ATP-binding cassette transporter ABCA4 and chemical isomerization protect photoreceptor cells from the toxic accumulation of excess 11-cis-retinal.

The visual cycle is a series of enzyme-catalyzed reactions which converts all-trans-retinal to 11-cis-retinal for the regeneration of visual pigments in rod and cone photoreceptor cells. Although essential for vision, 11-cis-retinal like all-trans-retinal is highly toxic due to its highly reactive aldehyde group and has to be detoxified by either reduction to retinol or sequestration within retinal-binding proteins. Previous studies have focused on the role of the ATP-binding cassette transporter ABCA4 associated with Stargardt macular degeneration and retinol dehydrogenases (RDH) in the clearance of all-trans-retinal from photoreceptors following photoexcitation. How rod and cone cells prevent the accumulation of 11-cis-retinal in photoreceptor disk membranes in excess of what is required for visual pigment regeneration is not known. Here we show that ABCA4 can transport N-11-cis-retinylidene-phosphatidylethanolamine (PE), the Schiff-base conjugate of 11-cis-retinal and PE, from the lumen to the cytoplasmic leaflet of disk membranes. This transport function together with chemical isomerization to its all-trans isomer and reduction to all-trans-retinol by RDH can prevent the accumulation of excess 11-cis-retinal and its Schiff-base conjugate and the formation of toxic bisretinoid compounds as found in ABCA4-deficient mice and individuals with Stargardt macular degeneration. This segment of the visual cycle in which excess 11-cis-retinal is converted to all-trans-retinol provides a rationale for the unusually high content of PE and its long-chain unsaturated docosahexaenoyl group in photoreceptor membranes and adds insight into the molecular mechanisms responsible for Stargardt macular degeneration.

Differential phospholipid substrates and directional transport by ATP-binding cassette proteins ABCA1, ABCA7, and ABCA4 and disease-causing mutants.

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.

Mammalian P4-ATPases and ABC transporters and their role in phospholipid transport.

Transport of phospholipids across cell membranes plays a key role in a wide variety of biological processes. These include membrane biosynthesis, generation and maintenance of membrane asymmetry, cell and organelle shape determination, phagocytosis, vesicle trafficking, blood coagulation, lipid homeostasis, regulation of membrane protein function, apoptosis, etc. P(4)-ATPases and ATP binding cassette (ABC) transporters are the two principal classes of membrane proteins that actively transport phospholipids across cellular membranes. P(4)-ATPases utilize the energy from ATP hydrolysis to flip aminophospholipids from the exocytoplasmic (extracellular/lumen) to the cytoplasmic leaflet of cell membranes generating membrane lipid asymmetry and lipid imbalance which can induce membrane curvature. Many ABC transporters play crucial roles in lipid homeostasis by actively transporting phospholipids from the cytoplasmic to the exocytoplasmic leaflet of cell membranes or exporting phospholipids to protein acceptors or micelles. Recent studies indicate that some ABC proteins can also transport phospholipids in the opposite direction. The importance of P(4)-ATPases and ABC transporters is evident from the findings that mutations in many of these transporters are responsible for severe human genetic diseases linked to defective phospholipid transport. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.

Posttranslational modifications of the photoreceptor-specific ABC transporter ABCA4.

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 mammalian cells, we showed that the Stargardt disease-associated alanine mutation in the phosphorylation site at position 901 led to protein misfolding and degradation. Furthermore, replacing the S1317 phosphorylation site reduced the basal ATPase activity of ABCA4, whereas an alanine mutation in either the S1185 or T1313 phosphorylation site resulted in a significant decrease in the all-trans-retinal-stimulated ATPase activity without affecting the basal activity, protein expression, or localization. In agreement with this observation, partial dephosphorylation of native bovine ABCA4 led to reduction of both basal and stimulated ATPase activity. Thus, we present the first evidence that phosphorylation of ABCA4 can regulate its function.

Characterization of the S289A,D mutants of yeast cystathionine beta-synthase.

Cystathionine beta-synthase (CBS) catalyzes the pyridoxal 5'-phosphate (PLP)-dependent condensation of l-serine and l-homocysteine to form l-cystathionine in the first step of the reverse transsulfuration pathway. Residue S289 of yeast CBS, predicted to form a hydrogen bond with the pyridine nitrogen of the PLP cofactor, was mutated to alanine and aspartate. The k(cat)/K(m)(l-Ser) of the S289A mutant is reduced by a factor of approximately 800 and the beta-replacement activity of the S289D mutant is undetectable. Fluorescence energy transfer between tryptophan residue(s) of the enzyme and the PLP cofactor, observed in the wild-type enzyme and diminished in the S289A mutant, is absent in S289D. These results demonstrate that residue S289 is essential in maintaining the properties and orientation of the pyridine ring of the PLP cofactor. The reduction in activity of ytCBS-S289A suggests that ytCBS catalyzes the alpha,beta-elimination of l-Ser via an E1cB mechanism.

The role of the photoreceptor ABC transporter ABCA4 in lipid transport and Stargardt macular degeneration.

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 2,273 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.

Kinetic characterization of recombinant human cystathionine beta-synthase purified from E. coli.

