Identification of a sequence within the C-terminal 26 amino acids of cholesteryl ester transfer protein responsible for binding a neutralizing monoclonal antibody and necessary for neutral lipid transfer activity.
The cholesteryl ester transfer protein (CETP; 476 amino acids) mediates the transfer of neutral lipids and phospholipids between plasma lipoproteins. Previous studies showed that the epitope of a neutralizing monoclonal antibody (TP2) was located within the C-terminal 26 amino acids (aa) of CETP. To determine possible involvement of this region in lipid transfer activities, we generated six deletion mutants between Arg-451 and Leu-475 by in vitro mutagenesis and expressed mutant proteins in mammalian cells. Only deletion mutants between aa Phe-463 and Leu-475 failed to bind TP2; these mutant proteins were well secreted by cells but showed markedly reduced cholesteryl ester transfer activity. One of the deletion mutants (delta 470-475) showed similar reductions in cholesteryl ester and triglyceride transfer activities but normal or increased phospholipid transfer activity. Limited proteolysis of this mutant protein indicated a similar overall folding pattern to the wild-type protein. Thus, aa between Phe-463 and Leu-475 are necessary for binding TP2. Deletions within this sequence selectively impair neutral lipid transfer activity, suggesting a direct involvement in neutral lipid transfer.
Wang
S
S
Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New York 10032.
Deng
L
L
Milne
R W
RW
Tall
A R
AR
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D016466:CHO Cells; D002352:Carrier Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002460:Cell Line; D053480:Cholesterol Ester Transfer Proteins; D002788:Cholesterol Esters / Q000378:metabolism*; D002872:Chromosome Deletion; D006224:Cricetinae; D000939:Epitopes / Q000032:analysis*; D006023:Glycoproteins; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009500:Neutralization Tests; D010446:Peptide Fragments / Q000302:isolation & purification; D010743:Phospholipids / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D053480:Cholesterol Ester Transfer Proteins; D002788:Cholesterol Esters; D000939:Epitopes; D006023:Glycoproteins; D010446:Peptide Fragments; D010743:Phospholipids; D011994:Recombinant Proteins