Expression, secretion, and lipid-binding characterization of the N-terminal 17% 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, 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.
Herscovitz
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Department of Biophysics, Boston University School of Medicine, Housman Medical Research Center, MA 02118.
Hadzopoulou-Cladaras
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Walsh
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Cladaras
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Zannis
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Small
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D000595:Amino Acid Sequence; D000818:Animals; D001055:Apolipoproteins B / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D001483:Base Sequence; D002460:Cell Line; D002942:Circular Dichroism; D004134:Dimyristoylphosphatidylcholine / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D007231:Infant, Newborn; D007700:Kinetics; D051379:Mice; D008854:Microscopy, Electron; D008961:Models, Structural; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019301:Oleic Acid; D009829:Oleic Acids / Q000494:pharmacology; D010957:Plasmids; D011485:Protein Binding; D011487:Protein Conformation; D015183:Restriction Mapping; D014162:Transfection
D001055:Apolipoproteins B; D009829:Oleic Acids; D019301:Oleic Acid; D004134:Dimyristoylphosphatidylcholine