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• W2014078875 abstract "The retina-specific ATP binding cassette transporter, ABCA4 protein, is associated with a broad range of inherited macular degenerations, including Stargardt disease, autosomal recessive cone rod dystrophy, and fundus flavimaculatus. In order to understand its role in retinal transport in rod out segment discs, we have investigated the interactions of the soluble domains of ABCA4 with both 11-cis- and all-trans-retinal. Using fluorescence anisotropy-based binding analysis and recombinant polypeptides derived from the amino acid sequences of the four soluble domains of ABCA4, we demonstrated that the nucleotide binding domain 1 (NBD1) specifically bound 11-cis-retinal. Its affinity for all-trans-retinal was markedly reduced. Stargardt disease-associated mutations in this domain resulted in attenuation of 11-cis-retinal binding. Significant differences in 11-cis-retinal binding affinities were observed between NBD1 and other cytoplasmic and lumenal domains of ABCA4. The results suggest a possible role of ABCA4 and, in particular, the NBD1 domain in 11-cis-retinal binding. These results also correlate well with a recent report on the in vivo role of ABCA4 in 11-cis-retinal transport. The retina-specific ATP binding cassette transporter, ABCA4 protein, is associated with a broad range of inherited macular degenerations, including Stargardt disease, autosomal recessive cone rod dystrophy, and fundus flavimaculatus. In order to understand its role in retinal transport in rod out segment discs, we have investigated the interactions of the soluble domains of ABCA4 with both 11-cis- and all-trans-retinal. Using fluorescence anisotropy-based binding analysis and recombinant polypeptides derived from the amino acid sequences of the four soluble domains of ABCA4, we demonstrated that the nucleotide binding domain 1 (NBD1) specifically bound 11-cis-retinal. Its affinity for all-trans-retinal was markedly reduced. Stargardt disease-associated mutations in this domain resulted in attenuation of 11-cis-retinal binding. Significant differences in 11-cis-retinal binding affinities were observed between NBD1 and other cytoplasmic and lumenal domains of ABCA4. The results suggest a possible role of ABCA4 and, in particular, the NBD1 domain in 11-cis-retinal binding. These results also correlate well with a recent report on the in vivo role of ABCA4 in 11-cis-retinal transport. The retina-specific ABC 2The abbreviations used are:ABCATP binding cassetteROSrod outer segmentECDextracellular domainNBDnucleotide binding domainaaamino acid(s)AMP-PNP5′-adenylyl-β,γ-imidodiphosphateAanisotropy. transporter, ABCA4 protein, has been linked through genetic studies to a number of inherited visual diseases, including Stargardt macular dystrophy (1.Zhang K. Kniazeva M. Hutchinson A. Han M. Dean M. Allikmets R. The ABCR gene in recessive and dominant Stargardt diseases. A genetic pathway in macular degeneration.Genomics. 1999; 60: 234-237Crossref PubMed Scopus (45) Google Scholar, 2.Koenekoop R.K. The gene for Stargardt disease, ABCA4, is a major retinal gene. A mini-review.Ophthalmic Genet. 2003; 24: 75-80Crossref PubMed Scopus (69) Google Scholar), fundus flavimaculatus (3.Souied E.H. Ducroq D. Rozet J.M. Gerber S. Perrault I. Sterkers M. Benhamou N. Munnich A. Coscas G. Soubrane G. Kaplan J. A novel ABCR nonsense mutation responsible for late-onset fundus flavimaculatus.Invest. Ophthalmol. Vis. Sci. 1999; 40: 2740-2744PubMed Google Scholar, 4.Papaioannou M. Ocaka L. Bessant D. Lois N. Bird A. Payne A. Bhattacharya S. An analysis of ABCR mutations in British patients with recessive retinal dystrophies.Invest. Ophthalmol. Vis. Sci. 2000; 41: 16-19PubMed Google Scholar, 5.Lois N. Holder G.E. Fitzke F.W. Plant C. Bird A.C. Intrafamilial variation of phenotype in Stargardt macular dystrophy-Fundus flavimaculatus.Invest. Ophthalmol. Vis. Sci. 1999; 40: 2668-2675PubMed Google Scholar, 6.Klevering B.J. Deutman A.F. Maugeri A. Cremers F.P. Hoyng C.B. The spectrum of retinal phenotypes caused by mutations in the ABCA4 gene.Graefes Arch. Clin. Exp. Ophthalmol. 