The long-term goal of this project is to study the chemistry of apolipoprotein B (ApoB) and ApoB-containing lipoproteins. The short-term objective is to study the post-translational crosslinking with focus on thiolester linkage. ApoB and its lipoproteins play very important roles in lipid transport, in regulation of cholesterol synthesis, and in pathogenesis of atherosclerosis. Therefore, a better understanding of the chemistry of ApoB is more than desirable. The main difficulties with ApoB are its insolubility in aqueous buffers and strong tendency to aggregate. By eliminating the oxidative conditions for low density lipoproteins (LDL) and by providing opportunity for proper folding of ApoB to attain a favorable conformation, we have developed isolation and solubilization methods which overcome these difficulties and yielded water-soluble ApoB. This has offered a good opportunity to study its chemistry. ApoB is a peculiar protein. It shows unusual behavior. These behaviors can be best explained by the presence of labile thiolester crosslinkage(s).
The specific aims and methods for this research plan are as follows: identification of the presence of thiolester bond in reduced and carboxymethylated (RCM-)LDL2 and in RCM-ApoB by specific incorporation of [14C]-methylamine and [3H]-iodoacetic acid; establishment of optimal pH, temperature and methylamine concentration for cleaving the thiolester bond; establishing the form of ApoB and conditions under which ApoB will give the highest yield of incorporation of 14C-methylamine from comparison of ApoB in aqueous buffer, in urea, in intact LDL2 and in whole plasma for the cleavage of the labile bond(s); establishing the stoichiometry of thiolester in ApoB by performing the cleavage experiment under the optimal condition with the selected form of ApoB; establishing thiolester bond as being an inter- or an intra-molecular crosslinkage by SDS-PAGE of the double labeled samples; establishing the number of tryptic or chymotryptic peptides containing the thiolester linkage by autoradiography of the peptide maps or from the number of radiolabeled fractions purified by high-performance liquid chromatography (HPLC); purification of the tryptic or chymotryptic peptides which contain the thiolester linkage with activated thiol-Sepharose and HPLC; amino acid analyses of the labeled peptides; sequence determination of the labeled peptides by automated Edman degradation and identification of the labeled amino acids. Thus, the identity, the nature, the stoichiometry of thiolester and the primary structure proximal to the thiolester sites in ApoB will be established.
| Lee, D M (1991) Inter- and intramolecular thiolester linkages in apolipoprotein B. Prog Lipid Res 30:245-52 |
| Huang, G; Lee, D M; Singh, S (1988) Identification of the thiol ester linked lipids in apolipoprotein B. Biochemistry 27:1395-400 |
