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This study elucidates the factors underlying the enhancement in efflux of human fibroblast unesterified cholesterol and phospholipid (PL) by lipid-free apolipoprotein (apo) A-I that is induced by cholesterol enrichment of the cells. Doubling the unesterified cholesterol content of the plasma membrane by incubation for 24 h with low density lipoprotein and lipid/cholesterol dispersions increases the pools of PL and cholesterol available for removal by apoA-I from about 0.8–5%; the initial rates of mass release of cholesterol and PL are both increased about 6-fold. Expression of the ATP binding cassette transporter A1 (ABCA1) is critical for this increased efflux of lipids, and cholesterol loading of the fibroblasts over 24 h increases ABCA1 mRNA about 12-fold. The presence of more ABCA1 and cholesterol in the plasma membrane results in a 2-fold increase in the level of specific binding of apoA-I to the cells with no change in binding affinity. Characterization of the species released from either control or cholesterol-enriched cells indicates that the plasma membrane domains from which lipids are removed are cholesterol-enriched with respect to the average plasma membrane composition. Cholesterol enrichment of fibroblasts also affects PL synthesis, and this leads to enhanced release of phosphatidylcholine (PC) relative to sphingomyelin (SM); the ratios of PC to SM solubilized from control and cholesterol-enriched fibroblasts are ∼2/1 and 5/1, respectively. Biosynthesis of PC is critical for this preferential release of PC and the enhanced cholesterol efflux because inhibition of PC synthesis by choline depletion reduces cholesterol efflux from cholesterol-enriched cells. Overall, it is clear that enrichment of fibroblasts with unesterified cholesterol enhances efflux of cholesterol and PL to apoA-I because of three effects, 1) increased PC biosynthesis, 2) increased PC transport via ABCA1, and 3) increased cholesterol in the plasma membrane.

Lipid-free apolipoprotein (apo) A-I contributes to the reverse transport of cholesterol from the periphery to the liver by solubilizing plasma membrane phospholipid and cholesterol. The features of the apolipoprotein required for this process are not understood and are addressed in the current study. Membrane microsolubilization of human fibroblasts is not specific for apo A-I; unlipidated apos A-II, C, and E incubated with the fibroblast monolayers at a saturating concentration of 50 micrograms/ml are all able to release cholesterol and phospholipid similarly. To determine the properties of the apolipoprotein that drive the process, apo A-I peptides spanning the entire sequence of the protein were utilized; the peptides correspond to the 11- and 22-residue amphipathic alpha-helical segments, as well as adjacent combinations of the helices. Of the 20 helical peptides examined, only peptides representing the N-and C-terminal portions of the protein had the ability to solubilize phospholipid and cholesterol. Cholesterol efflux to the most effective peptides, 44-65 and 209-241, was approximately 50 and 70%, respectively, of that to intact apo A-I. Deletion mutants of apo E and apo A-I were constructed that have reduced lipid binding affinities as compared with the intact molecule. The proteins, apo A-I (Delta222-243), apo A-I (Delta190-243), apo E3 (Delta192-299) and apo E4 (Delta192-299) all exhibited a decreased ability to remove cellular cholesterol and phospholipid. These decreases correlated with the reduced ability of these proteins to penetrate into a phospholipid monomolecular film. Overall, the results indicate that insertion of amphipathic alpha-helices between the plasma membrane phospholipid molecules is a required step in the mechanism of apolipoprotein-mediated cellular lipid efflux. Therefore the lipid binding ability of the apolipoprotein is critical for efficient membrane microsolubilization.

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.

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.

Human plasminogen, a glycoprotein with NH2-terminal Glu, is rapidly converted by traces of plasmin to proteolytic derivatives with NH2-terminal Met 68, Lys 77, or Val 78 (“Lys-plasminogen”), which are much more readily activated to plasmin than is Glu-plasminogen. It has, therefore, been proposed that physiological activation of Glu-plasminogen occurs mainly via Lys-plasminogen intermediates (Wiman, B., and Wallén, P. (1973) Eur. J. Biochem. 36, 25-31). In the present study we have characterized a murine monoclonal antibody (LPm1) directed against an epitope exposed in Lys-plasminogen but not in Glu-plasminogen. The antibody was secreted by a hybridoma obtained by fusion of mouse myeloma cells (P3X63-Ag8-6.5.3) with spleen cells of a mouse immunized with purified Lys-plasmin-alpha 2-antiplasmin complex. Coupling of the alpha-amino groups of Lys-plasminogen with phenylisothiocyanate resulted in complete loss of immunoreactivity for LPm1, which was, however, fully restored by cleavage of the derivatized NH2-terminal amino acid. After a second cycle, immunoreactivity was not restored, indicating that the LPm1 antibody-binding site depends on the presence of Lys 77 and/or Val 78 as NH2-terminal amino acids. The immunoreactivity of Lys-plasminogen with LPm1 is abolished by reduction of the protein, suggesting that conversion of Glu-plasminogen to Lys-plasminogen is associated with a conformational alteration exposing the epitope for the LPm1 monoclonal antibody. In order to investigate the pathways of plasminogen activation in vivo, total plasmin-alpha 2-antiplasmin and Lys-plasmin-alpha 2-antiplasmin complexes were measured with sandwich-type micro enzyme-linked immunosorbent assays. Therefore, microtiter plates were coated with monoclonal antibodies against alpha 2-antiplasmin, and bound antigen was quantitated with horseradish peroxidase-conjugated LPm1 or a monoclonal antibody reacting equally well with Glu-plasmin as with Lys-plasmin. In 25 healthy subjects the plasmin-alpha 2-antiplasmin levels in plasma were undetectable (less than 0.1 nM). Infusion of tissue-type plasminogen activator in patients with thromboembolic disease resulted in generation of high concentrations of Glu-plasmin-alpha 2-antiplasmin complex (620 +/- 150 nM, n = 7) whereas neither Lys-plasmin-alpha 2-antiplasmin complex nor Lys-plasminogen were consistently detected. It is, therefore, concluded that activation of the fibrinolytic system in vivo occurs by direct cleavage of the Arg 560-Val 561 bond in Glu-plasminogen and not via formation of the Lys-plasminogen intermediates.

The Arg123–Tyr166central and Ala190–Gln243carboxyl-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 coliand 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 significantversusapoA-I) nmol of cholesteryl ester/h/μm, respectively. The lower LCAT activity of the central domain chimera was due to a 27-fold reducedVmaxwith unaltered Km. 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–Tyr166central or Ala190–Gln243carboxyl-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.