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Robert S Molday - One of the best experts on this subject based on the ideXlab platform.
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Localization and functional characterization of the p.Asn965Ser (N965S) ABCA4 variant in mice reveal pathogenic mechanisms underlying Stargardt macular degeneration.
Human Molecular Genetics, 2017Co-Authors: Laurie L Molday, Daniel J. Wahl, Marinko V. Sarunic, Robert S MoldayAbstract:ABCA4 is a member of the superfamily of ATP-binding cassette (ABC) proteins that transports N-retinylidene-phosphatidylethanolamine (N-Ret-PE) across outer segment disc membranes thereby facilitating the removal of potentially toxic retinoid compounds from photoreceptor cells. Mutations in the gene encoding ABCA4 are responsible for Stargardt disease (STGD1), an autosomal recessive retinal degenerative disease that causes severe vision loss. To define the molecular basis for STGD1 associated with the p.Asn965Ser (N965S) mutation in the Walker A motif of nucleotide binding domain 1 (NBD1), we generated a p.Asn965Ser knockin mouse and compared the subcellular localization and molecular properties of the disease variant with wild-type (WT) ABCA4. Here, we show that the p.Asn965Ser ABCA4 variant expresses at half the level of WT ABCA4, partially mislocalizes to the endoplasmic reticulum (ER) of photoreceptors, is devoid of N-Ret-PE activated ATPase activity, and causes an increase in autofluorescence and the bisretinoid A2E associated with lipofuscin deposits in retinal pigment epithelial cells as found in Stargardt patients and ABCA4 knockout mice. We also show for the first time that a significant fraction of WT ABCA4 is retained in the inner segment of photoreceptors. On the basis of these studies we conclude that loss in substrate-dependent ATPase activity and protein misfolding are mechanisms underlying STGD1 associated with the p.Asn965Ser mutation in ABCA4. Functional and molecular modeling studies further suggest that similar pathogenic mechanisms are responsible for Tangiers disease associated with the p.Asn935Ser (N935S) mutation in the NBD1 Walker A motif of ABCA1.
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differential phospholipid substrates and directional transport by atp binding cassette proteins abca1 abca7 and ABCA4 and disease causing mutants
Journal of Biological Chemistry, 2013Co-Authors: Faraz Quazi, Robert S MoldayAbstract:Abstract ABCA1, ABCA7, and ABCA4 are members of the ABCA subfamily of ATP-binding cassette transporters which share extensive sequence and structural similarity. Mutations in ABCA1 cause Tangier disease characterized by defective cholesterol homeostasis and high-density lipoprotein (HDL) deficiency. Mutations in ABCA4 are responsible for Stargardt disease, a degenerative disorder associated with severe loss in central vision. Although cell-based studies have implicated ABCA proteins in lipid transport, the substrates and direction of transport have not been firmly established. We have purified and reconstituted ABCA1, ABCA7 and ABCA4 into liposomes for fluorescent-lipid transport studies. ABCA1 actively exported or flipped phosphatidylcholine (PC), phosphatidylserine (PS), and sphingomyelin (SM) from the cytoplasmic to the exocytoplasmic leaflet of membranes, whereas ABCA7 preferentially exported PS. In contrast ABCA4 transported phosphatidylethanolamine (PE) in the reverse direction. The same phospholipids stimulated the ATPase activity of these ABCA transporters. The transport activity and ATPase activity of ABCA1 and ABCA4 was reduced by 25% in the presence of 20% cholesterol. Nine ABCA1 Tangier mutants and corresponding ABCA4 Stargardt mutants showed significantly reduced phospholipid transport activity and subcellular mislocalization. These studies provide the first direct evidence for ABCA1 and ABCA7 functioning as phospholipid transporters and suggest that this activity is an essential step in the loading of ApoA-1 with phospholipids for HDL formation.
