Apolipoprotein (apo) B-100 is a physiological ligand for the low density lipoprotein (LDL) apoB,E receptor and an integral component of lipoprotein (a). It is therefore a central molecule in lipoprotein metabolism and atherosclerosis. Although the complete sequence of apoB-100 was deduced from its cDNA sequence over four years ago, we still know very little about the structure-function relationships of the protein. ApoB-100 contains 4536 residues and is one of the largest monomeric proteins known. It is an extremely difficult protein to study because of its large size and tendency to aggregate. It has two major functions: lipid binding and LDL receptor binding. A number of naturally occurring truncated apoBs have been described. While they show differential association with different lipoprotein particles, information on lipid binding requirements is limited to C-terminal truncations, and the contribution of individual parts of the molecule (i.e., N-terminal and middle regions) is unknown. For the receptor binding function, again little is known. Two domains, domain A (residues 3147-3157) and domain B (residues 3359-3367) have been postulated to be important for binding. Only a single natural human apoB-100 variant that contains an Arg 3500 -> Gln mutation has been found to be strongly associated with impairment of LDL receptor binding. To date, there is no direct proof for the importance of domains A or B, or Arg 3500 in apoB- 100 - LDL receptor interaction. We propose to generate transgenic animals that express partial and full length copies of apoB-100 and study its structure-function relationships. Initially we will generate transgenic mice that produce partial and full-length apoB-100. Then we will generate transgenic rabbits that express the protein. Both wildtype and site- specific mutants will be studied. Mutations will be targeted to domains A and B, Arg 3500 and some other potentially important regions. The effect of expression of the various constructs on the serum lipid profiles of the transgenic animal will be analyzed. The LDLs containing the human recombinant apoB-100 protein will be isolated from the transgenic rabbits by a combination of zonal ultracentrifugation and immunoaffinity chromatography. The LDL receptor binding characteristics of the recombinant human apoB-100 containing LDL particles will be examined. ApoB-100 is an extremely complex molecule. We believe that the approach we have proposed represents the best method currently available to examine the structure-function relationships of apoB-100 especially with respect to its receptor-binding activity.
