Binding characteristics of a panel of monoclonal antibodies against the ligand binding domain of the human LDLr.
To obtain a panel of monoclonal antibodies (MAbs) to study the folding and conformation of the low density lipoprotein receptor (LDLr), we have generated hybridomas from LDLr-deficient mice that had been immunized with the extracellular domain of the human LDLr. The 12 MAbs were specific for the ligand binding domain of the LDLr, with individual MAbs recognizing epitopes in ligand binding repeats 1, 2, 3, 5, and 7. A subset of the MAbs failed to react with the LDLr when disulfide bonds were reduced, and one MAb, specific for an epitope that spans ligand binding repeats 1 and 2, recognized two conformational forms of the LDLr with different affinities. Antibodies specific for ligand binding repeats 3, 5, and 7 completely blocked the binding of LDL particles to the LDLr on cultured human fibroblasts, whereas MAbs with epitopes in ligand binding repeats 1 and 2 partially blocked the binding of LDL to the LDLr. These anti-LDLr MAbs will serve as useful probes for further analysis of LDLr conformation and LDLr-mediated lipoprotein binding.
Nguyen
Anh T
AT
Lipoprotein and Atherosclerosis Research Group, University of Ottawa Heart Institute, Ottawa, Ontario, Canada.
Hirama
Tomoko
T
Chauhan
Vinita
V
Mackenzie
Roger
R
Milne
Ross
R
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001665:Binding Sites; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D066298:In Vitro Techniques; D008024:Ligands; D008077:Lipoproteins, LDL / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011973:Receptors, LDL / Q000737:chemistry* / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D020349:Surface Plasmon Resonance
D000911:Antibodies, Monoclonal; D000939:Epitopes; D008024:Ligands; D008077:Lipoproteins, LDL; D010446:Peptide Fragments; D011973:Receptors, LDL; D011994:Recombinant Proteins