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differential phospholipid substrates and directional transport by atp binding cassette proteins abca1 abca7 and ABCA4 and disease causing mutants
Journal of Biological Chemistry, 2013Co-Authors: Faraz Quazi, Robert S MoldayAbstract:ABCA1, ABCA7, and ABCA4 are members of the ABCA subfamily of ATP-binding cassette transporters that share extensive sequence and structural similarity. Mutations in ABCA1 cause Tangier disease characterized by defective cholesterol homeostasis and high density lipoprotein (HDL) deficiency. Mutations in ABCA4 are responsible for Stargardt disease, a degenerative disorder associated with severe loss in central vision. Although cell-based studies have implicated ABCA proteins in lipid transport, the substrates and direction of transport have not been firmly established. We have purified and reconstituted ABCA1, ABCA7, and ABCA4 into liposomes for fluorescent-lipid transport studies. ABCA1 actively exported or flipped phosphatidylcholine, phosphatidylserine, and sphingomyelin from the cytoplasmic to the exocytoplasmic leaflet of membranes, whereas ABCA7 preferentially exported phosphatidylserine. In contrast, ABCA4 transported phosphatidylethanolamine in the reverse direction. The same phospholipids stimulated the ATPase activity of these ABCA transporters. The transport and ATPase activities of ABCA1 and ABCA4 were reduced by 25% in the presence of 20% cholesterol. Nine ABCA1 Tangier mutants and the corresponding ABCA4 Stargardt mutants showed significantly reduced phospholipid transport activity and subcellular mislocalization. These studies provide the first direct evidence for ABCA1 and ABCA7 functioning as phospholipid transporters and suggest that this activity is an essential step in the loading of apoA-1 with phospholipids for HDL formation.
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molecular organization and atp induced conformational changes of ABCA4 the photoreceptor specific abc transporter
Structure, 2013Co-Authors: Yaroslav Tsybovsky, Robert S Molday, Tivadar Orban, Derek J Taylor, Krzysztof PalczewskiAbstract:ATP-binding cassette (ABC) transporters use ATP to translocate various substrates across cellular membranes. Several members of subfamily A of mammalian ABC transporters are associated with severe health disorders, but their unusual complexity and large size have so far precluded structural characterization. ABCA4 is localized to the discs of vertebrate photoreceptor outer segments. This protein transports N-retinylidene-phosphatidylethanolamine to the outer side of disc membranes to prevent formation of toxic compounds causing macular degeneration. An 18 A-resolution structure of ABCA4 isolated from bovine rod outer segments was determined using electron microscopy and single-particle reconstruction. Significant conformational changes in the cytoplasmic and transmembrane regions were observed upon binding of a nonhydrolyzable ATP analog and accompanied by altered hydrogen/deuterium exchange in the Walker A motif of one of the nucleotide-binding domains. These findings provide an initial view of the molecular organization and functional rearrangements for any member of the ABCA subfamily of ABC transporters.
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Posttranslational Modifications of the Photoreceptor-Specific ABC Transporter ABCA4
Biochemistry, 2011Co-Authors: Yaroslav Tsybovsky, Faraz Quazi, Robert S Molday, Benlian Wang, Krzysztof PalczewskiAbstract:ABCA4 is a photoreceptor-specific ATP-binding cassette transporter implicated in the clearance of all-trans-retinal produced in the retina during light perception. Multiple mutations in this protein have been linked to Stargardt disease and other visual disorders. Here we report the first systematic study of posttranslational modifications in native ABCA4 purified from bovine rod outer segments. Seven N-glycosylation sites were detected in exocytoplasmic domains 1 and 2 by mass spectrometry, confirming the topological model of ABCA4 proposed previously. The modifying oligosaccharides were relatively short and homogeneous, predominantly representing a high-mannose type of N-glycosylation. Five phosphorylation sites were detected in cytoplasmic domain 1, with four of them located in the linker “regulatory-like” region conserved among ABCA subfamily members. Contrary to published results, phosphorylation of ABCA4 was found to be independent of light. Using human ABCA4 mutants heterologously expressed in mamm...
Silvia C Finnemann - One of the best experts on this subject based on the ideXlab platform.
