Lipid-free apolipoproteins A-I and A-II promote remodeling of reconstituted high density lipoproteins and alter their reactivity with lecithin:cholesterol acyltransferase

Abstract

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.

Role of apoA-II in lipid metabolism and atherosclerosis: advances in the study of an enigmatic protein

“…LCAT reactivity is higher in HDL reconstituted with apoA-I than in those reconstituted with apoA-II (42). This is due to the ability of apoA-II or its C-terminal end to displace apoA-I (at high apoA-II/apoA-I ratios) or to destabilize apoA-I (at the physiological apoA-II/apoA-I ratio of 0.5:1), which, in the latter case, becomes more easily exchangeable although this would not result in HDL particle rearrangement (41,42,65). This has been attributed, depending on the study, to a decrease in K m or to a de-crease in V max (41,42,65).…”

“…As analyzed in detail below, HDL remodeling may be determined in part by the influence of apoA-II on the reactivity toward lipid transfer proteins, enzymes, and the receptors involved in HDL metabolism and, also, by its ability to displace apoA-I from the HDL surface even though this changes HDL reactivity toward one of the above-mentioned enzymes, LCAT. The latter effect has been described in vitro in both isolated and reconstituted HDL (40)(41)(42). Studying the effect of overexpression, or knocking out, of apoA-II in genetically modified mice has provided insight into the speciesspecific role of this apolipoprotein in HDL remodeling.…”

“…This is due to the ability of apoA-II or its C-terminal end to displace apoA-I (at high apoA-II/apoA-I ratios) or to destabilize apoA-I (at the physiological apoA-II/apoA-I ratio of 0.5:1), which, in the latter case, becomes more easily exchangeable although this would not result in HDL particle rearrangement (41,42,65). This has been attributed, depending on the study, to a decrease in K m or to a de-crease in V max (41,42,65). This could indicate that LCAT binding is sterically hindered by portions of apoA-I and apoA-II and would not bind to HDL lipids (41,42,65).…”

“…This has been attributed, depending on the study, to a decrease in K m or to a de-crease in V max (41,42,65). This could indicate that LCAT binding is sterically hindered by portions of apoA-I and apoA-II and would not bind to HDL lipids (41,42,65). Therefore, apoA-II could modulate the reaction of HDL with LCAT by decreasing binding to LpA-I/A-II and making the enzyme available for reaction with other lipoprotein particles (65).…”

Novel Changes in Discoidal High Density Lipoprotein Morphology: A Molecular Dynamics Study

“…This interaction results in remodeling of the spheroidal HDL particle, a process that regenerates lipidpoor HDL. Also, plasma enzymes and transfer proteins remodel lipid-poor and discoidal HDL particles (11,12). Thus a fuller understanding of reverse cholesterol transport demands knowledge of the detailed structure of the various HDL particles and the intermediates in their assembly.…”