Arg123-Tyr166 domain of human ApoA-I is critical for HDL-mediated inhibition of macrophage homing and early atherosclerosis in mice.

Atherosclerosis was studied in apolipoprotein E (apoE) knockout mice expressing human apolipoprotein A-I (apoA-I) or an apoA-I/apolipoprotein A-II (apoA-II) chimera in which the Arg123-Tyr166 central domain of apoA-I was substituted with the Ser12-Ala75 segment of apoA-II. High density lipoprotein (HDL) cholesterol levels were identical in apoA-I and apoA-I/apoA-II mice, but at 4 months, plaques were 2.7-fold larger in the aortic root of the apoA-I/apoA-II mice (P<0.01). The macrophage-to-smooth muscle cell ratio of lesions was 2.1-fold higher in apo-I/apoA-II mice than in apoA-I mice (P<0.01). This was due to a 2.7-fold higher (P<0.001) in vivo macrophage homing in the aortic root of apoA-I/apoA-II mice. Plasma platelet-activating factor acetyl hydrolase activity was lower (P<0.01) in apoA-I/apoA-II mice, resulting in increased oxidative stress, as evidenced by the higher titer of antibodies against oxidized low density lipoprotein (P<0.01). Increased oxidative stress resulted in increased stimulation of ex vivo macrophage adhesion by apoA-I/apoA-II beta-very low density lipoprotein and decreased inhibition of beta-very low density lipoprotein-induced adhesion by HDL from apoA-I/apoA-II mice. The cellular cholesterol efflux capacity of HDL from apoA-I/apoA-II mice was very similar to that of apoA-I mice. Thus, the Arg123-Tyr166 central domain of apoA-I is critical for reducing oxidative stress, macrophage homing, and early atherosclerosis in apoE knockout mice independent of its role in HDL production and cholesterol efflux.

Substitution of the carboxyl-terminal domain of apo AI with apo AII sequences restores the potential of HDL to reduce the progression of atherosclerosis in apo E knockout mice.

HDL metabolism and atherosclerosis were studied in apo E knockout (KO) mice overexpressing human apo AI, a des- (190-243)-apo AI carboxyl-terminal deletion mutant of human apo AI or an apo AI-(1-189)-apo AII-(12-77) chimera in which the carboxyl-terminal domain of apo AI was substituted with the pair of helices of apo AII. HDL cholesterol levels ranked: apo AI/apo E KO approximately apo AI-(1-189)-apo AII- (12-77)/apo E KO > > des-(190-243)-apo AI/apo E KO > apo E KO mice. Progression of atherosclerosis ranked: apo E KO > des-(190-243)-apo AI/apo E KO > > apo AI-(1-189)- apo AII-(12-77)/apo E KO approximately apo AI/apo E KO mice. Whereas the total capacity to induce cholesterol efflux from lipid-loaded THP-1 macrophages was higher for HDL of mice overexpressing human apo AI or the apo AI/apo AII chimera, the fractional cholesterol efflux rate, expressed in percent cholesterol efflux/microg apolipoprotein/h, for HDL of these mice was similar to that for HDL of mice overexpressing the deletion mutant and for HDL of apo E KO mice. This study demonstrates that the tertiary structure of apo AI, e.g., the number and organization of its helices, and not its amino sequence is essential for protection against atherosclerosis because it determines HDL cholesterol levels and not cholesterol efflux. Amino acid sequences of apo AII, which is considered to be less antiatherogenic, can be used to restore the structure of apo AI and thereby its antiatherogenicity.

Role of the Arg123-Tyr166 paired helix of apolipoprotein A-I in lecithin:cholesterol acyltransferase activation.

The Arg123-Tyr166 central 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(Delta(Arg123-Tyr166), nablaA-II(Ser12-Ala75)) and apoA-I(Delta(Ala190-Gln243), nablaA-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 alpha-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 versus apoA-I), and 1.3 +/- 0.32 (p = not significant versus apoA-I) nmol of cholesteryl ester/h/microM, respectively. The lower LCAT activity of the central domain chimera was due to a 27-fold reduced Vmax with unaltered Km. Binding of radiolabeled LCAT to rHDL of apoA-I and apoA-I(Delta(Arg123-Tyr166), nablaA-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.

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.

In the present study, the lipoprotein association of apoA-I, an apoA-I (DeltaAla190-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 microg/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.

Phospholipid binding and lecithin-cholesterol acyltransferase activation properties of apolipoprotein A-I mutants.

Recombinant human apolipoprotein A-I (apo A-I) and three deletion mutants: apo A-I(delta Leu44-Leu126), apo A-I(delta Glu139-Leu170), and apo A-I(delta Ala190-Gln243), purified from the periplasmic space of Escherichia coli, were studied. The rate of turbidity decrease following mixing of apo A-I(delta Ala190-Gln243) with dimyristoylphosphatidylcholine (DMPC) vesicles at 23 degrees C was 10-fold lower than that of the other apo A-I proteins, confirming that the carboxy-terminal region of apo A-I plays a role in rapid lipid binding. The Stokes radii of reconstituted high-density lipoproteins (rHDL), containing dipalmitoylphosphatidylcholine and cholesterol, were larger for the three apo A-I mutants [6.3 nm for apo A-I(delta Leu44-Leu126), 6.1 nm for apo A-I(delta Glu139-Leu170), and 6.5 nm for apo A-I(delta Ala190-Gln243)] than for intact apo A-I (5.0 nm). The mutant rHDL all contained 4 apo A-I molecules per particle as compared to 2 for intact apo A-I. Circular dichroism measurements revealed 8 alpha-helices per apo A-I molecule, 5 per apo A-I(delta Leu44-Leu126), 6 per apo A-I(delta Glu139-Leu170), and 4 per apo A-I(delta Ala190-Gln243) molecule as compared to predicted values of 8, 5, 6, and 6 alpha-helices, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)