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expression of ABCA4 in the retinal pigment epithelium and its implications for stargardt macular degeneration
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Tamara L Lenis, Jane Hu, Sze Yin Ng, Zhichun Jiang, Shanta Sarfare, Marcia Lloyd, Nicholas J Esposito, William Samuel, Cynthia Jaworski, Silvia C FinnemannAbstract:Recessive Stargardt disease (STGD1) is an inherited blinding disorder caused by mutations in the ABCA4 gene. ABCA4 is a flippase in photoreceptor outer segments (OS) that translocates retinaldehyde conjugated to phosphatidylethanolamine across OS disc membranes. Loss of ABCA4 in ABCA4−/− mice and STGD1 patients causes buildup of lipofuscin in the retinal pigment epithelium (RPE) and degeneration of photoreceptors, leading to blindness. No effective treatment currently exists for STGD1. Here we show by several approaches that ABCA4 is additionally expressed in RPE cells. (i) By in situ hybridization analysis and by RNA-sequencing analysis, we show the ABCA4 mRNA is expressed in human and mouse RPE cells. (ii) By quantitative immunoblotting, we show that the level of ABCA4 protein in homogenates of wild-type mouse RPE is about 1% of the level in neural retina homogenates. (iii) ABCA4 immunofluorescence is present in RPE cells of wild-type and Mertk−/− but not ABCA4−/− mouse retina sections, where it colocalizes with endolysosomal proteins. To elucidate the role of ABCA4 in RPE cells, we generated a line of genetically modified mice that express ABCA4 in RPE cells but not in photoreceptors. Mice from this line on the ABCA4−/− background showed partial rescue of photoreceptor degeneration and decreased lipofuscin accumulation compared with nontransgenic ABCA4−/− mice. We propose that ABCA4 functions to recycle retinaldehyde released during proteolysis of rhodopsin in RPE endolysosomes following daily phagocytosis of distal photoreceptor OS. ABCA4 deficiency in the RPE may play a role in the pathogenesis of STGD1.
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expression of ABCA4 in the retinal pigment epithelium and its implications for stargardt macular degeneration
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Tamara L Lenis, Jane Hu, Sze Yin Ng, Zhichun Jiang, Shanta Sarfare, Marcia Lloyd, Nicholas J Esposito, William Samuel, Cynthia Jaworski, Silvia C FinnemannAbstract:Recessive Stargardt disease (STGD1) is an inherited blinding disorder caused by mutations in the ABCA4 gene. ABCA4 is a flippase in photoreceptor outer segments (OS) that translocates retinaldehyde conjugated to phosphatidylethanolamine across OS disc membranes. Loss of ABCA4 in ABCA4−/− mice and STGD1 patients causes buildup of lipofuscin in the retinal pigment epithelium (RPE) and degeneration of photoreceptors, leading to blindness. No effective treatment currently exists for STGD1. Here we show by several approaches that ABCA4 is additionally expressed in RPE cells. (i) By in situ hybridization analysis and by RNA-sequencing analysis, we show the ABCA4 mRNA is expressed in human and mouse RPE cells. (ii) By quantitative immunoblotting, we show that the level of ABCA4 protein in homogenates of wild-type mouse RPE is about 1% of the level in neural retina homogenates. (iii) ABCA4 immunofluorescence is present in RPE cells of wild-type and Mertk−/− but not ABCA4−/− mouse retina sections, where it colocalizes with endolysosomal proteins. To elucidate the role of ABCA4 in RPE cells, we generated a line of genetically modified mice that express ABCA4 in RPE cells but not in photoreceptors. Mice from this line on the ABCA4−/− background showed partial rescue of photoreceptor degeneration and decreased lipofuscin accumulation compared with nontransgenic ABCA4−/− mice. We propose that ABCA4 functions to recycle retinaldehyde released during proteolysis of rhodopsin in RPE endolysosomes following daily phagocytosis of distal photoreceptor OS. ABCA4 deficiency in the RPE may play a role in the pathogenesis of STGD1.
Faraz Quazi - One of the best experts on this subject based on the ideXlab platform.
