1.92, 3.84, 7.68, or 15.36 µg of apoA-I, deletion mutant, or chimera. After 1 h of incubation at 37°C, free apolipoproteins were separated from the HDL3 by filtration. The HDL3-associated radioactivity and the radioactivity in the filtrate were measured. The percentage of HDL3-associated radiolabeled apoA-I was determined as a function of the amount of competing apolipoprotein in the incubation mixture. The predicted amphipathic helical regions in apoA-I, the apoA-I (ΔAla190-Gln243) carboxyl-terminal deletion mutant, apoA-II, and the apoA-I (Asp1-Leu189)/apoA-II (Ser12-Gln77) chimera, according to Brasseur et al. (37Brasseur R. De Meutter J. Vanloo B. Goormaghtigh E. Ruysschaert J.M. Rosseneu M. Biochim. Biophys. Acta. 1990; 1043: 245-252Google Scholar, 38Brasseur R. J. Biol. Chem. 1991; 266: 16120-16127Google Scholar), are illustrated in Fig. 1. Edmundson wheel diagrams confirmed that the aligned carboxyl-terminal segments in apoA-I and the chimera indeed may form amphipathic helices and that the orientation of their respective hydrophilic and hydrophobic surfaces is very similar (Fig. 2). The hydrophobicity of the carboxyl-terminal helix of the chimera was higher than that of the original ninth helix of apoA-I (mean residue hydrophobicities were 0.038 for apoA-I and 0.060 for the chimera).Fig. 2Edmundson wheel diagrams of the Ala190-Ala207 (A) and Pro220-Thr237 (B) α-helical segments of apoA-I and the Ser12-Val29 (C) and Pro51-Val68 of apoA-II (D). Shown are α-helical segments of apoA-II that have been inserted in apoA-I (Asp1-Leu89)/apoA-II (Ser12-Gln77) chimera as indicated in Fig. 1. These segments are indicated as A1-A18, P1-T18, S1-V18, and P1-V18, respectively. Positively (+) and negatively (−) charged amino acids are shown. Hydrophobic residues are shown by thick circles.View Large Image Figure ViewerDownload (PPT) Apolipoproteins were expressed in the periplasmic space of E. coli cells and purified to homogeneity as revealed by SDS-polyacrylamide gel electrophoresis (Fig. 3). Each of the proteins migrated as a single band with the expected molecular masses on polyacrylamide gels: 28.3 kDa for wild-type recombinant apoA-I, 29.8 kDa for the chimera, and 22.0 kDa for the deletion mutant. The identity of each band was confirmed by immunoblot analysis, using polyclonal sheep anti-human apoA-I and sheep anti-human apoA-II antibodies (data not shown). As previously shown, the molecular masses and the in vitro phospholipid binding properties of wild-type recombinant apoA-I and plasma apoA-I are identical (25Holvoet 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: 13334-13342Google Scholar). To assess the impact of the deletion or the substitution of the carboxyl-terminal domain on protein self-association, the lipid-free apolipoproteins were solubilized in phosphate-buffered saline at three concentrations (0.125, 0.25, and 0.50 mg/ml) and treated with the cross-linking agent bis(sulfosuccinimidyl)suberate. Fig. 4 illustrates the oligomerization patterns of the different apolipoproteins. At the lowest concentration all apolipoproteins existed predominantly as monomers. At the highest concentration, apoA-I exhibited monomeric (38% of total protein), dimeric (22%), trimeric (16%), and tetrameric (14%) forms (Fig. 4). At the same concentration, the percentage of apoA-I (ΔAla190-Gln243) that existed as monomer was higher (monomer, 75%; dimer, 16%; trimer, 6%; and tetramer, 3%). At all concentrations, >90% of the chimera existed as monomer (Fig. 4). Radiolabeled apolipoproteins with specific radioactivities of 3 × 106 cpm/µg of protein were used within 24 h after radiolabeling and analyzed by SDS-polyacrylamide gel electrophoresis to exclude degradation and self-association (not shown). The association of radiolabeled apolipoproteins with lipoprotein particles was studied following in vitro incubation in rabbit plasma for 60 min. Fractions of added apolipoproteins that were associated with HDL2,3 particles were 0.60 ± 0.05 for apoA-I (mean ± S.E.; n = 6), 0.32 ± 0.02 for the chimera (p = 0.007 versus apoA-I), and 0.14 ± 0.005 for the deletion mutant (p < 0.001 