VLDL lipolysis products increase VLDL fluidity and convert apolipoprotein E4 into a more expanded conformation
2009; Elsevier BV; Volume: 51; Issue: 6 Linguagem: Inglês
10.1194/jlr.m000406
ISSN1539-7262
AutoresSarada D. Tetali, Madhu S. Budamagunta, Catalina Simion, Laura J. den Hartigh, Tamás Kálai, Kálmán Hideg, Danny M. Hatters, Karl H. Weisgraber, John C. Voss, John C. Rutledge,
Tópico(s)Diabetes, Cardiovascular Risks, and Lipoproteins
ResumoOur 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 fluidity, 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 fluidity. Our data revealed that the order of lipoprotein fluidity 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 fluidity, VLDL lipolysis products significantly altered apoE4 into a more expanded conformation. Our studies indicate that after every meal, VLDL fluidity is increased causing apoE4 associated with VLDL to assume a more expanded conformation, potentially enhancing the pathogenicity of apoE4 in vascular tissue. 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 fluidity, 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 fluidity. Our data revealed that the order of lipoprotein fluidity 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 fluidity, VLDL lipolysis products significantly altered apoE4 into a more expanded conformation. Our studies indicate that after every meal, VLDL fluidity is increased causing apoE4 associated with VLDL to assume a more expanded conformation, potentially enhancing the pathogenicity of apoE4 in vascular tissue. apolipoprotein dimyristoylphosphatidylcholine doxyl stearic acid electron paramagnetic resonance lipoprotein lipase triglyceride Apolipoprotein E (apoE), a 34 kDa protein that is important in lipid metabolism and cholesterol transport, has three common alleles (ε2, ε3, and ε4). ApoE polymorphisms influence the risk of atherosclerotic cardiovascular disease and neurodegenerative disorders (1Davignon J. Apolipoprotein E and atherosclerosis: beyond lipid effect.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 267-269Crossref PubMed Scopus (81) Google Scholar). ApoE3 binds preferentially to HDL and apoE4 to VLDL (2Weisgraber K.H. Apolipoprotein E distribution among human plasma lipoproteins: role of the cysteine-arginine interchange at residue 112.J. Lipid Res. 1990; 31: 1503-1511Abstract Full Text PDF PubMed Google Scholar). 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 (3Wetterau J.R. Aggerbeck L.P. Rall Jr., S.C. Weisgraber K.H. Human apolipoprotein E3 in aqueous solution. I. Evidence for two structural domains.J. Biol. Chem. 1988; 263: 6240-6248Abstract Full Text PDF PubMed Google Scholar). 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 (4Dong L.M. Weisgraber K.H. Human apolipoprotein E4 domain interaction. Arginine 61 and glutamic acid 255 interact to direct the preference for very low density lipoproteins.J. Biol. Chem. 1996; 271: 19053-19057Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 5Saito H. Dhanasekaran P. Baldwin F. Weisgraber K.H. Phillips M.C. Lund-Katz S. Effects of polymorphism on the lipid interaction of human apolipoprotein E.J. Biol. Chem. 2003; 278: 40723-40729Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Upon binding to lipid, apolipoproteins undergo conformational rearrangements (6Saito H. Lund-Katz S. Phillips M.C. Contributions of domain structure and lipid interaction to the functionality of exchangeable human apolipoproteins.Prog. Lipid Res. 2004; 43: 350-380Crossref PubMed Scopus (188) Google Scholar, 7Oda M.N. Forte T.M. Ryan R.O. Voss J.C. The C-terminal domain of apolipoprotein A-I contains a lipid-sensitive conformational trigger.Nat. Struct. Biol. 2003; 10: 455-460Crossref PubMed Scopus (114) Google Scholar) 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 interaction of apoE4, i.e., the self-association of apoE4 via its C terminus (8Tetali S.D. Budamagunta M.S. Voss J.C. Rutledge J.C. C-terminal interactions of apolipoprotein