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

Abstract

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 A, apoB-32.5 = 88 A, and apoB-37 = 101 A. Immunoprecipitated particles labeled with [(3)H]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 approximately 1 TAG per 2 amino acids. Cryoelectron micrographs of isolated apoB-37 particles revealed mostly spherical particles of approximately 110 A (11.0 nm) with an electron lucent center, consistent with these particles having a TAG core. We suggest that the predicted amphipathic beta-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.

Microsomal Triglyceride Transfer Protein Activity Is Not Required for the Initiation of Apolipoprotein B-containing Lipoprotein Assembly in McA-RH7777 Cells

“…At present, the structural elements within the apoB polypeptide that govern requirement for MTP are not known. Available evidence suggest the following: (i) segments equal to or greater than apoB48 containing the N-terminal 17% of the protein respond to MTP activity (14); (ii) sequences in the C terminus of apoB29 bind PL (69); (iii) sequences between apoB29 and apoB32.5 augment TAG binding (69); (iv) sequences between apoB32.5 and apoB41 account for the marked incorporation of TAG (69); and (v) the domain between apoB51 and apoB53 has a high requirement for MTP (66), and this region is within the ␣ 2 domain of apoB100 (53). Our results provide a compelling argument that the addition of phospholipids to apoB:1000 (apoB22.05), initiation of apoB particle assembly, and the formation of the primordial PL-rich apoB-containing lipoproteins are independent of MTP lipid transfer activity.…”

Apolipoprotein B is conformationally flexible but anchored at a triolein/water interface: A possible model for lipoprotein surfaces

“…The second region is high in A␤S. This motif appears to be related to the recruitment of lipids (30,31), and the region from the N-terminal 32% of apoB (B32) to B41 recruits TAGs to form nascent microemulsion particles in microsomal triglyceride transfer protein-deficient C127 cells (32). Truncated apoB longer than ϷB30 must be assembled with lipids to be secreted if not it is degraded and fails to appear in plasma (1).…”

Defining Lipid-Interacting Domains in the N-Terminal Region of Apolipoprotein B

“…It has been proposed that this region has a strong affinity for phospholipids (28,29,31). In this study, we use a "divide and conquer" approach to map the phospholipid recruiting elements in the βα1 domain of apoB.…”

“…Although the exact length required for apoB to form a lipoprotein is still under debate (26,27,31), these seemingly inconsistent observations might reflect an intrinsic property of apoB during the assembly of apoB-containing lipoproteins. There may be flexibility in the assembly of these lipoproteins in response to the environment, in particular the lipid profile and MTP availability.…”

“…All apoB domains were cloned from B37 initially described by Carraway et al (31). The following constructs were generated for this study: B5.9 (residues 1−264), B6.4−10 (residues 292−469), B6.4−13 (residues 292−593), B6.4−15 (residues 292−680), B6.4−17 (residues 292 −782), B9−13 (residues 430−593), B13−15 (residues 611−680) and B13−17 (residues 611 −782).…”