Endoplasmic reticulum microenvironment and conserved histidines govern ELOVL4 fatty acid elongase activity
2014; Elsevier BV; Volume: 55; Issue: 4 Linguagem: Inglês
10.1194/jlr.m045443
ISSN1539-7262
AutoresSreemathi Logan, Martin‐Paul Agbaga, Michael D. Chan, Richard S. Brush, Robert E. Anderson,
Tópico(s)Pancreatic function and diabetes
ResumoAutosomal dominant Stargardt-like macular dystrophy (STGD3) in humans results from mutations in elongation of very long chain FAs-like 4 (ELOVL4), which leads to vision loss in young adults. ELOVL4 is an integral endoplasmic reticulum (ER) protein that mediates the elongation of very long chain (VLC) FAs. Mutations in ELOVL4 lead to truncation and mislocalization of the translated protein from the ER, the site of FA elongation. Little is known about the enzymatic elongation of VLC-FAs by ELOVL4. We over-expressed full-length mouse ELOVL4, an N-glycosylation-deficient mutant, an ER-retention mutant, and mutants of active site histidines to parse their individual roles in VLC-FA elongation. ELOVL4 elongated appropriate precursors to the corresponding VLC-FA species ≥28 carbons. Active site histidine mutants of ELOVL4 did not elongate appropriate precursors, establishing ELOVL4 as the elongase. Displacing ELOVL4 from the ER was sufficient to cause loss of condensation activity, while absence of N-glycosylation was irrelevant for enzyme function. This study shows that ELOVL4 enzymatic activity is governed by individual histidines in its active site and the ER microenvironment, both of which are essential for elongation of VLC-FAs. Autosomal dominant Stargardt-like macular dystrophy (STGD3) in humans results from mutations in elongation of very long chain FAs-like 4 (ELOVL4), which leads to vision loss in young adults. ELOVL4 is an integral endoplasmic reticulum (ER) protein that mediates the elongation of very long chain (VLC) FAs. Mutations in ELOVL4 lead to truncation and mislocalization of the translated protein from the ER, the site of FA elongation. Little is known about the enzymatic elongation of VLC-FAs by ELOVL4. We over-expressed full-length mouse ELOVL4, an N-glycosylation-deficient mutant, an ER-retention mutant, and mutants of active site histidines to parse their individual roles in VLC-FA elongation. ELOVL4 elongated appropriate precursors to the corresponding VLC-FA species ≥28 carbons. Active site histidine mutants of ELOVL4 did not elongate appropriate precursors, establishing ELOVL4 as the elongase. Displacing ELOVL4 from the ER was sufficient to cause loss of condensation activity, while absence of N-glycosylation was irrelevant for enzyme function. This study shows that ELOVL4 enzymatic activity is governed by individual histidines in its active site and the ER microenvironment, both of which are essential for elongation of VLC-FAs. The etiology of autosomal dominant Stargardt-like macular dystrophy (STGD3) in humans was described by three independent research groups as resulting from mutations in the elongation of very long chain fatty acids-like 4 (ELOVL4) gene (1Bernstein P.S. Tammur J. Singh N. Hutchinson A. Dixon M. Pappas C.M. Zabriskie N.A. Zhang K. Petrukhin K. Leppert M. et al.Diverse macular dystrophy phenotype caused by a novel complex mutation in the ELOVL4 gene.Invest. Ophthalmol. Vis. Sci. 2001; 42: 3331-3336PubMed Google Scholar, 2Edwards A.O. Donoso L.A. Ritter III, R. A novel gene for autosomal dominant Stargardt-like macular dystrophy with homology to the SUR4 protein family.Invest. Ophthalmol. Vis. Sci. 2001; 42: 2652-2663PubMed Google Scholar, 3Zhang K. Kniazeva M. Han M. Li W. Yu Z. Yang Z. Li Y. Metzker M.L. Allikmets R. Zack D.J. et al.A 5-bp deletion in ELOVL4 is associated with two related forms of autosomal dominant macular dystrophy.Nat. Genet. 