Relation of the size and intracellular sorting of apoB to the formation of VLDL 1 and VLDL 2 (original) (raw)
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Journal of Lipid Research, 2002
We determined the role of N-linked glycosylation of apolipoprotein B (apoB) in the assembly and secretion of lipoproteins using transfected rat hepatoma McA-RH7777 cells expressing human apoB-17, apoB-37, and apoB-50, three apoB variants with different ability to recruit neutral lipids. Substituting Asn residue with Gln at the single glycosylation site within apoB-17 (N 158) decreased its secretion efficiency to a level equivalent to that of wild-type apoB-17 treated with tunicamycin, but had little effect on its synthesis or intracellular distribution. When selective N-to-Q substitution was introduced at one or more of the five N-linked glycosylation sites within apoB-37 (N 158 , N 956 , N 1341 , N 1350 , and N 1496), secretion efficiency of apoB-37 from transiently transfected cells was variably affected. When all five N-linked glycosylation sites were mutated within apoB-37, the secretion efficiency and association with lipoproteins were decreased by Ͼ 50% as compared with wild-type apoB-37. Similarly, mutant apoB-50 with all of its N-linked glycosylation sites mutagenized showed decreased secretion efficiency and decreased lipoprotein association in both d Ͻ 1.02 and d Ͼ 1.02 g/ml fractions. The inability of mutant apoB-37 and apoB-50 to associate with very low-density lipoproteins was attributable to impaired assembly and was not due to the limitation of lipid availability. The decreased secretion of mutant apoB-17 and apoB-37 was not accompanied by accumulation within the cells, suggesting that the proportion of mutant apoB not secreted was rapidly degraded. However unlike apoB-17 or apoB-37, accumulation of mutant apoB-50 was observed within the endoplasmic reticulum and Golgi compartments. These data imply that the N-glycans at the amino terminus of apoB play an important role in the assembly and secretion of lipoproteins containing the carboxyl terminally truncated apoB.-Vukmirica, J.
The Journal of Lipid Research, 2010
Supplementary key words lipid fl uidity • postprandial state • structural conformation • very low density lipoprotein Apolipoprotein E (apoE), a 34 kDa protein that is important in lipid metabolism and cholesterol transport, has three common alleles (2, 3, and 4). ApoE polymorphisms infl uence the risk of atherosclerotic cardiovascular disease and neurodegenerative disorders (1). ApoE3 binds preferentially to HDL and apoE4 to VLDL (2). ApoE contains a 22 kDa N-terminal domain (residues 1-191) and a 10 kDa C-terminal domain (residues 222-299) separated by a protease-sensitive loop (3). ApoE4 shows a more pronounced domain interaction or closed conformation than the other apoE isoforms because it has Arg-112, which enables Arg-61 in the N-terminal domain to interact with Glu-255 in the C-terminal domain, a feature responsible for the preferential association of apoE4 with VLDL (4, 5). Upon binding to lipid, apolipoproteins undergo conformational rearrangements (6, 7) that affect their function. The association of apoE isoform-dependant postprandial lipoprotein metabolism with vascular disease is not well understood. Previously, we reported that lipolytic products of VLDL reduce the intermolecular inter-Abstract Our previous work indicated that apolipoprotein (apo) E4 assumes a more expanded conformation in the postprandial period. The postprandial state is characterized by increased VLDL lipolysis. In this article, we tested the hypothesis that VLDL lipolysis products increase VLDL particle fl uidity, which mediates expansion of apoE4 on the VLDL particle. Plasma from healthy subjects was collected before and after a moderately high-fat meal and incubated with nitroxyl-spin labeled apoE. ApoE conformation was examined by electron paramagnetic resonance spectroscopy using targeted spin probes on cysteines introduced in the N-terminal (S76C) and C-terminal (A241C) domains. Further, we synthesized a novel nitroxyl spin-labeled cholesterol analog, which gave insight into lipoprotein particle fl uidity. Our data revealed that the order of lipoprotein fl uidity was HDL~LDL<VLDL<VLDL+lipoprotein lipase. Moreover, the conformation of apoE4 depended on the lipoprotein fraction: VLDL-associated apoE4 had a more linear conformation than apoE4 associated with LDL or HDL. Further, by changing VLDL fl uidity, VLDL lipolysis products significantly altered apoE4 into a more expanded conformation. Our studies indicate that after every meal, VLDL fl uidity is increased causing apoE4 associated with VLDL to assume a more expanded conformation, potentially enhancing the pathogenicity of apoE4 in vascular tissue.-Tetali, S.
