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• W1985176073 abstract "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 ∼50% 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. 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 ∼50% 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, fat-soluble vitamins, and other hydrophobic molecules in the plasma. Apolipoprotein B (apoB) 2The abbreviations used are: apoB, apolipoprotein B; BSA, bovine serum albumin; MTP, microsomal triglyceride transfer protein; dMTP, Drosophila MTP; hMTP, human MTP; OA, oleic acid; PDI, protein disulfide isomerase; SREBP, sterol regulatory element-binding protein; ELISA, enzyme-linked immunosorbent assay. 2The abbreviations used are: apoB, apolipoprotein B; BSA, bovine serum albumin; MTP, microsomal triglyceride transfer protein; dMTP, Drosophila MTP; hMTP, human MTP; OA, oleic acid; PDI, protein disulfide isomerase; SREBP, sterol regulatory element-binding protein; ELISA, enzyme-linked immunosorbent assay. is a structural protein embedded in the phospholipid monolayer on the surface of triglyceride-rich lipoproteins. It has been hypothesized that apoB contains amphipathic α-helical and β-sheet domains (1Segrest J.P. Jones M.K. Mishra V.K. Anantharamaiah G.M. Garber D.W. Arterioscler. Thromb. 1994; 14: 1674-1685Crossref PubMed Scopus (167) Google Scholar). Lipidation of the β-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. 2Hussain M.M. Shi J. Dreizen P. J. Lipid. Res. 2003; 44: 22-32Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar, 3Hussain M.M. Iqbal J. Anwar K. Rava P. Dai K. Front. Biosci. 2003; 8: S500-S506Crossref PubMed Scopus (72) Google Scholar, 4Shelness G.S. Sellers J.A. Curr. Opin. Lipidol. 2001; 12: 151-157Crossref PubMed Scopus (228) Google Scholar, 5Wetterau J.R. Lin M.C.M. Jamil H. Biochim. Biophys. Acta. 1997; 1345: 136-150Crossref PubMed Scopus (283) Google Scholar, 6Gordon D.A. Jamil H. Biochim. Biophys. Acta. 2000; 1486: 72-83Crossref PubMed Scopus (191) Google Scholar). MTP is absent in abetalipoproteinemia, a disease characterized by the deficit of plasma apoB lipoproteins and low plasma cholesterol levels (7Wetterau J.R. Aggerbeck L.P. Bouma M.-E. Eisenberg C. Munck A. Hermier M. Schmitz J. Gay G. Rader D.J. Gregg R.E. Science. 1992; 258: 999-1001Crossref PubMed Scopus (629) Google Scholar, 8Sharp D. Blinderman L. Combs K.A. Kienzle B. Ricci B. Wager-Smith K. Gil C.M. Turck C.W. Bouma M.-E. Rader D.J. Aggerbeck L.P. Gregg R.E. Gordon D.A. Wetterau J.R. Nature. 1993; 365: 65-69Crossref PubMed Scopus (399) Google Scholar). Reconstitution of MTP in heterologous systems rescues apoB secretion, whereas tissue-specific liver knock-out models recreate the lipoprotein deficiency present in abetalipoproteinemia (9Raabe M. Véniant M.M. Sullivan M.A. Zlot C.H. Björkegren J. Nielsen L.B. Wong J.S. Hamilton R.L. Young S.G. J. Clin. Invest. 1999; 103: 1287-1298Crossref PubMed Scopus (357) Google Scholar, 10Gordon D.A. Jamil H. Sharp D. Mullaney D. Yao Z. Gregg R.E. Wetterau J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7628-7632Crossref PubMed Scopus (189) Google Scholar). 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. 2Hussain M.M. Shi J. Dreizen P. J. Lipid. Res. 2003; 44: 22-32Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar, 3Hussain M.M. Iqbal J. Anwar K. Rava P. Dai K. Front. Biosci. 2003; 8: S500-S506Crossref PubMed Scopus (72) Google Scholar, 4Shelness G.S. Sellers J.A. Curr. Opin. Lipidol. 2001; 12: 151-157Crossref PubMed Scopus (228) Google Scholar, 5Wetterau J.R. Lin M.C.M. Jamil H. Biochim. Biophys. Acta. 1997; 1345: 136-150Crossref PubMed Scopus (283) Google Scholar, 6Gordon D.A. Jamil H. Biochim. Biophys. Acta. 2000; 1486: 72-83Crossref PubMed Scopus (191) Google Scholar). 