Abstract
Bisretinoid adducts accumulate as lipofuscin in retinal pigment epithelial (RPE) cells of the eye and are implicated in the pathology of inherited and age-related macular degeneration. Characterization of the bisretinoids A2E and the all-trans-retinal dimer series has shown that these pigments form from reactions in photoreceptor cell outer segments that involve all-trans-retinal, the product of photoisomerization of the visual chromophore 11-cis-retinal. Here we have identified two related but previously unknown RPE lipofuscin compounds. By high performance liquid chromatography-electrospray ionization-tandem mass spectrometry, we determined that the first of these compounds is a phosphatidyl-dihydropyridine bisretinoid; to indicate this structure and its formation from two vitamin A-aldehyde (A2), we will refer to it as A2-dihydropyridine-phosphatidylethanolamine (A2-DHP-PE). The second pigment, A2-dihydropyridine-ethanolamine, forms from phosphate hydrolysis of A2-DHP-PE. The structure of A2-DHP-PE was corroborated by Fourier transform infrared spectroscopy, and density functional theory confirmed the presence of a dihydropyridine ring. This lipofuscin pigment is a fluorescent compound with absorbance maxima at approximately 490 and 330 nm, and it was identified in human, mouse, and bovine eyes. We found that A2-DHP-PE forms in reaction mixtures of all-trans-retinal and phosphatidylethanolamine, and in mouse eyecups we observed an age-related accumulation. As compared with wild-type mice, A2-DHP-PE is more abundant in mice with a null mutation in Abca4 (ATP-binding cassette transporter 4), the gene causative for recessive Stargardt macular degeneration. Efforts to clarify the composition of RPE lipofuscin are important because these compounds are targets of gene-based and drug therapies that aim to alleviate ABCA4-related retinal disease.
Highlights
A Pigment with Absorbance Maxima at 490 and 331 nm Is Detectable in Abca4Ϫ/Ϫ Mouse Eyecups—The Abca4 null mutant mouse serves as a model of autosomal recessive Stargardt disease and has been shown to accumulate the bisretinoid compounds of retinal pigment epithelial (RPE) lipofuscin in abundance [8, 10, 17, 18, 28, 29]
After reaction with a second molecule of all-trans-retinal, we suggested that an iminium salt would form and following electrocyclization, a phosphatidyl dihydropyridinium molecule
Mass spectrometry was utilized to confirm the structure of NRPE
Summary
20156 JOURNAL OF BIOLOGICAL CHEMISTRY the protonation state of the Schiff base linkage in atRAL dimer-PE and atRAL dimer-E is pH-dependent [18]. The moieties generated by the addition of oxygens at CϭC bonds of these bisretinoid compounds include endoperoxides, furanoid oxides, and epoxides [25,26,27] These oxidized products are more polar than the parent compound, and mono- and bis-oxidized forms of A2E and atRAL dimer have been detected in RPE from human eyes and in eyecups from mice with null mutations in Abca4Ϫ/Ϫ [18, 25], the gene responsible for recessive Stargardt macular degeneration. We report that a previously unrecognized bisretinoid molecule absorbing with maxima at 490 and 331 nm is detected at elevated levels in Abca4Ϫ/Ϫ mice, a model of recessive Stargardt macular degeneration This compound is present in human RPE. We propose a biosynthetic pathway by which this pigment may form and demonstrate that enzymatic removal of the phosphatidic acid portion of the molecule generates a second novel component of RPE lipofuscin
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