Measurement of apolipoprotein B in various cell lines: Correlation between intracellular levels and rates of secretion

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Human microsomal triacylglycerol transfer protein (hMTP) is essential for apolipoprotein B (apoB)-lipoprotein assembly and secretion and is known to transfer triacylglycerols, cholesterol esters, and phospholipids. To understand the relative importance of each lipid transfer activity, we compared the ability of hMTP and its Drosophila ortholog (dMTP) to assemble apoB lipoproteins and to transfer various lipids. apoB48 secretion was induced when co-expressed with either hMTP or dMTP in COS cells, and oleic acid supplementation further augmented secretion without altering particle density. C-terminal epitope-tagged dMTP (dMTP-FLAG) facilitated the secretion of apoB polypeptides in the range of apoB48 to apoB72 but was ϳ50% as efficient as hMTP-FLAG. Comparison of lipid transfer activities revealed that although phospholipid transfer was similar in both orthologs, dMTP was unable to transfer neutral lipids. We conclude that the phospholipid transfer activity of MTP is sufficient for the assembly and secretion of primordial apoB lipoproteins and may represent its earliest function evolved for the mobilization of lipid in invertebrates. Identification of MTP inhibitors, which selectively affect transfer of a specific lipid class, may have therapeutic potential.Lipoproteins are lipid-protein complexes that transport lipids, fatsoluble vitamins, and other hydrophobic molecules in the plasma. Apolipoprotein B (apoB) 2 is a structural protein embedded in the phospholipid monolayer on the surface of triglyceride-rich lipoproteins. It has been hypothesized that apoB contains amphipathic ␣-helical and ␤-sheet domains (1). Lipidation of the ␤-sheets is necessary for the assembly of larger apoB polypeptides into lipoprotein emulsion particles. Lipoprotein assembly begins co-translationally and microsomal triglyceride transfer protein (MTP) plays a pivotal role in this process (for reviews, see Refs. 2-6). MTP is absent in abetalipoproteinemia, a disease characterized by the deficit of plasma apoB lipoproteins and low plasma cholesterol levels (7,8). Reconstitution of MTP in heterologous systems rescues apoB secretion, whereas tissue-specific liver knock-out models recreate the lipoprotein deficiency present in abetalipoproteinemia (9, 10). The signature activity of MTP is its ability to transfer lipids between membranes. It has been demonstrated that MTP lipid transfer activity is necessary for apoB lipoprotein assembly and secretion (for reviews, see Refs. 2-6). More recent evidence suggests that MTP lipid transfer activity is also responsible for lipid accretion within the secretory pathway and that MTP potentially stabilizes lipid vesicles in the endoplasmic reticulum (reviewed in Refs. 2 and 3).MTP transfers several lipids including triacylglycerols, cholesterol esters, and phospholipids between vesicles in vitro (11)(12)(13)(14). It has been suggested that MTP transiently interacts with a membrane, extracts lipids, and then delivers them to another lipid acceptor or to nascent apoB lipoproteins (15). Kinetic studies in...

Section: Methodsmentioning

confidence: 99%

Phospholipid Transfer Activity of Microsomal Triacylglycerol Transfer Protein Is Sufficient for the Assembly and Secretion of Apolipoprotein B Lipoproteins

Rava

Ojakian

Shelness

et al. 2006

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“…For experiments, 80% confluent monolayers were incubated with serum-free medium containing 0.2% bovine serum albumin for 48 h. The concentrated conditioned medium was used to determine the amount of apoB polypeptides present and to study the binding of these peptides to the immobilized MTP in the presence and the absence of phospholipid vesicles. The apoB polypeptides that interacted with MTP were quantified by ELISA (27,34,35). COS-7 cells were transiently transfected with plasmids expressing apoB17 and apoB:270 -570 using Fugene-6 reagent according to the manufacturer's protocol.…”

Section: Methodsmentioning

confidence: 99%

Binding of Microsomal Triglyceride Transfer Protein to Lipids Results in Increased Affinity for Apolipoprotein B

