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. 1991 Aug 15;88(16):7313–7317. doi: 10.1073/pnas.88.16.7313

Expression, secretion, and lipid-binding characterization of the N-terminal 17% of apolipoprotein B.

H Herscovitz 1, M Hadzopoulou-Cladaras 1, M T Walsh 1, C Cladaras 1, V I Zannis 1, D M Small 1
PMCID: PMC52285  PMID: 1871138

Abstract

The N-terminal 17% of human apolipoprotein B (apoB-17) was expressed in murine C127 cells following transfection with a bovine papilloma virus-based expression vector. A permanent cell line overexpressing the expected 89-kDa protein was selected and characterized. Pulse-chase experiments showed that the depletion of intracellular apoB-17 follows an apparent first-order kinetics with t1/2 = 51 min. Under conditions of continuous labeling, greater than 60% of the total synthesized apoB-17 was secreted in a soluble form, approximately 98% lipid-poor and approximately 2% lipid-bound. Inclusion of 1.2 mM oleate resulted in 5- and 2.5-fold increases in the amount of labeled apoB-17 in the p less than 1.063 g/ml and 1.063 less than p less than 1.21 g/ml fractions, respectively, which was coordinated with increased secretion of radiolabeled core lipids, triacylglycerols, and cholesteryl esters. Thus under conditions in which lipid pools are enriched a greater fraction of apoB-17 may be secreted on lipoprotein-like particles. The lipid-poor apoB-17 present in p greater than 1.21 g/ml readily associates with exogenously added dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles to form discoidal particles. Discs formed with DMPC/apoB-17, 7:1 (wt/wt), are 239 +/- 43 A in diameter and 61 +/- 4 A thick and contain approximately 2 molecules of apoB-17 and 2250 molecules of DMPC per disc. Based on volume calculations we conclude that apoB-17 forms an annulus about one bilayer high and 10 A thick surrounding the DMPC disc. Circular dichroic spectra of apoB-17 on DMPC discs showed apoB-17 to contain 39% alpha-helix, 36% beta-sheet, 9% beta-turn, and 16% random coil. To be consistent with this model greater than 70% of apoB-17 on DMPC discs must bind to lipid. These data suggest that the N-terminal 17% of apoB-100 can bind lipid and may contribute to some extent to the stabilization of triglyceride-rich lipoproteins.

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