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Volume 272, Number 28, Issue of July 11, 1997 pp. 17511-17522
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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

(Received for publication, March 3, 1997, and in revised form, April 25, 1997)

Maria Laccotripe , Savvas C. Makrides , Ana Jonas ^§ and Vassilis I. Zannis

From the  Section of Molecular Genetics, Center for Advanced Biomedical Research, Departments of Medicine and Biochemistry, Boston University Medical Center, Boston, Massachusetts 02118-2394 and the ^§ Department of Biochemistry, College of Medicine at Urbana-Champaign, University of Illinois, Urbana, Illinois 61801

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 Leu²²², Phe²²⁵, and Phe²²⁹ with lysines, as well as replacement of Leu²¹¹, Leu²¹⁴, Leu²¹⁸, and Leu²¹⁹ with valines, diminished the ability of apoA-I to bind to HDL and to lyse dimyristoyl phosphatidylcholine liposomes. The findings indicate that Leu²²², and Phe²²⁵, Phe²²⁹ located in the putative random coil region, and Leu²¹¹, Leu²¹⁴, Leu²¹⁸, and Leu²¹⁹ 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 185-243 and 209-243 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 (Pro¹⁶⁵  Ala,Gln¹⁷³  Glu) (Leu³¹¹  Val,Leu²¹⁴  Val,Leu³¹⁸  Val,Leu³¹⁹  Val), Leu²²²  Lys,Phe²⁵⁵  Lys,Phe²⁹⁰  Lys) and 209-243 reduced LCAT activation (38-68%). Mutant (Glu¹⁹¹  Ala,His¹⁹⁵  Ala,Lys¹⁹⁶  Ala) enhanced LCAT activation (131%), and mutant (Ala¹⁶²  Leu,Leu¹⁸⁹  Trp) exhibited normal LCAT activation as compared with the wild type proapoA-I and plasma apoA-I forms. The apparent catalytic efficiency (V_max(app)/K_m(app)) of the apoA-I mutants ranged from 17.8 to 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 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.

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