Structure-function relationships of human cholesteryl ester transfer protein: analysis using monoclonal antibodies.
Cholesteryl ester transfer protein (CETP), a 476 amino acid glycoprotein, mediates cholesteryl ester (CE), triglyceride, and phospholipid transfer among plasma lipoproteins. A monoclonal antibody (mAb), TP2, specific for an epitope within the last 26 amino acids of CETP has been shown to block all CETP-mediated lipid transfer, apparently by limiting access to lipid-binding sites in the carboxy terminal of CETP. A new panel of 16 anti-human CETP mAbs has now been used to further probe the structure-function relationships of CETP. Of the new mAbs, 9 partially inhibit CETP-mediated CE transfer (24-43%) from HDL to LDL. The corresponding epitopes were mapped within the CETP primary structure by the reactivity of the mAbs with CETP variants having deletions or amino acid substitutions. Of the 9 new, neutralizing mAbs, 6 are specific for epitopes situated between residues 410-450 and two others for epitopes between residues 184-260 and 332-366, respectively. The epitope of one neutralizing mAbs could not be mapped. Therefore, binding of mAbs to epitopes situated in four non-overlapping regions within CETP primary structure that are separated by as many as 280 residues can neutralize CETP-mediated CE transfer. Epitopes of mAbs that do not influence CE transfer activity map to the regions 184-260, 261-331, and 367-409, respectively. When pairs of mAbs were tested for their abilities to mutually compete for binding to immobilized CETP, competition was observed for mAbs specific for epitopes that are distant in CETP primary structure. The cross-competition patterns demonstrate that the carboxy terminal 60% of CETP adopts a compact structure. Together with previous mutagenesis studies, the data suggests that a carboxy terminal neutral lipid binding domain may be in close proximity to a lipoprotein binding region within native CETP.
Roy
P
P
Departement des Sciences Biologiques, Universite du Quebec a Montreal, Canada.
MacKenzie
R
R
Hirama
T
T
Jiang
X C
XC
Kussie
P
P
Tall
A
A
Rassart
E
E
Milne
R
R
D000818:Animals; D000911:Antibodies, Monoclonal; D001667:Binding, Competitive; D001692:Biological Transport; D015374:Biosensing Techniques; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D002478:Cells, Cultured; D053480:Cholesterol Ester Transfer Proteins; D002788:Cholesterol Esters / Q000378:metabolism*; D018604:Epitope Mapping; D006023:Glycoproteins; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; C506642:CETP protein, human; D002352:Carrier Proteins; D053480:Cholesterol Ester Transfer Proteins; D002788:Cholesterol Esters; D006023:Glycoproteins; D011993:Recombinant Fusion Proteins