Journal of Biological Chemistry, 2007
The assembly of very low density lipoproteins involves the formation of a primordial, poorly lipidated apoB-containing particle in the endoplasmic reticulum, followed by the addition of neutral lipid from luminal lipid droplets (LLD). However, the lipid and protein compositions of LLD have not been determined. We have isolated LLD from mouse liver microsomes and analyzed their lipid and protein compositions. LLD are variably sized particles relatively poor in triacylglycerol (TG) content when compared with the lipid composition of cytosolic lipid droplets (CLD). They are devoid of apoB, adipophilin, and albumin but contain numerous proteins different from those found on CLD, including TG hydrolase (TGH), carboxylesterase 1 (Ces1), microsomal triglyceride transfer protein (MTP), and apoE. Ectopic expression of TGH in McArdle RH7777 hepatoma cells resulted in decreased cellular TG levels, demonstrating a role for TGH in the mobilization of hepatic neutral lipid stores. The isolation and characterization of LLD provide new supporting evidence for the two-step assembly of very low density lipoproteins.
The liver plays a primary role in lipid metabolism. Important functions include the synthesis and incorporation of hydrophobic lipids, triacylglycerols and cholesteryl esters into the core of water-miscible particles called lipoproteins and the secretion of these particles into the circulation for transport to distant tissues. In this article, we present a brief overview of one aspect of the assembly process of very low density lipoproteins, namely, possible mechanisms for combining core lipids with apolipoprotein B. This is a complex process in which apolipoprotein B interacts with core lipids to form very low density lipoproteins by a two-step process that can be dissociated biochemically. J. Nutr. 129: 463S-466S, 1999. KEY WORDS: • apolipoproteinB-HDL • Brefeldin A • hepatoma cells • microsomal triglyceride transfer protein • secretion
Journal of Lipid Research, 2004
Electronegative low density lipoprotein (LDL ؊) formation that structurally resembles LDL ؊ isolated from plasma was evaluated after LDL treatment with snake venom phospholipase A 2 (PLA 2). PLA 2 treatment of LDL increased its electrophoretic mobility in proportion to the amount of LDL ؊ formed without evidence of lipid peroxidation. These changes dose-dependently correlated with the degree of phospholipid hydrolysis. Strong immunoreactivity of LDL ؊ subfraction from plasma and PLA 2-treated LDL (PLA 2-LDL) to amyloid oligomer-specific antibody was observed. Higher -strand structural content and unfolding proportionate to the loss of ␣-helical structure of apolipoprotein B-100 (apoB-100) of LDL ؊ isolated from both native and PLA 2-LDLs was demonstrated by circular dichroism (CD) spectropolarimetry. These structural changes resembled the characteristics of some oxidatively modified LDLs and soluble oligomeric aggregates of amyloidogenic proteins. PLA 2-LDL was also more susceptible to nitration by peroxynitrite, likely because of exposure of otherwise inaccessible hydrophilic and hydrophobic domains arising from apoB-100 unfolding. This was also demonstrated for plasma LDL ؊. In contrast, PLA 2-LDL was more resistant to coppermediated oxidation that was reversed upon the addition of small amounts of unsaturated fatty acids. The observed similarities between PLA 2-LDL ؊-derived LDL ؊ and plasma LDL ؊ implicate a role for secretory PLA 2 in producing modified LDL ؊ that is facilitated by unfolding of apoB-100.
Structure of apolipoprotein B100 in low density lipoproteins
There is general consensus that amphipathic ␣ -helices and  sheets represent the major lipid-associating motifs of apolipoprotein (apo)B-100. In this review, we examine the existing experimental and computational evidence for the pentapartite domain structure of apoB. In the pentapartite nomenclature presented in this review (NH 2 -␣ 1 - 1 -␣ 2 - 2 -␣ 3 -COOH), the original ␣ 1 globular domain (Segrest, J. P. et al. 1994. Arterioscler. Thromb. 14: 1674-1685) is expanded to include residues 1-1,000 and renamed the ␣ 1 domain. This change reflects the likelihood that the ␣ 1 domain, like lamprey lipovitellin, is a globular composite of ␣ -helical and  -sheet secondary structures that participates in lipid accumulation in the co-translationally assembled prenascent triglyceride-rich lipoprotein particles. Evidence is presented that the hydrophobic faces of the amphipathic  sheets of the  1 and  2 domains of apoB-100 are in direct contact with the neutral lipid core of apoB-containing lipoproteins and play a role in core lipid organization. Evidence is also presented that these  sheets largely determine LDL particle diameter. Analysis of published data shows that with a reduction in particle size, there is an increase in the number of amphipathic helices of the ␣ 2 and ␣ 3 domains associated with the surface lipids of the LDL particle; these increases modulate the surface pressure decreases caused by a reduction in radius of curvature. The properties of the LDL receptor-binding region within the overall domain structure of apoB-100 are also discussed. Finally, recent three-dimensional models of LDL obtained by cryoelectron microscopy and X-ray crystallography are discussed. These models show three common features: a semidiscoidal shape, a surface knob with the dimensions of the  C globular domain of lipovitellin, and planar multilayers in the lipid core that are approximately 35 Å apart; the multilayers are thought to represent cholesteryl ester in the smectic phase. These models present a conundrum: are LDL particles circulating at 37 Њ C spheroidal in shape, as generally assumed, or are they semidiscoidal in shape, as suggested by the models? The limited evidence available supports a spheroidal shape. -Segrest, J. P., M. K. Jones, H. De Loof, and N. Dashti. Structure of apolipoprotein B-100 in low density lipoproteins.
