Apolipoprotein A-I (apoA-I) accepts cholesterol and phospholipids from ATP-binding cassette transporter A1 (ABCA1)-expressing cells to form high-density lipoprotein (HDL). Human apoA-I has two tertiary structural domains and the C-terminal domain (approximately amino acids 190-243) plays a key role in lipid binding. Although the high lipid affinity region of the C-terminal domain of apoA-I (residues 223-243) is essential for the HDL formation, the function of low lipid affinity region (residues 191-220) remains unclear. To evaluate the role of residues 191-220, we analyzed the structure, lipid binding properties, and HDL formation activity of Δ191-220 apoA-I, in comparison to wild-type and Δ223-243 apoA-I. Although deletion of residues 191-220 has a slight effect on the tertiary structure of apoA-I, the Δ191-220 variant showed intermediate behavior between wild-type and Δ223-243 regarding the formation of hydrophobic sites and lipid interaction through the C-terminal domain. Physicochemical analysis demonstrated that defective lipid binding of Δ191-220 apoA-I is due to the decreased ability to form α-helix structure which provides the energetic source for lipid binding. In addition, the ability to form HDL particles in vitro and induce cholesterol efflux from ABCA1-expressing cells of Δ191-220 apoA-I was also intermediate between wild-type and Δ223-243 apoA-I. These results suggest that despite possessing low lipid affinity, residues 191-220 play a role in enhancing the ability of apoA-I to bind to and solubilize lipids by forming α-helix upon lipid interaction. Our results demonstrate that the combination of low lipid affinity region and high lipid affinity region of apoA-I is required for efficient ABCA1-dependent HDL formation.
Keywords: 8-anilino-1-naphthalenesulfonic acid; ABC; ABCA1; ANS; ATP-binding cassette; ApoA-I; BHK; BSA; CD; Cholesterol; DMEM; DMPC; Dulbecco's modified Eagle's medium; FBS; GdnHCl; HDL; ITC; MLV; PBS; PC; SUV; UV; apo; apolipoprotein; baby hamster kidney; bovine serum albumin; circular dichroism; dimyristoyl phosphatidylcholine; fetal bovine serum; guanidine hydrochloride; high-density lipoprotein; isothermal titration calorimetry; multilamellar vesicle; phosphate-buffered saline; phosphatidylcholine; small unilamellar vesicle; ultraviolet.
© 2013.