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differential phospholipid substrates and directional transport by atp binding cassette proteins abca1 abca7 and ABCA4 and disease causing mutants
Journal of Biological Chemistry, 2013Co-Authors: Faraz Quazi, Robert S MoldayAbstract:Abstract ABCA1, ABCA7, and ABCA4 are members of the ABCA subfamily of ATP-binding cassette transporters which share extensive sequence and structural similarity. Mutations in ABCA1 cause Tangier disease characterized by defective cholesterol homeostasis and high-density lipoprotein (HDL) deficiency. Mutations in ABCA4 are responsible for Stargardt disease, a degenerative disorder associated with severe loss in central vision. Although cell-based studies have implicated ABCA proteins in lipid transport, the substrates and direction of transport have not been firmly established. We have purified and reconstituted ABCA1, ABCA7 and ABCA4 into liposomes for fluorescent-lipid transport studies. ABCA1 actively exported or flipped phosphatidylcholine (PC), phosphatidylserine (PS), and sphingomyelin (SM) from the cytoplasmic to the exocytoplasmic leaflet of membranes, whereas ABCA7 preferentially exported PS. In contrast ABCA4 transported phosphatidylethanolamine (PE) in the reverse direction. The same phospholipids stimulated the ATPase activity of these ABCA transporters. The transport activity and ATPase activity of ABCA1 and ABCA4 was reduced by 25% in the presence of 20% cholesterol. Nine ABCA1 Tangier mutants and corresponding ABCA4 Stargardt mutants showed significantly reduced phospholipid transport activity and subcellular mislocalization. These studies provide the first direct evidence for ABCA1 and ABCA7 functioning as phospholipid transporters and suggest that this activity is an essential step in the loading of ApoA-1 with phospholipids for HDL formation.
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differential phospholipid substrates and directional transport by atp binding cassette proteins abca1 abca7 and ABCA4 and disease causing mutants
Journal of Biological Chemistry, 2013Co-Authors: Faraz Quazi, Robert S MoldayAbstract:ABCA1, ABCA7, and ABCA4 are members of the ABCA subfamily of ATP-binding cassette transporters that share extensive sequence and structural similarity. Mutations in ABCA1 cause Tangier disease characterized by defective cholesterol homeostasis and high density lipoprotein (HDL) deficiency. Mutations in ABCA4 are responsible for Stargardt disease, a degenerative disorder associated with severe loss in central vision. Although cell-based studies have implicated ABCA proteins in lipid transport, the substrates and direction of transport have not been firmly established. We have purified and reconstituted ABCA1, ABCA7, and ABCA4 into liposomes for fluorescent-lipid transport studies. ABCA1 actively exported or flipped phosphatidylcholine, phosphatidylserine, and sphingomyelin from the cytoplasmic to the exocytoplasmic leaflet of membranes, whereas ABCA7 preferentially exported phosphatidylserine. In contrast, ABCA4 transported phosphatidylethanolamine in the reverse direction. The same phospholipids stimulated the ATPase activity of these ABCA transporters. The transport and ATPase activities of ABCA1 and ABCA4 were reduced by 25% in the presence of 20% cholesterol. Nine ABCA1 Tangier mutants and the corresponding ABCA4 Stargardt mutants showed significantly reduced phospholipid transport activity and subcellular mislocalization. These studies provide the first direct evidence for ABCA1 and ABCA7 functioning as phospholipid transporters and suggest that this activity is an essential step in the loading of apoA-1 with phospholipids for HDL formation.
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Posttranslational Modifications of the Photoreceptor-Specific ABC Transporter ABCA4
Biochemistry, 2011Co-Authors: Yaroslav Tsybovsky, Faraz Quazi, Robert S Molday, Benlian Wang, Krzysztof PalczewskiAbstract:ABCA4 is a photoreceptor-specific ATP-binding cassette transporter implicated in the clearance of all-trans-retinal produced in the retina during light perception. Multiple mutations in this protein have been linked to Stargardt disease and other visual disorders. Here we report the first systematic study of posttranslational modifications in native ABCA4 purified from bovine rod outer segments. Seven N-glycosylation sites were detected in exocytoplasmic domains 1 and 2 by mass spectrometry, confirming the topological model of ABCA4 proposed previously. The modifying oligosaccharides were relatively short and homogeneous, predominantly representing a high-mannose type of N-glycosylation. Five phosphorylation sites were detected in cytoplasmic domain 1, with four of them located in the linker “regulatory-like” region conserved among ABCA subfamily members. Contrary to published results, phosphorylation of ABCA4 was found to be independent of light. Using human ABCA4 mutants heterologously expressed in mamm...
