Abstract
The principal protein of high density lipoprotein (HDL), apolipoprotein (apo) A-I, in the lipid-free state contains two tertiary structure domains comprising an N-terminal helix bundle and a less organized C-terminal domain. It is not known how the properties of these domains modulate the formation and size distribution of apoA-I-containing nascent HDL particles created by ATP-binding cassette transporter A1 (ABCA1)-mediated efflux of cellular phospholipid and cholesterol. To address this issue, proteins corresponding to the two domains of human apoA-I (residues 1-189 and 190-243) and mouse apoA-I (residues 1-186 and 187-240) together with some human/mouse domain hybrids were examined for their abilities to form HDL particles when incubated with either ABCA1-expressing cells or phospholipid multilamellar vesicles. Incubation of human apoA-I with cells gave rise to two sizes of HDL particles (hydrodynamic diameter, 8 and 10 nm), and removal or disruption of the C-terminal domain eliminated the formation of the smaller particle. Variations in apoA-I domain structure and physical properties exerted similar effects on the rates of formation and sizes of HDL particles created by either spontaneous solubilization of phospholipid multilamellar vesicles or the ABCA1-mediated efflux of cellular lipids. It follows that the sizes of nascent HDL particles are determined at the point at which cellular phospholipid and cholesterol are solubilized by apoA-I; apparently, this is the rate-determining step in the overall ABCA1-mediated cellular lipid efflux process. The stability of the apoA-I N-terminal helix bundle domain and the hydrophobicity of the C-terminal domain are important determinants of both nascent HDL particle size and their rate of formation.
Highlights
high density lipoprotein (HDL) particle biogenesis involves an interaction between the ATP-binding cassette transporter A1 (ABCA1)2 and apolipoprotein A-I, the principal protein of HDL (10 –12)
Contributions of ApoA-I Tertiary Structure Domains to free (unesterified) cholesterol (FC) Efflux—As we have reported previously [28], the ABCA1-mediated efflux of FC from ABCA1-expressing J774 cells exhibits a hyperbolic dependence on the concentration of human apoA-I in the extracellular medium and conforms to the MichaelisMenten equation (Fig. 1A)
In the three-step reaction by which apoA-I and ABCA1 promote the efflux of cellular lipid and create nascent HDL particles, the last step involving the solubilization by apoA-I of plasma membrane PL and FC is apparently rate-limiting [15]
Summary
HDL particle biogenesis involves an interaction between the ATP-binding cassette transporter A1 (ABCA1) and apolipoprotein (apo) A-I, the principal protein of HDL (10 –12) This reaction involves the recruitment of cellular phospholipids (PLs) and free (unesterified) cholesterol (FC) to interact with apoA-I and form nascent HDL particles. The size of a discoidal HDL particle is dependent upon the number of apoA-I molecules located in it; smaller discs contain two apoA-I molecules, and larger particles contain up to four apoA-I molecules In addition to these quaternary structure effects, the tertiary structure of apoA-I (and other apolipoproteins that interact with ABCA1 to form HDL particles) is likely to play a role. We exploit the known differences in the tertiary structure domain properties of human and mouse apoA-I [24] to elucidate the contributions of the N-terminal helix bundle domain and the separately folded C-terminal domain [25] to the ABCA1-mediated efflux of cellular cholesterol and the determination of nascent HDL particle size heterogeneity
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