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

We examined the effect of lipid-free apolipoprotein A-I (apoA-I) and apoA-I I on the structure of reconstituted high density lipoproteins (rHDL) and on their reactivity as substrates for lecithin:cholesterol acyltransferase (L CAT), First, homogeneous rHDL were prepared with either apoA-I or apoA-II u sing palmitoyloleoylphosphatidyl-choline (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 th e lipid-free apolipoproteins to the rHDL particles in a 1:1 molar ratio. Fo r both apolipoproteins, the binding to rHDL was rapid, occurring within 5 m in. Next, the effect on rHDL structure and particle size was determined aft er incubations of lipid-free apolipoproteins with homogeneous rHDL at 37 de grees C from 0.5 to 24 h. The products were analyzed by nondenaturing gradi ent gel electrophoresis followed by Western blotting, The effect of apoA-I or apoA-II on 103 Angstrom A-I-POPCrHDL was a rearrangement into 78 Angstro m particles containing apoA-I and/or apoA-II, and 90 Angstrom particles con taining only apoA-II, The effect of apoA-I or apoA-II on 98 Angstrom A-I-PO PCrHDL was a rearrangement into complexes ranging in size from 78 Angstrom to 105 Angstrom containing apoA-I and/or apoA-II, with main particles of 78 Angstrom, 88 Angstrom, and 98 Angstrom. Finally, the effect of lipid-free apoA-I and apoA-II on rHDL as substrates for LCAT was determined. The addit ion 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 a pparent K-m, while the addition of apoA-II to A-II-POPCrHDL had no effect o n 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 inhib ition was due to a 2-fold increase in apparent K-m; there was no significan t change in apparent V-max. Likewise, the addition of apoA-I to A-I-POPCrHD L 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 p romote the remodeling of rHDL into hybrid particles of primarily smaller si ze. Both apoA-I and apoA-II affect the reactivity of rHDL with LCAT, when a dded to the reaction in Lipid-free form. These results have important impli cations for the roles of Lipid-free apoA-I and apoA-II in HDL maturation an d metabolism.