June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Retinal-phospholipid Schiff-base conjugates and their interaction with ABCA4, the ABC transporter involved in the visual cycle and Stargardt disease
Author Affiliations & Notes
  • Tongzhou Xu
    Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, British Columbia, Canada
  • Laurie L. Molday
    Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, British Columbia, Canada
  • Robert S Molday
    Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, British Columbia, Canada
  • Footnotes
    Commercial Relationships   Tongzhou Xu None; Laurie Molday None; Robert Molday None
  • Footnotes
    Support  The Canadian Institutes of Health Research (CIHR): PJT 148649 and PJT 175118
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 4433. doi:
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      Tongzhou Xu, Laurie L. Molday, Robert S Molday; Retinal-phospholipid Schiff-base conjugates and their interaction with ABCA4, the ABC transporter involved in the visual cycle and Stargardt disease. Invest. Ophthalmol. Vis. Sci. 2023;64(8):4433.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : The Schiff base conjugates formed through the reaction of retinal (vitamin A aldehyde) with phospholipids, and in particular N-retinylidene-phosphatidylethanolamine (N-Ret-PE), play an important role in visual pigment regeneration and the visual cycle. However, N-Ret-PE is also reactive and has to be cleared from photoreceptors through its transport across disc membranes by ABCA4, to prevent the accumulation of toxic bisretinoids, a pathological hallmark of Stargardt disease (STGD1). In this study, we have characterized the formation of retinal-phospholipid conjugates and investigated their interaction with ABCA4, in order to further define their role in the visual process and the pathogenesis of STGD1.

Methods : All-trans-retinal was added to liposomes composed of different phospholipid contents (varying percentages of phosphatidylcholine, phosphatidylethanolamine or phosphatidylserine), phospholipid concentrations, and pH. All-trans-retinal was also added to photoreceptor membranes deprived of endogenous retinal. The amounts of retinal and Schiff base conjugates were quantified spectrophotometrically. ABCA4 substrate binding assays were conducted in the presence of different phospholipids. ATPase activity assays were carried out with ABCA4 variants containing mutations in amino acid residues within the substrate binding pocket (SBP).

Results : The formation of N-Ret-PE increased with increasing pH, PE percentage and concentration. At the endpoint of reactions at pH 7.2, 40-60% of the total added retinal existed as N-Ret-PE. Phosphatidylserine (PS) also reacted with retinal to form its Schiff-base conjugate (N-Ret-PS), but at a significantly reduced level. N-Ret-PS did not bind to ABCA4 or react efficiently with retinal to form bisretinoids. Substitution of amino acid residues within the SBP of ABCA4 with alanine or residues implicated in STGD1 reduced or eliminated ABCA4 substrate-dependent ATPase activity.

Conclusions : N-Ret-PE forms in a considerable amount in the presence of free retinal and PE, highlighting its importance in vision. N-Ret-PS is not a substrate for ABCA4. Arginine and hydrophobic residues within the SBP facilitate the interaction of ABCA4 with N-Ret-PE. Mutations in a number of these residues cause STGD1 through the loss in substrate binding and transport.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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