Missense Mutations in APOB within the βα1 Domain of Human APOB-100 Result in Impaired Secretion of ApoB and ApoB-containing Lipoproteins in Familial Hypobetalipoproteinemia
2007; Elsevier BV; Volume: 282; Issue: 33 Linguagem: Inglês
10.1074/jbc.m702442200
ISSN1083-351X
AutoresJohn R. Burnett, Shumei Zhong, Z. Gordon Jiang, Amanda J. Hooper, Eric A. Fisher, Roger S. McLeod, Yang Zhao, P. Hugh R. Barrett, Robert A. Hegele, Frank M. van Bockxmeer, Hongyu Zhang, Dennis E. Vance, C. James McKnight, Zemin Yao,
Tópico(s)Lipoproteins and Cardiovascular Health
ResumoFamilial hypobetalipoproteinemia (FHBL) is associated with mutations in the APOB gene. We reported the first missense APOB mutation, R463W, in an FHBL kindred (Burnett, J. R., Shan, J., Miskie, B. A., Whitfield, A. J., Yuan, J., Tran, K., Mc-Knight, C. J., Hegele, R. A., and Yao, Z. (2003) J. Biol. Chem. 278, 13442-13452). Here we identified a second nonsynonymous APOB mutation, L343V, in another FHBL kindred. Heterozygotes for L343V (n = 10) had a mean plasma apoB at 0.31 g/liter as compared with 0.80 g/liter in unaffected family members (n = 22). The L343V mutation impaired secretion of apoB-100 and very low density lipoproteins. The secretion efficiency was 20% for B100wt and 10% for B100LV and B100RW. Decreased secretion of mutant apoB-100 was associated with increased endoplasmic reticulum retention and increased binding to microsomal triglyceride transfer protein and BiP. Reduced secretion efficiency was also observed with B48LV and B17LV. Biochemical and biophysical analyses of apoB domain constructs showed that L343V and R463W altered folding of the α-helical domain within the N terminus of apoB. Thus, proper folding of the α-helical domain of apoB-100 is essential for efficient secretion. Familial hypobetalipoproteinemia (FHBL) is associated with mutations in the APOB gene. We reported the first missense APOB mutation, R463W, in an FHBL kindred (Burnett, J. R., Shan, J., Miskie, B. A., Whitfield, A. J., Yuan, J., Tran, K., Mc-Knight, C. J., Hegele, R. A., and Yao, Z. (2003) J. Biol. Chem. 278, 13442-13452). Here we identified a second nonsynonymous APOB mutation, L343V, in another FHBL kindred. Heterozygotes for L343V (n = 10) had a mean plasma apoB at 0.31 g/liter as compared with 0.80 g/liter in unaffected family members (n = 22). The L343V mutation impaired secretion of apoB-100 and very low density lipoproteins. The secretion efficiency was 20% for B100wt and 10% for B100LV and B100RW. Decreased secretion of mutant apoB-100 was associated with increased endoplasmic reticulum retention and increased binding to microsomal triglyceride transfer protein and BiP. Reduced secretion efficiency was also observed with B48LV and B17LV. Biochemical and biophysical analyses of apoB domain constructs showed that L343V and R463W altered folding of the α-helical domain within the N terminus of apoB. Thus, proper folding of the α-helical domain of apoB-100 is essential for efficient secretion. Apolipoprotein (apo) 6The abbreviations used are: apo, apolipoprotein; DMEM, Dulbecco's modified Eagle's medium; ER, endoplasmic reticulum; FBS, fetal bovine serum; FHBL, familial hypobetalipoproteinemia; IDL, intermediate density lipoproteins; LDL, low density lipoprotein; mAb, monoclonal antibody; MTP, microsomal triglyceride transfer protein; VLDL, very low density lipoprotein; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; LV, B100LV. 6The abbreviations used are: apo, apolipoprotein; DMEM, Dulbecco's modified Eagle's medium; ER, endoplasmic reticulum; FBS, fetal bovine serum; FHBL, familial hypobetalipoproteinemia; IDL, intermediate density lipoproteins; LDL, low density lipoprotein; mAb, monoclonal antibody; MTP, microsomal triglyceride transfer protein; VLDL, very low density lipoprotein; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; LV, B100LV. B, a large amphipathic glycoprotein, plays a central role in human lipoprotein metabolism (1Chan L. J. Biol. Chem. 1992; 267: 25621-25624Abstract Full Text PDF PubMed Google Scholar, 2Young S.G. Circulation. 