The risk of coronary heart disease is correlated with plasma levels of low density lipoprotein (LDL) and possibly with genetic polymorphisms in the structure of LDL. Although mutations in the LDL receptor have been discovered, there has not been much progress in identifying mutations in LDL. We have a herd of pigs carrying eight immunologically- characterized alleles for the predominant protein of LDL, apolipoprotein-B. Our studies will focus on two mutant strains: 1) A strain which has an apo-B allele associated with LDL that stimulates cholesterol ester synthesis in macrophages in vitro. Pigs with this apo-B allele have significant lipid infiltration in their coronary arteries. 2) A strain which has an apo-B allele, that, in the presence of two other lipoprotein-borne markers, is associated with hypercholesterolemia and accelerated atherosclerosis. Our project is aimed at identifying functional domains of apo-B and elucidating the mechanisms by which mutations in apo-B produce coronary heart disease in our pigs.
The specific aims of the proposal are: 1) Immunological probing of the mutant apo-B's. 2) Cloning and sequencing of the variant segments of potential interest. 3) Cloning of the variant segments into an E. coli expression vector. 4) Production of antibodies to the fusion proteins. 5) Purification of fusion proteins from bacterial inclusion bodies. 6) Incorporation of the fusion peptides into methylated, trypsinized HDLc. 7) Test modified HDLc for ability to bind LDL receptor and to block normal LDL binding. 8) Test of antibodies for ability to inhibit binding of LDL to cell receptors.
Showing the most recent 10 out of 16 publications