Bakillah¹,

Hussain²

2001

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Apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) are known to interact with each other. We evaluated the effect of different lipids on the protein-protein interactions between MTP and apoB100 or its C-terminally truncated forms. Negatively charged lipids decreased protein-protein interactions between apoB and MTP. In contrast, zwitterionic phospholipids enhanced (2-4-fold) the binding of apoB100 to MTP by increasing affinity (1.5-3-fold) between these proteins without affecting the number of binding sites. Similarly, phospholipids augmented (1.5-4-fold) the binding of various C-terminally truncated apoB peptides to MTP. The increased binding was greater for apoB peptides containing lipidbinding domains, such as apoB28 and apoB42. Surprisingly, preincubation of apoB28 with lipid vesicles had no effect on MTP binding. In contrast, incubation of MTP with lipid vesicles resulted in a stable association of MTP with vesicles, and MTP-lipid vesicles bound better (5-fold increase) to LDL than did lipid-free MTP. To determine whether MTP exists stably associated with lipids in cells, microsomal contents from COS cells expressing MTP, HepG2 cells, and mouse liver were ultracentrifuged, and MTP was visualized in different density fractions. MTP was found associated and unassociated with lipids. In contrast, apoB17 and apoB:270 -570 were present unassociated with lipids in COS cells. These studies show that the binding of MTP to lipids results in increased affinity for apoB and that stable MTP-lipid complexes exist in the lumen of the endoplasmic reticulum. Protein-protein interactions between apoB and MTP may juxtapose lipids associated with MTP to lipid-binding domains of apoB and facilitate hydrophobic interactions leading to enhance affinity. We speculate that MTP-lipid complexes may serve as nuclei to form "primordial lipoproteins" and may also play a role in the bulk addition of lipids during the "core expansion" of these lipoproteins. Apolipoprotein B (apoB)1 is essential for the transport of lipids packaged into lipid-protein complexes called lipoproteins. Assembly of these lipoproteins requires, in addition to apoB, microsomal triglyceride transfer protein (MTP). In vitro kinetic studies suggest that MTP may catalyze transfer of lipids between vesicles by a shuttle mechanism (1). In this process, each MTP molecule is proposed to interact transiently with donor vesicles, extract up to 2 mol of PC and 0.25 mol of triglycerides, dissociate from these vesicles, interact transiently with acceptor vesicles, and rapidly deliver lipid molecules to these vesicles (1, 2). The importance of this lipid transfer activity in lipoprotein assembly was established by identifying mutations in MTP gene that result in loss of lipid transfer activity in patients that lack apoB-containing lipoproteins in their plasma (3-5), by identifying compounds that inhibit in vitro lipid transfer activity and decrease apoB secretion (6 -9), and by manipulating gene expression and correlating it to changes in apoB secretion...

“…All the assays were performed with the purified immobilized homodimeric LPL. Antibodies used in ELISA for the detection of apoB have been described (12,15,16).…”

Section: Methodsmentioning

confidence: 99%

“…Microtiter wells were washed three times with PBS and 0.3% BSA. A sandwich ELISA (15,16) quantitated apoB bound to immobilized LPL. In parallel, a standard curve for the apoB was generated by coating wells with a monoclonal antibody, 1D1, and incubating with different concentrations of LDL (0 -14 ng/well) as described before (15,16).…”

Section: Methodsmentioning

confidence: 99%

“…Microtiter wells were coated with M2 (3 g/well), a monoclonal antibody that recognizes FLAG, or purified, dimeric LPL (0.5 g/well). Wells were incubated in triplicate with 100 l of different conditioned media and washed and the bound peptides were quantitated using polyclonal sheep anti-human apoB antibodies and alkaline phosphatase-labeled anti-sheep IgG as described before (15,16,21). The optical densities were determined at 405 nm.…”

Section: Table Imentioning

confidence: 99%

See 1 more Smart Citation

High Affinity Binding between Lipoprotein Lipase and Lipoproteins Involves Multiple Ionic and Hydrophobic Interactions, Does Not Require Enzyme Activity, and Is Modulated by Glycosaminoglycans

Hussain

Obunike

Shaheen

et al. 2000

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Lipoprotein lipase (LPL) physically associates with lipoproteins and hydrolyzes triglycerides. To characterize the binding of LPL to lipoproteins, we studied the binding of low density lipoproteins (LDL), apolipoprotein (apo) B17, and various apoB-FLAG (DYKDDDDK octapeptide) chimeras to purified LPL. LDL bound to LPL with high affinity (K d values of 10 ؊12 M) similar to that observed for the binding of LDL to its receptors and 1D1, a monoclonal antibody to LDL, and was greater than its affinity for microsomal triglyceride transfer protein. LDL-LPL binding was sensitive to both salt and detergents, indicating the involvement of both hydrophobic and hydrophilic interactions. In contrast, the N-terminal 17% of apoB interacted with LPL mainly via ionic interactions. Binding of various apoB fusion peptides suggested that LPL bound to apoB at multiple sites within apoB17. Tetrahydrolipstatin, a potent enzyme activity inhibitor, had no effect on apoB-LPL binding, indicating that the enzyme activity was not required for apoB binding. LDL-LPL binding was inhibited by monoclonal antibodies that recognize amino acids 380 -410 in the C-terminal region of LPL, a region also shown to interact with heparin and LDL receptor-related protein.The LDL-LPL binding was also inhibited by glycosaminoglycans (GAGs); heparin inhibited the interactions by ϳ50% and removal of trace amounts of heparin from LPL preparations increased LDL binding. Thus, we conclude that the high affinity binding between LPL and lipoproteins involves multiple ionic and hydrophobic interactions, does not require enzyme activity and is modulated by GAGs. It is proposed that LPL contains a surface exposed positively charged amino acid cluster that may be important for various physiological interactions of LPL with different biologically important molecules. Moreover, we postulate that by binding to this cluster, GAGs modulate the association between LDL and LPL and the in vivo metabolism of LPL.