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. 2Hussain M.M. Shi J. Dreizen P. J. Lipid. Res. 2003; 44: 22-32Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar and 3Hussain M.M. Iqbal J. Anwar K. Rava P. Dai K. Front. Biosci. 2003; 8: S500-S506Crossref PubMed Scopus (72) Google Scholar).MTP transfers several lipids including triacylglycerols, cholesterol esters, and phospholipids between vesicles in vitro (11Wetterau J.R. Zilversmit D.B. J. Biol. Chem. 1984; 259: 10863-10866Abstract Full Text PDF PubMed Google Scholar, 12Wetterau J.R. Zilversmit D.B. Chem. Phys. Lipids. 1985; 38: 205-222Crossref PubMed Scopus (99) Google Scholar, 13Athar H. Iqbal J. Jiang X.C. Hussain M.M. J. Lipid Res. 2004; 45: 764-772Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 14Rava P. Athar H. Johnson C. Hussain M.M. J. Lipid Res. 2005; 46: 1779-1785Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). 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 (15Atzel A. Wetterau J.R. Biochemistry. 1993; 32: 10444-10450Crossref PubMed Scopus (80) Google Scholar). Kinetic studies indicate the presence of two lipid-binding sites: one site binds triacylglycerols, cholesterol esters, and phospholipids with a preference for neutral lipids, whereas a second site binds only phospholipids (16Atzel A. Wetterau J.R. Biochemistry. 1994; 33: 15382-15388Crossref PubMed Scopus (50) Google Scholar). It is not clear whether all MTP lipid transfer activities are required for lipoprotein assembly.Recently, we identified a human MTP (hMTP) ortholog from the genome of the fruit fly, Drosophila melanogaster, that supported the secretion of human apoB34 and apoB41 (17Sellers J.A. Hou L. Athar H. Hussain M.M. Shelness G.S. J. Biol. Chem. 2003; 278: 20367-20373Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). However, the mechanisms involved in the secretion of apoB lipoproteins by the Drosophila MTP (dMTP) have not been elucidated. In this paper, we show that although dMTP is defective in triacylglycerol transfer activity, the phospholipid transfer activity is equivalent to that of hMTP. Thus, phospholipid, but not the triacylglycerol transfer, activity may be necessary for the assembly of primordial lipoproteins.MATERIALS AND METHODSConstruction of MTP-FLAG Chimeras—hMTP and dMTP expression vectors have been previously described (17Sellers J.A. Hou L. Athar H. Hussain M.M. Shelness G.S. J. Biol. Chem. 2003; 278: 20367-20373Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). C-terminal FLAG-tagged forms of Drosophila and hMTP were produced by PCR, utilizing 3′ antisense primers encoding the FLAG sequence (DYKDDDDK) followed by an in-frame termination codon.Cell Culture and apoB Secretion—COS-7 cells were grown in Dulbecco's modified Eagle's medium (CellGrow) containing 10% fetal bovine serum (BSA) supplemented with l-glutamine and antibiotics. The cells were initially dislodged from the plate with trypsin and seeded in six-well plates (400,000 cells/well). Transfections were performed using FuGENE 6 transfection reagent (Roche Applied Sciences) according to the manufacturer's instructions. For sequential transfections, apoB expression vectors were initially introduced into COS cells using FuGENE 6 in T175 flasks (7.2 × 106 cells). After 8 h, the cells were detached by trypsin treatment, seeded in six-well plates, and transfected with MTP expression plasmids. At 48 h post-transfection, the media were aspirated and either 1 ml of Dulbecco's modified Eagle's medium or 1 ml of lipid-containing medium (Dulbecco's modified Eagle's medium including 0.4 mm oleic acid complexed with 1.5% BSA and 1 mm glycerol) were added. Following additional 18 h of incubation, the media were collected, protease inhibitors (Sigma) were added, the samples were centrifuged (2,500 rpm, 4 °C, 10 min) to pellet cell debris, and the apoB contents were measured in the supernatants by ELISA (18Hussain M.M. Zhao Y. Kancha R.K. Blackhart B.D. Yao Z. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 485-494Crossref PubMed Scopus (63) Google Scholar, 19Bakillah A. Zhou Z. Luchoomun J. Hussain M.M. Lipids. 1997; 32: 1113-1118Crossref PubMed Scopus (38) Google Scholar). The media were also subjected to ultracentrifugation to separate lipoproteins (20Iqbal J. Hussain M.M. J. Lipid Res. 2005; 46: 1491-1501Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Conditioned media (4 ml) were adjusted to 1.30 g/ml with KBr and overlaid with 2 ml each of 1.24, 1.15, and 1.063 g/ml KBr solutions and 1 ml each of 1.019 and 1.006 g/ml. After ultracentrifugation (SW41 rotor, 40,000 rpm, 17 h, 15 °C), 1-ml fractions were collected, and their apoB content was determined by ELISA. The density in each fraction was measured using a refractometer (Fisher).Immunofluorescence—COS-7 cells grown on coverslips in 24-well tissue culture plates were transfected with apoB48 and either hMTP- or dMTP-FLAG expression plasmids. Forty-eight hours post-transfection, the cells were fixed and permeabilized in methanol for 15 min at -20 °C. The fixed cells were blocked with phosphate-buffered saline containing 1 mm MgCl2, 0.5 mm CaCl2, 3% BSA, and 1% goat serum and incubated with antibodies diluted 1:100 (unless stated otherwise) in the same buffer at room temperature for 1 h. The cells were incubated with mouse anti-FLAG M2 (Sigma) and either rabbit anti-calnexin (Stressgen) or anti-α-mannosidase II (USBiological; 1:25) followed by treatment with Alexa Fluor 488 (green fluorescence)-conjugated goat anti-mouse IgG1 and Alexa Fluor 594 (red fluorescence)-conjugated goat anti-rabbit IgG1 (Molecular Probes). The coverslips were mounted in phosphate-buffered saline containing 10% glycerol and 12% triethyldiamine (Sigma) to prevent fluorescent bleaching and visualized using a Bio-Rad Radiance 2000 confocal microscope.Affinity Purification of MTP-FLAG—COS-7 cells in 150-mm dishes were transiently transfected with either hMTP- or dMTP-FLAG expression plasmids, washed with phosphate-buffered saline, and lysed by incubating in hypotonic buffer (1 mm Tris, pH 7.4, 1 mm MgCl2, 1 mm EGTA, and protease inhibitor mixture) as previously described (13Athar H. Iqbal J. Jiang X.C. Hussain M.M. J. Lipid Res. 2004; 45: 764-772Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 14Rava P. Athar H. Johnson C. Hussain M.M. J. Lipid Res. 2005; 46: 1779-1785Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The lysates were then centrifuged (SW55 Ti rotor, 50,000 rpm, 1 h, 4 °C), and the supernatants were adjusted to 10 mm Tris, pH 7.4, 150 mm NaCl and mixed by rotation with M2-agarose beads (Sigma) for 3 h at 4 °C. The beads were pelleted by centrifugation (10,000 rpm, 4 °C, 10 s) and washed three times with 10 mm Tris, pH 7.4, 150 mm NaCl, and protease inhibitor. To elute the bound proteins, the beads were rotated with 300 ng/μl of FLAG peptide (DYKDDDDK) in the same buffer for 2 h at 4 °C. The samples were centrifuged (10,000 rpm, 4 °C, 10 s), and the supernatants containing MTP-FLAG were collected.Lipid Transfer Assays—Radiolabeled lipid transfer assays were performed according to the method described by Wetterau and Zilversmit (11Wetterau J.R. Zilversmit D.B. J. Biol. Chem. 1984; 259: 10863-10866Abstract Full Text PDF PubMed Google Scholar, 12Wetterau J.R. Zilversmit D.B. Chem. Phys. Lipids. 