versus apoA-I and versus the chimera). Fractions of added apolipoproteins associated with VHDL particles were 0.20 ± 0.01 for apoA-I, 0.48 ± 0.05 for the chimera (p > 0.001 versus apoA-I), and 0.65 ± 0.06 for the deletion mutant (p < 0.001 versus apoA-I and p = NS versus the chimera). Fractions present as free apolipoproteins in the plasma were 0.20 for all three compounds (data not shown). The density distributions of radiolabeled apolipoproteins associated with lipoprotein particles in human plasma are illustrated in Fig. 5. Fractions that were associated with HDL2,3 particles were 0.55 ± 0.06 for apoA-I, 0.30 ± 0.04 for the chimera (p = 0.006 versus apoA-I), and 0.15 ± 0.02 for the deletion mutant (p < 0.001 versus apoA-I, p = 0.007 versus the chimera). Fractions that were associated with VHDL particles were 0.25 ± 0.02 for apoA-I, 0.50 ± 0.06 for the chimera (p < 0.003 versus apoA-I), and 0.65 ± 0.05 for the deletion mutant (p < 0.001 versus apoA-I and p = NS versus the chimera). Fractions that were present as free apolipoproteins in the plasma again were 0.20 for all three compounds. The plasma clearance of radiolabeled wild-type apoA-I, apoA-I (ΔAla190-Gln243), and apoA-I (Asp1-Leu189)/apoA-II (Ser12-Gln77) was analyzed in rabbits (Fig. 6). The disappearance rates of all proteins could be described by a sum of three exponential terms by graphical curve peeling. The calculated pharmacokinetic parameters are summarized in Table I. The plasma clearance rate was 0.028 ± 0.0012 ml·min−1 for 125I-labeled apoA-I as compared with 0.025 ± 0.0011 ml·min−1 for apoA-I antigen, as determined in an enzyme-linked immunosorbent assay based on monoclonal antibodies specific for apoA-I following injection of human apoA-I. These data suggest that clearance of apoA-I was not affected by the labeling procedures. Plasma clearance rates of the chimera and of the deletion mutant, respectively, were 3.6-fold and 13.6-fold higher than that of apoA-I (Table I). Values of t1/2α, t1/2β, and t1/2γ were, however, very similar for all apolipoproteins: 3, 220, and 2,300 min, respectively, suggesting that the differences in clearance resulted from differences in the lipoprotein profiles. This is illustrated in Fig. 7, which shows the distributions of cholesterol and phospholipids (upper panel) and of radiolabeled apolipoproteins (lower panel) in the different lipoprotein fractions at 20 min postinjection. Ninety percent of 125I-labeled apoA-I was associated with HDL2 and HDL3 particles (fractions 15-25), whereas only 10% was associated with smaller, phospholipid-rich VHDL particles (fractions 26-35). Corresponding values were 60 and 40% for the chimera and 30 and 70% for the deletion mutant.Table I.Pharmacokinetic parameters of the clearance of 125I-labeled wild-type apoA-I, apoA-I (Asp1-Leu189)/apo A-II (Ser12-Gln77), and apoA-I (ΔAla190-Gln243) from blood following bolus injection of 3 µg/kg in normolipemic New Zealand White rabbitsParameterApolipoproteinApoA-IApoA-I (Asp1-Leu189)/ApoA-II (Ser12-Gln77)ApoA-I (ΔAla190-Gln243)VD (ml)170 ± 10160 ± 6.1180 ± 8.2AUC (µg ·; min ·; ml−1360 ± 1386 ± 4.9ap < 0.001.26 ± 2.5ap < 0.001.Clp (ml ·; min−10.028 ± 0.00120.10 ± 0.008ap < 0.001.0.38 ± 0.022ap < 0.001.a p < 0.001. Open table in a new tab Fig. 7Lipoprotein analysis of rabbit plasma by gel filtration chromatography. Levels of cholesterol (Δ) and of phospholipids (○) are shown in the upper panel. Elution profiles of radiolabeled apoA-I (•), apoA-I (ΔAla190-Gln243) (▴), and apoA-I (Asp1-Leu189)/apoA-II (Ser12-Gln77) (▪) are shown in the lower panel. Blood samples were collected at 20 min after bolus injection of radiolabeled apolipoproteins in normolipemic New Zealand White rabbits. Data represent mean values of six independent experiments.View Large Image Figure ViewerDownload (PPT) The lipoprotein binding properties of the three apolipoproteins were further investigated by analyzing the density distribution of radiolabeled apolipoproteins. The distribution patterns at 20 min postinjection are illustrated in Fig. 8. At 20 min postinjection, HDL2,3-associated fractions were 0.55 ± 0.06 for apoA-I, 0.25 ± 0.03 for the chimera (p = 0.001 versus apoA-I) and 0.09 ± 0.01 for the deletion mutant (p < 0.001 versus apoA-I and versus the chimera) (Fig. 9). At 4 h postinjection HDL2,3- associated fractions were 0.43 ± 0.04, 0.19 ± 0.02 (p < 0.001 versus apoA-I) and 0.08 ± 0.006 (p < 0.001 versus apoA-I and versus the chimera), respectively (Fig. 9). At 24 h postinjection, HDL2,3- associated fractions were 0.34 ± 0.05, 0.14 ± 0.02 (p = 0.004 versus apoA-I), and 0.06 ± 0.008 (p < 0.001 versus apoA-I and p = 0.004 versus the chimera), respectively (Fig. 9). Estimated half-lives of HDL2,3-associated protein were 2,200 ± 130 min for apoA-I, 2,500 ± 300 min (p not significant) for the chimera and 2,400 ± 240 min (p not significant) for the deletion mutant, which are very similar to the values of t1/2γ. At 20 min postinjection, VHDL- associated fractions were 0.15 ± 0.006, 0.25 ± 0.03 (p = 0.008 versus apoA-I) and 0.27 ± 0.03 (p = 0.003 versus apoA-I, and p not significant versus the chimera), respectively (Fig. 9). At 4 h postinjection, corresponding fractions were 0.05 ± 0.005, 0.08 ± 0.01 (p = 0.002 versus apoA-I), and 0.10 ± 0.012 (p = 0.003 versus apoA-I and p not significant versus the chimera) (Fig. 9). At 24 h postinjection, fractions were 0.02 ± 0.002, 0.04 ± 0.007 (p = 0.02 versus apoA-I), and 0.04 ± 0.008 (p = 0.04 versus apoA-I and p not significant versus the chimera) (Fig. 9). Estimated half-lives of VHDL-associated proteins were 260 ± 45 min for apoA-I, 240 ± 35 min (p not significant) for the chimera, and 280 ± 25 min (p not significant) for the deletion mutant, which are very similar to the values of t1/2β. Fractions that were not associated with lipoproteins at 20 min postinjection and that most probably were cleared as free apolipoproteins were 0.30 ± 0.04 for apoA-I, 0.50 ± 0.06 (p = 0.02 versus apoA-I) for the chimera, and 0.64 ± 0.07 (p = 0.002 versus apoA-I and p not significant versus the chimera) for the deletion mutant.Fig. 9HDL2,3-associated (right panel) and VHDL-associated fractions (left panel) as a function of time. Black bars, ApoA-I; white bars, apoA-I (Ala190-Gln243); gray bars, apoA-I (Asp1-Leu189)/apoA-II (Ser12-Gln77).View Large Image Figure ViewerDownload (PPT) Incubation of 50-µl aliquots of radiolabeled HDL3, containing 0.8 µg of radiolabeled apoA-I, with 50 µl aliquots containing increasing amounts (0.24, 0.48, 0.96, 1.92, 3.84, 7.68, or 15.36 µg) of apoA-I, the chimera, or the deletion mutant resulted in a concentration-dependent displacement of radiolabeled apoA-I from the surface of HDL. Fifty percent displacement was obtained with 0.9 µg of apoA-I, 3.0 µg of the chimera, and 8.7 µg of the deletion mutant, thus at 1:1, 3:1, and 12:1 molar ratios of added protein to HDL3-associated 125I-labeled apoA-I, respectively (Fig. 10). These data indicate that the affinity for HDL3 of 125I-labeled apoA-I was identical to that of nonlabeled apoA-I, whereas the affinities of, respectively, the chimera and the deletion mutant were 3- and 12-fold lower. The variability in HDL cholesterol levels is largely determined by differences in the fractional catabolic rate of apoA-I, which is inversely correlated with HDL particle size (39Ikewaki K. Rader D.J. Schaeffer J.R. Fairwell T. Zech L.A. Brewer Jr., H.B. J. Lipid Res. 1993; 34: 2207-2215Google Scholar). In order to analyze the role of the carboxyl-terminal domain of apoA-I in phospholipid binding, lipoprotein association, HDL particle size distribution, and clearance, mutants of apoA-I have been generated. In the present study, the in vitro and in vivo lipoprotein binding properties of apoA-I, an apoA-I (ΔAla190-Gln243) deletion mutant, and an apoA-I (Asp1-Leu189)/apoA-II (Ser12-Gln77) chimera were compared. In the apoA-I (Asp1-Leu189)/apoA-II (Ser12-Gln77) chimera, the Ala190-Gln243 carboxyl-terminal domain of apoA-I was substituted with the Ser12-Lys28 and Pro51-Val68α-helical segments of apoA-II. Previously, it has been shown that synthetic peptides overlapping with these apoA-II sequences associated with phospholipids, suggesting that these helical segments constitute phospholipid binding domains (40Chen T.C. Sparrow J.T. Gotto Jr., A.M. Morrisett J.D. Biochemistry. 