June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Investigation of ABCA4 missense variant plasma membrane trafficking in cell models.
Author Affiliations & Notes
  • Davide Piccolo
    University College London, London, London, United Kingdom
  • Christina Zarouchlioti
    University College London, London, London, United Kingdom
  • Jim Bellingham
    University College London, London, London, United Kingdom
  • Michael E Cheetham
    University College London, London, London, United Kingdom
  • Footnotes
    Commercial Relationships   Davide Piccolo None; Christina Zarouchlioti None; Jim Bellingham None; Michael Cheetham ProQR, Code C (Consultant/Contractor), Alia Therapeutics, Code C (Consultant/Contractor), PYC, Code C (Consultant/Contractor)
  • Footnotes
    Support  EU Grant 548691
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 672 – F0126. doi:
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      Davide Piccolo, Christina Zarouchlioti, Jim Bellingham, Michael E Cheetham; Investigation of ABCA4 missense variant plasma membrane trafficking in cell models.. Invest. Ophthalmol. Vis. Sci. 2022;63(7):672 – F0126.

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

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Purpose : A potential strategy to treat inherited diseases that are associated with protein misfolding is to correct folding, restore the three-dimensional structure and enhance traffic. Here we investigate the plasma membrane localisation of ABCA4 protein using an in vitro cell model, which could be used as a screen for drugs that positively affect folding.

Methods : The trafficking of the ABCA4 was investigated at 370C in vitro by transient transfection of HEK293 and CHO cells using plasmids encoding for the wild-type (WT)-ABCA4 protein and four missense variants, provided by Professor Molday, UBC, Canada. The expression levels of the proteins was assessed by Western Blot (WB) and the cell surface expression was evaluated by immunocytochemistry (ICC) using an antibody against an extracellular epitope. In order to determine whether the traffic and folding were temperature-sensitive, the same experiments were performed at reduced temperature (300C). A quantitative assay was developed to monitor the membrane protein plasma membrane traffic. Statistical analysis was performed with two-tailed Student’s t-test.

Results : At 370C the expression level of the WT-ABCA4 assessed by WB did not show a significant difference to the missense variants. Plasma membrane staining was observed only in few cells expressing WT-ABCA4. When cells were grown at 300C, the steady-state protein levels and the plasma membrane signal of the WT and the missense variants were increased. The plasma membrane specificity of the quantitative assay and the ability to detect pharmacological rescue of protein folding was confirmed using WT rhodopsin (RHO) and the misfolding P23H-RHO variant. In this assay WT-ABCA4 showed an increase in plasma membrane signal compared to all the missense variants, except A1038V, suggesting it is not associated with mistrafficking.

Conclusions : Reduced growth temperature can enhance the plasma membrane traffic of WT and missense variants of ABCA4 in cultured cells, suggesting that some of these variants are temperature sensitive misfolding variants that might be amenable to pharmacological rescue. The assay we developed is able to quickly and robustly detect plasma membrane localization, which could be used as a surrogate marker for correct folding and trafficking, and has potential use for high-throughput screening of small molecules able to restore ABCA4 folding.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.


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