Apolipoprotein B-containing lipoprotein particle assembly: Lipid capacity of the nascent lipoprotein particle

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We previously proposed that the N-terminal 1000 residue {beta}{alpha}{sub 1} domain of apolipoprotein B (apoB) forms a bulk lipid pocket homologous to that of lamprey lipovitellin (LV). In support of this ''lipid pocket'' hypothesis, apoB:1000 (residues 1-1000) was shown to be secreted by a stable transformant of McA-RH7777 cells as a monodisperse particle with HDL{sub 3} density and Stokes diameter of 112 {angstrom}. In contrast, apoB:931 (residues 1-931), missing only 69 residues of the sequence homologous to LV, was secreted as a particle considerably more dense than HDL with Stokes diameter of 110 {angstrom}. The purpose of the present study ... continued below

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48 pages

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Manchekar, Medha; Forte, Trudy M.; Datta, Geeta; Richardson, Paul E.; Segrest, Jere P. & Dashti, Nassrin December 1, 2003.

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We previously proposed that the N-terminal 1000 residue {beta}{alpha}{sub 1} domain of apolipoprotein B (apoB) forms a bulk lipid pocket homologous to that of lamprey lipovitellin (LV). In support of this ''lipid pocket'' hypothesis, apoB:1000 (residues 1-1000) was shown to be secreted by a stable transformant of McA-RH7777 cells as a monodisperse particle with HDL{sub 3} density and Stokes diameter of 112 {angstrom}. In contrast, apoB:931 (residues 1-931), missing only 69 residues of the sequence homologous to LV, was secreted as a particle considerably more dense than HDL with Stokes diameter of 110 {angstrom}. The purpose of the present study was to determine the stoichiometry of the lipid component of the apoB:931 and apoB:1000 particles. This was accomplished by metabolic labeling of cells with either [{sup 14}C]oleic acid or [{sup 3}H]glycerol followed by immunoprecipitation (IP) or nondenaturing gradient gel electrophoresis (NDGGE) of secreted lipoproteins and by immunoaffinity chromatography of secreted unlabeled lipoproteins. The [{sup 3}H]-labeled apoB:1000-containing particles, isolated by NDGGE, contained 50 phospholipids (PL) and 11 triacylglycerols (TAG) molecules per particle. In contrast, apoB:931-containing particles contained only a few molecules of PL and were devoid of TAG. The unlabeled apoB:1000-containing particles isolated by immunoaffinity chromatography and analyzed for lipid mass, contained 56 PL, 8 TAG, and 7 cholesteryl ester molecules per particle. The surface:core lipid ratio of apoB:1000-containing particles was approximately 4:1 and was not affected by incubation of cells with oleate. Although small amounts of microsomal triglyceride transfer protein (MTP) were associated with apoB:1000-containing particles, it never approached a 1:1 molar ratio of MTP to apoB. These results support a model in which: (1) the first 1000 amino acid residues of apoB are competent to complete the ''lipid pocket'' without a structural requirement for MTP; (2) amino acids between 931 to 1000 of apoB-100 are critical for the formation of a nascent lipoprotein particle, and (3) the ''lipid pocket'' created by the first 1000 amino acid residues of apoB-100 is PL-rich, suggesting a small bilayer type organization and has a maximum capacity on the order of 70 molecules of lipid. This model is supported by the allatom molecular model of the {beta}{alpha}{sub 1} lipid pocket presented in the accompanying paper.

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48 pages

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OSTI as DE00836379

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  • Journal Name: Journal of Biological Chemistry; Journal Volume: 279; Journal Issue: 38; Other Information: Submitted to Journal of Biological Chemistry: Volume 279, No.38; Journal Publication Date: 09/17/2004

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  • Report No.: LBNL--54502
  • Grant Number: AC03-76SF00098
  • Office of Scientific & Technical Information Report Number: 836379
  • Archival Resource Key: ark:/67531/metadc785255

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  • December 1, 2003

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  • Dec. 3, 2015, 9:30 a.m.

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  • Oct. 3, 2017, 7:32 p.m.

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Manchekar, Medha; Forte, Trudy M.; Datta, Geeta; Richardson, Paul E.; Segrest, Jere P. & Dashti, Nassrin. Apolipoprotein B-containing lipoprotein particle assembly: Lipid capacity of the nascent lipoprotein particle, article, December 1, 2003; Berkeley, California. (digital.library.unt.edu/ark:/67531/metadc785255/: accessed November 18, 2017), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.