Point mutagenesis of carboxyl-terminal amino acids of cholesteryl ester transfer protein. Opposite faces of an amphipathic helix important for cholesteryl ester transfer or for binding neutralizing antibody.
The cholesteryl ester transfer protein (CETP) mediates the transfer of neutral lipids between the plasma lipoproteins. A carboxyl-terminal sequence of CETP was recently shown to form the epitope of a neutralizing monoclonal antibody (TP2) and to be necessary for neutral lipid transfer activity. To determine the role of specific amino acids in the epitope and/or in lipid transfer activity, we made single amino acid substitution mutants between Pro446 and Ser476 by in vitro mutagenesis and expressed the mutants in mammalian cells. The binding of TP2 to CETP was abolished by mutations primarily of polar or charged amino acids that occurred periodically within the sequence between His466 and Leu475 and at amino acid Asp460; however, these mutants had well preserved cholesteryl ester (CE) transfer activity. By contrast, mutants of bulky hydrophobic amino acids in this region (particularly Leu475, Phe471, Leu468, Phe461, and Phe454) showed markedly decreased CE transfer specific activity, but essentially normal binding of TP2. The paradoxical effects on antibody binding and activity could be explained if amino acids determining monoclonal antibody binding and activity are disposed on opposite faces of an amphipathic helix between amino acids 465 and 476. This model was tested by substituting alanine residues for pairs of nonpolar amino acids which would be contiguous on a helix, resulting in low activity mutants equivalent to those produced by deletion of this region. We conclude that the hydrophobic face of a carboxyl-terminal helix of CETP is directly involved in the mechanism of CE transfer, and that TP2 inhibits activity by local sterical hindrance. The general hydrophobic character of this region, imparted by the bulky hydrophobic amino acids Leu and Phe, is important for normal CE transfer.
Wang
S
S
Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New York 10032.
Wang
X
X
Deng
L
L
Rassart
E
E
Milne
R W
RW
Tall
A R
AR
D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001666:Binding Sites, Antibody; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D053480:Cholesterol Ester Transfer Proteins; D002788:Cholesterol Esters / Q000378:metabolism*; D004262:DNA Restriction Enzymes; D000939:Epitopes / Q000276:immunology; D006023:Glycoproteins; D016296:Mutagenesis; D017354:Point Mutation; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D014162:Transfection
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D053480:Cholesterol Ester Transfer Proteins; D002788:Cholesterol Esters; D000939:Epitopes; D006023:Glycoproteins; D004262:DNA Restriction Enzymes