The Carboxyl-terminal Hydrophobic Residues of Apolipoprotein A-I Affect Its Rate of Phospholipid Binding and Its Association with High Density Lipoprotein (1997)

We performed a series of mutations in the human apolipoprotein A-I (apoA-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 stable cell lines, and developed strategies for the large scale production and purification of the mutated apoA-I proteins from conditioned media. HDL and dimyristoyl phosphatidylcholine binding assays using the variant apoA-I forms have shown that replacement of specific carboxyl-terminal hydrophobic residues Leu<sup>222</sup>, Phe<sup>225</sup>, and Phe<sup>229</sup> with lysines, as well as replacement of Leu<sup>211</sup>, Leu<sup>214</sup>, Leu<sup>218</sup>, and Leu<sup>219</sup> with valines, diminished the ability of apoA-I to bind to HDL and to lyse dimyristoyl phosphatidylcholine liposomes. The findings indicate that Leu<sup>222</sup>, and Phe<sup>225</sup>, Phe<sup>229</sup> located in the putative random coil region, and Leu<sup>211</sup>, Leu<sup>214</sup>, Leu<sup>218</sup>, and Leu<sup>219</sup> located in the putative helix 8, are important for lipid binding. In contrast, substitutions of alanines for specific charged residues in putative helices 7, 8, or 9 as well as various point mutations in other regions of apoA-I, did not affect the ability of the variant apoA-I forms to bind to HDL or to lyse dimyristoyl phosphatidylcholine liposomes. Cross-linking experiments confirmed that the carboxyl-terminal domain of apoA-I participates in the self-association of the protein, as demonstrated by the inability of the carboxyl-terminal deletion mutants Δ185–243 and Δ209–243 to form higher order aggregates in solution. Lecithin:cholesterol acyltransferase analysis, using reconstituted HDL particles prepared by the sodium cholate dialysis method, has shown that mutants (Pro<sup>165</sup> → Ala,Gln<sup>173</sup> → Glu) (Leu<sup>311</sup> → Val,Leu<sup>214</sup> → Val,Leu<sup>318</sup> → Val,Leu<sup>319</sup> → Val), Leu<sup>222</sup> → Lys,Phe<sup>255</sup> → Lys,Phe<sup>290</sup> → Lys) and Δ209–243 reduced LCAT activation (38–68%). Mutant (Glu<sup>191</sup> → Ala,His<sup>195</sup> → Ala,Lys<sup>196</sup> → Ala) enhanced LCAT activation (131%), and mutant (Ala<sup>162</sup> → Leu,Leu<sup>189</sup> → Trp) exhibited normal LCAT activation as compared with the wild type proapoA-I and plasma apoA-I forms. The apparent catalytic efficiency (<i>V</i> <sub>max(app)</sub>/<i>K</i> <sub>m</sub> <sub>(app)</sub>) of the apoA-I mutants ranged from 17.8 to 107.2% of the control and was the result of variations in both the <i>K</i> <sub>m</sub> and the<i>V</i> <sub>max</sub> in the different mutants. These findings indicate that putative helices 6 and 7, and the carboxyl-terminal helices 8 and 9 contribute to the optimum activation of lecithin:cholesterol acyltransferase. In addition to their use in the present study, the variant apoA-I forms generated will serve as valuable reagents for the identification of the domains and residues of apoA-I involved in binding the scavenger receptor BI, and facilitating cholesterol efflux from cells as well as aid in the structural analysis of apoA-I.