1979; 18: 1617-1622Google Scholar, 41Mao S.J.T. Sparrow J.T. Gilliam F.B. Gotto Jr., A.M. Jackson R.L. Biochemistry. 1977; 16: 4150-4156Google Scholar). Furthermore, epitope-mapping studies showed that antibodies to the carboxyl-terminal domain of apoA-I bound to an epitope in the Gln36-Gln77 segment of apoA-II, demonstrating significant structural homology between these domains (42Allan C.M. Fidge N.H. Kanellos J. J. Biol. Chem. 1992; 267: 13257-13261Google Scholar). Finally, apoA-II can displace apoA-I from the surface of recombinant HDL particles without loss of phospholipids or major change in particle size (43Rye K.A. Biochim. Biophys. Acta. 1990; 1042: 227-236Google Scholar). 125I-Labeled apoA-I associated preferentially (60%) with HDL2 and HDL3 particles both in rabbit and human plasma. Deletion of the Ala190-Gln243 carboxyl-terminal domain of apoA-I did not reduce the extent (80%) of in vitro lipoprotein association in rabbit and human plasma under equilibrium conditions but altered its distribution profile. Only 15% of the apoA-I (Ala190-Gln243) deletion mutant associated with HDL2,3 particles. Although the predicted secondary structure and the amphipathicity of the chimera were very similar to those of apoA-I, only 30% of the chimera associated with HDL2,3 particles. Because lipoprotein distribution profiles in human and rabbit plasma were very similar, rabbits were used as model animals to investigate the effects of changed lipoprotein distribution of both the chimera and the deletion mutant on their pharmacokinetic properties. Following bolus injection of 125I-labeled apoA-I in rabbits, it was cleared with a rate of 0.028 ml·min−1. This value is very similar to that determined by Ikewaki et al. (39Ikewaki K. Rader D.J. Schaeffer J.R. Fairwell T. Zech L.A. Brewer Jr., H.B. J. Lipid Res. 1993; 34: 2207-2215Google Scholar) following bolus injection of either exogenously or endogenously labeled apoA-I in humans. Deletion of the carboxyl-terminal domain of apoA-I resulted in a 13.6-fold increased clearance rate. This was most probably due to an enhanced clearance in the α-phase (64% as compared with 30% for apoA-I) of free apolipoprotein in solution, suggesting a slower rate of in vivo lipoprotein association of the deletion mutant. These data are in agreement with earlier findings that deletion of the carboxyl-terminal domain of apoA-I reduced the rate of in vitro phospholipid binding (25Holvoet 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: 13334-13342Google Scholar). Accelerated clearance may further result from a decreased association with HDL2,3 particles (9% as compared with 55% for apoA-I) and from an increased association with smaller, phospholipid-rich VHDL particles (30% as compared with 15% for apoA-I) that were cleared more rapidly from the circulation. Indeed, estimated half-lives of HDL2,3 particles were approximately 2,200 min as compared with approximately 260 min for VHDL particles. These data are in agreement with earlier findings of Schmidt et al. (30Schmidt H.H.J. Remaley A.T. Stonik J.A. Ronan R. Wellmann A. Thomas F. Zech L.A. Brewer Jr., H.B. Hoeg J.M. J. Biol. Chem. 1995; 270: 5469-5475Google Scholar) that progressive carboxyl-terminal domain truncation of apoA-I resulted in a progressive increase of its clearance rate, resulting from an increased association with VHDL. In aggregate, these data suggest that deletion of the carboxyl-terminal domain of apoA-I correlated with a decreased rate of lipoprotein association and/or increased rate of dissociation and a preferential association with smaller, phospholipid-rich VHDL particles that are cleared more rapidly. The plasma clearance rate of the chimera was 3.6-fold faster than that of apoA-I but was 3.8-fold slower than that of the deletion mutant. The differences between the chimera and apoA-I could be explained by a decreased rate of lipoprotein association, decreased association with HDL2,3 particles (25% as compared with 55% for apoA-I), and increased association with VHDL (25% as compared with 15%). The