2001; 27: 89-93Crossref PubMed Scopus (375) Google Scholar). Dominant mutations in patients with STGD3 lead to early onset loss of central vision with progressive degeneration of the macula and subsequently the peripheral retina. Patients with homozygous mutations in ELOVL4 exhibit severe skin and brain dysfunction (4Aldahmesh M.A. Mohamed J.Y. Alkuraya H.S. Verma I.C. Puri R.D. Alaiya A.A. Rizzo W.B. Alkuraya F.S. Recessive mutations in ELOVL4 cause ichthyosis, intellectual disability, and spastic quadriplegia.Am. J. Hum. Genet. 2011; 89: 745-750Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Truncating mutations in the last exon of ELOVL4 cause a frame shift, leading to premature termination of the encoded protein and loss of its C-terminal endoplasmic reticulum (ER) retention signal. The truncated gene product is subsequently mislocalized to other cellular compartments, resulting in protein aggregation (5Grayson C. Molday R.S. Dominant negative mechanism underlies autosomal dominant Stargardt-like macular dystrophy linked to mutations in ELOVL4.J. Biol. Chem. 2005; 280: 32521-32530Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 6Karan G. Lillo C. Yang Z. Cameron D.J. Locke K.G. Zhao Y. Thirumalaichary S. Li C. Birch D.G. Vollmer-Snarr H.R. et al.Lipofuscin accumulation, abnormal electrophysiology, and photoreceptor degeneration in mutant ELOVL4 transgenic mice: a model for macular degeneration.Proc. Natl. Acad. Sci. USA. 2005; 102: 4164-4169Crossref PubMed Scopus (155) Google Scholar, 7Karan G. Yang Z. Zhang K. Expression of wild type and mutant ELOVL4 in cell culture: subcellular localization and cell viability.Mol. Vis. 2004; 10: 248-253PubMed Google Scholar). Bearing sequence homology with the yeast Elo family of FA elongases, mammalian ELOVL4 has been shown to be involved in the biosynthesis of very long chain (VLC) FAs greater than 26 carbons in length (8Agbaga M.P. Brush R.S. Mandal M.N. Henry K. Elliott M.H. Anderson R.E. Role of Stargardt-3 macular dystrophy protein (ELOVL4) in the biosynthesis of very long chain fatty acids.Proc. Natl. Acad. Sci. USA. 2008; 105: 12843-12848Crossref PubMed Scopus (202) Google Scholar, 9Logan S. Agbaga M.P. Chan M.D. Kabir N. Mandal N.A. Brush R.S. Anderson R.E. Deciphering mutant ELOVL4 activity in autosomal-dominant Stargardt macular dystrophy.Proc. Natl. Acad. Sci. USA. 2013; 110: 5446-5451Crossref PubMed Scopus (36) Google Scholar). Elongation of FAs occurs through the cooperation of several ER-resident enzymes. The initial rate-limiting condensation reaction determining the chain length of the FA is catalyzed by an elongase, yielding a 3-keto-acyl-CoA intermediate (10Paul S. Gable K. Beaudoin F. Cahoon E. Jaworski J. Napier J.A. Dunn T.M. Members of the Arabidopsis FAE1-like 3-ketoacyl-CoA synthase gene family substitute for the Elop proteins of Saccharomyces cerevisiae.J. Biol. Chem. 2006; 281: 9018-9029Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 11Jakobsson A. Westerberg R. Jacobsson A. Fatty acid elongases in mammals: their regulation and roles in metabolism.Prog. Lipid Res. 2006; 45: 237-249Crossref PubMed Scopus (635) Google Scholar, 12Bernert Jr, J.T. Sprecher H. An analysis of partial reactions in the overall chain elongation of saturated and unsaturated fatty acids by rat liver microsomes.J. Biol. Chem. 1977; 252: 6736-6744Abstract Full Text PDF PubMed Google Scholar). This intermediate product subsequently undergoes reduction catalyzed by 3-keto-acyl-CoA reductase (KAR), followed by dehydration by any of four different 3-hydroxylacyl-CoA dehydratases (HACD1, HACD2, HACD3, and HACD4) (13Ikeda M. Kanao Y. Yamanaka M. Sakuraba H. Mizutani Y. Igarashi Y. Kihara A. Characterization of four mammalian 3-hydroxyacyl-CoA dehydratases involved in very long-chain fatty acid synthesis.FEBS Lett. 2008; 582: 2435-2440Crossref PubMed Scopus (79) Google Scholar). The final reduction step yielding the elongated FA product is catalyzed by trans-2,3-enoyl-CoA reductase (TER) (14Moon Y.A. Horton J.D. Identification of two mammalian reductases involved in the two-carbon fatty acyl elongation cascade.J. Biol. Chem. 2003; 278: 7335-7343Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). ELOVL4 has been shown to associate with KAR and TER (15Okuda A. Naganuma T. Ohno Y. Abe K. Yamagata M. Igarashi Y. Kihara A. Hetero-oligomeric interactions of an ELOVL4 mutant protein: implications in the molecular mechanism of Stargardt-3 macular dystrophy.Mol. Vis. 2010; 16: 2438-2445PubMed Google Scholar) and HACD1 (16Konishi H. Okuda A. Ohno Y. Kihara A. Characterization of HACD1 K64Q mutant found in arrhythmogenic right ventricular dysplasia patients.J. Biochem. 2010; 148: 617-622Crossref PubMed Scopus (14) Google Scholar) when coexpressed in culture. HACD1 has also been shown to coimmunoprecipitate with KAR and TER, thereby supporting the existence of an elongase complex (16Konishi H. Okuda A. Ohno Y. Kihara A. Characterization of HACD1 K64Q mutant found in arrhythmogenic right ventricular dysplasia patients.J. Biochem. 2010; 148: 617-622Crossref PubMed Scopus (14) Google Scholar). VLC-FAs greater than 26 carbons (>C26) are unique in that they are found only in tissues expressing ELOVL4 such as retina, brain, testes, and skin (17Mandal M.N. Ambasudhan R. Wong P.W. Gage P.J. Sieving P.A. Ayyagari R. Characterization of mouse orthologue of ELOVL4: genomic organization and spatial and temporal expression.Genomics. 2004; 83: 626-635Crossref PubMed Scopus (84) Google Scholar), with the highest expression in the retina. VLC-PUFAs are enriched in phosphatidylcholine of retina (18Aveldaño M.I. A novel group of very long chain polyenoic fatty acids in dipolyunsaturated phosphatidylcholines from vertebrate retina.J. Biol. Chem. 1987; 262: 1172-1179Abstract Full Text PDF PubMed Google Scholar) and brain (19Poulos A. Sharp P. Singh H. Johnson D. Fellenberg A. Pollard A. Detection of a homologous series of C26-C38 polyenoic fatty acids in the brain of patients without peroxisomes (Zellweger's syndrome).Biochem. J. 1986; 235: 607-610Crossref PubMed Scopus (54) Google Scholar) lipids, and in the SM and ceramide in testis and sperm (20Poulos A. Sharp P. Johnson D. White I. Fellenberg A. The occurrence of polyenoic fatty acids with greater than 22 carbon atoms in mammalian spermatozoa.Biochem. J. 1986; 240: 891-895Crossref PubMed Scopus (81) Google Scholar). The VLC saturated FAs, on the other hand, are found in the SM and ceramide fractions in skin (21Vasireddy V. Uchida Y. Salem Jr, N. Kim S.Y. Mandal M.N. Reddy G.B. Bodepudi R. Alderson N.L. Brown J.C. Hama H. et al.Loss of functional ELOVL4 depletes very long-chain fatty acids (> or =C28) and the unique omega-O-acylceramides in skin leading to neonatal death.Hum. Mol. Genet. 