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.