Apolipoprotein b-containing lipoprotein particle assembly - Lipid capacity of the nascent lipoprotein particle

We previously proposed that the N-terminal 1000-residue betaalpha(1) domain of apolipoprotein B ( apoB) forms a bulk lipid pocket homologous to that of lamprey lipovitellin. In support of this lipid pocket hypothesis, we demonstrated that apoB: 1000 ( residues 1 - 1000) is secreted by a stable transformant of McA-RH7777 cells as a monodisperse particle with high density lipoprotein 3 (HDL3) density. In contrast, apoB: 931 ( residues 1 - 931), missing only 69 residues of the sequence homologous to lipovitellin, was secreted as a particle considerably more dense than HDL3. In the present study we have determined the stoichiometry of the lipid component of the apoB: 931 and apoB: 1000 particles. The secreted [H-3]glycerol- labeled apoB: 1000 particles, isolated by nondenaturing gradient gel electrophoresis, contained 50 phospholipid (PL) and 11 triacylglycerol ( TAG) molecules/ particle. In contrast, apoB: 931 particles contained only a few molecules of PL and were devoid of TAG. The unlabeled apoB: 1000 particles, isolated by immunoaffinity chromatography, contained 56 PL, 8 TAG, and 7 cholesteryl ester molecules/ particle. The surface to core lipid ratio of apoB: 1000-containing particles was similar to4: 1 and was not affected by oleate supplementation. Although very small amounts of microsomal triglyceride transfer protein (MTP) were associated with apoB: 1000 particles, it never approached a 1: 1 molar ratio of MTP to apoB. These results support a model in which (i) the first 1000 amino acid residues of apoB are competent to complete the lipid pocket without a structural requirement for MTP; (ii) a portion, or perhaps all, of the amino acid residues between 931 and 1000 of apoB-100 are critical for the formation of a stable, bulk lipid-containing nascent lipoprotein particle, and (iii) the lipid pocket created by the first 1000 residues of apoB-100 is PL-rich, suggesting a small bilayer type organization and has a maximum capacity on the order of 50 molecules of phospholipid.