Specificity of Lipid Incorporation Is Determined by Sequences in the N-Terminal 37 of ApoB

• Published in: Biochemistry (Easton) 2000-08, Vol.39 (32), p.9737-9745
• Main Authors: Carraway, Margaretha, Herscovitz, Haya, Zannis, Vassilis, Small, Donald M
• Format: Article
• Language: English
• Subjects: Apolipoproteins B - chemistry; Apolipoproteins B - genetics; Apolipoproteins B - metabolism; Binding Sites; Lipoproteins - metabolism; Models, Biological; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - metabolism; Protein Biosynthesis; Protein Structure, Secondary; Triglycerides - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi000791h

The N-terminal 17% of apolipoprotein B (apoB-17) is secreted lipid-poor while apoB-41 particles are secreted with a triacylglycerol (TAG)-rich core. Thus, the sequence between apoB-17 and apoB-41 is necessary for the assembly of TAG-rich lipoproteins. To delineate this region, C127 cells were permanently transfected to secrete the N-terminal 29, 32.5, or 37% of apoB. Density gradient centrifugation showed that secreted apoB-29, apoB-32.5, and apoB-37 had peak densities of 1.25, 1.22, and 1.16 g/mL and percent lipid of particle weights of 30, 37, and 49%, respectively. Calculated anhydrous particle diameters were:  apoB-29 = 81 Å, apoB-32.5 = 88 Å, and apoB-37 = 101 Å. Immunoprecipitated particles labeled with [3H]oleate showed that, as apoB length increased from apoB-29 to apoB-32.5 and apoB-37, the number of TAG (core) molecules per apoB particle increased almost 16-fold from 8 to 32 to 124, while phospholipids and diacylglycerols (surface lipids) increased only slightly from 71 to 87 to 97 molecules, respectively. Thus, sequences in the C-terminus of apoB-29 bind phospholipids and diacylglycerols, sequences between apoB-29 and apoB-32.5 augment TAG binding and sequences between apoB-32.5 and apoB-41 account for the marked incorporation of TAG at a rate of ∼1 TAG per 2 amino acids. Cryoelectron micrographs of isolated apoB-37 particles revealed mostly spherical particles of ∼110 Å (11.0 nm) with an electron lucent center, consistent with these particles having a TAG core. We suggest that the predicted amphipathic β-sheets beginning at apoB-29, starts to preferentially recruit core lipids into apoB and propose that the consistent presence of DAG in the secreted particles may have a role in fission of the nascent lipoprotein particles from the endoplasmic reticulum membrane.