1990; 82: 1574-1594Crossref PubMed Scopus (330) Google Scholar). The human apoB gene (APOB) is located on chromosome 2 and produces, via a unique mRNA editing process (3Chen S.H. Habib G. Yang C.Y. Gu Z.W. Lee R. Wang S.A. Silberman S.R. Cai S.J. Deslypere J.P. Rosseneu M. Gotto Jr., A.M. Li W.H. Chan L. Science. 1987; 238: 363-366Crossref PubMed Scopus (530) Google Scholar), two forms of apoB, namely apoB-48 (2152 amino acids) and apoB-100 (4536 amino acids) (4Blackhart B.D. Ludwig E.M. Pierotti V.R. Caiati L. Onasch M.A. Wallis S.C. Powell L. Pease R. Knott T.J. Chu M-L. Mahley R.W Scott J. McCarthy B.J. Levy-Wilson B. J. Biol. Chem. 1986; 261: 15364-15367Abstract Full Text PDF PubMed Google Scholar, 5Knott T.J. Pease R. Powell L.M. Wallis S.C. Rall Jr., S.C. Innerarity T.L. Blackhart B. Taylor W.H. Marcel Y.L. Milne R. Johnson D. Fuller M. Lusis A.J. McCarthy B.J. Mahley R.W. Levy-Wilson B. Scott J. Nature. 1986; 323: 734-738Crossref PubMed Scopus (402) Google Scholar). ApoB-48 is the truncated form of apoB-100 consisting of the N-terminal 48% of full-length apoB-100. ApoB-48 is synthesized in the intestine and is essential for the formation and secretion of chylomicrons. ApoB-100 is synthesized in the liver and is an essential structural component of very low density lipoprotein (VLDL) and its metabolic products, intermediate density lipoprotein (IDL) and low density lipoprotein (LDL), and is also a ligand for the LDL receptor. Unlike humans, the rat liver produces both apoB-100 and apoB-48, and both forms can assemble VLDL (6Elovson J. Chatterton J.E. Bell G.T. Schumaker V.N. Reuben M.A. Puppione D.L. Reeve Jr., J.R. Young N.L. J. Lipid Res. 1988; 29: 1461-1473Abstract Full Text PDF PubMed Google Scholar). A pentapartite model for human apoB-100 has been proposed, which depicts a five-domain structure composed of alternating amphipathic α-helices and amphipathic β-strands, namely βα1-β1-α2-β2-α3 (7Segrest J.P. Jones M.K. De Loof H. Dashti N. J. Lipid Res. 2001; 42: 1346-1367Abstract Full Text Full Text PDF PubMed Google Scholar). The βα1 domain is a mixture of 13 amphipathic β-strands and 17 amphipathic α-helices, whose amino acids share extensive sequence homologies to the yolk protein lipovitellin (7Segrest J.P. Jones M.K. De Loof H. Dashti N. J. Lipid Res. 2001; 42: 1346-1367Abstract Full Text Full Text PDF PubMed Google Scholar, 8Baker M.E. Biochem. J. 1988; 255: 1057-1060Crossref PubMed Scopus (136) Google Scholar, 9Mann C.J. Anderson T.A. Read J. Chester S.A. Harrison G.B. Kochl S. Ritchie P.J. Bradbury P. Hussain F.S. Amey J. Vanloo B. Rosseneu M. Infante J. Hancock J.M. Levitt D.G. Banaszak L.J. Scott J. Shoulders C.C. J. Mol. Biol. 1999; 285: 391-408Crossref PubMed Scopus (170) Google Scholar). The apoB βα1 domain has been modeled on the structure of silver lamprey lipovitellin, in which the 13 β-strands (amino acids 21-263) form a β-barrel, whereas the 17 α-helices (amino acids 440-592) form a two-layered helical bundle (10Anderson T.A. Levitt D.G. Banaszak L.J. Structure (Lond.). 