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J. Lipid Res.
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Originally published In Press as doi:10.1194/jlr.M600130-JLR200 on April 6, 2006

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Journal of Lipid Research, Vol. 47, 1399-1405, July 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology

Binding characteristics of a panel of monoclonal antibodies against the ligand binding domain of the human LDLr

Anh T. Nguyen*, Tomoko Hirama{dagger}, Vinita Chauhan*, Roger MacKenzie{dagger} and Ross Milne1,*,§

* Lipoprotein and Atherosclerosis Research Group, University of Ottawa Heart Institute, Ottawa, Ontario, Canada
{dagger} Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada
§ Department of Pathology and Laboratory Medicine, University of Ottawa, Ottawa, Ontario, Canada

Published, JLR Papers in Press, April 6, 2006.

1 To whom correspondence should be addressed. e-mail: rmilne{at}ottawaheart.ca

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.

Abbreviations: apoE, apolipoprotein E; CETP, cholesteryl ester transfer protein; EGF, epidermal growth factor; EGFPHD, epidermal growth factor precursor homology domain; LBD, ligand binding domain; LDLr, low density lipoprotein receptor; MAb, monoclonal antibody

Supplementary key words low density lipoprotein receptor • knockout mice • structure-function analysis


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