E4 respond to the postprandial state.J. Lipid Res. 2006; 47: 1358-1365Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar); however, the mechanism for such effect was unknown. In this study, we used electron paramagnetic resonance (EPR) spectroscopy to investigate intramolecular interaction, i.e., domain interaction in apoE4 affected by VLDL lipolysis products. Our findings show that the conformation of apoE4 is modulated postprandially and is mediated by VLDL particle fluidity. These conformational changes may be important in lipoprotein-vascular cell binding and possibly in vascular injury. Materials for this work are as follows: streptokinase, ICN Pharmaceuticals (Costa Mesa, CA); vacutainers and venipuncture supplies, Fisher Scientific International (Hampton, NH); glass capillaries, VitroCom (Mountain Lakes, NJ); flat cell (catalog no. ES-LC11), JEOL (Tokyo, Japan); lipoprotein lipase, 5-doxy and 16-doxylstearic acid spin probes, and guanidine thiocyanate, Sigma-Aldrich (St. Louis, MO); (1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl) methanethiosulfonate and 12-doxylstearic acid, Toronto Research Chemicals (Toronto, Canada); titan gel electrophoresis supplies, Helena Laboratories (Beaumont, TX); dimyristoylphosphatidylcholine (DMPC), egg yolk phosphatidylcholine, extruder were from Avanti Polar Lipids (Alabaster, AL); and trioelin, cholesteryl oleate, cholesterol, and free fatty acids were from Nu-Chek-Prep (Elysian, MN). α-Thrombin was from Hematologic Technologies (Essex Junction, VT), and Slide-A-Lyzer dialysis cassettes (MWCO 10,000) were from Pierce (Rockford, IL). Recombinant plasmids containing thioredoxin-his tagged apoE sequences in pET32a-NT were expressed in BL21 (DE3) Escherichi coli cells as described previously but with modifications (9Morrow J.A. Arnold K.S. Weisgraber K.H. Functional characterization of apolipoprotein E isoforms overexpressed in Escherichia coli.Protein Expr. Purif. 1999; 16: 224-230Crossref PubMed Scopus (91) Google Scholar, 10Hatters D.M. Budamagunta M.S. Voss J.C. Weisgraber K.H. Modulation of apolipoprotein E structure by domain interaction: differences in lipid-bound and lipid-free forms.J. Biol. Chem. 2005; 280: 34288-34295Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The protein was purified with a Ni-affinity column, complexed with DMPC vesicles, and extruded through 100-nm polycarbonate membrane filters. The thioredoxin-his tag was cleaved from apoE with human α-thrombin. The apoE/DMPC complex was lyophilized, dispersed in 50 ml of methanol, and centrifuged at 6,000 g for 20 min at 4°C to remove the DMPC. The pelleted protein was dissolved in denaturation buffer (6 M guanidine and 2× TBS), purified by a second Ni-affinity column step to remove the N-terminal tag, labeled with a methanethiosulfonate spin label, renatured, and assayed for protein content as described (10Hatters D.M. Budamagunta M.S. Voss J.C. Weisgraber K.H. Modulation of apolipoprotein E structure by domain interaction: differences in lipid-bound and lipid-free forms.J. Biol. Chem. 2005; 280: 34288-34295Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The purity and integrity of the protein was tested by SDS-PAGE. The apoE mutants used for this study were apoE4 with two cysteine moieties substituted at amino acid positions 76 and 241 (76C-241C) and apoE3-like protein (R61T-76C-241C). Healthy human subjects were recruited from the University of California, Davis. The study was approved by the Human Subjects Research Committee of the University of California, Davis. Written informed consent was obtained from each participant. The volunteers were fed a moderately high-fat meal (40% calories from fat), and blood was obtained by venipuncture into Vacutainer tubes containing streptokinase (1,500 units) or EDTA, before (fasting, 0 h) and 3.5 and 6 h after ingestion of the test meal (8Tetali S.D. Budamagunta M.S. Voss J.C. Rutledge J.C. C-terminal interactions of apolipoprotein E4 respond to the postprandial state.J. Lipid Res. 2006; 47: 1358-1365Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Blood samples were centrifuged at 1,750 g for 10 min at 4°C to separate plasma from cellular blood constituents. Lipoproteins were isolated by sequential flotation with minor modifications (11Cohn J.S. Wagner D.A. Cohn S.D. Millar J.S. Schaefer E.J. Measurement of very low density and low density lipoprotein apolipoprotein (Apo) B-100 and high density lipoprotein Apo A-I production in human subjects using deuterated leucine. Effect of fasting and feeding.J. Clin. Invest. 