2005; 243: 90-100Crossref PubMed Scopus (99) Google Scholar), autosomal recessive retinitis pigmentosa (6.Klevering B.J. Deutman A.F. Maugeri A. Cremers F.P. Hoyng C.B. The spectrum of retinal phenotypes caused by mutations in the ABCA4 gene.Graefes Arch. Clin. Exp. Ophthalmol. 2005; 243: 90-100Crossref PubMed Scopus (99) Google Scholar, 7.Martínez-Mir A. Paloma E. Allikmets R. Ayuso C. del Rio T. Dean M. Vilageliu L. Gonzàlez-Duarte R. Balcells S. Retinitis pigmentosa caused by a homozygous mutation in the Stargardt disease gene ABCR.Nat. Genet. 1998; 18: 11-12Crossref PubMed Scopus (331) Google Scholar, 8.Klevering B.J. Yzer S. Rohrschneider K. Zonneveld M. Allikmets R. van den Born L.I. Maugeri A. Hoyng C.B. Cremers F.P. Microarray-based mutation analysis of the ABCA4 (ABCR) gene in autosomal recessive cone-rod dystrophy and retinitis pigmentosa.Eur. J. Hum. Genet. 2004; 12: 1024-1032Crossref PubMed Scopus (85) Google Scholar, 9.Klevering B.J. van Driel M. van de Pol D.J. Pinckers A.J. Cremers F.P. Hoyng C.B. Phenotypic variations in a family with retinal dystrophy as result of different mutations in the ABCR gene.Br. J. Ophthalmol. 1999; 83: 914-918Crossref PubMed Scopus (47) Google Scholar, 10.Cremers F.P. Maugeri A. Klevering B.J. Hoefsloot L.H. Hoyng C.B. [From gene to disease. From the ABCA4 gene to Stargardt disease, cone-rod dystrophy and retinitis pigmentosa].Ned. Tijdschr Geneeskd. 2002; 146: 1581-1584PubMed Google Scholar, 11.Allikmets R. Simple and complex ABCR. Genetic predisposition to retinal disease.Am. J. Hum. Genet. 2000; 67: 793-799Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), cone-rod dystrophy (8.Klevering B.J. Yzer S. Rohrschneider K. Zonneveld M. Allikmets R. van den Born L.I. Maugeri A. Hoyng C.B. Cremers F.P. Microarray-based mutation analysis of the ABCA4 (ABCR) gene in autosomal recessive cone-rod dystrophy and retinitis pigmentosa.Eur. J. Hum. Genet. 2004; 12: 1024-1032Crossref PubMed Scopus (85) Google Scholar, 12.van Driel M.A. Maugeri A. Klevering B.J. Hoyng C.B. Cremers F.P. ABCR unites what ophthalmologists divide(s).Ophthalmic Genet. 1998; 19: 117-122Crossref PubMed Scopus (101) Google Scholar, 13.Simonelli F. Testa F. Zernant J. Nesti A. Rossi S. Rinaldi E. Allikmets R. Association of a homozygous nonsense mutation in the ABCA4 (ABCR) gene with cone-rod dystrophy phenotype in an Italian family.Ophthalmic Res. 2004; 36: 82-88Crossref PubMed Scopus (18) Google Scholar, 14.Maugeri A. Klevering B.J. Rohrschneider K. Blankenagel A. Brunner H.G. Deutman A.F. Hoyng C.B. Cremers F.P. Mutations in the ABCA4 (ABCR) gene are the major cause of autosomal recessive cone-rod dystrophy.Am. J. Hum. Genet. 2000; 67: 960-966Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 15.Klevering B.J. Blankenagel A. Maugeri A. Cremers F.P. Hoyng C.B. Rohrschneider K. Phenotypic spectrum of autosomal recessive cone-rod dystrophies caused by mutations in the ABCA4 (ABCR) gene.Invest. Ophthalmol. Vis. Sci. 2002; 43: 1980-1985PubMed Google Scholar, 16.Jaakson K. Zernant J. Külm M. Hutchinson A. Tonisson N. Glavac D. Ravnik-Glavac M. Hawlina M. Meltzer M.R. Caruso R.C. Testa F. Maugeri A. Hoyng C.B. Gouras P. Simonelli F. Lewis R.A. Lupski J.R. Cremers F.P. Allikmets R. Genotyping microarray (gene chip) for the ABCR (ABCA4) gene.Hum. Mutat. 