2007; 16: 471-482Crossref PubMed Scopus (202) Google Scholar, 22Cameron D.J. Tong Z. Yang Z. Kaminoh J. Kamiyah S. Chen H. Zeng J. Chen Y. Luo L. Zhang K. Essential role of Elovl4 in very long chain fatty acid synthesis, skin permeability barrier function, and neonatal survival.Int. J. Biol. Sci. 2007; 3: 111-119Crossref PubMed Scopus (109) Google Scholar, 23Li W. Chen Y. Cameron D.J. Wang C. Karan G. Yang Z. Zhao Y. Pearson E. Chen H. Deng C. et al.Elovl4 haploinsufficiency does not induce early onset retinal degeneration in mice.Vision Res. 2007; 47: 714-722Crossref PubMed Scopus (29) Google Scholar, 24McMahon A. Butovich I.A. Mata N.L. Klein M. Ritter III, R. Richardson J. Birch D.G. Edwards A.O. Kedzierski W. Retinal pathology and skin barrier defect in mice carrying a Stargardt disease-3 mutation in elongase of very long chain fatty acids-4.Mol. Vis. 2007; 13: 258-272PubMed Google Scholar), where they function to prevent dehydration. Interestingly, these FAs are not normally detectable in the blood and liver, suggesting that they are produced locally from long chain FA precursors. However, they have been found in the blood of patients with adrenoleukodystrophy (25Ofman R. Dijkstra I.M. van Roermund C.W. Burger N. Turkenburg M. van Cruchten A. van Engen C.E. Wanders R.J. Kemp S. The role of ELOVL1 in very long-chain fatty acid homeostasis and X-linked adrenoleukodystrophy.EMBO molecular medicine. 2010; 2: 90-97Crossref PubMed Scopus (115) Google Scholar) and Zellweger syndrome (19Poulos A. Sharp P. Singh H. Johnson D. Fellenberg A. Pollard A. Detection of a homologous series of C26-C38 polyenoic fatty acids in the brain of patients without peroxisomes (Zellweger's syndrome).Biochem. J. 1986; 235: 607-610Crossref PubMed Scopus (54) Google Scholar, 26Poulos A. Sharp P. Johnson D. Easton C. The occurrence of polyenoic very long chain fatty acids with greater than 32 carbon atoms in molecular species of phosphatidylcholine in normal and peroxisome-deficient (Zellweger's syndrome) brain.Biochem. J. 1988; 253: 645-650Crossref PubMed Scopus (54) Google Scholar, 27Sharp P. Poulos A. Fellenberg A. Johnson D. Structure and lipid distribution of polyenoic very-long-chain fatty acids in the brain of peroxisome-deficient patients (Zellweger syndrome).Biochem. J. 1987; 248: 61-67Crossref PubMed Scopus (31) Google Scholar, 28Poulos A. Lipid metabolism in Zellweger's syndrome.Prog. Lipid Res. 1989; 28: 35-51Crossref PubMed Scopus (22) Google Scholar), both of which are peroxisomal disorders. In the retina, VLC-PUFAs have been shown to be tightly associated with rhodopsin, and were suggested to possibly aid in phototransduction (29Aveldaño M.I. Phospholipid species containing long and very long polyenoic fatty acids remain with rhodopsin after hexane extraction of photoreceptor membranes.Biochemistry. 1988; 27: 1229-1239Crossref PubMed Scopus (126) Google Scholar). Due to similarities in the pathologies of STGD3 and autosomal recessive Stargardt's disease (STGD1), it was proposed that VLC-PUFAs in the retina may influence the flippase activity by the ABCA4 protein, the STGD1 disease gene product (30Agbaga M.P. Mandal M.N. Anderson R.E. Retinal very long-chain PUFAs: new insights from studies on ELOVL4 protein.J. Lipid Res. 2010; 51: 1624-1642Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Thus, it is crucial to have a better understanding of the activity of ELOVL4 and the VLC products it generates, which are important for skin, brain, and retinal function. We have confirmed that ELOVL4 mediates the rate-limiting condensation reaction in VLC-FA elongation (9Logan S. Agbaga M.P. Chan M.D. Kabir N. Mandal N.A. Brush R.S. Anderson R.E. Deciphering mutant ELOVL4 activity in autosomal-dominant Stargardt macular dystrophy.Proc. Natl. Acad. Sci. USA. 2013; 110: 5446-5451Crossref PubMed Scopus (36) Google Scholar), specifically 26:0 to 28:0 (8Agbaga M.P. Brush R.S. Mandal M.N. Henry K. Elliott M.H. Anderson R.E. Role of Stargardt-3 macular dystrophy protein (ELOVL4) in the biosynthesis of very long chain fatty acids.Proc. Natl. Acad. Sci. USA. 2008; 105: 12843-12848Crossref PubMed Scopus (202) Google Scholar). However, it is not known