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the role of the photoreceptor abc transporter ABCA4 in lipid transport and stargardt macular degeneration
Biochimica et Biophysica Acta, 2009Co-Authors: Robert S Molday, Ming Zhong, Faraz QuaziAbstract:Abstract ABCA4 is a member of the ABCA subfamily of ATP binding cassette (ABC) transporters that is expressed in rod and cone photoreceptors of the vertebrate retina. ABCA4, also known as the Rim protein and ABCR, is a large 2273 amino acid glycoprotein organized as two tandem halves, each containing a single membrane spanning segment followed sequentially by a large exocytoplasmic domain, a multispanning membrane domain and a nucleotide binding domain. Over 500 mutations in the gene encoding ABCA4 are associated with a spectrum of related autosomal recessive retinal degenerative diseases including Stargardt macular degeneration, cone–rod dystrophy and a subset of retinitis pigmentosa. Biochemical studies on the purified ABCA4 together with analysis of ABCA4 knockout mice and patients with Stargardt disease have implicated ABCA4 as a retinylidene-phosphatidylethanolamine transporter that facilitates the removal of potentially reactive retinal derivatives from photoreceptors following photoexcitation. Knowledge of the genetic and molecular basis for ABCA4 related retinal degenerative diseases is being used to develop rationale therapeutic treatments for this set of disorders.
Ming Zhong - One of the best experts on this subject based on the ideXlab platform.
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Binding of retinoids to ABCA4, the photoreceptor ABC transporter associated with Stargardt macular degeneration.
Methods of Molecular Biology, 2010Co-Authors: Ming Zhong, Robert S MoldayAbstract:ABCA4 is a member of the superfamily of ATP-binding cassette (ABC) transporters, which has been implicated in the clearance of all-trans retinal derivatives from rod and cone photoreceptor cells following photoexcitation as part of the visual cycle. Mutations in ABCA4 are known to cause Stargardt macular degeneration and related disorders, associated with a severe loss in vision. Recently, a solid-phase binding assay has been developed to identify retinoids that likely serve as substrates for this transporter. In this procedure, monoclonal antibodies directed either against an epitope within ABCA4 (Rim 3F4 antibody) or against the 9 amino acid 1D4 epitope tag engineered onto the C-terminus of expressed ABCA4 (Rho 1D4 antibody) are covalently bound to a Sepharose matrix. This immunoaffinity matrix is then used to isolate ABCA4 from photoreceptor outer segments or transfected cells. All-trans retinal is added to immobilized ABCA4 in the presence of a phospholipid mixture containing phosphatidylethanolamine. The bound retinoid is then analyzed directly by spectrophotometry or identified by HPLC and/or mass spectrometry following extraction with organic solvents. Using this procedure, it has been shown that unprotonated N-retinylidene-phosphatidylethanolamine binds with high affinity to ABCA4 and is released by the addition of ATP. These procedures and related radiometric assays using titrated retinal have been used to study the binding of N-retinylidene-PE to wild-type and mutant ABCA4 in the absence and presence of nucleotides for structure-function studies.
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the role of the photoreceptor abc transporter ABCA4 in lipid transport and stargardt macular degeneration
Biochimica et Biophysica Acta, 2009Co-Authors: Robert S Molday, Ming Zhong, Faraz QuaziAbstract:Abstract ABCA4 is a member of the ABCA subfamily of ATP binding cassette (ABC) transporters that is expressed in rod and cone photoreceptors of the vertebrate retina. ABCA4, also known as the Rim protein and ABCR, is a large 2273 amino acid glycoprotein organized as two tandem halves, each containing a single membrane spanning segment followed sequentially by a large exocytoplasmic domain, a multispanning membrane domain and a nucleotide binding domain. Over 500 mutations in the gene encoding ABCA4 are associated with a spectrum of related autosomal recessive retinal degenerative diseases including Stargardt macular degeneration, cone–rod dystrophy and a subset of retinitis pigmentosa. Biochemical studies on the purified ABCA4 together with analysis of ABCA4 knockout mice and patients with Stargardt disease have implicated ABCA4 as a retinylidene-phosphatidylethanolamine transporter that facilitates the removal of potentially reactive retinal derivatives from photoreceptors following photoexcitation. Knowledge of the genetic and molecular basis for ABCA4 related retinal degenerative diseases is being used to develop rationale therapeutic treatments for this set of disorders.