2003; 22: 395-403Crossref PubMed Scopus (196) Google Scholar, 17.Birch D.G. Peters A.Y. Locke K.L. Spencer R. Megarity C.F. Travis G.H. Visual function in patients with cone-rod dystrophy (CRD) associated with mutations in the ABCA4 (ABCR) gene.Exp. Eye Res. 2001; 73: 877-886Crossref PubMed Scopus (56) Google Scholar), and perhaps increased susceptibility to age-related macular degeneration (1.Zhang K. Kniazeva M. Hutchinson A. Han M. Dean M. Allikmets R. The ABCR gene in recessive and dominant Stargardt diseases. A genetic pathway in macular degeneration.Genomics. 1999; 60: 234-237Crossref PubMed Scopus (45) Google Scholar, 2.Koenekoop R.K. The gene for Stargardt disease, ABCA4, is a major retinal gene. A mini-review.Ophthalmic Genet. 2003; 24: 75-80Crossref PubMed Scopus (69) Google Scholar, 12.van Driel M.A. Maugeri A. Klevering B.J. Hoyng C.B. Cremers F.P. ABCR unites what ophthalmologists divide(s).Ophthalmic Genet. 1998; 19: 117-122Crossref PubMed Scopus (101) Google Scholar, 18.Yates J.R. Moore A.T. Genetic susceptibility to age related macular degeneration.J. Med. Genet. 2000; 37: 83-87Crossref PubMed Scopus (82) Google Scholar, 19.De La Paz M.A. Guy V.K. Abou-Donia S. Heinis R. Bracken B. Vance J.M. Gilbert J.R. Gass J.D. Haines J.L. Pericak-Vance M.A. Analysis of the Stargardt disease gene (ABCR) in age-related macular degeneration.Ophthalmology. 1999; 106: 1531-1536Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Stargardt disease and its adult onset variant, fundus flavimaculatus, are autosomal recessive disorders affecting ∼1 in 10,000 in the population. These diseases are characterized by a progressive loss of central vision and atrophy of the retinal pigment epithelium, ultimately leading to blindness. Disease-associated ABCA4 alleles show an extraordinary genetic heterogeneity, now numbering over 700 variants, and are found throughout the entire open reading frame of ABCA4 (20.Zernant J. Schubert C. Im K.M. Burke T. Brown C.M. Fishman G.A. Tsang S.H. Gouras P. Dean M. Allikmets R. Analysis of the ABCA4 gene by next-generation sequencing.Invest. Ophthalmol. Vis. Sci. 2011; 52: 8479-8487Crossref PubMed Scopus (118) Google Scholar). Currently, there are no therapeutics (small molecules or biologics) that target the ABCA4 transporter, although gene therapy holds great promise for afflicted individuals (21.Bennett J. Chung D.C. Maguire A. Gene delivery to the retina. From mouse to man.Methods Enzymol. 2012; 507: 255-274Crossref PubMed Scopus (12) Google Scholar). Immunohistological studies demonstrate that the ABCA4 protein is localized primarily to the rod and cone outer segment discs (22.Azarian S.M. Travis G.H. The photoreceptor rim protein is an ABC transporter encoded by the gene for recessive Stargardt's disease (ABCR).FEBS Lett. 1997; 409: 247-252Crossref PubMed Scopus (150) Google Scholar, 23.Molday L.L. Rabin A.R. Molday R.S. ABCR expression in foveal cone photoreceptors and its role in Stargardt macular dystrophy.Am. J. Ophthalmol. 2000; 130: 689Abstract Full Text Full Text PDF PubMed Google Scholar) (supplemental Fig. S1). ATP binding cassette rod outer segment extracellular domain nucleotide binding domain amino acid(s) 5′-adenylyl-β,γ-imidodiphosphate anisotropy. The historically accepted model of ABCA4 function has been simply its role in exporting all-trans-retinal from the ROS to the cytoplasm. In the context of the visual transduction, this is important because in order to maintain the retinoid cycle, all-trans-retinal released from light-activated rhodopsin must be recycled from the ROS disc back to the retinal pigment epithelium, where it is enzymatically converted to the 11-cis isomer, to again serve as the chromophore of rhodopsin. Defective ABCA4 function is believed to contribute to