Identification of a sequence of apolipoprotein A-I associated with the efflux of intracellular cholesterol to human serum and apolipoprotein A-I containing particles.
The effect of monoclonal antibodies against apolipoprotein A-I (apoA-I) on the efflux of intracellular and plasma membrane cholesterol from HepG2 cells to human serum, high-density lipoprotein (HDL), apoA-I, and apoA-I/phosphatidylcholine complex (apoA-I/PC) was studied. Fab fragments of two monoclonal antibodies, AI-3 (residues 140-147) and Al-4.2 (residues 149-150), inhibited the efflux of intracellular cholesterol to serum in a dose-dependent manner. In combination, these antibodies were twice as effective than when used alone. None of the antibodies tested inhibited efflux of the plasma membrane cholesterol. When different types of acceptors were compared for their ability to promote intracellular cholesterol efflux, they were effective in the following order: serum > HDL > apoA-I/PC > pure apoA-I. Antibody AI-3 inhibited efflux of intracellular cholesterol to serum, HDL, and pure apoA-I, but not to apoA-I/PC. Antibody AI-4.2 inhibited efflux to serum, apoA-I/PC, and pure apoA-I, but not to HDL. An explanation for this is that antibody AI-4.2 reacts poorly with isolated alpha-HDL in an immunoprecipitation assay and has higher affinity for pre beta 2-HDL and pre beta 3-HDL particles than antibody AI-3 in nondenaturing two-dimensional electrophoresis. In conclusion, we have demonstrated that a region of apoA-I within or adjacent to residues 140-150 determines the ability of apoA-I to promote intracellular cholesterol efflux.
Sviridov
D
D
Baker Medical Research Institute, Prahran, Australia.
Pyle
L
L
Fidge
N
N
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D001120:Arginine; D001224:Aspartic Acid; D001665:Binding Sites; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000494:pharmacology; D007700:Kinetics; D008075:Lipoproteins, HDL / Q000097:blood*; D008081:Liposomes; D008113:Liver Neoplasms; D008563:Membrane Lipids / Q000378:metabolism; D010713:Phosphatidylcholines; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D016632:Apolipoprotein A-I; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D008075:Lipoproteins, HDL; D008081:Liposomes; D008563:Membrane Lipids; D010713:Phosphatidylcholines; D001224:Aspartic Acid; D001120:Arginine; D002784:Cholesterol