Defective Binding of Neutral Lipids by a Carboxyl-terminal Deletion Mutant of Cholesteryl Ester Transfer Protein

The plasma cholesteryl ester transfer protein (CETP, 476 amino acids) transfers cholesteryl ester (CE) from high density lipoprotein (HDL) to triglyceride-rich lipoproteins and plays a major role in HDL catabolism. Using deletional and site-directed mutagenesis, we previously showed that the carboxyl terminus of human CETP comprises the epitope of a neutralizing monoclonal antibody and is necessary for neutral lipid transfer activity. To assess the nature of the involvement of the COOH terminus in cholesteryl ester transfer activity, we characterized a deletion mutant of CETP lacking amino acid residues 470-475 in terms of CE transfer kinetics, association with HDL, and capacity to bind CE, triglyceride (TG), and phosphatidylcholine (PC). Kinetic analysis indicated a major catalytic defect of the deletion mutant, as shown by markedly decreased maximum cholesteryl ester transfer activities (apparent Vmax) for donor (HDL) and acceptor (low density lipoprotein (LDL)) lipoproteins but there were no significant changes of concentrations of the donor and acceptor at 50% Vmax (apparent K). The binding of CETP to HDL, as determined by native gel electrophoresis, was similar for wild-type and mutant protein. When egg PC/CE vesicles were incubated with wild type CETP and then separated by gel filtration chromatography, there was maximum binding of about 1 mol of CE/mol of CETP. Under similar conditions the mutant CETP bound 0.09-0.37 mol of CE/mol of protein. Similarly, when egg PC/TG vesicles were incubated with the CETP proteins, there was a maximum binding of 0.5 mol of triglyceride/mol of wild-type CETP, whereas there was only 0.00-0.07 mol of TG/mol of deletion mutant. The binding of phosphatidylcholine was similar for wild-type and the deletion mutant. The studies suggest that amino acids 470-475 (forming part of a COOH-terminal amphipathic helix) are involved in CE and TG binding by CETP but are not required either for the binding of PC by CETP or the association of CETP with HDL. The COOH terminus of CETP may comprise a neutral lipid binding site directly involved in the lipid transfer mechanism.