Journal of Biological Chemistry, 2010
The principal protein of high density lipoprotein (HDL), apolipoprotein (apo) A-I, in the lipid-free state contains two tertiary structure domains comprising an N-terminal helix bundle and a less organized C-terminal domain. It is not known how the properties of these domains modulate the formation and size distribution of apoA-I-containing nascent HDL particles created by ATP-binding cassette transporter A1 (ABCA1)-mediated efflux of cellular phospholipid and cholesterol. To address this issue, proteins corresponding to the two domains of human apoA-I (residues 1-189 and 190 -243) and mouse apoA-I (residues 1-186 and 187-240) together with some human/mouse domain hybrids were examined for their abilities to form HDL particles when incubated with either ABCA1-expressing cells or phospholipid multilamellar vesicles. Incubation of human apoA-I with cells gave rise to two sizes of HDL particles (hydrodynamic diameter, 8 and 10 nm), and removal or disruption of the C-terminal domain eliminated the formation of the smaller particle. Variations in apoA-I domain structure and physical properties exerted similar effects on the rates of formation and sizes of HDL particles created by either spontaneous solubilization of phospholipid multilamellar vesicles or the ABCA1-mediated efflux of cellular lipids. It follows that the sizes of nascent HDL particles are determined at the point at which cellular phospholipid and cholesterol are solubilized by apoA-I; apparently, this is the rate-determining step in the overall ABCA1mediated cellular lipid efflux process. The stability of the apoA-I N-terminal helix bundle domain and the hydrophobicity of the C-terminal domain are important determinants of both nascent HDL particle size and their rate of formation.
Journal of lipid research, 1993
Very low density lipoproteins (VLDL) are a heterogenous population of particles differing in size and composition. Heparin-Sepharose chromatography yields three VLDL subfractions. Two subfractions, VLDLNR-1 and VLDLNR-2, which are not retained by heparin, contain little or no detectable apolipoprotein (apo)E. According to negative stain electron microscopy, VLDLNR-1 is slightly larger than VLDLNR-2. The third fraction, VLDLR, is composed of smaller particles that are retained by the heparin-Sepharose and contain apoE. The C apolipoproteins of the respective VLDL subfractions transfer to 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) single bilayer vesicles giving three subfractions designated VLDLNR-1-C, VLDLNR-2-C, and VLDLR-C. The protein, phospholipid, and cholesterol (free + esterified) contents decrease in the order VLDLR > VLDLNR-2 > VLDLNR-1. Triglyceride content decreases in the opposite order. POPC treatment of each VLDL subfraction increases the phospholipid and dec...
Molecular Biology of the Cell, 2006
In the present study, by using Huh7 cells, derived from human hepatoma and competent for VLDL secretion, we found that ApoB is highly concentrated around CLDs to make "ApoB-crescents." ApoB-crescents were seen in <10% of Huh7 cells under normal conditions, but the ratio increased to nearly 50% after 12 h of proteasomal inhibition by N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal. Electron microscopy showed ApoB to be localized to a cluster of electron-lucent particles 50 -100 nm in diameter adhering to CLDs. ApoB, proteasome subunits, and ubiquitinated proteins were detected in the CLD fraction, and this ApoB was ubiquitinated. Interestingly, proteasome inhibition also caused increases in autophagic vacuoles and ApoB in lysosomes. ApoB-crescents began to decrease after 12-24 h of proteasomal inhibition, but the decrease was blocked by an autophagy inhibitor, 3-methyladenine. Inhibition of autophagy alone caused an increase in ApoB-crescents. These observations indicate that both proteasomal and autophagy/lysosomal degradation of ApoB occur around CLDs and that the CLD surface functions as a unique platform for convergence of the two pathways.