the accumulation of cytotoxic lipofuscin that underlies the pathology of the macular diseases leading to photoreceptor cell death. Excess all-trans-retinal can lead to the formation of all-trans-retinylidene-phosphatidylethanolamine, a component of lipofuscin (24.Kiser P.D. Golczak M. Maeda A. Palczewski K. Key enzymes of the retinoid (visual) cycle in vertebrate retina.Biochim. Biophys. Acta. 2012; 1821: 137-151Crossref PubMed Scopus (121) Google Scholar). This reasoning has helped to support the hypothesis that ABCA4 functions as an exporter of all-trans-retinal. However, using ABCA4(−/−) mice, Boyer et al. (25.Boyer N.P. Higbee D. Currin M.B. Blakeley L.R. Chen C. Ablonczy Z. Crouch R.K. Koutalos Y. Lipofuscin and N-retinylidene-N-retinylethanolamine (A2E) accumulate in retinal pigment epithelium in absence of light exposure. Their origin is 11-cis-retinal.J. Biol. Chem. 2012; 287: 22276-22286Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) recently showed that 11-cis-retinal can also support the formation of lipofuscin and proposed that 11-cis-retinal may be imported into the ROS discs by ABCA4. Currently, a mechanism of 11-cis-retinal import into the ROS discs is not known except through membrane diffusion. A transporter for 11-cis-retinal has not yet been identified. ABCA4 possesses a characteristic ABC protein family structure consisting of two membrane-spanning domains (transmembrane domains), with each containing six α-helices, and two cytosolic nucleotide-binding domains (NBDs) (Fig. 1). The primary sequences of ABC transporter transmembrane domains are highly variable, whereas the NBDs contain the conserved Walker A and Walker B consensus motifs. In addition, the NBDs harbor the LSGGQ signature sequence of ABC proteins, albeit diminished to SGG in the NBD2 domain of ABCA4 (26.Walker J.E. Saraste M. Runswick M.J. Gay N.J. Distantly related sequences in the α and β subunits of ATP synthase, myosin, kinases, and other ATP requiring enzymes and a common nucleotide binding fold.EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4248) Google Scholar). Unique to the ABCA subfamily is the presence of large extracellular loops, characteristic of this subfamily (Fig. 1). In ABCA4, these loops are thought to project into the lumen of the rod outer segment discs. Tysbovsky et al. identified the presence of the EAA motif in the transmembrane region of the N-terminal half of ABCA4 (27.Mourez M. Hofnung M. Dassa E. Subunit interactions in ABC transporters. A conserved sequence in hydrophobic membrane proteins of periplasmic permeases defines an important site of interaction with the ATPase subunits.EMBO J. 1997; 16: 3066-3077Crossref PubMed Scopus (165) Google Scholar, 28.Tsybovsky Y. Molday R.S. Palczewski K. The ATP-binding cassette transporter ABCA4. Structural and functional properties and role in retinal disease.Adv. Exp. Med. Biol. 2010; 703: 105-125Crossref PubMed Scopus (133) Google Scholar). This motif is characteristic of ABC proteins that are known to be importers (29.Rees D.C. Johnson E. Lewinson O. ABC transporters. The power to change.Nat. Rev. Mol. Cell Biol. 2009; 10: 218-227Crossref PubMed Scopus (881) Google Scholar). The presence of the EAA motif in ABCA4 is unusual, because in general, eukaryotic ABC transporters are exporters. The EAA motif is absent in the corresponding C-terminal half of ABCA4. The significance of the EAA motif in ABCA4 remains unknown; however, it may point to a 11-cis-retinal import function of the protein. The biochemical and kinetic properties of the nucleotide binding domains have been studied in individual recombinant polypeptides as well as in native and recombinant full-length ABCA4 protein (30.Sun H. Smallwood P.M. Nathans J. Biochemical defects in ABCR protein variants associated with human retinopathies.Nat. Genet. 