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.
Binding of lecithin cholesterol acyltransferase (LCAT) to lipoprotein surfaces is a key step in the reverse cholesterol transport process, as the subsequent cholesterol esterification reaction drives the removal of cholesterol from tissues into plasma. In this study, the surface plasmon resonance method was used to investigate the binding kinetics and affinity of LCAT for lipoproteins. Reconstituted high-density lipoproteins (rHDL) containing apolipoprotein A-I or A-II, (apoA-I or apoA-II), low-density lipoproteins (LDL), and small unilamellar phosphatidylcholine vesicles, with biotin tags, were immobilized on biosensor chips containing streptavidin, and the binding kinetics of pure recombinant LCAT were examined as a function of LCAT concentration. In addition, three mutants of LCAT (T123I, N228K, and (Delta53-71) were examined in their interactions with LDL. For the wild-type LCAT, binding to all lipid surfaces had the same association rate constant, k(a), but different dissociation rate constants, k(d), that depended on the presence of apoA-I (k(d) decreased) and different lipids in LDL. Furthermore, increased ionic strength of the buffer decreased k(a) for the binding of LCAT to apoA-I rHDL. For the LCAT mutants, the Delta53-71 (lid-deletion mutant) exhibited no binding to LDL, while the LCAT-deficiency mutants (T123I and N228K) had nearly normal binding to LDL. In conclusion, the association of LCAT to lipoprotein surfaces is essentially independent of their composition but has a small electrostatic contribution, while dissociation of LCAT from lipoproteins is decreased due to the presence of apoA-I, suggesting protein-protein interactions. Also, the region of LCAT between residues 53 and 71 is essential for interfacial binding.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.