title: The carboxyl-terminal hydrophobic residues of apolipoprotein A-I affect its rate of phospholipid binding and its association with high density lipoprotein.
abstract: 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 delta185-243 and delta209-243 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,Gln172-->Glu) (Leu211-->Val,Leu214-->Val, Leu218-->Val,Leu219-->Val), Leu222-->Lys,Phe225-->Lys, Phe229-->Lys) and delta209-243 reduced LCAT activation (38-68%). Mutant (Glu191-->Ala,His193-->Ala,Lys195-->Ala) enhanced LCAT activation (131%), and mutant (Ala152-->Leu, Leu159-->Trp) exhibited normal LCAT activation as compared with the wild type proapoA-I and plasma apoA-I forms [corrected]. The apparent catalytic efficiency (Vmax(app)/Km(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 Km and the Vmax 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.
authors.affiliation: Section of Molecular Genetics, Center for Advanced Biomedical Research, Department of Medicine, Boston University Medical Center, Boston, Massachusetts 02118-2394, USA.
authors.forename: M
authors.initials: M
authors.lastname: Laccotripe
authors.forename: S C
authors.initials: SC
authors.lastname: Makrides
authors.forename: A
authors.initials: A
authors.lastname: Jonas
authors.forename: V I
authors.initials: VI
authors.lastname: Zannis
mesh_terms: D016632:Apolipoprotein A-I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D015180:Electrophoresis, Gel, Two-Dimensional; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D050356:Lipid Metabolism; D008075:Lipoproteins, HDL / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D013329:Structure-Activity Relationship
chemical_list: D016632:Apolipoprotein A-I; D008075:Lipoproteins, HDL; D010743:Phospholipids; D007862:Phosphatidylcholine-Sterol O-Acyltransferase