1985; 38: 205-222Crossref PubMed Scopus (99) Google Scholar). To prepare unilamellar donor vesicles 1800 nmol of phosphatidylcholine (Avanti Polar Lipids), 3.6 nmol of either [14C]triacylglycerol, [14C]phosphatidylcholine (New England Nuclear), or [3H]cholesterol ester (Amersham Biosciences), and 135 nmol of cardiolipin (Sigma) were dried under nitrogen, resuspended in 4.5 ml of buffer (15 mm Tris, 40 mm NaCl, 1 mm EDTA, and 0.02% sodium azide, pH 7.4), and sonicated. Acceptor vesicles containing 10,800 nmol of phosphatidylcholine were prepared similarly. The vesicles were then centrifuged (50,000 rpm, 15 °C, 1 h, SW55 Ti rotor), and the top 4-ml fraction was collected for activity determinations. The reaction mixture (final volume, 0.5 ml) contained 100 μl each of donor and acceptor vesicles in 10 mm Tris-HCl, pH 7.4, 0.1% BSA, and 150 mm NaCl. M2 affinity-purified MTP (100 μl) was added to the reaction mixture and incubated for 2 h at 37 °C. The reaction was stopped by the addition of 0.5 ml of DE52 anion exchange resin (1:1, v/v, suspension in 15 mm Tris, 1 mm EDTA, 0.02% sodium azide, pH 7.4.), rotated at 4 °C for 5 min, and centrifuged (12,000 rpm, 4 °C, 10 min). Radiolabeled lipids in supernatants (500 μl) were measured by scintillation counting. Blank assays were performed in the absence of purified proteins. Total radioactivity present in the assays was measured by omitting DE52 from the stop buffer. Lipid transfer (% transfer/2 h) was determined.Transfer of fluorescently labeled lipids was assayed as described previously (13Athar H. Iqbal J. Jiang X.C. Hussain M.M. J. Lipid Res. 2004; 45: 764-772Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 14Rava P. Athar H. Johnson C. Hussain M.M. J. Lipid Res. 2005; 46: 1779-1785Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Briefly, donor vesicles containing nitrobenzoaxadiazole-labeled triacylglycerols (Chylos, Inc.) and phosphatidylethanolamine (14Rava P. Athar H. Johnson C. Hussain M.M. J. Lipid Res. 2005; 46: 1779-1785Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) were incubated with acceptor vesicles in the presence of equal amounts of total cell protein lysates or purified hMTP- and dMTP-FLAG proteins for the indicated times. Increases in fluorescence during transfer were recorded, and the percentage of transfer was calculated.MTP Gene Deletion Studies—MTPfl/flMx1Cre mice (21Lieu H.D. Withycombe S.K. Walker Q. Rong J.X. Walzem R.L. Wong J.S. Hamilton R.L. Fisher E.A. Young S.G. Circulation. 2003; 107: 1315-1321Crossref PubMed Scopus (106) Google Scholar) were obtained from Jackson Laboratories and bred at SUNY Downstate Medical Center. The mice were injected once with either phosphate-buffered saline or pIpC (250 μg). After 48 h, the livers were collected and used to measure lipid transfer activities of MTP as described before (13Athar H. Iqbal J. Jiang X.C. Hussain M.M. J. Lipid Res. 2004; 45: 764-772Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 14Rava P. Athar H. Johnson C. Hussain M.M. J. Lipid Res. 2005; 46: 1779-1785Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar).RESULTSWe previously showed that dMTP promotes human apoB34 and apoB41 secretion (17Sellers J.A. Hou L. Athar H. Hussain M.M. Shelness G.S. J. Biol. Chem. 2003; 278: 20367-20373Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Here we explored how dMTP aids in the secretion of apoB. First, we asked whether apoB secretion supported by dMTP is modulated by oleic acid (OA) supplementation. Transfection of COS cells with apoB48 expressing plasmids resulted in barely detectable apoB levels in the media with or without OA supplementation (Fig. 1, A and B). However, co-transfection with hMTP significantly increased (∼20-fold) the secretion of apoB48, and OA supplementation further enhanced its secretion by 25.6 ± 6.9% (p = 0.003) (Fig. 1A). Similar to hMTP, co-expression of dMTP and apoB48 resulted in increased secretion of apoB48 (∼7-fold) that was further augmented (80.8 ± 21.6%, p = 0.004) by OA supplementation (Fig. 1B). Next, we explored the effect