2000; 26: 242-246Crossref PubMed Scopus (164) Google Scholar, 31.Sun H. Molday R.S. Nathans J. Retinal stimulates ATP hydrolysis by purified and reconstituted ABCR, the photoreceptor-specific ATP-binding cassette transporter responsible for Stargardt disease.J. Biol. Chem. 1999; 274: 8269-8281Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 32.Molday R.S. ATP-binding cassette transporter ABCA4. Molecular properties and role in vision and macular degeneration.J. Bioenerg. Biomembr. 2007; 39: 507-517Crossref PubMed Scopus (70) Google Scholar, 33.Biswas-Fiss E.E. Functional analysis of genetic mutations in nucleotide binding domain 2 of the human retina specific ABC transporter.Biochemistry. 2003; 42: 10683-10696Crossref PubMed Scopus (11) Google Scholar, 34.Ahn J. Molday R.S. Purification and characterization of ABCR from bovine rod outer segments.Methods Enzymol. 2000; 315: 864-879Crossref PubMed Google Scholar, 35.Suárez T. Biswas S.B. Biswas E.E. Biochemical defects in retina-specific human ATP binding cassette transporter nucleotide binding domain 1 mutants associated with macular degeneration.J. Biol. Chem. 2002; 277: 21759-21767Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Through a variety of independent experimental approaches, the NBD1 and NBD2 domains have been shown to have distinct enzymatic and kinetic properties. NBD1 possesses a low general ribonucleotidase activity, whereas NBD2 has a higher rate of hydrolysis and is specific for adenine nucleotides (36.Biswas E.E. Nucleotide binding domain 1 of the human retinal ABC transporter functions as a general ribonucleotidase.Biochemistry. 2001; 40: 8181-8187Crossref PubMed Scopus (24) Google Scholar). Thus, NBD1 and NBD2, although both important, probably play distinct mechanistic roles, consistent with that found for other ABC transporters, such as ABCB1 and ABCG2 (37.Vasiliou V. Vasiliou K. Nebert D.W. Human ATP-binding cassette (ABC) transporter family.Hum. Genomics. 2009; 3: 281-290Crossref PubMed Scopus (492) Google Scholar). Although half the size of NBD1, based on its kinetic properties, NBD2 appears to provide the energy required for translocation, whereas NBD1 serves a yet undefined cellular function. Several studies demonstrate that many disease-associated mutations mapping to the NBD1 of ABCA4 do not affect its function as an ATPase (15.Klevering B.J. Blankenagel A. Maugeri A. Cremers F.P. Hoyng C.B. Rohrschneider K. Phenotypic spectrum of autosomal recessive cone-rod dystrophies caused by mutations in the ABCA4 (ABCR) gene.Invest. Ophthalmol. Vis. Sci. 2002; 43: 1980-1985PubMed Google Scholar, 28.Tsybovsky Y. Molday R.S. Palczewski K. The ATP-binding cassette transporter ABCA4. Structural and functional properties and role in retinal disease.Adv. Exp. Med. Biol. 2010; 703: 105-125Crossref PubMed Scopus (133) Google Scholar, 30.Sun H. Smallwood P.M. Nathans J. Biochemical defects in ABCR protein variants associated with human retinopathies.Nat. Genet. 2000; 26: 242-246Crossref PubMed Scopus (164) Google Scholar, 32.Molday R.S. ATP-binding cassette transporter ABCA4. Molecular properties and role in vision and macular degeneration.J. Bioenerg. Biomembr. 