1990; 85: 804-811Crossref PubMed Scopus (157) Google Scholar). Plasma was transferred into ultracentrifuge tubes (Beckman-Coulter), and 0.01% (w/v) NaN3 was added as a preservative. Plasma was diluted 1:2 in 196 mM NaCl and 0.25 mM EDTA (ρ = 1.0063) and centrifuged at 63,000 g for 30 min at 14°C to allow chylomicron flotation. The remaining plasma was spun at 285,000 g for 18 h at 14°C to isolate VLDL. The density of the remaining plasma was adjusted to 1.063 to isolate LDL and to 1.21 to isolate HDL and spun at 285,000 g for 18 h at 14°C for each lipoprotein fraction. Lipid fractions were dialyzed against 150 mM NaCl + 0.25 mM EDTA overnight at 4°C. Plasma and lipid fractions were stored at 4°C and used within 3 days after isolation. Triglyceride rich lipoprotein (TGRL) containing both chylomicrons and VLDL were isolated by centrifuging the plasma samples at 285,000 g for 18 h at 14°C. Lipoproteins were lipolyzed by incubation with bovine milk lipoprotein lipase (LpL) (3 U/ml) at 37°C for 30 min and assayed for nonesterified fatty acids. LDL cholesterol, HDL cholesterol, total cholesterol, triglycerides (TGs), and nonesterified fatty acids were quantified with an autoanalyzer. Equal amounts of spin-labeled apoE4 or apoE3-like protein (0.2 mg/ml) were incubated with fasting plasma, postprandial VLDL, or LpL-treated postprandial VLDL at 37°C for 1 h and applied (2 µl/lane) to Titan-agarose precasted gels. To assay the association of apoE with each lipoprotein class, lipoprotein bands on the gel were visualized by staining and destaining (8Tetali S.D. Budamagunta M.S. Voss J.C. Rutledge J.C. C-terminal interactions of apolipoprotein E4 respond to the postprandial state.J. Lipid Res. 2006; 47: 1358-1365Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). HDL, LDL, and VLDL bands were excised from the gel, solubilized in 4.5 mol/l guanidine isothiocyanate at 65°C, and analyzed by EPR spectroscopy, and the quantity of spin-labeled apoE associated with the VLDL fraction was determined (8Tetali S.D. Budamagunta M.S. Voss J.C. Rutledge J.C. C-terminal interactions of apolipoprotein E4 respond to the postprandial state.J. Lipid Res. 2006; 47: 1358-1365Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). The 5 doxyl-, 12 doxyl-, and 16-doxyl stearic acid (DSA) spin probes solubilized in ethanol were added to lipoproteins and mixed extensively by pipetting to ensure uniform equilibration of the probe with the sample. The final concentration of the 5-, 12-, and 16-DSA probes in the lipoprotein samples was 2 µmol/l. Spin-labeled probe/total cholesterol ratios were maintained the at 1:650. The synthesis of SL-cholesterol 3-[17-(1,5dimethyl-hexyl)-3- hydroxy-10,13-dimethyl-hexadecahydro-cyclopenta[a]phenantren- 2-ylidenethyl]2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-1-yloxyl radical cholestanol (Fig. 1A) is described as follows. To a solution of 5α-cholestan-3-one (3.86 g, 0.01 mol) in methanol (30 ml), 10% NaOH solution (2 ml), and aldehyde (1.68 g, 0.01 mol) were added. The mixture was stirred at room temperature for 3 h, acidified with 5% H2SO4, and extracted with CHCl3 (3 × 20 ml). The organic phase was washed with brine (2 × 20 ml), dried (MgSO4), and evaporated. The residue was purified by flash chromatography with hexane/ethanol to give the ketone 17-(1,5-dimethyl-hexyl)- 