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role of the c terminus of the photoreceptor ABCA4 transporter in protein folding function and retinal degenerative diseases
Journal of Biological Chemistry, 2009Co-Authors: Ming Zhong, Laurie L Molday, Robert S MoldayAbstract:ABCA4 is an ATP-binding cassette transporter that is expressed in rod and cone photoreceptor cells and implicated in the removal of retinal derivatives from outer segments following photoexcitation. Mutations in the ABCA4 gene are responsible for a number of related retinal degenerative diseases, including Stargardt macular degeneration, cone-rod dystrophy, retinitis pigmentosa, and age-related macular degeneration. In order to determine the role of the C terminus of ABCA4 in protein structure and function and understand mechanisms by which C-terminal mutations cause retinal degenerative diseases, we have expressed and purified a series of deletion and substitution mutants of ABCA4 and ABCA1 in HEK 293T cells for analysis of their cellular localization and biochemical properties. Removal of the C-terminal 30 amino acids of ABCA4, including a conserved VFVNFA motif, resulted in a loss in N-retinylidene-phosphatidylethanolamine substrate binding, ATP photoaffinity labeling, and retinal-stimulated ATPase activity. This mutant was also retained in the endoplasmic reticulum of cells. Replacement of the VFVNFA motif with alanine residues also resulted in loss in function and cellular mislocalization. In contrast, C-terminal deletion mutants that retain the VFVNFA motif were functionally active and localized to intracellular vesicles similar to wild-type ABCA4. Our studies indicated that the VFVNFA motif is required for the proper folding of ABCA4 into a functionally active protein. This motif also contributes to the efficient folding of ABCA1 into an active protein. Our results provide a molecular based rationale for the disease phenotype displayed by individuals with mutations in the C terminus of ABCA4.
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Photoreceptor ABC Transporter ABCA4: Its Role in the Visual Cycle and Retinal Degenerative Diseases
Biophysical Journal, 2009Co-Authors: Robert S Molday, Ming ZhongAbstract:ABCA4 is a member of the superfamily of ATP binding cassette (ABC) proteins that is localized in outer segment disc membranes of rod and cone photoreceptor cells. Mutations in the ABCA4 gene are responsible for Stargardt macular dystrophy, cone-rod dystrophy and retinitis pigmentosa. Biochemical studies together with analysis of ABCA4 knockout mice implicate ABCA4 in the transport of N-retinylidene-phosphatidylethanolamine across disk membranes. This transport process facilitates the complete removal of retinal derivatives from photoreceptors following the photobleaching of rhodopsin and cone opsin as part of the visual cycle. Loss in the activity of ABCA4 leads to the production of diretinal derivatives in disc membranes which accumulate in adjacent retinal pigment epithelial (RPE) cells as lipofuscin deposits following phagocytosis of outer segments. Progressive buildup of these toxic diretinal compounds causes the degeneration of RPE and photoreceptors and a loss in vision. Recently, we have investigated the effect of C-terminal deletion, including several disease related mutations, on the structural and functional properties of ABCA4. Our studies indicate that ABCA4 contains a conserved motif near the C-terminus that is crucial for proper protein folding and functional activity of ABCA4. Individuals missing this motif due to C-terminal truncation of ABCA4 exhibit a severe form of retinal degeneration known as cone-rod dystrophy.
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N-retinylidene-phosphatidylethanolamine is the preferred retinoid substrate for the photoreceptor-specific ABC transporter ABCA4 (ABCR).
Journal of Biological Chemistry, 2004Co-Authors: Seelochan Beharry, Ming Zhong, Robert S MoldayAbstract:Abstract ABCA4, a member of the family of ATP binding cassette (ABC) proteins found in rod and cone photoreceptors, has been implicated in the transport of retinoid compounds across the outer segment disk membrane following the photoactivation of rhodopsin. Mutations in the ABCA4 gene are responsible for Stargardt macular dystrophy and related retinal degenerative diseases that cause a loss in vision. To identify the retinoid substrate that interacts with ABCA4, we have isolated ABCA4 from rod outer segment disk membranes on an immunoaffinity matrix and analyzed retinoid compounds that bind to ABCA4 using high performance liquid chromatography and radiolabeling methods. When all-trans-retinal was added to ABCA4 in the presence of phosphatidylethanolamine, ∼0.9 mol of N-retinylidene-phosphatidylethanolamine and 0.3 mol of all-trans-retinal were bound per mol of ABCA4 with an apparent Kd of 2–5 μm. ATP and GTP released these retinoids from ABCA4, whereas ADP, GDP, and nonhydrolyzable derivatives, adenosine 5′-(β,γ-imido)triphosphate and guanosine 5′-(β,γ-imido)triphosphate, were ineffective. One mole of N-retinyl-phosphatidylethanolamine, the reduced form of N-retinylidene-phosphatidylethanolamine, bound per mol of ABCA4, whereas 0.3 mol of all-trans-retinal were bound in the absence of phosphatidylethanolamine. No binding of all-trans-retinol to ABCA4 was observed. Our results indicate that ABCA4 preferentially binds N-retinylidene-phosphatidylethanolamine with high affinity in the absence of ATP. Our studies further suggest that ATP binding and hydrolysis induces a protein conformational change that causes N-retinylidene-phosphatidylethanolamine to dissociate from ABCA4.