of OA supplementation on the density of secreted lipoproteins. In the absence of OA, apoB secreted from COS cells expressing hMTP or dMTP was present in fractions 3-6 corresponding to a density of 1.1-1.15 g/ml (Fig. 1, C-E). Although OA increased the total apoB secreted, it did not change the density of the secreted lipoproteins (Fig. 1, compare C and D). These studies indicate that apoB secretion driven by dMTP expression is responsive to OA supplementation and produces lipoproteins with properties similar to those secreted in the presence of hMTP.We observed that dMTP can render apoB48 secretion-competent, but cells expressing dMTP consistently secreted less apoB than those expressing hMTP (Fig. 1). To understand why dMTP was less efficient in promoting apoB secretion, we considered two possibilities. First, the reduction might be due to the lower expression of dMTP compared with hMTP in COS cells. Second, different levels of apoB expression might be due to variations in co-transfection efficiency. To obtain equal expression, the cells were transfected with expression plasmids containing sequences for apoB18 through apoB72 and subsequently transfected with expression vectors containing either hMTP-FLAG or dMTP-FLAG. The relative expression of the different MTPs was determined by Western blotting. apoB18-expressing cells synthesized similar amounts of hMTP- and dMTP-FLAG (Fig. 2A, inset), and apoB18 secretion was not affected by the co-expression of either MTP (Fig. 2A) consistent, with previous studies (22McLeod R.S. Zhao Y. Selby S.L. Westerlund J. Yao Z. J. Biol. Chem. 1994; 269: 2852-2862Abstract Full Text PDF PubMed Google Scholar, 23Graham D.L. Knott T.J. Jones T.C. Pease R.J. Pullinger C.R. Scott J. Biochemistry. 1991; 30: 5616-5621Crossref PubMed Scopus (54) Google Scholar). Next, COS cells were transfected with apoB48 and subsequently transfected with either hMTP- or dMTP-FLAG. The amount of apoB48 secreted by dMTP-transfected cells was ∼33% of the level observed in hMTP-expressing cells (Fig. 2B). However, after correcting for MTP protein levels (Fig. 2B, inset), we calculated that dMTP was ∼50% as effective as hMTP in promoting apoB48 secretion. Utilizing similar correction methods it was calculated that dMTP-FLAG was ∼50 and ∼40% as efficient as the hMTP-FLAG in assisting apoB53 (Fig. 2C) and apoB72 (Fig. 2D) secretion, respectively. These studies suggest that dMTP can support the secretion of longer apoB polypeptides; however, it is less efficient compared with hMTP.FIGURE 2Drosophila MTP is less efficient than human MTP at supporting secretion of various apoB polypeptides. COS-7 cells were first transfected with either apoB18 (A), apoB48 (B), apoB53 (C), or apoB72 (D) and then with FuGENE 6 alone (no MTP), hMTP-FLAG (hMTP), or dMTP-FLAG (dMTP) expression plasmids. Following 48 h incubation, growth media was aspirated and replaced with oleate-containing media as described in the legend to Fig. 1. After a further 18-h incubation, the media were collected, and apoB content was determined by ELISA. The mean concentration of apoB is depicted by bar graphs ± S.D. (n = 3). The cells were then lysed, and equal amounts of protein were applied to SDS-polyacrylamide gels and subjected to Western blotting using M2 anti-FLAG antibody. The protein bands are shown in insets. Band intensities were quantified using NIH Image software and used to normalize apoB secretion to cellular MTP content.