whether ELOVL4 can mediate successive elongation of other VLC-FAs, such as those generated in culture (8Agbaga M.P. Brush R.S. Mandal M.N. Henry K. Elliott M.H. Anderson R.E. Role of Stargardt-3 macular dystrophy protein (ELOVL4) in the biosynthesis of very long chain fatty acids.Proc. Natl. Acad. Sci. USA. 2008; 105: 12843-12848Crossref PubMed Scopus (202) Google Scholar), as well as those present in the retina (18Aveldaño M.I. A novel group of very long chain polyenoic fatty acids in dipolyunsaturated phosphatidylcholines from vertebrate retina.J. Biol. Chem. 1987; 262: 1172-1179Abstract Full Text PDF PubMed Google Scholar, 31Aveldaño M.I. Sprecher H. Very long chain (C24 to C36) polyenoic fatty acids of the n-3 and n-6 series in dipolyunsaturated phosphatidylcholines from bovine retina.J. Biol. Chem. 1987; 262: 1180-1186Abstract Full Text PDF PubMed Google Scholar). Furthermore, little is known about the individual protein motifs in ELOVL4 and their influence on VLC-FA elongation. Characteristic of other elongase and desaturase enzymes, ELOVL4 has three distinctive motifs along the length of the protein that may participate in VLC-FA biosynthesis: 1) ELOVL4 has an N-glycosylation consensus site at the N terminus (5Grayson C. Molday R.S. Dominant negative mechanism underlies autosomal dominant Stargardt-like macular dystrophy linked to mutations in ELOVL4.J. Biol. Chem. 2005; 280: 32521-32530Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), possibly aiding proper folding and structural stability of the protein; 2) ELOVL4 contains an active site comprising a histidine-rich motif (HXXHH) proposed to chelate iron, which acts as an electron transfer moiety during O2-dependent redox reactions (32Chertemps T. Duportets L. Labeur C. Ueda R. Takahashi K. Saigo K. Wicker-Thomas C. A female-biased expressed elongase involved in long-chain hydrocarbon biosynthesis and courtship behavior in Drosophila melanogaster.Proc. Natl. Acad. Sci. USA. 2007; 104: 4273-4278Crossref PubMed Scopus (111) Google Scholar); and 3) a dilysine ER retention motif at the C terminus that localizes ELOVL4 to the ER, where KAR, TER, and HACD1–4 are also located. Lysine residues juxtaposed to the C terminus of transmembrane proteins provide a recognition signal for the ER retention machinery to allow retrieval and retention of ER-resident proteins (33Jackson M.R. Nilsson T. Peterson P.A. Identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum.EMBO J. 1990; 9: 3153-3162Crossref PubMed Scopus (724) Google Scholar). The dependence of ELOVL4-mediated elongation of VLC-FAs on the histidine residues, N-glycosylation status, or retention to the ER is unclear. Hence, in this study we sought to characterize the enzymatic activity of the ELOVL4 protein and the contribution of each of the protein motifs toward the synthesis of VLC-FAs. We hypothesized that ELOVL4 is indeed the elongase that mediates successive elongation steps generating FA chain lengths ≥28 carbons, and that these individual motifs are critical for enzyme function. Using a gain-of-function approach, we show conclusively that ELOVL4 is the elongase responsible for elongation of FAs to C28, C30, C32, and C36 FAs. We also show that this activity is dependent on the coordination of the histidine residues in the active site and requires localization to the ER, while N-glycosylation is dispensable for enzyme function. ARPE19 cells were cultured in 10 cm2 plates with DMEM-nutrient mixture F-12 (DMEM-F-12; Invitrogen, Grand Island, NY) supplemented with 10% heat-inactivated FBS (v/v; Aleken Biologicals, Nash, TX) and antibiotics [100 units/ml each of penicillin and streptomycin (Invitrogen)]. HEK293T and