Carel B. Hoyng - One of the best experts on this subject based on the ideXlab platform.
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Retinal Dystrophies: Functional Genomics to Gene Therapy: Novartis Foundation Symposium 255 - The expanding roles of ABCA4 and CRB1 in inherited blindness.
Novartis Foundation Symposium, 2008Co-Authors: Frans P. M. Cremers, Alessandra Maugeri, A.i. Den Hollander, Carel B. HoyngAbstract:Mutations in the ABCA4 gene cause Stargardt disease (STGD), most cases with autosomal recessive (ar) cone-rod dystrophy (CRD), and some cases with atypical ar retinitis pigmentosa (arRP). We found compound heterozygous ABCA4 mutations in two unrelated patients with STGD and homozygous splice site mutations in their 2nd and 4th degree cousins with RP. Some ABCA4 mutations display strong founder effects. In Dutch and German STGD patients, the 768G > T mutation is present in 8% and 0.6% of ABCA4 alleles respectively. Vice versa, the complex L541P;A1038V allele is found in 70% of ABCA4 alleles in German STGD patients but absent in Dutch patients. As approximately 70% of ABCA4 mutations are known, a microarray-based analysis of known ABCA4 gene variants allows routine DNA diagnostics in Caucasian patients. Mutations in the CRB1 gene underlie RP12, some cases with classic arRP, 55% of cases with RP and Coats-like exudative vasculopathy, and 13% of patients with Leber congenital amaurosis (LCA), rendering CRB1 a significant cause of autosomal recessive retinal dystrophy. Different combinations of mutations in ABCA4 or CRB1 can be correlated with disease severity, suggesting that small increments of protein activities in patients might have significant therapeutic effects. Mouse and Drosophila studies strongly suggest that both patient groups might benefit from reduced light exposure and therefore should be detected as early as possible using molecular techniques.
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The spectrum of retinal phenotypes caused by mutations in the ABCA4 gene
Graefe's Archive for Clinical and Experimental Ophthalmology, 2005Co-Authors: B. Jeroen Klevering, August F. Deutman, Frans P. M. Cremers, Alessandra Maugeri, Carel B. HoyngAbstract:Background The majority of studies on the retina-specific ATP-binding cassette transporter ( ABCA4 ) gene have focussed on molecular genetic analysis; comparatively few studies have described the clinical aspects of ABCA4 -associated retinal disorders. In this study, we demonstrate the spectrum of retinal dystrophies associated with ABCA4 gene mutations. Methods Nine well-documented patients representing distinct phenotypes in the continuum of ABCA4 -related disorders were selected. All patients received an extensive ophthalmologic evaluation, including kinetic perimetry, fluorescein angiography, and electroretinography (ERG). Mutation analysis had been performed previously with the genotyping microarray (ABCR400 chip) and/or single-strand conformation polymorphism analysis in combination with direct DNA sequencing. Results In all patients, at least one pathologic ABCA4 mutation was identified. Patient 10034 represented the mild end of the phenotypic spectrum, demonstrating exudative age-related macular degeneration (AMD). Patient 24481 received the diagnosis of late-onset fundus flavimaculatus (FFM), patient 15168 demonstrated the typical FFM phenotype, and patient 19504 had autosomal recessive Stargardt disease (STGD1). Patients 11302 and 7608 exhibited progression from FFM/STGD1 to cone–rod dystrophy (CRD). A more typical CRD phenotype was found in patients 15680 and 12608. Finally, the most severe ABCA4 -associated phenotype was retinitis pigmentosa (RP) in patient 11366. This phenotype was characterised by extensive atrophy with almost complete loss of peripheral and central retinal functions. Conclusion We describe nine patients during different stages of disease progression; together, these patients form a continuum of ABCA4 -associated phenotypes. Besides characteristic disorders such as FFM/STGD1, CRD and RP, intermediate phenotypes may be encountered. Moreover, as the disease progresses, marked differences may be observed between initially comparable phenotypes. In contrast, distinctly different phenotypes may converge to a similar final stage, characterised by extensive chorioretinal atrophy and very low visual functions. The identified ABCA4 mutations in most, but not all, patients were compatible with the resulting phenotypes, as predicted by the genotype–phenotype model for ABCA4 -associated disorders. With the advent of therapeutic options, recognition by the general ophthalmologist of the various retinal phenotypes associated with ABCA4 mutations is becoming increasingly important.