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Consideration was given to the possibility that dMTP is less proficient in supporting apoB secretion because of incorrect subcellular distribution. MTP is present in the endoplasmic reticulum due in part to association with protein disulfide isomerase (PDI) (24Rehberg E.F. Samson-Bouma M.E. Kienzle B. Blinderman L. Jamil H. Wetterau J.R. Aggerbeck L.P. Gordon D.A. J. Biol. Chem. 1996; 271: 29945-29952Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 25Ricci B. Sharp D. O'Rourke E. Kienzle B. Blinderman L. Gordon D. Smith-Monroy C. Robinson G. Gregg R.E. Rader D.J. Wetterau J.R. J. Biol. Chem. 1995; 270: 14281-14285Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) and has also been localized to the Golgi apparatus (26Levy E. Stan S. Delvin E. Menard D. Shoulders C. Garofalo C. Slight I. Seidman E. Mayer G. Bendayan M. J. Biol. Chem. 2002; 277: 16470-16477Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 27Swift L.L. Zhu M.Y. Kakkad B. Jovanovska A. Neely M.D. Valyi-Nagy K. Roberts R.L. Ong D.E. Jerome W.G. J. Lipid Res. 2003; 44: 1841-1849Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The subcellular distribution of the hMTP- and dMTP-FLAG proteins was determined using confocal microscopy (Fig. 3). No staining with anti-FLAG antibodies was seen in nontransfected cells (Fig. 3A, panel a). However, punctate staining was observed in cells transfected with hMTP- and dMTP-FLAG expression plasmids (Fig. 3A, panels d and g). Calnexin, also detected as punctate cytoplasmic staining (red), provides a marker for endoplasmic reticulum localization (Fig. 3A, panels b, e, and h). In transfected cells, both hMTP- and dMTP-FLAG staining showed extensive co-localization with calnexin as illustrated in the merged images (Fig. 3A, panels c, f, and i, yellow fluorescence). In addition to endoplasmic reticulum, hMTP- and dMTP-FLAG (Fig. 3B, panels a and d) co-localized with the Golgi marker protein α-mannosidase II (Fig. 3B, panels b and e). This was confirmed in the merged images (Fig. 3B, c and f, yellow fluorescence). These studies indicate similar distribution of dMTP- and hMTP-FLAG chimeras in the endoplasmic reticulum and Golgi apparatus.FIGURE 3Subcellular localization of human and Drosophila MTP-FLAG. COS cells transfected with apoB48 and either hMTP-FLAG (d-f) or dMTP-FLAG (g-i) were grown on coverslips in 24-well tissue culture dishes. After 48 h, the cells were fixed and treated as described under “Materials and Methods.” A, cells were stained with anti-FLAG M2 to label MTP (panels a, d, and g; green fluorescence) and anti-calnexin (panels b, e, and h; red fluorescence) as a marker for endoplasmic reticulum. Co-localization of MTP with the endoplasmic reticulum was demonstrated in the merged images (panels c, f, and i; yellow fluorescence). B, cells were incubated with anti-FLAG (panels a and d; green) and anti-α-mannosidase II (panels b and e; red) to label the Golgi. The merged images (panels c and f; yellow) illustrated co-localization of MTP with the Golgi marker.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Mammalian MTP is a heterodimeric complex of a 97-kDa “M” subunit and a 55-kDa PDI subunit (28Wetterau J.R. Combs K.A. Spinner S.N. Joiner B.J. J. Biol. Chem. 1990; 265: 9800-9807Abstract Full Text PDF PubMed Google Scholar, 29Wetterau J.R. Combs K.A. McLean L.R. Spinner S.N. Aggerbeck L.P. Biochemistry. 1991; 30: 9728-9735Crossref PubMed Scopus (154) Google Scholar). We therefore determined whether dMTP also associates with endogenous PDI. hMTP-FLAG and dMTP-FLAG were affinity-purified using M2 (anti-FLAG monoclonal antibody)-agarose from transiently transfected COS cell lysates (Fig. 4). Polyacrylamide gel electrophoresis followed by silver staining demonstrated two predominant protein bands of ∼95 and 55 kDa in dMTP- and hMTP-transfected cells, but not in control cells (Fig. 4A, compare control lane 2 with lanes 3 and 4). These were shown to be the M subunit and PDI by Western blotting using specific antisera (Fig. 4B), indicating that dMTP interacts with endogenous PDI similarly to