2007; 39: 507-517Crossref PubMed Scopus (70) Google Scholar, 38.Fukui T. Yamamoto S. Nakano K. Tsujikawa M. Morimura H. Nishida K. Ohguro N. Fujikado T. Irifune M. Kuniyoshi K. Okada A.A. Hirakata A. Miyake Y. Tano Y. ABCA4 gene mutations in Japanese patients with Stargardt disease and retinitis pigmentosa.Invest. Ophthalmol. Vis. Sci. 2002; 43: 2819-2824PubMed Google Scholar). Using fluorescence anisotropy, we have shown that the ECD2 domain of ABCA4 interacts specifically and with high affinity to all-trans-retinal (39.Biswas-Fiss E.E. Kurpad D.S. Joshi K. Biswas S.B. Interaction of extracellular domain 2 of the human retina-specific ATP-binding cassette transporter (ABCA4) with all-trans-retinal.J. Biol. Chem. 2010; 285: 19372-19383Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). Analysis of the interaction of the other soluble domains of ABCA4 (ECD1, NBD1, and NBD2) showed that these domains do not bind all-trans-retinal with appreciable affinity. The ability of all-trans- and 11-cis-retinal to stimulate the ATPase of ABCA4 indicates that the protein engages in a physical interaction with both geometric isomers. However, it is not known which domain of ABCA4 mediates interaction with the 11-cis isomer of retinal. Based on recent studies proposing a physiological role of ABCA4 that may include translocation of 11-cis-retinal across the ROS disc membrane (25.Boyer N.P. Higbee D. Currin M.B. Blakeley L.R. Chen C. Ablonczy Z. Crouch R.K. Koutalos Y. Lipofuscin and N-retinylidene-N-retinylethanolamine (A2E) accumulate in retinal pigment epithelium in absence of light exposure. Their origin is 11-cis-retinal.J. Biol. Chem. 2012; 287: 22276-22286Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) as well as preliminary studies pointing to a specific interaction of NBD1 with 11-cis-retinal (40.Biswas-Fiss E.E. Ha M. Kurpad D Biswas S.B. Retinoid binding properties of the nucleotide binding domains of human ABCA4 protein.Invest. Ophthalmol. Vis. Sci. 2011; 52 (E-Abstract 51)Google Scholar), we posed the following question. Do the nucleotide binding domains of ABCA4 interact with retinal, and if so, how is this interaction influenced by disease-associated mutations in these domains? In this report, we have investigated the retinal binding properties of the first nucleotide binding domain of ABCA4 and examined the effects of disease-associated mutations on retinal binding. The pRK5 plasmid containing the full-length, wild-type cDNA of the human ABCA4 gene was obtained as a generous gift from Drs. J. Nathans and Michael Dean of Johns Hopkins University (Baltimore, MD) and NCBI (Frederick, MD), respectively. The T7 expression system vector pET30b, Bug Buster protein extraction reagent, Benzonase nuclease, and the S-protein-agarose affinity resin were from Novagen (EMD Sciences, Briggstown, NJ). All-trans-retinal was from Sigma-Aldrich, whereas 11-cis-retinal was received through Dr. R. Crouch (Medical University of South Carolina) under the auspices of the NEI, National Institutes of Health, resource program for vision researchers. Buffer A was 20 mm Tris-HCl (pH 8.0), 100 mm NaCl, 2 mm dithiothreitol, and 15% (v/v) glycerol. Buffer B contained 20 mm Tris-HCl (pH 7.5), 1 mm MgCl2, 50 mm NaCl, 5% glycerol, and 0.01% Nonidet P-40. Buffer C was 6 m guanidine hydrochloride, 0.1 m Tris-HCl (pH 8.0), and 2 mm EDTA. Buffer D contained 0.1 m Tris-HCl (pH 8.0), 0.5 m l-arginine, and 2 mm EDTA. Buffer E contained 10 mm NaPO4 (pH 7.4), 25 mm NaCl, and 2 mm dithiothreitol. The NBD1 construct was amplified from full-length ABCA4 cDNA and cloned into pET30b T7 expression vector (EMD Sciences) using standard recombinant DNA technology (41.