1998; 6: 895-909Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). There is an interface between the α-helical bundle and the extended amphipathic β-sheets (termed C-sheet and A-sheet in the lipovitellin structure). Recent studies have shown that the ability of apoB to initiate lipid binding resides in a region within the amphipathic C-sheet (i.e. between apoB-19 (residues 1-862) and apoB20.1 (residues 1-912)) (11Shelness G.S. Hou L. Ledford A.S. Parks J.S. Weinberg R.B. J. Biol. Chem. 2003; 278: 44702-44707Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 12Ledford A.S. Weinberg R.B. Cook V.R. Hantgan R.R. Shelness G.S. J. Biol. Chem. 2006; 281: 8871-8876Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Several lines of evidence indicate that the βα1 domain of apoB is of particular importance in lipoprotein assembly. Transfection studies show that apoB segments lacking the βα1 domain are either unable to be secreted (13Gretch D.G. Sturley S.L. Wang L. Lipton B.A. Dunning A. Grunwald K.A.A. Wetterau J.R. Yao Z. Talmud P. Attie A.D. J. Biol. Chem. 1996; 271: 8682-8691Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) or poorly secreted (14McLeod R.S. Wang Y. Wang S. Rusiñol A. Links P. Yao Z. J. Biol. Chem. 1996; 271: 18445-18455Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Mutagenesis experiments show that correct disulfide bond formation within the βα1 domain is required for efficient secretion of apoB (15Huang X.F. Shelness G.S. J. Biol. Chem. 1997; 272: 31872-31876Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 16Tran K. Bóren J. Macri J. Wang Y. McLeod R.S. Avramoglu R.K. Adeli K. Yao Z. J. Biol. Chem. 1998; 273: 7244-7251Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 17Rustaeus S. Stillemark P. Lindberg K. Gordon D. Olofsson S-O. J. Biol. Chem. 1998; 273: 5196-5203Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), and this requirement is independent of the lipidation state of apoB (18Burch W.L. Herscovitz H. J. Biol. Chem. 2000; 275: 16267-16274Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The sequence elements involved in the physical interaction between apoB and its molecular chaperone, the microsomal triglyceride transfer protein (MTP), have been located to the βα1 domain (19Hussain M.M. Shi J. Dreizen P. J. Lipid Res. 2003; 44: 22-32Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). MTP, the product of the abetalipoproteinemia (OMIM 200100) gene (20Sharp 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 (403) Google Scholar, 21Shoulders C.C. Brett D.J. Bayliss J.D. Narcisi T.M. Jarmuz A. Grantham T.T. Leoni P.R. Bhattacharya S. Pease R.J. Cullen P.M. Levy S. Byfield P.G.H. Purkiss P. Scott J. Hum. Mol. Genet. 1993; 2: 2109-2116Crossref PubMed Scopus (226) Google Scholar), is known to transfer triglyceride, cholesteryl esters, and phospholipids and is essential for apoB-containing lipoprotein assembly and secretion (22Wetterau 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 (635) Google Scholar). In vitro experiments suggest the presence of multiple MTP-binding sites within apoB (9Mann C.J. Anderson T.A. Read J. Chester S.A. Harrison G.B. Kochl S. Ritchie P.J. Bradbury P. Hussain F.S. Amey J. Vanloo B. Rosseneu M. Infante J. Hancock J.M. Levitt D.G. Banaszak L.J. Scott J. Shoulders C.C. J. Mol. Biol. 1999; 285: 391-408Crossref PubMed Scopus (170) Google Scholar, 23Bradbury P. Mann C.J. Kochl S. Anderson T.A. Chester S.A. Hancock J.M. Ritchie P.J. Amey J. Harrison G.B. Levitt D.G. Banaszak L.J. Scott J. Shoulders C.C. J. Biol. Chem. 1999; 274: 3159-3164Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 24Hussain M.M. Bakillah A. Nayak N. Shelness