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The spectrum of retinal phenotypes caused by mutations in the ABCA4 gene.
Graefes Archive for Clinical and Experimental Ophthalmology, 2004Co-Authors: B. Jeroen Klevering, August F. Deutman, Frans P. M. Cremers, Alessandra Maugeri, Carel B. HoyngAbstract:Background The majority of studies on the retina-specific ATP-binding cassette transporter (ABCA4) gene have focussed on molecular genetic analysis; comparatively few studies have described the clinical aspects of ABCA4-associated retinal disorders. In this study, we demonstrate the spectrum of retinal dystrophies associated with ABCA4 gene mutations.
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From gene to disease: from the ABCA4 gene to Stargardt disease, cone-rod dystrophy and retinitis pigmentosa
Nederlands Tijdschrift voor Geneeskunde, 2002Co-Authors: Frans P. M. Cremers, B. Jeroen Klevering, Alessandra Maugeri, L.h. Hoefsloot, Carel B. HoyngAbstract:Autosomal recessive Stargardt disease is caused by mutations in the ABCA4 gene. Mutations in ABCA4 are also found in two-thirds of cases with autosomal recessive cone-rod dystrophy, and a small fraction of patients with autosomal recessive retinitis pigmentosa. Patients with autosomal recessive retinitis pigmentosa, the most severe of these three phenotypes, invariably carry ABCA4 inactivating mutations; patients with autosomal recessive cone-rod dystrophy and Stargardt disease carry combinations of mutations that do not completely inactivate the retina specific 'ATP-binding cassette transporter' (ABCR) protein. DNA diagnostics is complicated by the high allelic heterogeneity and the uncertainty as to whether some ABCA4 variants are pathological. Nevertheless, ABCA4 mutation analysis is particularly important for patients with cone-rod dystrophy to confirm the autosomal recessive mode of inheritance.
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Mutations in the ABCA4 (ABCR) gene are the major cause of autosomal recessive cone-rod dystrophy.
American Journal of Human Genetics, 2000Co-Authors: Alessandra Maugeri, B. Jeroen Klevering, August F. Deutman, Carel B. Hoyng, Klaus Rohrschneider, Anita Blankenagel, Han G. Brunner, Frans P. M. CremersAbstract:The photoreceptor cell–specific ATP-binding cassette transporter gene (ABCA4; previously denoted “ABCR”) is mutated in most patients with autosomal recessive (AR) Stargardt disease (STGD1) or fundus flavimaculatus (FFM). In addition, a few cases with AR retinitis pigmentosa (RP) and AR cone-rod dystrophy (CRD) have been found to have ABCA4 mutations. To evaluate the importance of the ABCA4 gene as a cause of AR CRD, we selected 5 patients with AR CRD and 15 patients with isolated CRD, all from Germany and The Netherlands . Single-strand conformation–polymorphism analysis and sequencing revealed 19 ABCA4 mutations in 13 (65%) of 20 patients. In six patients, mutations were identified in both ABCA4 alleles; in seven patients, mutations were detected in one allele. One complex ABCA4 allele (L541P;A1038V) was found exclusively in German patients with CRD; one patient carried this complex allele homozygously, and five others were compound heterozygous. These findings suggest that mutations in the ABCA4 gene are the major cause of AR CRD. A primary role of the ABCA4 gene in STGD1/FFM and AR CRD, together with the gene's involvement in an as-yet-unknown proportion of cases with AR RP, strengthens the idea that mutations in the ABCA4 gene could be the most frequent cause of inherited retinal dystrophy in humans.
