A long term objective has been and will be to understand the structure: function relationships of the various apolipoproteins that participate in lipoprotein and cholesterol metabolism. Apolipoprotein B100 plays a central role in cholesterol metabolism in that it is the ligand on low density lipoproteins responsible for the receptor-mediated catabolism of these lipoproteins, which transport and distribute more than 60% of total plasma cholesterol. Low density lipoproteins and apo-B have also been implicated in the formation of atherosclerotic lesions. It is therefore important to understand the mechanisms by which this protein carries out its functions.
The specific aim i s to identify those regions of the apo-B polypeptide that are the functional sites for 1) receptor binding, 2) heparin binding, and 3) lipid binding. The receptor binding domain(s) will be identified by a combination techniques involving a) apo-B fragment recombination, b) monoclonal antibody inhibition of binding and epitope determination, and c) identification of dysfunctional apo-B mutants. Apo-B fragments will be obtained from proteolyzed LDL (thrombin, trypsin, V8 protease) and from apo-B CNBr digests. Polyclonal and monoclonal antibodies will be obtained using as antigens: LDL, apo-B, specific apo-B peptide fragments, synthetic peptides from known apo-B sequences, and fusion proteins expressed after insertion of apo-B cDNA fragments into expression vectors. The receptor binding domain will be characterized by identification of receptor-active fragments and by identification of the epitopes of inhibitory monoclonal antibodies. Dysfunctional apo-B mutants will be identified by a functional assay and their site of mutation(s) used to confirm receptor binding regions. The heparin binding domain(s) will be identified by heparin affinity chromatography using CNBr peptides, as well as proteolyzed LDL peptides. Lipid binding domains will be identified from lipid recombination studies, as well as by predictive structural analysis of known apo-B sequences.
| Weisgraber, K H; Innerarity, T L; Newhouse, Y M et al. (1988) Familial defective apolipoprotein B-100: enhanced binding of monoclonal antibody MB47 to abnormal low density lipoproteins. Proc Natl Acad Sci U S A 85:9758-62 |
| Innerarity, T L; Young, S G; Poksay, K S et al. (1987) Structural relationship of human apolipoprotein B48 to apolipoprotein B100. J Clin Invest 80:1794-8 |
| Innerarity, T L; Weisgraber, K H; Arnold, K S et al. (1987) Familial defective apolipoprotein B-100: low density lipoproteins with abnormal receptor binding. Proc Natl Acad Sci U S A 84:6919-23 |
| Corsini, A; Spilman, C H; Innerarity, T L et al. (1987) Receptor binding activity of lipid recombinants of apolipoprotein B-100 thrombolytic fragments. J Lipid Res 28:1410-23 |
| Weisgraber, K H; Rall Jr, S C (1987) Human apolipoprotein B-100 heparin-binding sites. J Biol Chem 262:11097-103 |
