Vesicle-binding properties of wild-type and cysteine mutant forms of α1 domain of apolipoprotein B

DeLozier, Jeanine A.; Parks, John S.; Shelness, Gregory S.

doi:10.1016/s0022-2275(20)31664-3

Cited by 8 publications

(2 citation statements)

References 35 publications

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“…The N-terminal β1 superdomain in apoB is the very first region that enters the ER and initiates lipoprotein assembly. 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 βR1 domain of apoB.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2006

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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

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Section: Discussionmentioning

confidence: 99%

“…We hypothesize that the N-terminal βR1 superdomain of apoB contains phospholipid recruiting sequences that nucleate lipoprotein assembly. In fact, B17 binds both phospholipid vesicles and phospholipid-coated triglyceride emulsions in vitro (28)(29)(30). It also has the potential to convert multilamellar DMPC into smaller particles (29).…”

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

et al. 2006

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Identification of the Lipoprotein Initiating Domain of Apolipoprotein B

Shelness

Ledford

et al. 2003

Journal of Biological Chemistry

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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.

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Self-association and Lipid Binding Properties of the Lipoprotein Initiating Domain of Apolipoprotein B

Ledford

Weinberg

Cook

et al. 2006

Journal of Biological Chemistry

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The amino-terminal 20.1% of apolipoprotein B (apoB20.1; residues 1-912) is sufficient to initiate and direct the formation of nascent apoB-containing lipoprotein particles. To investigate the mechanism of initial lipid acquisition by apoB, we examined the lipid binding and interfacial properties of a carboxyl-terminal His 6 -tagged form of apoB20.1 (apoB20.1H). ApoB20.1H was expressed in Sf9 cells and purified by nickel affinity chromatography. ApoB20.1H was produced in a folded state as characterized by formation of intramolecular disulfide bonds and resistance to chemical reduction. Dynamic light scattering in physiological buffer indicated that purified apoB20.1H formed multimers, which were readily dissociable upon the addition of nonionic detergent (0.1% Triton X-100). ApoB20.1H was incapable of binding dimyristoylphosphatidylcholine multilamellar vesicles, unless its multimeric structure was first disrupted by guanidine hydrochloride. However, apoB20.1H multimers spontaneously dissociated and bound to the interface of naked and phospholipid-coated triolein droplets. These data reveal that the initiating domain of apoB contains solvent-accessible hydrophobic sequences, which, in the absence of a hydrophobic lipid interface or detergent, engage in self-association. The high affinity of apoB20.1H for neutral lipid is consistent with the membrane binding and desorption model of apoB-containing lipoprotein assembly. ApoB3 is the major structural protein of hepatic very low density lipoproteins and intestinal chylomicrons (1, 2). The assembly of apoBcontaining lipoproteins proceeds in two steps: cotranslational incorporation of the nascent apoB polypeptide into a precursor lipoprotein particle in the rough ER followed by the additional posttranslational lipidation via fusion with lipid droplets in the ER and/or the Golgi (3-6). Both steps of apoB-containing lipoprotein assembly require MTP, a dedicated cofactor composed of a unique 97-kDa subunit complexed with the ubiquitous ER-localized folding enzyme, protein-disulfide isomerase (7,8). During the first step of lipoprotein assembly, MTP may transfer lipid molecules from the ER membrane or other donor sites directly to apoB and/or may bind to apoB and promote its proper folding and autonomous lipid acquisition (7, 9). MTP also participates in bulk transmembrane lipid transport from the cytosol into the ER-Golgi, a process critical for second step apoB particle expansion (10 -12).Several models of the first initiating step of lipoprotein assembly have been proposed. The earliest and most commonly accepted theory posits that the apoB polypeptide interacts with the inner leaflet of the ER membrane during translation, causing the nucleation and desorption of an ϳ20-nm diameter emulsion particle upon the completion of translation (13-17). A variation of this model proposes that the interfacial binding and lipoprotein desorption process is completed upon translation of only the first ϳ1000 amino-terminal residues of apoB, giving rise to a small, high density lipoprot...

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Vesicle-binding properties of wild-type and cysteine mutant forms of α1 domain of apolipoprotein B

Cited by 8 publications

References 35 publications

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

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

Identification of the Lipoprotein Initiating Domain of Apolipoprotein B

Self-association and Lipid Binding Properties of the Lipoprotein Initiating Domain of Apolipoprotein B

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