The Effect of Apolipoprotein A-II on the Structure and Function of Apolipoprotein A-I in a Homogeneous Reconstituted High Density Lipoprotein Particle

Abstract

In this study we examined the effects of apoA-II on the structure and function of apoA-I in homogeneous reconstituted HDL (rHDL). First, we measured the binding of apoA-II to apoA-I-rHDL, containing dipalmitoylphosphatidylcholine or palmitoyloleoylphosphatidylcholine, and the degree of apoA-I displacement at various ratios of apolipoproteins. Using fluorescence methods, we determined that apoA-II binding is rapid, irreversible, and associated with apoA-I displacement only when the molar ratio of apoA-II/apoA-I is greater than 1:2. Next, we used the stable apoA-II/apoA-I-rHDL complex at the apoA-II/apoA-I ratio of 1:2 to examine its physical properties, apoA-I structure, and reactivity with lecithin: cholesterol acyltransferase (LCAT). Using chemical cross-linking in conjunction with fluorescence and electrophoretic methods, we demonstrated that the conformation of apoA-I must be flexible to allow apoA-II binding to the apoA-I-rHDL particles and showed that the hybrid particles have an unchanged Stokes diameter. Fluorescence and circular dichroism measurements revealed little or no change in the secondary structure or in the N-terminal domain of apoA-I, but showed a marked destabilization of apoA-I to denaturation by guanidine hydrochloride. Limited tryptic digestion indicated that the central region of apoA-I becomes accessible to proteolysis in the hybrid particles. Together, these results suggest that amphipathic ␣-helices of apoA-II replace four central helices of one apoA-I molecule (residues ϳ99 -187) in the complex and in the process destabilize apoA-I. Thus, apoA-II binding at physiologic ratios may not completely displace apoA-I from HDL, but may provide a reservoir of easily exchangeable apoA-I. Finally, we showed that the reaction of the hybrid HDL with LCAT was inhibited 2-5-fold, relative to apoA-I-rHDL, due to a corresponding increase in the apparent K m value. This suggests that LCAT binding to the hybrid particles is sterically hindered by the excess protein (portions of apoA-I and apoA-II not bound to lipid). Therefore, apoA-II can modulate the reaction of HDL with LCAT by decreasing LCAT binding to hybrid particles and making the enzyme available for reaction with other substrates.

A Reconstituted High Density Lipoprotein Containing the V156E Mutant of Apolipoprotein A-I Exhibits Anti-Atherosclerotic Activity in Apo-E Deficient Mice

“…Although the exact mechanism remains to be elucidated, this result suggests that V156E-rHDL might be more resistant to protein degradation, with a putative interaction with LDL or displacement of apoA-by the addition of the apoA-helix domain into apoAhelix. Similarly, Durbin and Jonas 36) proposed that 4 helices of apoA-between residues 99 and 187 are displaced from phospholipid acyl chains by insertion of 4 helical domains of apoA-. This result is in good agreement with our previous observation that V156E-rHDL was resistant to particle rearrangement in the presence of LDL and showed different cleavage patterns by mild proteolysis 14,15) .…”

Graded Effects of Proteinuria on HDL Structure in Nephrotic Rats

“…ApoA-II also inhibits LCAT activity (42,43) and cholesterol ester transfer protein (44) but increases hepatic lipase activity (45). Whereas increased hepatic lipase activity is associated with reduced atherogenic risk, reduced LCAT is pivotal in the maturation of HDL in this model.…”

Helical domains that mediate lipid solubilization and ABCA1-specific cholesterol efflux in apolipoproteins C-I and A-II

“…A background scan of phosphate buffer was subtracted from each sample scan. Plasma apoA-II from normolipidemic patients was isolated and purifi ed as previously described for apoA-I ( 20 ).…”