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY1989Google Scholar). This domain corresponds to a 57.8-kDa (522-amino acid (aa)) polypeptide. The cloning was designed such that the polypeptide was produced as an S-tagged fusion protein, leading to a predicted mass of 62 kDa for the recombinant NBD1. For subsequent recombinant protein expression, the plasmid was used to transform Escherichia coli strain BL21-CodonPlus(DE3)-RILP competent cells (Stratagene, La Jolla, CA). Site-directed mutagenesis was carried out using a PCR-based mutagenesis kit (Stratagene, La Jolla, CA) (35.Suárez T. Biswas S.B. Biswas E.E. Biochemical defects in retina-specific human ATP binding cassette transporter nucleotide binding domain 1 mutants associated with macular degeneration.J. Biol. Chem. 2002; 277: 21759-21767Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), pET30-NBD1 plasmid as template, and allele-specific primers as described previously (35.Suárez T. Biswas S.B. Biswas E.E. Biochemical defects in retina-specific human ATP binding cassette transporter nucleotide binding domain 1 mutants associated with macular degeneration.J. Biol. Chem. 2002; 277: 21759-21767Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The authenticity of the mutations and the absence of other fortuitous mutations were confirmed by DNA sequencing carried out by Eurofins MWG/Operon (Huntsville, AL). E. coli cells (strain BL21-CodonPlus(DE3)-RIPL, Stratagene (La Jolla, CA)) harboring pET30b-NBD1 plasmid were used to produce the recombinant NBD1 polypeptide following the manufacturer's instructions. The expressed recombinant polypeptide appeared to be of the anticipated size (62 kDa), as determined by SDS-PAGE. Extraction and purification of wild-type NBD1 protein carrying the S-tag was performed using immobilized S-protein-agarose affinity resin (EMD Chemicals, Gibbstown, NJ) following the manufacturer's recommendations as described previously (36.Biswas E.E. Nucleotide binding domain 1 of the human retinal ABC transporter functions as a general ribonucleotidase.Biochemistry. 2001; 40: 8181-8187Crossref PubMed Scopus (24) Google Scholar). Introduction of mutations into wild-type NBD1 polypeptide appeared to decrease the solubility of the expressed proteins as determined by SDS-PAGE and a Western blot procedure (data not shown). Consequently, we explored the extraction of recombinant proteins (wild type and mutants) from the inclusion bodies followed by refolding (42.Winterfeld S. Imhof N. Roos T. Bär G. Kuhn A. Gerken U. Substrate-induced conformational change of the Escherichia coli membrane insertase YidC.Biochemistry. 2009; 48: 6684-6691Crossref PubMed Scopus (20) Google Scholar). This approach has been shown to be highly successful in the purification of a number of ABC transporters (35.Suárez T. Biswas S.B. Biswas E.E. Biochemical defects in retina-specific human ATP binding cassette transporter nucleotide binding domain 1 mutants associated with macular degeneration.J. Biol. Chem. 2002; 277: 21759-21767Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 43.Booth C.L. Pulaski L. Gottesman M.M. Pastan I. Analysis of the properties of the N-terminal nucleotide-binding domain of human P-glycoprotein.Biochemistry. 2000; 39: 5518-5526Crossref PubMed Scopus (27) Google Scholar, 44.Balan A. Ferreira R.C. Ferreira L.C. Production of the refolded oligopeptide-binding protein (OppA) encoded by the citrus pathogen Xanthomonas axonopodis pv. Citri.Genet. Mol. Res. 2008; 7: 117-126Crossref PubMed Scopus (3) Google Scholar, 45.Brunkhorst C. Wehmeier U.F. Piepersberg W. Schneider E. The acbH gene of Actinoplanes sp. encodes a solute receptor with binding activities for acarbose and longer homologs.Res. Microbiol. 2005; 156: 322-327Crossref PubMed Scopus (15) Google Scholar). The wild-type and mutant NBD1 proteins were extracted from inclusion bodies using a protocol that combines the use of BugBuster protein extraction reagent (Novagen, Madison, WI) to process the insoluble fraction and yield purified inclusion bodies with the method described by Booth et al. (35.Suárez T. Biswas S.B. Biswas E.E. Biochemical defects in retina-specific human ATP binding cassette transporter nucleotide binding domain 1 mutants associated with macular degeneration.J. Biol. Chem. 2002; 277: 21759-21767Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 43.Booth C.L. Pulaski L. Gottesman M.M. Pastan I. Analysis of the properties of the N-terminal nucleotide-binding domain of human P-glycoprotein.Biochemistry. 