HeLa cells were grown in DMEM medium supplemented with 10% FBS and antibiotics. Mouse Elovl4 (WT and 5 bp deletion) were PCR amplified and cloned in frame with a triple hemagglutinin (HA) tag in pKH3 BSENX vector (kindly provided by Dr. Scott M. Plafker). Active site mutants of WT ELOVL4 were constructed by mutating single histidines to glutamine or by mutating all three histidines to glutamines. N-glycosylation mutant was constructed by mutating the NDTV consensus site to NDAV and the lysine mutant was constructed by mutating the two lysines in the ER retention signal to arginines. All mutagenesis was performed using QuickChange II site directed mutagenesis kits (Agilent Technologies Inc., Santa Clara, CA). Adenovirus particles (Ad5) were generated as previously described (9Logan S. Agbaga M.P. Chan M.D. Kabir N. Mandal N.A. Brush R.S. Anderson R.E. Deciphering mutant ELOVL4 activity in autosomal-dominant Stargardt macular dystrophy.Proc. Natl. Acad. Sci. USA. 2013; 110: 5446-5451Crossref PubMed Scopus (36) Google Scholar) using the RAPAd CMV adenoviral expression system (Cell Biolabs, Inc., San Diego, CA). We used the C-terminal rabbit polyclonal ELOVL4 antibody (C-ELOVL4) at 1:1,000 dilution to detect untagged ELOVL4 as reported in Agbaga et al. (8Agbaga M.P. Brush R.S. Mandal M.N. Henry K. Elliott M.H. Anderson R.E. Role of Stargardt-3 macular dystrophy protein (ELOVL4) in the biosynthesis of very long chain fatty acids.Proc. Natl. Acad. Sci. USA. 2008; 105: 12843-12848Crossref PubMed Scopus (202) Google Scholar). In this study, we used mouse anti-HA (Cell Signaling Technology, Inc., Danvers, MA), rabbit anti-HA (Clonetech, Mountain View, CA), mouse anti-β actin (Abcam, Cambridge, MA), mouse anti-calnexin (Abcam), and rabbit anti-Green Fluorescent Protein (GFP) (Sigma-Aldrich, St. Louis, MO) antibodies. Cell pellets were lysed in lysis buffer containing 20 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA, complete protease inhibitors (EDTA free) (Roche, Mannheim, Germany), 1 mM PMSF, and 1% Triton X-100, and processed as previously described (9Logan S. Agbaga M.P. Chan M.D. Kabir N. Mandal N.A. Brush R.S. Anderson R.E. Deciphering mutant ELOVL4 activity in autosomal-dominant Stargardt macular dystrophy.Proc. Natl. Acad. Sci. USA. 2013; 110: 5446-5451Crossref PubMed Scopus (36) Google Scholar). Equal amounts of protein were separated on 12% polyacrylamide gels by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat dry milk and incubated with primary antibody overnight, followed by horseradish peroxidase-conjugated secondary goat anti-mouse or donkey anti-rabbit IgG for 1 h at room temperature. Immunoreactivity was detected by chemiluminescence using Super-Signal West Femto maximum sensitivity substrate (Pierce, Rockford, IL). Membranes were reprobed as necessary for the various markers. HeLa cells were grown on Labtek chamber slides and transiently transfected with ELOVL4 constructs. After 48 h, slides were rinsed and fixed as per Logan et al. (9Logan S. Agbaga M.P. Chan M.D. Kabir N. Mandal N.A. Brush R.S. Anderson R.E. Deciphering mutant ELOVL4 activity in autosomal-dominant Stargardt macular dystrophy.Proc. Natl. Acad. Sci. USA. 2013; 110: 5446-5451Crossref PubMed Scopus (36) Google Scholar). The slides were blocked with 5% nonfat dry milk and incubated with primary rabbit anti-HA antibody (Clonetech) and mouse anti-calnexin antibody (Abcam) overnight at 4°C. The following day, cells were washed and incubated with secondary anti-rabbit antibody conjugated with Alexa Fluor® 488 dye (Invitrogen) and anti-mouse antibody conjugated with Alexa Fluor® 568 dye (Invitrogen). The slides were then washed and coverslipped with