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

Abstract

Apolipoprotein B (ApoB) is a nonexchangeable apolipoprotein that dictates the synthesis of chylomicrons and very low density lipoproteins. ApoB is the major protein in low density lipoprotein, also known as the "bad cholesterol" that is directly implicated in atherosclerosis. It has been suggested that the N-terminal domain of apoB plays a critical role in the formation of apoB-containing lipoproteins through the initial recruitment of phospholipids in the endoplasmic reticulum. However, very little is known about the mechanism of lipoprotein nucleation by apoB. Here we demonstrate that a strong phospholipid remodeling function is associated with the predicted α-helical and C-sheet domains in the N-terminal 17% of apoB (B17). Using dimyristoylphosphatidylcholine (DMPC) as a model lipid, these domains can convert multilamellar DMPC vesicles into discoidal-shaped particles. The nascent particles reconstituted from different apoB domains are distinctive and compositionally homogenous. This phospholipid remodeling activity is also observed with egg phosphatidylcholine (egg PC) and is therefore not DMPC dependent. Using kinetic analysis of the DMPC clearance assay, we show that the identified phospholipid binding sequences all map to the surface of the lipid binding pocket in the B17 model based on the homologous protein, lipovitellin. Since both B17 and microsomal triglyceride transfer protein (MTP), a critical chaperone during lipoprotein assembly, are homologous to lipovitellin, the identification of these phospholipid remodeling sequences in B17 provides important insights into the potential mechanism that initiates the assembly of apoB-containing lipoproteins.Apolipoprotein B (apoB) is the only known nonexchangeable apolipoprotein in humans. It is the major protein component of low density lipoprotein (LDL), a major risk factor for atherosclerosis. Two forms of apoB are synthesized in humans, B48 from in small intestine and B100 in the liver (1). The production of B48 is the result of a tissue specific mRNA processing occurring in the small intestine (2,3). The translation of B48 and B100 in the endoplasmic reticulum (ER) is directly coupled with the assembly of two major classes of lipoproteins: chylomicrons and very low density lipoproteins (VLDL) (4).Although an apoB structure at atomic resolution is still unavailable, it is generally accepted that the full length apoB has an βα1-β1-α2-β2-α3 pentapartite domain organization, in which α represents predominantly α-helical structures and β corresponds to β-sheets (5). According to this model, apoB is depicted as a belt wrapped around a lipoprotein particle (6). Evidence from electron microscopy indicates that the N-terminus of apoB, largely the βα1 domain, is globularly folded and protrudes away from the LDL particle (7,8). These findings are consistent

Surface Tensiometry of Apolipoprotein B Domains at Lipid Interfaces Suggests a New Model for the Initial Steps in Triglyceride-rich Lipoprotein Assembly

“…Putting these findings into context with other studies of the ␤␣1 superdomain, such as the LV structure of the ␤␣1 superdomain (8 -11, 13, 28), in vitro lipid binding assays (12,14,17,23,29), the conserved sequence motifs (9,30), and apoB C-terminal truncation secretion (31-33), we have developed a more detailed mechanism of initial co-translational apoB lipid binding (see Fig. 14), using the TPs we report here as a road map.…”

“…The hydrophilic face is predicted to form salt-bridge interactions with the C-terminal half of the ␣HD (8,9). By itself, the C-sheet sequence clears 1,2-dimyristoyl-snglycero-3-phosphocholine vesicles extremely rapidly (12). The A-sheet is less homologous to LV than the rest of the ␤␣1 domain and is only partially modeled.…”

“…The N-terminal half of the ␣H domain containing helices 1-10 (H1-10) clears 1,2-dimyristoyl-sn-glycero-3-phosphocholine at the same rate as the entire ␣HD. The C-terminal helices 9 -17 (H9 -17) do not, suggesting a globular structure with hydrophobic residues sequestered in the core (12).…”

“…The A-sheet is less homologous to LV than the rest of the ␤␣1 domain and is only partially modeled. It is predicted to form eight amphipathic ␤-strands but is not soluble in solution without detergent (12). The A-sheet is predicted to interact with the ␤-barrel and ␣-helical domains and is considered the beginning of the ␤1 domain.…”

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

“…Third, Rava et al (60) demonstrated that MTP is not required for the secretion of apoB18. Relevant to this finding are studies demonstrating that apoB17 readily associates with phospholipids (61)(62)(63). Assuming that PL transfer activity of MTP is sufficient for the formation of primordial apoB particles, it is reasonable to expect that the secretion of apoB18 in COS cells would increase, at least partially, by the expression of Drosophila MTP; this was not observed by Rava et al (60).…”