G.S. J. Biol. Chem. 1998; 273: 25612-25615Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Segrest et al. (25Segrest J.P. Jones M.K. Dashti N. J. Lipid Res. 1999; 40: 1401-1416Abstract Full Text Full Text PDF PubMed Google Scholar) have proposed that the interaction of MTP with the apoB βα1 domain forms a lipid pocket that facilitates lipid recruitment during lipoprotein formation. Recently, the same group has postulated a hairpin-bridge mechanism for lipid pocket completion (26Richardson P.E. Manchekar M. Dashti N. Jones M.K. Beigneux A. Young S.G. Harvey S.C. Segrest J.P. Biophys. J. 2005; 88: 2789-2800Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Familial hypobetalipoproteinemia (FHBL; OMIM 107730) is a genetically heterogeneous autosomal co-dominant disorder characterized by low levels (<5th percentile for age and sex) of plasma apoB-containing lipoproteins (27Schonfeld G. J. Lipid Res. 2003; 44: 878-883Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 28Whitfield A.J. Barrett P.H.R. van Bockxmeer F.M. Burnett J.R. Clin. Chem. 2004; 50: 1725-1732Crossref PubMed Scopus (153) Google Scholar, 29Schonfeld G. Lin X. Yue P. Cell. Mol. Life Sci. 2005; 62: 1372-1378Crossref PubMed Scopus (131) Google Scholar, 30Hooper A.J. van Bockxmeer F.M. Burnett J.R. Crit. Rev. Clin. Lab. Sci. 2005; 42: 515-545Crossref PubMed Scopus (83) Google Scholar). It has been suggested that FHBL represents a longevity syndrome (31Glueck C.J. Gartside P. Fallat R.W. Sielski J. Steiner P.M. J. Lab. Clin. Med. 1976; 88: 941-957PubMed Google Scholar) and might be associated with cardiovascular protection because of resistance to atherosclerosis (32Sankatsing R.R. Fouchier S.W. de Haan S. Hutten B.A. de Groot E. Kastelein J.J.P. Stroes E.S.G. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 1979-1984Crossref PubMed Scopus (90) Google Scholar). Heterozygotes are usually asymptomatic with LDL cholesterol and apoB-100 concentrations 100), VLDL2 (Sf 20-100), and other lipoproteins by rate flotation ultracentrifugation. The 35S-apoB proteins in each fraction were recovered by immunoprecipitation, resolved by SDS-PAGE, and visualized by fluorography as described above. Co-immunoprecipitation—Cells were treated with MG132 (25 μm) for 1 h, washed twice with cold PBS, and harvested in 1 ml of lysis buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1% Nonidet P-40, 5 mm EDTA, 20% sucrose, protease inhibitor mixture). The samples were incubated for 1 h at 4 °C by gentle mixing, and the insoluble materials were removed by centrifugation (13,000 rpm, 4 °C, 15 min). Aliquot of the supernatant (500 μg of protein) was mixed with lysis buffer to a final volume of 1 ml. The samples were pre-cleared by incubation with non-immune rabbit serum (2 h, 4 °C) prior to incubation with an anti-human LDL antiserum (16 h, 4 °C). Immunocomplexes were captured with protein A-Sepharose CL-4B beads, and proteins were eluted with SDS-PAGE sample buffer, resolved by SDS-PAGE, and analyzed by immunoblotting for apoB, MTP (using anti-MTP antibody that was a gift of Dr. C. C. Shoulders, MRC Clinical Sciences Centre, Hammersmith Hospital, London, UK), and BiP (the anti-Grp78 (BiP) antibody was purchased from StressGen, Ann Arbor, MI). Assessment of ApoB Polyubiquitination—Cells (∼80% confluence in 10-cm culture dishes) were incubated in media ±MG132 (25 μm) for 1 h and lysed with 1% SDS in RIPA buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% w/v phenylmethylsulfonyl fluoride, 1 mm dithiothreitol) at 80 °C. The samples were incubated for additional 16 h (4 °C) and diluted