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.

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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.

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The central region of apolipoprotein A-I (apoA-I), spanning residues 143-165, has been implicated in lecithin:cholesterol acyltransferase (LCAT) activation and also in high density lipoprotein (HDL) structural rearrangements. To examine the role of individual amino acids in these functions, we constructed, overexpressed, and purified two additional point mutants of apoA-I (P143R and R160L) and compared them with the previously studied V156E mutant. These mutants have been reported to occur naturally and to affect HDL cholesterol levels and cholesterol esterification in plasma. The P143R and R160L mutants were effectively expressed in Escherichia coli as fusion proteins and were isolated in at least 95% purity. In the lipid-free state, the mutants self-associated similarly to wildtype protein. All the mutants, including V156E, were able to lyse dimyristoylphosphatidylcholine liposomes. In the lipidbound state, the major reconstituted HDL (rHDL) of the mutants had diameters similar to wild type (96-98 Å). Circular dichroism and fluorescence methods revealed no major differences among the structures of the lipid-free or lipid-bound mutants and wild type. In contrast, the V156E mutant had exhibited significant structural, stability, and self-association differences compared with wild-type apoA-I in the lipid-free state, and formed rHDL particles with larger diameters. In this study, limited proteolytic digestion with chymotrypsin showed that the V156E mutant, in lipidfree form, has a distinct digestion pattern and surface exposure of the central region, compared with wild type and the other mutants. Reactivity of rHDL with LCAT was highest for wild type (100%), followed by P143R (39%) and R160L (0.6%). Tested for their ability to rearrange into 78-Å particles, the rHDL of the two mutants (P143R and R160L) behaved normally, compared with the rHDL of V156E, which showed no rearrangement after the 24-h incubation with low density lipoprotein (LDL). Similarly, the rHDL of V156E was resistant to rearrangement in the presence of apoA-I or apoA-II. These results indicate that structural changes are absent or modest for the P143R and R160L mutants, especially in rHDL form; that these mutants have normal conformational adaptability; and that LCAT activation is obliterated for R160L.Thus, individual amino acid changes may have markedly different structural and functional consequences in the 143-165 region of apoA-I. The R160L mutation appears to have a direct effect in LCAT activation, while the P143R mutation results in only minor structural and functional effects. Also, the processes for LCAT activation and hinge mobility appear to be distinct even if the same region of apoA-I is involved. -Cho, K-H., D. M. Durbin, and A. Jonas. Role of individual amino acids of apolipoprotein A-I in the activation of lecithin:cholesterol acyltransferase and in HDL rearrangements . J. Lipid Res. 2001. 42: 379-389. Supplementary key words lipoproteins • lipid binding • phospholipid • apoA-I mutants • circular dichroism • fluorescence • LCAT A...

We examined the effect of lipid-free apolipoprotein A-I (apoA-I) and apoA-II on the structure of reconstituted high density lipoproteins (rHDL) and on their reactivity as substrates for lecithin:cholesterol acyltransferase (LCAT). First, homogeneous rHDL were prepared with either apoA-I or apoA-II using palmitoyloleoylphosphatidylcholine (POPC) and cholesterol. Lipid-free apoA-I and apoA-II were labeled with the fluorescent probe dansyl chloride (DNS). The binding kinetics of apoA-I-DNS to A-II-POPCrHDL and of apoA-II-DNS to A-I-POPCrHDL were monitored by fluorescence polarization, adding the lipidfree apolipoproteins to the rHDL particles in a 1:1 molar ratio. For both apolipoproteins, the binding to rHDL was rapid, occurring within 5 min. Next, the effect on rHDL structure and particle size was determined after incubations of lipid-free apolipoproteins with homogeneous rHDL at 37 ؇ C from 0.5 to 24 h. The products were analyzed by nondenaturing gradient gel electrophoresis followed by Western blotting. The effect of apoA-I or apoA-II on 103 Å A-II-POPCrHDL was a rearrangement into 78 Å particles containing apoA-I and/or apoA-II, and 90 Å particles containing only apoA-II. The effect of apoA-I or apoA-II on 98 Å A-I-POPCrHDL was a rearrangement into complexes ranging in size from 78 Å to 105 Å containing apoA-I and/or apoA-II, with main particles of 78 Å, 88 Å, and 98 Å. Finally, the effect of lipid-free apoA-I and apoA-II on rHDL as substrates for LCAT was determined. The addition of apoA-I to A-II-POPCrHDL increased its reactivity with LCAT 24-fold, reflected by a 4-fold increase in apparent V max and a 6-fold decrease in apparent K m , while the addition of apoA-II to A-II-POPCrHDL had no effect on its minimal reactivity with LCAT. In contrast, the addition of apoA-II to A-I-POPCrHDL decreased the reaction with LCAT by about one-half. The inhibition was due to a 2-fold increase in apparent K m ; there was no significant change in apparent V max . Likewise, the addition of apoA-I to A-I-POPCrHDL inhibited the reaction with LCAT to about two-thirds that of A-I-POPCrHDL without added apoA-I. In summary, both lipid-free apoA-I and apoA-II can promote the remodeling of rHDL into hybrid particles of primarily smaller size. Both apoA-I and apoA-II affect the reactivity of rHDL with LCAT, when added to the reaction in lipid-free form. These results have important implications for the roles of lipid-free apoA-I and apoA-II in HDL maturation and metabolism. -Durbin, D. M., and A. Jonas. Lipid-free apolipoprotein A-I and A-II promote remodeling of reconstituted high density lipoproteins and alter their reactivity with lecithin:cholesterol acyltransferase.

Interactions of apolipoprotein A-I (apoA-I) with cell membranes appear to be important in the initial steps of reverse cholesterol transport. The objective of this work was to examine the effect of three distinct conformations of apoA-I (lipid-free and in 78 Å or 96 Å reconstituted high density lipoproteins, rHDL) on its ability to bind to, and abstract lipids from, palmitoyl oleoyl phosphatidylcholine membrane vesicles (small unilamellar vesicles, SUV, and giant unilamellar vesicles, GUV). The molecular interactions were observed by two-photon fluorescence microscopy, and the binding parameters were quantified by gel-permeation chromatography or isothermal titration microcalorimetry. Rearrangement of apoA-I-containing particles after exposure to SUVs was examined by native gel electrophoresis. The results indicate that lipid-free apoA-I binds reversibly, with high affinity, to the vesicles but does not abstract a significant amount of lipid nor perturb the vesicle structure. The 96 Å rHDL, where all the amphipathic helices of apoA-I are saturated with lipid within the particles, do not bind to vesicles or perturb their structure. In contrast, the 78 Å rHDL have a region of apoA-I, corresponding to a few amphipathic helical segments, which is available for external or internal phospholipid binding. These particles bind to vesicles with measurable affinity (lower than lipid-free apoA-I), abstract lipids from the membranes, and form particles of larger diameters, including 96 Å rHDL. We conclude that the conformation of apoA-I regulates its binding affinity for phospholipid membranes and its ability to abstract lipids from the membranes. -Tricerri, M. A