2-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-ylmethylene)-10,13-dimethyl-hexadecahydro-cyclopenta[a]phenantren-3-one radical. To a solution of ketone (2.69 g, 5.0 mmol) in absolute ethanol (20 ml), NaBH4 (0.19 g, 5 mmol) was added at 0°C. The reaction mixture was allowed to warm up to room temperature. After 1 h at room temperature, the mixture was quenched with water (10 ml), the alcohol was evaporated, and the aqueous phase was extracted with CHCl3 (3 × 20 ml). The organic phase was dried (MgSO4), evaporated, and purified by flash chromatography to give the final product. The yield was 1.93 g (72%); mp 187–188°C; Rf 0.60 (CHCl3/Et2O 2:1); MS, m/z (%) = 538 (M+, 7), 508 (7), 465 (10), and 43 (100). Elemental analysis calculated for C36H60NO2 (538.88), C 80.24, H 11.22, N 2.60% found C 80.20, H 11.25, and N 2.58%. To label lipoproteins, the spin-labeled cholestanol probe of 8 mmol/l was prepared in dimethyl formamide and slowly stirred into the lipoprotein sample to a final concentration of 20 μmol/l in a flat-bottom glass vial with continuous moderate stirring at 37°C for 2 h before the EPR study. In this case, the spin-labeled probe/total cholesterol ratio was 1:65. Significant signal-to-noise issues arise if the spin-labeled probe/total cholesterol ratio is <1:65. The final concentration of the vehicle in the sample did not exceed 0.5% and was kept constant across the samples. The TG-rich emulsions were prepared as previously described (12Almeida K.A. Schreiber R. Amancio R.F. Bydlowski S.P. Debes-Bravo A. Issa J.S. Strunz C.M. Maranhao R.C. Metabolism of chylomicron-like emulsions in carriers of the S447X lipoprotein lipase polymorphism.Clin. Chim. Acta. 2003; 335: 157-163Crossref PubMed Scopus (9) Google Scholar). Briefly, lipids composed of 69% triolein, 22% egg yolk phosphatidylcholine, 6% cholesteryl oleate, and varied concentrations of cholesterol (2–8%) were mixed together and then dried under a stream of nitrogen. The dried mixtures were resuspended in a small aliquot of 10 mM Tris-HCl buffer (pH 8.0) containing 0.1 M KCl and 1 mM EDTA and then emulsified in a bath sonicator at 50°C for an hour or until the homogenous emulsion was formed. Then, the emulsions were extruded using an extruder to form particles by passing 15 to 20 times through polycarbonate membrane of 100 nm pore size. After the extrusion, particles were assayed for their total triglyceride and cholesterol content. Further, the extruded particles were used for apoE binding assay on the day of preparation. EPR measurements of apoE4 were performed with a JEOL X-band spectrometer fitted with a loop-gap resonator (8Tetali S.D. Budamagunta M.S. Voss J.C. Rutledge J.C. C-terminal interactions of apolipoprotein E4 respond to the postprandial state.J. Lipid Res. 2006; 47: 1358-1365Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Spin-labeled apoE in TBS (10 mM Tris, pH 7.4, 150 mM sodium chloride, and 0.005% sodium azide) to a final concentration of 0.2 mg/ml protein was added to plasma or lipoprotein fractions or VLDL spiked, as described in Ref. 8Tetali S.D. Budamagunta M.S. Voss J.C. Rutledge J.C. C-terminal interactions of apolipoprotein E4 respond to the postprandial state.J. Lipid Res. 2006; 47: 1358-1365Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, with 0.5 mM of fatty acids [stearic (18:0), oleic (18:1), linoleic (18:2), or linolenic (18:3) acids], and the ratio between fatty acid and cholesterol content of lipoprotein was maintained at 1:2.6. The samples were loaded into one-sided sealed glass capillaries, incubated at 37°C for 1 h, and scanned by EPR. For all the samples, vehicle controls were used. The spectra were obtained by an average of three scans (2 min each) over 100 G at a microwave power of 2 mW and a modulation amplitude of 1 G at room temperature (20–22°C) or at 37°C. To assess the signal content in guanidine-extracted gel samples, a quartz flat cell was used, and the signal-averaged spectra from six 20-G scans (20 s each) over the central (mI = 0) line were