to 0.1% SDS with RIPA buffer, and apoB proteins were immunoprecipitated with the rabbit anti-human apoB antiserum as described above. After repeated washing with 0.1% SDS in RIPA buffer, the captured proteins were released into SDS-PAGE sample buffer, divided into 2 equal aliquots, and resolved by SDS-PAGE. Proteins were transferred to nitrocellulose membranes, probed by mAb 1D1 or anti-ubiquitin (SPA-203: StressGen, Ann Arbor, MI), and visualized by enhanced chemiluminescence (Roche Diagnostics) using horseradish peroxidase-conjugated goat anti-mouse IgG (Bio-Rad). Protease Protection Analysis of ApoB-100—Cells (80% confluence in 10-cm dishes) were incubated in media +MG132 (25 μm) for 1 h. The media were removed, and the cells (combined from two dishes) were harvested in ice-cold PBS. The cells were collected after centrifugation (50 × g, 2 min), resuspended in microsome buffer (10 mm Tris-HCl, pH 7.4, 250 mm sucrose, 100 μm leupeptin, 100 μm phenylmethylsulfonyl fluoride, 25 μm MG132, 10 kilounits/ml aprotinin), and homogenized using ball-bearing homogenizer as described previously (44Lapierre L.R. Currie D.L. Zao Z. Wang J. McLeod R.S. J. Lipid Res. 2004; 45: 366-377Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Nuclei were pelleted by centrifugation (9,500 rpm, 4 °C, 10 min, in an SS34 rotor), and the postnuclear supernatant was further centrifuged (100,000 rpm, 4 °C, 16 min, in a MLA-130 rotor) to obtain microsomes. The microsomes were resuspended in the microsome buffer (without protease inhibitors) and subjected to protease protection analysis as described previously (44Lapierre L.R. Currie D.L. Zao Z. Wang J. McLeod R.S. J. Lipid Res. 2004; 45: 366-377Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 45Cavallo D. McLeod R.S. Rudy D. Aiton A. Yao Z. Adeli K. J. Biol. Chem. 1998; 273: 33397-33405Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Immunoblotting for human apoB (1D1), protein-disulfide isomerase (SPA-891, StressGen, Ann Arbor, MI), Hsp70 (SPA-820, StressGen), p97 (RDI-PRO6527, Research Diagnostics, Inc., Concord, MA), and the N-terminal epitope of calnexin (SPA-865, StressGen) was performed using respective antibodies. Preparation of Bacterially Expressed ApoB Domain Constructs—For protein expression, plasmids coding for apoB domain constructs, namely B6.4-13, B6.4-17, and B13-17, were transformed into BL21 (DE3) competent cells. Transformed cells were grown at 37 °C to an absorbance of 0.6-0.8 at 600 nm in Luria broth and treated with isopropyl-β-d-thiogalactopyranoside (1 mm) for 3 h. Cell pellets were collected, treated with lysozyme (1 mg/ml, 30 min, 25 °C), and disintegrated with a probe sonicator (Branson, Danbury, CT). Inclusion bodies were dissolved in 8 m urea after washing with 1% Triton X-100 and 1 m urea. For protein preparation, denatured proteins were loaded onto a nickel-nitrilotriacetic acid-Sepharose column (Qiagen, Valencia, CA) and eluted with 250 mm imidazole in 6 m guanidine hydrochloride (GdnHCl) at pH 8.0. Purified proteins were refolded by slowly adding the denatured protein into a refolding buffer (50 mm Tris, 800 mm arginine, 10 mm reduced glutathione, 2 mm oxidized glutathione, 0.02% sodium azide, pH 8.0.) The proteins (1-2 μm in the refolding buffer) were incubated at 4 °C for 16 h and dialyzed extensively against 10 mm Tris, 150 mm sodium chloride, pH 7.5 (TS buffer). The final protein volume and concentration were adjusted using an Amicon Ultra concentrating apparatus (Millipore, Billerica, MA), and the protein concentration
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