EXPRESSION, SECRETION, AND LIPID-BINDING CHARACTERIZATION OF THE N-TERMINAL 17-PERCENT OF APOLIPOPROTEIN-B

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, > 60% of the total synthesized apoB-17 was secreted in a soluble form, almost-equal-to 98% lipid-poor and almost-equal-to 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 rho < 1.063 g/ml and 1.063 < rho < 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 rho > 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 angstrom in diameter and 61 +/- 4 angstrom thick and contain almost-equal-to 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 angstrom 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 > 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.