Identification of the Lipoprotein Initiating Domain of Apolipoprotein B

Abstract

We have explored the minimum sequence requirement for the initiation of apolipoprotein B (apoB)-mediated triglyceride-rich lipoprotein assembly. A series of apoB COOH-terminal truncation mutants, spanning a range from apoB34 (amino acid residues 1-1544 of apoB100) to apoB19 (residues 1-862) were transfected into COS cells with and without coexpression of the microsomal triglyceride transfer protein (MTP). ApoB34, -25, -23, -21, -20.5, and -20.1 underwent efficient conversion to buoyant lipoproteins when coexpressed with MTP. ApoB19.5 (amino acids 1-884) also directed MTP-dependent particle assembly, although at reduced efficiency. When apoB19.5 was truncated by another 22 amino acids to form apoB19, MTP-dependent lipoprotein assembly was abolished. Analysis of the lipid stoichiometry of secreted lipoproteins revealed that all apoB truncation mutants formed spherical particles containing a hydrophobic core. Even highly truncated assembly-competent forms of apoB, such as apoB19.5 and 20.1, formed lipoproteins with surface:core lipid ratios of <1. We conclude that the translation of the first ϳ884 amino acids of apoB completes a domain capable of initiating nascent lipoprotein assembly. The composition of lipids recruited into lipoproteins by this initiating domain is consistent with formation of small emulsion particles, perhaps by simultaneous desorption of both polar and neutral lipids from a saturated bilayer.

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

“…Our results supported a model in which the first 1000 amino acid residues of apoB are competent to complete the lipid pocket without a structural requirement for MTP, and that this lipid pocket has a fixed lipid capacity on the order of 50 phospholipids (PL) for a total stoichiometry of 70 lipid molecules, a number in close agreement with that reported for the LV complex (29 -31). We propose that the initiation complex in the assembly of apoB-containing lipoprotein particle is PL-rich suggesting a small bilayer type organization rather than a TAG-rich emulsion proposed by others (32). Although our results (27,28) demonstrated that MTP is not a structural requirement for the formation of the lipid pocket, they did not rule out its potential functional role, i.e.…”

Microsomal Triglyceride Transfer Protein Promotes the Secretion of Xenopus laevis Vitellogenin A1

“…As a positive control, apoB20.1 was analyzed. This form of apoB was shown previously to form a small emulsion particle with a density of ϳ1.24 g/ml when coexpressed with MTP (24). Under the same conditions used for the Vtg analysis, MTP coexpression converted ϳ20% of the total secreted apoB20.1 into a form that floated at d Ͻ 1.25 g/ml (Fig.…”

“…To explore whether MTP-mediated stimulation of Vtg secretion is accompanied by its acquisition of lipid, equilibrium density gradient centrifugation at d ϭ 1.25 g/ml KBr was performed as described (24). Vtg recovered from AP-or MTPtransfected cells was recovered in the d Ͼ 1.25 g/ml, lipid-poor bottom gradient fraction (Fig.…”

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

“…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.…”