differences between the chimera and the deletion mutant could be explained essentially by an increased association of the chimera with HDL2,3 particles (25% as compared with 9%). The finding that α-, β- and γ-half-lives of all apolipoproteins were very similar and corresponded with half-lives of free apolipoprotein, VHDL, and HDL2,3 particles, respectively, indicated that the differences in clearance rate indeed could be explained by differences in distribution profile. These differences in distribution profile could be explained by a 3-fold (for the chimera) and a 12-fold (for the deletion mutant) lower affinity for HDL3 particles. Cross-linking experiments demonstrated that the self-association of both the deletion mutant and the chimera were lower than that of apoA-I. Human apoA-II self-associates in lipid-free solution (44Vitello L.B. Scanu A.M. J. Biol. Chem. 1976; 251: 1131-1136Google Scholar, 45Pownall H.J. Hickson D. Gotto Jr., A.M. J. Biol. Chem. 1981; 256: 9849-9854Google Scholar), resulting in dimerization of the disulfide-linked monomer, but it does not seem to aggregate in a monomer-dimer-tetramer fashion as does apoA-I. It has been suggested that this greater ability of apoA-I to self-associate may shield hydrophobic surfaces within the molecule better and allow apoA-I to be thermodynamically stable when not bound to lipid (46Loeb J. Dawson G. Mol. Cell. Biochem. 1983; 52: 161-176Google Scholar). On the other hand, apoA-II must bind to lipid in order to form a stable entity and will therefore bind to a more diverse group of lipids (46Loeb J. Dawson G. Mol. Cell. Biochem. 1983; 52: 161-176Google Scholar). Despite the lack of self-association of the chimera (Fig. 3), the lipoprotein affinity of this molecule was lower than that of apoA-I (Fig. 10). It seems unlikely that the decreased lipoprotein affinity of the chimera results from a stronger intramolecular association of the apoA-II-(51-68) helix with the apoA-II-(12-28) helix relative to that between the apoA-I-(223-239) and apoA-I-(190-206) helices because this putative association does not affect the lipoprotein association of apoA-II. An alternative explanation is that although the carboxyl-terminal domain of apoA-I is important for its initial rapid binding to the lipid surface of HDL, cooperative interactions with the middle six amphipathic helices of apoA-I may be important for its HDL subspecies distribution. Recently such a model has been proposed (47Palgunachari M. Mishra V.K. Lund-Katz S. Phillips M.C. Adeyeye S.O. Anantharamaiah G.M. Segrest J.P. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 328-338Google Scholar) on the basis of the finding that although only the end 22-residue helices of apoA-I have significant lipid affinity, apoA-I is, on a molar basis, about 10 times more effective than the most effective 22-residue peptide in reducing the enthalpy of the gel-to-liquid crystalline phase transition of L-α-dimyristoylphosphatidylcholine multilamellar vesicles. In summary, deletion of the carboxyl-terminal domain of apoA-I results in a significant decrease of its lipoprotein affinity, a reduction of its HDL3 association, and thus a more rapid clearance. Although substitution of this domain with helices of apoA-II restores the number of amphipathic helices and increases the hydrophobicity of the carboxyl-terminal domain, it does not restore completely its lipoprotein affinity and its HDL3 association, possibly because this substitution affects cooperative interactions with the middle amphipathic helices of apoA-I that are critical for its specific distribution over the HDL subspecies. We thank Ignace Lasters (Vlaams Instituut voor Biotechnologie) for helpful discussions and Els Brouwers, Frans De Cock, Eddy Demarsin, Michèle Landeloos, and Jean-Marie Stassen for technical assistance." @default.
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• W1964558380 title "Effects of Deletion of the Carboxyl-terminal Domain of ApoA-I or of Its Substitution with Helices of ApoA-II on in Vitro and in Vivo Lipoprotein Association" @default.
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