recorded. Order parameters for the spectra of samples containing spin-labeled lipids were calculated as described (13Tanaka T. Hidaka T. Ogura R. Sugiyama M. Changes of membrane fluidity and Na+,K+-ATPase activity during cellular differentiation in the guinea pig epidermis.Arch. Dermatol. Res. 1988; 280: 29-32Crossref PubMed Scopus (15) Google Scholar). For samples containing the 5-DSA label, the order parameter S was calculated as S = [(T‖ – T_ − C)/(T‖ + 2T_ + 2C)]*1.723, where C = 1.4 – 0.053 (T‖ – T_). For the unresolved T‖ in the 12- and 16-DSA spectra, T‖ was estimated from T‖ = 44.5 – 2T_. All statistical analyses were performed using ANOVA as guided by SigmaStat software, with pairwise comparisons made using the Holm-Sidak method. Where applicable, data are reported as mean ± SD. Statistical significance was reported for P < 0.001, as indicated. Mutagenesis studies (14Fan D. Li Q. Korando L. Jerome W.G. Wang J. A monomeric human apolipoprotein E carboxyl-terminal domain.Biochemistry. 2004; 43: 5055-5064Crossref PubMed Scopus (44) Google Scholar, 15Zhang Y. Vasudevan S. Sojitrawala R. Zhao W. Cui C. Xu C. Fan D. Newhouse Y. Balestra R. Jerome W.G. et al.A monomeric, biologically active, full-length human apolipoprotein E.Biochemistry. 2007; 46: 10722-10732Crossref PubMed Scopus (59) Google Scholar) have demonstrated that apoE self-associates via a hydrophobic face along the C-terminal domain helix. This intermolecular interface is largely disrupted when apoE4 binds lipids, as measured by the loss dipolar interaction of spin labels placed at position 264 (8Tetali S.D. Budamagunta M.S. Voss J.C. Rutledge J.C. C-terminal interactions of apolipoprotein E4 respond to the postprandial state.J. Lipid Res. 2006; 47: 1358-1365Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 10Hatters D.M. Budamagunta M.S. Voss J.C. Weisgraber K.H. Modulation of apolipoprotein E structure by domain interaction: differences in lipid-bound and lipid-free forms.J. Biol. Chem. 2005; 280: 34288-34295Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 16Hatters D.M. Voss J.C. Budamagunta M.S. Newhouse Y.N. Weisgraber K.H. Insight on the molecular envelope of lipid-bound apolipoprotein E from electron paramagnetic resonance spectroscopy.J. Mol. Biol. 2009; 386: 261-271Crossref PubMed Scopus (18) Google Scholar). The reduced intermolecular interaction as measured by spin-labeled apoE4(264C) proved to be a useful marker for apoE4 binding to plasma lipids collected pre- and postprandially (8Tetali S.D. Budamagunta M.S. Voss J.C. Rutledge J.C. C-terminal interactions of apolipoprotein E4 respond to the postprandial state.J. Lipid Res. 2006; 47: 1358-1365Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). To explore the influence of lipolysis on the conformation and distribution of apoE4, we used a previously developed intramolecular domain interaction indicator construct (76C-241C) to monitor conformational differences between apoE3 and apoE4 that are predicted to be important in mediating their functional differences (10Hatters D.M. Budamagunta M.S. Voss J.C. Weisgraber K.H. Modulation of apolipoprotein E structure by domain interaction: differences in lipid-bound and lipid-free forms.J. Biol. Chem. 2005; 280: 34288-34295Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). In artificial lipid systems [DMPC, emulsions, and dipalmitoylphosphatidylcholine (DPPC)], where elevated concentrations facilitate spin dilution, no evidence of intermolecular spin interaction (arising from labels within 2.0 nm of one another) is observed from either position 76 or position 241 (10Hatters D.M. Budamagunta M.S. Voss J.C. Weisgraber K.H. Modulation of apolipoprotein E structure by domain interaction: differences in lipid-bound and lipid-free forms.J. Biol. Chem. 2005; 280: 34288-34295Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 16Hatters D.M. Voss J.C. Budamagunta M.S. Newhouse Y.N. Weisgraber K.H. Insight on the molecular envelope of lipid-bound