Vectashield with DAPI mounting medium (Vector Labs) and imaged by confocal microscopy (Olympus FluoView 500, Olympus, Melville, NY). ARPE19 cells and HEK293T cells were transduced overnight with mouse ELOVL4 adenovirus and treated with FA precursors as described in Logan et al. (9Logan S. Agbaga M.P. Chan M.D. Kabir N. Mandal N.A. Brush R.S. Anderson R.E. Deciphering mutant ELOVL4 activity in autosomal-dominant Stargardt macular dystrophy.Proc. Natl. Acad. Sci. USA. 2013; 110: 5446-5451Crossref PubMed Scopus (36) Google Scholar). Sodium salts of the FAs were conjugated with BSA fraction V (Sigma) in a ratio 2:1 (w/w) BSA:FA for 20:5n3 (EPA) and 1:1 (w/w) BSA:FA for 26:0, 28:0, 30:0, and 34:5n3. Cells were treated with 30 μg/ml of the FA in media for a period of 48 h (HEK293T) or 72 h (ARPE19) unless otherwise stated. Following treatment, cells were harvested, washed once in 0.1 M PBS containing 50 μM of BSA fraction V (Sigma) to sequester excess free FAs, and washed with PBS only. The cell pellets were stored at −80°C until further processed for lipid analysis. Lipids were extracted from cells and other tissues according to the procedure described by Bligh and Dyer (34Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42666) Google Scholar). FA methyl esters (FAMEs) were generated by acid methanolysis with 16% HCl in methanol overnight. Following methanolysis, FAMEs were extracted into hexane and isolated by TLC using 80:20 hexane:ether mobile phase. The plate was stained with 2,7-dichlorofluorescein and the FAME band scraped and extracted into hexane. The FAME extract was resuspended in 20 μl of nonane and analyzed by GC-MS. Picomole values of saturated VLC-FAs (Fig. 1B–D, G–I) were measured via direct comparison to external standards as described in Agbaga et al. (8Agbaga M.P. Brush R.S. Mandal M.N. Henry K. Elliott M.H. Anderson R.E. Role of Stargardt-3 macular dystrophy protein (ELOVL4) in the biosynthesis of very long chain fatty acids.Proc. Natl. Acad. Sci. USA. 2008; 105: 12843-12848Crossref PubMed Scopus (202) Google Scholar). VLC-FAs (Fig. 1E) ≥C32 were measured as area counts due to the lack of external standards and were analyzed by GC-MS methodology described in Agbaga et al. (8Agbaga M.P. Brush R.S. Mandal M.N. Henry K. Elliott M.H. Anderson R.E. Role of Stargardt-3 macular dystrophy protein (ELOVL4) in the biosynthesis of very long chain fatty acids.Proc. Natl. Acad. Sci. USA. 2008; 105: 12843-12848Crossref PubMed Scopus (202) Google Scholar). VLC-PUFAs (Fig. 1F) represented as GC-MS tracings were normalized to the response of isocholesteryl methyl ether, which was shown to be consistent regardless of experimental differences, and were analyzed by GC-MS methodology described in Yu et al. (35Yu M. Benham A. Logan S. Brush R.S. Mandal M.N. Anderson R.E. Agbaga M.P. ELOVL4 protein preferentially elongates 20:5n3 to very long chain PUFAs over 20:4n6 and 22:6n3.J. Lipid Res. 2012; 53: 494-504Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). FA mole percentages (Figs. 4A–D; 5A, B; 6B) were measured directly by GC-flame ionization detector (FID) as described in Yu et al. (35Yu M. Benham A. Logan S. Brush R.S. Mandal M.N. Anderson R.E. Agbaga M.P. ELOVL4 protein preferentially elongates 20:5n3 to very long chain PUFAs over 20:4n6 and 22:6n3.J. Lipid Res. 2012; 53: 494-504Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar).Fig. 4ELOVL4 active site mutants are deficient in VLC-PUFA biosynthesis. A: Elongation of 20:5n3 in HEK293T cells to 32:5n3 and 34:5n3 in ELOVL4 and WT, but not in catalytic dead mutants (histidine mutants) or GFP-expressing and UT controls. B: Relative mole percent of 20:5n3, 22:5n3, and 