2000; 39: 5518-5526Crossref PubMed Scopus (27) Google Scholar, 46.Mace P.D. Cutfield J.F. Cutfield S.M. Bacterial expression and purification of the ovine type II bone morphogenetic protein receptor ectodomain.Protein Expr. Purif. 2007; 52: 40-49Crossref PubMed Scopus (4) Google Scholar). After harvesting the expressed proteins, the cell pellets were resuspended in room temperature BugBuster reagent, and protease inhibitors were added. After incubation on ice for 30 min, the cell suspension was centrifuged to collect purified inclusion bodies. Following cell lysis, the pellet of inclusion bodies was resuspended in buffer B and centrifuged once more. The inclusion body proteins were solubilized in Buffer C. Protein refolding was achieved by rapid dilution in Buffer D. The renatured protein was sequentially dialyzed in Buffer E. After overnight dialysis, proteins were concentrated to ∼0.5 mg/ml by ultrafiltration (Amicon/Millipore). Overall, the inclusion body protein purification methodology described here yielded highly concentrated, purified, and homogeneous preparations of protein (Fig. 2). The yield of NBD1 protein was >10 mg from 4 liters of induced cell culture. Purified proteins were stored at −80 °C until use. Fluorescence anisotropy was measured to investigate retinal binding by NBD1 protein in solution. Anisotropy measurements were carried out as described using a steady-state photon-counting spectrofluorometer, PC1, with Vinci software, from ISS Instruments (Champaign, IL) and Fluorolog 3 from Horiba Instruments Inc. (Edison, NJ) (39.Biswas-Fiss E.E. Kurpad D.S. Joshi K. Biswas S.B. Interaction of extracellular domain 2 of the human retina-specific ATP-binding cassette transporter (ABCA4) with all-trans-retinal.J. Biol. Chem. 2010; 285: 19372-19383Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). The temperature was maintained at 25 °C using a Peliter controlled cuvette holder. The excitation wavelength was set at 310 nm, and fluorescence anisotropy was recorded at an emission wavelength of 430 nm. Excitation and emission slits were adjusted to 8 nm to maximize intensity counts. The given retinal isomer was diluted to a concentration of 100 nm and titrated with NBD1 protein within a concentration range of 0.1 nm to 2 μm. The sample was incubated for 2 min after each addition. The S.D. for the anisotropy values was ≤±0.005 A. Anisotropy at each titration point was measured three times for 10 s and averaged. The total fluorescence intensity did not change significantly (≤10%) with increase in NBD1 concentration. Therefore, fluorescence lifetime changes or the scattered excitation light did not affect the anisotropy measurements. Anisotropy (A) is defined as follows, A=(Ivv−GIvh)/(Ivv−2GIvh)(Eq. 1) where G is the instrumental correction factor for the fluorometer, and it is defined by Equation 2, G=Ihv/Ihh(Eq. 2) where Ivv, Ivh, Ihv, and Ihh represent the fluorescence signal for excitation and emission with the polarizers set at (0°, 0°), (0°, 90°), (90°, 0°), and (90°, 90°), respectively. The interaction of NBD1 with ligand (all-trans-retinal or 11-cis-retinal) (L) can be represented as follows. At equilibrium, Ka, the equilibrium association constant, can be given as follows." @default.
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• W2014078875 title "Retinoid Binding Properties of Nucleotide Binding Domain 1 of the Stargardt Disease-associated ATP Binding Cassette (ABC) Transporter, ABCA4" @default.
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