Abstract
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 (HDL(3)) 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 HDL(3). In the present study we have determined the stoichiometry of the lipid component of the apoB:931 and apoB:1000 particles. The secreted [(3)H]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 approximately 4:1 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.
Highlights
We previously proposed that the N-terminal 1000-residue ␣1 domain of apolipoprotein B forms a bulk lipid pocket homologous to that of lamprey lipovitellin
We suggested [19, 20] that initiation of particle assembly occurs when the ␣1 domain folds into a three-sided LV-like lipid-binding cavity, or alternatively, the lipid pocket is formed by association of the region of the ␣1 domain homologous to the A and B sheets of LV with D-like amphipathic  sheet from microsomal triglyceride transfer protein (MTP)
We suggested that the 69-amino acid residues between apolipoprotein B (apoB): 931 and apoB:1000 are necessary for the formation of HDL3like lipoprotein particles
Summary
The full-length apoB, essentially the only protein component of the atherogenic LDL, has a pentapartite structure, NH2-␣11-␣2-2-␣3-COOH, the  domains containing multiple amserum; HDL, high density lipoprotein; LDL, low density lipoprotein; LV, lipovitellin; MTP, microsomal triglyceride transfer protein; NDGGE, nondenaturing gradient gel electrophoresis; PL, phospholipids; PBS, phosphate-buffered saline; TAG, triacylglycerol; VLDL, very low density lipoprotein; PVDF, polyvinylidene difluoride. The lack of a 1:1 molar ratio of apoB to MTP observed in this study supports a model in which the first 1000 amino acid residues of apoB are competent to complete the “lipid pocket” without a structural requirement for MTP This nascent lipoprotein intermediate has a relatively constant Stokes diameter of 112 Å, a mean density of 1.21 g/ml, and has a maximum lipid-transporting capacity on the order of 70 molecules of lipid/particle, primarily phospholipids (PL). The surface to core lipid ratio of ϳ4:1 supports a bilayer-like organization that is not responsive to the presence of oleate in the incubation medium
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