hMTP.FIGURE 4Purification and characterization of hMTP and dMTP. A, MTP-FLAG chimeras were affinity-purified from transiently expressing COS cells. The cell homogenates were applied to M2-agarose beads and washed, and bound proteins were eluted as described under “Materials and Methods.” Eluted proteins were applied to polyacrylamide gels, subjected to electrophoresis, and stained. Lane 1 (Marker), molecular weight markers; lane 2 (COS), proteins eluted from nontransfected cells; lane 3 (Drosophila), proteins eluted from cells expressing dMTP-FLAG; lane 4 (Human), proteins eluted from cells expressing hMTP-FLAG. The arrowhead and arrow point to the M and P subunits of MTP, respectively. Differences in the migration of M subunits are in agreement with previous studies (17Sellers J.A. Hou L. Athar H. Hussain M.M. Shelness G.S. J. Biol. Chem. 2003; 278: 20367-20373Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). B, Western blotting of eluted proteins. Eluted proteins were transferred to nitrocellulose membranes and probed using M2 anti-FLAG or rabbit anti-PDI followed by anti-IgG horseradish peroxidase conjugate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We then sought to understand which property of dMTP is critical for its ability to render apoB secretion competent by comparing the specificity of lipid transfer between hMTP and dMTP. Although the lysates from cells that transiently expressed hMTP demonstrated a measurable ability to transfer triacylglycerols (0.71 ± 0.04% triacylglycerol transfer/μg of protein/h), no significant increase was observed using lysates obtained from cells that expressed dMTP (0 ± 0.04% triacylglycerol transfer/μg of protein/h) (Fig. 5A). The lack of triacylglycerol transfer activity in dMTP is similar to what we previously reported (17Sellers J.A. Hou L. Athar H. Hussain M.M. Shelness G.S. J. Biol. Chem. 2003; 278: 20367-20373Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). To demonstrate that dMTP was expressed, we performed similar experiments using lysates from hMTP- or dMTP-FLAG-expressing cells (Fig. 5B). Once more, the lysates from cells expressing hMTP-FLAG, but not dMTP-FLAG transferred triacylglycerols. Furthermore, equal amounts of MTP were present in the lysates as assessed by Western blot (Fig. 5B, inset). Thus, dMTP appeared to lack the ability to transfer triacylglycerols.FIGURE 5Determination of lipid transfer activities of dMTP and hMTP using fluorescence assays. COS cells transiently expressing hMTP and dMTP (A) or hMTP- and dMTP-FLAG (B) were hypotonically lysed, and soluble proteins (30-36μg) were assayed for triacylglycerol transfer activity using the radiolabeled vesicle transfer assay. In B (inset), amounts of MTP present in the microsomal contents were visualized by Western blotting. Real time transfer of fluorescently labeled triacylglycerol (C), and phosphatidylethanolamine (D) was measured using equal amounts of the purified proteins (C, inset). Fluorescence at 550 nm was monitored over time after excitation at 485 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Next, we studied the phospholipid transfer activities of human and Drosophila MTP. We were unable to measure significant ph" @default.
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• W1985176073 creator A5023615795 @default.
• W1985176073 creator A5035074969 @default.
• W1985176073 creator A5040457461 @default.
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• W1985176073 date "2006-04-01" @default.
• W1985176073 modified "2023-10-10" @default.
• W1985176073 title "Phospholipid Transfer Activity of Microsomal Triacylglycerol Transfer Protein Is Sufficient for the Assembly and Secretion of Apolipoprotein B Lipoproteins" @default.
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