24:5n3 with and without (NT) supplementation showing comparable levels of these FAs across samples in transduced HEK293T cells. C: Elongation of 34:5n3 to 36:5n3 normalized to 22:0 in HEK293T cells expressing ELOVL4 and WT, but not in active site mutants, which were comparable to controls (GFP and UT). Data are represented as the mean ± SD (n = 3). Significance was assessed in comparison to WT; *P < 0.05; **P < 0.01. (Inset: adenoviral-mediated expression of HA-ELOVL4 proteins supplemented with either 20:5n3 or 34:5n3). D: Levels of 34:5n3 internalized across treated and NT samples showing comparable levels of the precursor.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5ELOVL4 active site mutants lack elongase activity in Arpe19 cells. A: Elongation of 20:5n3 to 34:5n3 and 36:5n3 in ARPE19 cells expressing ELOVL4 and WT, but not in active site mutants or controls. Data are represented as the mean ± SD (n = 3). Significance was assessed in comparison to WT activity (**P < 0.01). B: Relative mole percent of 20:5n3 and its immediate elongated products 22:5n3 and 24:5n3 in ARPE19 cells with and without supplementation. C: Representative Western blots of ARPE19 cells showing comparable levels of untagged (detected by C-ELOVL4 antibody) and HA-tagged (detected by both C-ELOVL4 and HA) ELOVL4 protein expression. Blots were reprobed for GFP and normalized to β-actin. IB, immunoblotting.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6ELOVL4-mediated biosynthesis of VLC-PUFAs is independent of N-glycosylation but requires retention in the ER. A: Western blot showing increased expression of ΔNG compared with WT (left panel) and comparable levels of Δ3His and ΔLys to WT (right panel) in ARPE19 cells. Blots were reprobed for GFP and β-actin. B: ΔNG showed increased levels of 34:5n3 and 36:5n3 elongation products compared with WT in ARPE19 cells supplemented with 20:5n3, correlating with increased expression levels of the protein. ΔLys and Δ3His did not show any detectable level of the elongated VLC-PUFA products. Data are represented as the mean ± SD (n = 3); significance was determined in comparison to WT; *P < 0.05; **P < 0.01; ***P < 0.001. C: Quantification of the condensation activity in the microsome elongase assay of WT, GFP, and ΔLys samples in the presence of 5 μM of 34:5n3-CoA shows a significant reduction in the generation of the 3-keto-acyl intermediate in the ΔLys microsomes. However, there was some residual activity above background (GFP). Data are represented as the mean ± SD (n = 3). Significance was assessed using Student's t-test (*P < 0.05; **P < 0.01). IB, immunoblotting.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Microsomes from HEK293T cells expressing ELOVL4 were prepared and elongase assays performed as per Logan et al. (9Logan S. Agbaga M.P. Chan M.D. Kabir N. Mandal N.A. Brush R.S. Anderson R.E. Deciphering mutant ELOVL4 activity in autosomal-dominant Stargardt macular dystrophy.Proc. Natl. Acad. Sci. USA. 2013; 110: 5446-5451Crossref PubMed Scopus (36) Google Scholar). Briefly, elongase activity was assayed in a total volume of 200 μl of TEGM reaction buffer [50 mM Tris (pH 7.5), 1 mM MgCl2, 150 μM Triton X-100, 1 mM NADPH, 1 mM NADH, 10 mM β-mercaptoethanol, and 1 mM dithiothreitol], acyl-CoA acceptor (34:5n3-CoA), and 10 μM 2-[14C]malonyl-CoA at 37°C for 1 h. Individual reactions were initiated with the addition of 200 μg of microsomal protein. To measure condensing activity alone, NADPH/NADH was omitted from the reaction buffer. FAMEs were spotted on C-18 reverse phase silica high-performance TLC plates (Analtech Inc., Newark, DE) and reso
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