apolipoprotein E from electron paramagnetic resonance spectroscopy.J. Mol. Biol. 2009; 386: 261-271Crossref PubMed Scopus (18) Google Scholar). In the lipid-free state, apoE4 is distinguished by domain interaction between the N-terminal bundle and the C-terminal helix, which is responsible for its binding preference for VLDL (10Hatters D.M. Budamagunta M.S. Voss J.C. Weisgraber K.H. Modulation of apolipoprotein E structure by domain interaction: differences in lipid-bound and lipid-free forms.J. Biol. Chem. 2005; 280: 34288-34295Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 16Hatters D.M. Voss J.C. Budamagunta M.S. Newhouse Y.N. Weisgraber K.H. Insight on the molecular envelope of lipid-bound apolipoprotein E from electron paramagnetic resonance spectroscopy.J. Mol. Biol. 2009; 386: 261-271Crossref PubMed Scopus (18) Google Scholar). Lipid-bound apoE has an extensive helical structure but adopts alternate conformations depending on lipid composition and particle size. For example, lipid-bound apoE3 with extended helical segments has been found on the edge of phospholipid (DMPC or 1-palmitoyl-2-oleoylphosphatidylcholine) particles (17Gupta V. Narayanaswami V. Budamagunta M.S. Yamamato T. Voss J.C. Ryan R.O. Lipid-induced extension of apolipoprotein E helix 4 correlates with low density lipoprotein receptor binding ability.J. Biol. Chem. 2006; 281: 39294-39299Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 18Narayanaswami V. Maiorano J.N. Dhanasekaran P. Ryan R.O. Phillips M.C. Lund-Katz S. Davidson W.S. Helix orientation of the functional domains in apolipoprotein e in discoidal high density lipoprotein particles.J. Biol. Chem. 2004; 279: 14273-14279Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). A similar conformation of apoE4 has been found on TG-rich emulsions (10Hatters D.M. Budamagunta M.S. Voss J.C. Weisgraber K.H. Modulation of apolipoprotein E structure by domain interaction: differences in lipid-bound and lipid-free forms.J. Biol. Chem. 2005; 280: 34288-34295Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). However, as shown by X-ray crystallography (19Peters-Libeu C.A. Newhouse Y. Hall S.C. Witkowska H.E. Weisgraber K.H. Apolipoprotein E*dipalmitoylphosphatidylcholine particles are ellipsoidal in solution.J. Lipid Res. 2007; 48: 1035-1044Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 20Peters-Libeu C.A. Newhouse Y. Hatters D.M. Weisgraber K.H. Model of biologically active apolipoprotein E bound to dipalmitoylphosphatidylcholine.J. Biol. Chem. 2006; 281: 1073-1079Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), apoE4 on DPPC particles has a double-helix structure with a hairpin near the midpoint of the protein sequence. EPR measurements showed that proximity of amino acids 76 and 241, indicating domain interaction in the lipid-free protein, is maintained in the DPPC-bound state (16Hatters D.M. Voss J.C. Budamagunta M.S. Newhouse Y.N. Weisgraber K.H. Insight on the molecular envelope of lipid-bound apolipoprotein E from electron paramagnetic resonance spectroscopy.J. Mol. Biol. 2009; 386: 261-271Crossref PubMed Scopus (18) Google Scholar). Thus, the 76-241 pair is useful for assessing domain interaction in the lipid-free protein and for distinguishing between a hairpin or extended helical conformation in the lipid-bound state. The indicator constructs, which contain cysteines at amino acids 76 and 241, detect the differences in the distance between the two regions of apoE when these sites are covalently modified with spin labels and assessed by EPR spectroscopy (10Hatters D.M. Budamagunta M.S. Voss J.C. Weisgraber K.H. Modulation of apolipoprotein E structure by domain interaction: differences in lipid-bound and lipid-free forms.J. Biol. Chem. 2005; 280: 34288-34295Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Because apoE3 has an endogenous cysteine that would react with a spin label, we used an apoE3 indicator construct with an R61T mutation (10Hatters