THE CARBOXYL-TERMINAL HYDROPHOBIC RESIDUES OF APOLIPOPROTEIN-A-I AFFECT ITS RATE OF PHOSPHOLIPID-BINDING AND ITS ASSOCIATION WITH HIGH-DENSITY-LIPOPROTEIN

We performed a series of mutations in the human apolipoprotein A-I (ap oA-I) gene designed to alter specific amino acid residues and domains implicated in lecithin: cholesterol acyltransferase (LCAT) activation or lipid binding. We used the mutant apoA-I forms to establish nine st able cell lines, and developed strategies for the large scale producti on and purification of the mutated apoA-I proteins from conditioned me dia. HDL and dimyristoyl phosphatidylcholine binding assays using the variant apoA-I forms have shown that replacement of specific carboxyl- terminal hydrophobic residues Leu(222), Phe(225), and Phe(229) with ly sines, as well as replacement of Leu(211), Leu(214), Leu(218), and Leu (219) with valines, diminished the ability of apoA-I to bind to HDL an d to lyse dimyristoyl phosphatidylcholine liposomes. The findings indi cate that Leu(222), and Phe(225), Phe(229) located in the putative ran dom coil region, and Leu(211), Leu(214), Leu(218), and Leu(219) locate d in the putative helix 8, are important for lipid binding. In contras t, substitutions of alanines for specific charged residues in putative helices 7, 8, or 9 as well as various point mutations in other region s of apoA-I, did not affect the ability of the variant apoA-I forms to bind to HDL or to lyse dimyristoyl phosphatidylcholine liposomes. Cro ss-linking experiments confirmed that the carboxyl-terminal domain of apoA-I participates in the self-association of the protein, as demonst rated by the inability of the carboxyl-terminal deletion mutants Delta 185-243 and Delta 209-243 to form higher order aggregates in solution . Lecithin:cholesterol acyltransferase analysis, using reconstituted H DL particles prepared by the sodium cholate dialysis method, has shown that mutants (Pro(165) --> Ala,Gln(173) --> Glu) (Leu(311) --> Val,Le u(214) --> Val,Leu(318) --> Val,Leu(319) --> Val), Leu(222) --> Lys,Ph e(255) --> Lys,Phe(290) --> Lys) and Delta 209-243 reduced LCAT activa tion (38-68%). Mutant (Glu(191) --> Ala,His(195) --> Ala,Lys(196) --> Ala) enhanced LCAT activation (131%), and mutant (Ala(162) --> Leu,Leu (189) --> Trp) exhibited normal LCAT activation as compared with the w ild type proapoA-I and plasma apoA-I forms. The apparent catalytic eff iciency (V-max(app)/K-m(app)) of the apoA-I mutants ranged from 17.8 t o 107.2% of the control and was the result of variations in both the K -m and the V-max in the different mutants. These findings indicate tha t putative helices 6 and 7, and the carboxyl-terminal helices 8 and 9 contribute to the optimum activation of lecithin:cholesterol acyltrans ferase. In addition to their use in the present study, the variant apo A-I forms generated will serve as valuable reagents for the identifica tion of the domains and residues of apoA-I involved in binding the sca venger receptor BI, and facilitating cholesterol efflux from cells as well as aid in the structural analysis of apoA-I.