An immunochemical analysis of human apolipoprotein B-100 structure-function relationships.

Abstract

Low density lipoproteins (LDL), the major cholesterol carriers in human plasma contain a single copy of apolipoprotein B-100 (apoB-100), a 4536 residue, water-insoluble polypeptide as their exclusive protein component. ApoB-100-containing lipoproteins (LpB) are secreted from the liver as large triglyceride-rich lipoproteins and are converted in the circulation to smaller cholesteryl ester-rich LDL. As a ligand for the cell surface LDL receptor, apoB-100 has an important role in cholesterol metabolism and elevated apoB-100 levels increase the risk of atherosclerosis. Genetic polymorphism, oxidative modification, non-enzymatic glycation and the lipid environment of apoB-100 can affect its ability to mediate binding to the LDL receptor. In my Ph.D. program, I have produced and employed a panel of anti-apoB-100 monoclonal antibodies (mAbs) to study apoB structure and function. To this end, I developed and evaluated 3 novel immunization strategies to generate apoB-100 isoform-specific mAbs. Immunization of human apoB-100 transgenic mice proved to be an effective method for production of anti-apoB-100 mAbs having unusual specificities. This strategy could also be applied to other antigens. Several mAbs produced by this protocol are shown to be useful probes of apoB-100 conformation. I have characterized the binding specificities of a large panel of anti-apoB-100 mAbs and have analyzed the expression of their corresponding epitopes on LpB as a function of LpB intravascular metabolism. I show that the apoB-100 carboxy-terminus undergoes a major conformational change as large LDL are converted to smaller particles, a conformational change that could allow the particle to become competent to mediate binding to the LDL receptor. Finally, I demonstrate that, when LDL undergoes non-enzymatic glycation, at least 6 sites in apoB-100 are modified, including 2 that flank the apoB LDL receptor-binding site. As the LDL receptor-binding site itself is not modified, the loss of binding activity may be secondary to a change in apoB conformation.