Retinal-phospholipid Schiff-base conjugates and their interaction with ABCA4, the ABC transporter associated with Stargardt disease

• Published in: The Journal of biological chemistry 2023-05, Vol.299 (5), p.104614-104614, Article 104614
• Main Authors: Xu, Tongzhou, Molday, Laurie L., Molday, Robert S.
• Format: Article
• Language: English
• Subjects: ABCA4; ATP-binding cassette transporters; ATP-Binding Cassette Transporters - metabolism; Humans; N-retinylidene-phosphatidylethanolamine; Phosphatidylserines; Phospholipids; photoreceptor cells; retinal; retinal disease; Retinoids - metabolism; Stargardt disease; Stargardt Disease - metabolism; vision; visual cycle
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/j.jbc.2023.104614

N-retinylidene-phosphatidylethanolamine (N-Ret-PE), the Schiff-base conjugate formed through the reversible reaction of retinal (Vitamin A-aldehyde) and phosphatidylethanolamine, plays a crucial role in the visual cycle and visual pigment photoregeneration. However, N-Ret-PE can react with another molecule of retinal to form toxic di-retinoids if not removed from photoreceptors through its transport across photoreceptor membranes by the ATP-binding-cassette transporter ABCA4. Loss-of-function mutations in ABCA4 are known to cause Stargardt disease (STGD1), an inherited retinal degenerative disease associated with the accumulation of fluorescent di-retinoids and severe loss in vision. A larger assessment of retinal-phospholipid Schiff-base conjugates in photoreceptors is needed, along with further investigation of ABCA4 residues important for N-Ret-PE binding. In this study we show that N-Ret-PE formation is dependent on pH and phospholipid content. When retinal is added to liposomes or photoreceptor membranes, 40 to 60% is converted to N-Ret-PE at physiological pH. Phosphatidylserine and taurine also react with retinal to form N-retinylidene-phosphatidylserine and N-retinylidene-taurine, respectively, but at significantly lower levels. N-retinylidene-phosphatidylserine is not a substrate for ABCA4 and reacts poorly with retinal to form di-retinoids. Additionally, amino acid residues within the binding pocket of ABCA4 that contribute to its interaction with N-Ret-PE were identified and characterized using site-directed mutagenesis together with functional and binding assays. Substitution of arginine residues and hydrophobic residues with alanine or residues implicated in STGD1 significantly reduced or eliminated substrate-activated ATPase activity and substrate binding. Collectively, this study provides important insight into conditions which affect retinal-phospholipid Schiff-base formation and mechanisms underlying the pathogenesis of STGD1.