Abstract
We have mapped the domains of lipid-free apoA-I that promote cAMP-dependent and cAMP-independent cholesterol and phospholipid efflux. The cAMP-dependent lipid efflux in J774 mouse macrophages was decreased by approximately 80-92% by apoA-I[delta(185-243)], only by 15% by apoA-I[delta(1-41)] or apoA-I[delta(1-59)], and was restored to 75-80% of the wild-type apoA-I control value by double deletion mutants apoA-I[delta(1-41)delta(185-243)] and apoA-I[delta(1-59)delta(185-243)]. Similar results were obtained in HEK293 cells transfected with an ATP-binding cassette transporter A1 (ABCA1) expression plasmid. The double deletion mutant of apoA-I had reduced thermal and chemical stability compared with wild-type apoA-I. Sequential carboxyl-terminal deletions showed that cAMP-dependent cholesterol efflux was diminished in all the mutants tested, except the apoA-I[delta(232-243)] which had normal cholesterol efflux. In cAMP-untreated or in mock-transfected cells, cholesterol efflux was not affected by the amino-terminal deletions, but decreased by 30-40% and 50-65% by the carboxyl-terminal and double deletions, respectively. After adenovirus-mediated gene transfer in apoA-I-deficient mice, wild-type apoA-I and apoA-I[delta(1-41)] formed spherical high density lipoprotein (HDL) particles, whereas apoA-I[delta(1-41)delta(185-243)] formed discoidal HDL. The findings suggest that although the central helices of apoA-I alone can promote ABCA1-mediated lipid efflux, residues 220-231 are necessary to allow functional interactions between the full-length apoA-I and ABCA1 that are required for lipid efflux and HDL biogenesis.
Highlights
We have mapped the domains of lipid-free Apolipoprotein A-I (apoA-I) that promote cAMP-dependent and cAMP-independent cholesterol and phospholipid efflux
Effect of Amino, Carboxyl-terminal and Double Deletion Mutants of ApoA-I on cAMP-dependent and cAMP-independent Efflux of Cellular Cholesterol from J774 Mouse Macrophages and the Influence of the His Tag—To determine the potential domains of apoA-I that contribute to cholesterol efflux, J774 mouse macrophages were labeled with [3H]cholesterol for 24 h and incubated in serum-free culture medium for 24 h in the presence or absence of 0.3 mM of the cAMP analog cptcAMP. cAMP analogs have been reported to enhance the expression of ATP-binding cassette transporter A1 (ABCA1) (18, 58, 59)
The apoA-I concentration used is higher than that required for saturable cholesterol or phospholipid efflux in untreated or cpt-cAMP-treated J774 macrophages (60) and in transiently or stably transfected cells expressing the ABCA1 transporter (27, 28)
Summary
We have mapped the domains of lipid-free apoA-I that promote cAMP-dependent and cAMP-independent cholesterol and phospholipid efflux. The findings suggest that the central helices of apoA-I alone can promote ABCA1-mediated lipid efflux, residues 220 – 231 are necessary to allow functional interactions between the full-length apoA-I and ABCA1 that are required for lipid efflux and HDL biogenesis. In the present study we have analyzed the ability of various recombinant apoA-I mutants lacking amino- and carboxyl-terminal domains to promote efflux of cellular cholesterol and phospholipid from J774 mouse macrophages in the presence of a cAMP analog (i.e. ABCA1-catalyzed) or in the absence of a cAMP analog (i.e. at least partially independent of ABCA1). The analysis of apoA-I mutants containing deletions in the amino-terminal region, carboxyl-terminal region, or both domains suggests that there is ligand specificity of the full-length apoA-I, the central helices of apoA-I alone are capable of promoting ABCA1-mediated lipid efflux and the formation of discoidal HDL in vitro and in vivo
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