D.M. Budamagunta M.S. Voss J.C. Weisgraber K.H. Modulation of apolipoprotein E structure by domain interaction: differences in lipid-bound and lipid-free forms.J. Biol. Chem. 2005; 280: 34288-34295Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). This construct behaves structurally and functionally like apoE3 because the mutation abolishes domain interaction (21Dong L.M. Wilson C. Wardell M.R. Simmons T. Mahley R.W. Weisgraber K.H. Agard D.A. Human apolipoprotein E. Role of arginine 61 in mediating the lipoprotein preferences of the E3 and E4 isoforms.J. Biol. Chem. 1994; 269: 22358-22365Abstract Full Text PDF PubMed Google Scholar). Specific labeling of apoE4 (which has no cysteines) is achieved by reacting the sulfhydryl-specific nitroxide label (MTS-SL) with apoE4 containing the substitutions S76C and A241C (Fig. 1B). Based on the level of dipolar interaction evident in the EPR spectrum in apoE4 containing these two spin-labeled side chains, the relative distance between the N- and C-terminal domains is evident. Because apoE3 has Cys-112, we used an apoE3-like protein (Fig. 1C) to determine the isoform-dependence of apoE's response to lipolysis (4Dong L.M. Weisgraber K.H. Human apolipoprotein E4 domain interaction. Arginine 61 and glutamic acid 255 interact to direct the preference for very low density lipoproteins.J. Biol. Chem. 1996; 271: 19053-19057Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 10Hatters D.M. Budamagunta M.S. Voss J.C. Weisgraber K.H. Modulation of apolipoprotein E structure by domain interaction: differences in lipid-bound and lipid-free forms.J. Biol. Chem. 2005; 280: 34288-34295Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The dipolar interaction between the spin labels at two sites can be qualitatively determined by the amount of spectral broadening. Therefore, we examined the double-spin-labeled apoE4 and apoE3-like proteins after incubation in postprandial plasma (Fig. 2A). The spectra in Fig. 2A are plotted so that each represents the same number of spins. The R61T mutation increased the distance between the domains in the lipid-free protein, resulting in less spectral broadening, as shown previously (10Hatters D.M. Budamagunta M.S. Voss J.C. Weisgraber K.H. Modulation of apolipoprotein E structure by domain interaction: differences in lipid-bound and lipid-free forms.J. Biol. Chem. 2005; 280: 34288-34295Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Postprandial plasma decreased the dipolar broadening of apoE4. The effect was greatest at 3.5 h, but the broadening was less than that in the apoE3-like sample. This suggests that this population of apoE4 retains some of the conformational features of its lipid-free state in plasma. In contrast, the apoE3-like protein maintained a more open conformation both in the lipid-free state and in plasma. This difference allows a systematic investigation of apoE isoform-specific behavior in the postprandial state. Fasting and postprandial serum lipid values of six of the healthy volunteers who participated in this study are listed in Table 1.TABLE 1Lipid values of healthy human volunteers recruited for the study (mg/dl)Preprandial (0 h)Postprandial (3.5 h)Postprandial (6 h)Volunteer No.TGTCLDLHDLTGTCLDLHDLTGTCLDLHDL71251852181404532423414546217222139419126153 ± 15203 ± 23134 ± 1839 ± 4311 ± 70207 ± 24134 ± 2040 ± 4191 ± 34206 ± 30136 ± 2540 ± 6265414317199571491779858781709858202078147754610615075495814673472206169 ± 6192 ± 1034.5 ± 0.5124 ± 7290 ± 40191 ± 5127 ± 835 ± 0208 ± 22198 ± 6133 ± 834.5 ± 0.56341158216130423762161273825723013841Volunteers 9126 and 2206 were reinvited for the study; therefore, the data from those volunteers are averages of two independent measurements. The data from volunteers 7125, 2654, 2020, and 6341 are from a single experiment. TG, total TGs; TC, total cholesterol; LDL, direct measurement of LDL-cholesterol; HDL, direct measurement of HDL-cholesterol.
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