During the past grant period, our laboratory characterized the lipoprotein phenotypes of mutant pigs with spontaneous hypercholesterolemia and the structures of their apoB alleles. The pigs were originally discovered and selected on the basis of allotypic epitopes on apoB. We discovered that LDL from the mutants pigs binds to the LDL receptor with reduced affinity and is sluggishly cleared from the circulation in vivo. Extensive sequencing of the mutant and normal apoB alleles revealed numerous point mutations. A unique haplotype was found in the mutant pig apoB, which includes an amino acid residue within a proposed LDL receptor binding domain. However, without a biological test, we could not ascertain which point mutations (or combinations thereof) are responsible for the dysfunctional apoB. To enable the study of the relationship between apoB structural variation and receptor binding activity, we developed the means to identify receptor binding domains of apoB. Two novel apolipoprotein expression systems were developed that for the first time enable systematic testing of candidate apoB domains for LDL receptor binding. In addition to our work ont he pig apoB genes, we uncovered evidence for a second major cholesterol-elevating gene locus. The mutant phenotype associated with this locus involves overproduction of cholesterol ester-enriched buoyant LDL particles and in vivo downregulation of the LDL receptor. The proposed work will yield new information about the receptor binding domains of apoB and how disruption of these domains produces dysfunctional LDL particles. This will result in an improved ability to recognize apoB mutations associated with significant clinical phenotypes. In addition, our work on a second cholesterol-elevating gene locus will provide clues to additional mechanisms underlying genetically-determined hypercholesterolemia.
The specific aims for the next phase of this project are: 1. To identify pig apoB mutations specifically responsible for the LDL receptor binding defect. To characterize apoB domains responsible for interaction with the LDL receptor. This approach will provide direct evidence of particular regions of the apoB protein that participate in binding to the LDL receptor. 2. To study the effects of an inhibitor of acyl- CoA:cholesterol acyltransferase (ACAT) on lipoprotein production in the mutant pigs. These experiments will test the hypothesis that an expanded cholesterol ester pool is responsible for overproduction of buoyant LDL particles. These experiments will provide information relevant to common human hyperlipidemias. 3. To test the hypothesis that a defect affecting the activity of cholesterol 7alpha-hydroxylase is associated with a defect in a second major cholesterol-elevating gene locus. We hypothesize that a defect affecting cholesterol 7alpha- hydroxylase activity underlies the overproduction of buoyant LDL and in vivo down regulation of the LDL receptor. These experiments will characterize hypercholesterolemia caused by a second cholesterol- elevating gene locus in our inbred pig population and potentially shed new light on genetically-determined hyperlipidemias in the human population.
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