Cystathionine beta-synthase (CBS) catalyzes the pyridoxal-50-phosphate-dependent condensation of L-serine and L-homocysteine to form L-cystathionine in the first step of the transsulfuration pathway. Although effective expression systems for recombinant human CBS (hCBS) have been developed, they require multiple chromatographic steps as well as proteolytic cleavage to remove the fusion partner. Therefore, a series of five expression constructs, each incorporating a 6-His tag, were developed to enable the efficient purification of hCBS via immobilized metal ion affinity chromatography. Two of the constructs express hCBS in fusion with a protein partner, while the others bear only the affinity tag. The addition of an amino-terminal, 6-His tag, in the absence of a protein fusion partner and in the absence or presence ofa protease-cleavable linker, was found to be sufficient for the purification of soluble hCBS and resulted in enzyme with 86-91% heme saturation and with activity similar to that reported for other hCBS expression constructs. The continuous assay for L-Cth production, employing cystathionine beta-lyase and L-lactate dehydrogenase as coupling enzymes, was employed here for the first time to determine the steady-state kinetic parameters of hCBS, via global analysis, and revealed previously unreported substrate inhibition by L-Hcys (K(i)(L-HCYS) = 2.1 +/- 0.2 mM). The kinetic parameters for the hCBS-catalyzed hydrolysis of L-Cth toL-Ser and L-Hcys were also determined and the k(cat)/K(m)(L-CTH) of this reaction is only approximately 2-fold lower than the k(cat)/K(m)(L-SER) of the physiological, condensation reaction.

Cholesterol transport via ABCA1: new insights from solid-phase binding assay.

It is now well established that the ATP-binding cassette transporter A1 (ABCA1) plays a pivotal role in HDL metabolism, reverse cholesterol transport and net efflux of cellular cholesterol and phospholipids. We aimed to resolve some uncertainties related to the putative function of ABCA1 as a mediator of lipid transport by using a methodology developed in the laboratory to isolate a protein and study its interactions with other compounds. ABCA1 was tagged with the 1D4 peptide at the C terminus and expressed in human HEK 293 cells. Preliminary experiments showed that the tag modified neither the protein expression/localization within the cells nor the ability of ABCA1 to promote cholesterol cellular efflux to apolipoprotein A-I. ABCA1-1D4 was then purified and reconstituted in liposomes. ABCA1 displayed an ATPase activity in phospholipid liposomes that was significantly decreased by cholesterol. Finally, interactions with either cholesterol or apolipoprotein A-I were assessed by binding experiments with protein immobilized on an immunoaffinity matrix. Solid-phase binding assays showed no direct binding of cholesterol or apolipoprotein A-I to ABCA1. Overall, our data support the hypothesis that ABCA1 is able to mediate the transport of cholesterol from cells without direct interaction and that apo A-I primarily binds to membrane surface or accessory protein(s).

ABCA4 is an N-retinylidene-phosphatidylethanolamine and phosphatidylethanolamine importer.

ATP-binding cassette (ABC) transporters comprise a superfamily of proteins, which actively transport a variety of compounds across cell membranes. Mammalian and most eukaryotic ABC transporters function as exporters, flipping or extruding substrates from the cytoplasmic to the extracellular or lumen side of cell membranes. Prokaryotic ABC transporters function either as exporters or importers. Here we show that ABCA4, an ABC transporter found in retinal photoreceptor cells and associated with Stargardt macular degeneration, is a novel importer that actively flips N-retinylidene-phosphatidylethanolamine from the lumen to the cytoplasmic leaflet of disc membranes, thereby facilitating the removal of potentially toxic retinoid compounds from photoreceptors. ABCA4 also actively transports phosphatidylethanolamine in the same direction. Mutations known to cause Stargardt disease decrease N-retinylidene-phosphatidylethanolamine and phosphatidylethanolamine transport activity of ABCA4. These studies provide the first direct evidence for a mammalian ABC transporter that functions as an importer and provide insight into mechanisms underlying substrate transport and the molecular basis of Stargardt disease.

Lipid transport by mammalian ABC proteins.

ABC (ATP-binding cassette) proteins actively transport a wide variety of substrates, including peptides, amino acids, sugars, metals, drugs, vitamins and lipids, across extracellular and intracellular membranes. Of the 49 hum an ABC proteins, a significant number are known to mediate the extrusion of lipids from membranes or the flipping of membrane lipids across the bilayer to generate and maintain membrane lipid asymmetry. Typical lipid substrates include phospholipids, sterols, sphingolipids, bile acids and related lipid conjugates. Members of the ABCA subfamily of ABC transporters and other ABC proteins such as ABCB4, ABCG1 and ABCG5/8 implicated in lipid transport play important roles in diverse biological processes such as cell signalling, membrane lipid asymmetry, removal of potentially toxic compounds and metabolites, and apoptosis. The importance of these ABC lipid transporters in cell physiology is evident from the finding that mutations in the genes encoding many of these proteins are responsible for severe inherited diseases. For example, mutations in ABCA1 cause Tangier disease associated with defective efflux of cholesterol and phosphatidylcholine from the plasma membrane to the lipid acceptor protein apoA1 (apolipoprotein AI), mutations in ABCA3 cause neonatal surfactant deficiency associated with a loss in secretion of the lipid pulmonary surfactants from lungs of newborns, mutations in ABCA4 cause Stargardt macular degeneration, a retinal degenerative disease linked to the reduced clearance of retinoid compounds from photoreceptor cells, mutations in ABCA12 cause harlequin and lamellar ichthyosis, skin diseases associated with defective lipid trafficking in keratinocytes, and mutations in ABCB4 and ABCG5/ABCG8 are responsible for progressive intrafamilial hepatic disease and sitosterolaemia associated with defective phospholipid and sterol transport respectively. This chapter highlights the involvement of various mammalian ABC transporters in lipid transport in the context of their role in cell signalling, cellular homoeostasis, apoptosis and inherited disorders.