RPGR^ORF15 glutamylation impaired by mutations in its C-terminal basic domain

Abstract

Purpose : Mutations in the RPGR gene represent a major contribution to retinitis pigmentosa. The photoreceptor-specific isoform, RPGR^ORF15, contributes to the intracellular cargo movement between inner and outer segments of rods and cones. Glutamylation of RPGR^ORF15 by the enzyme TTLL5 is essential for its normal function and mutations in the glutamic acid-glycine rich region have been shown to impair this post-translational modification. The aim of this study was to evaluate the effect of disease-causing mutations in the C-terminal basic domain of RPGR^ORF15 on its interaction with TTLL5 enzyme as a way of predicting the level of glutamylation in gene therapy vectors used in clinical trials.

Methods : RPGR^ORF15 mutant constructs were generated by site-directed mutagenesis using a wild type (WT) construct as a template. RPGR WT and mutant constructs were transfected into HEK293 cells. The level of RPGR^ORF15 glutamylation was assessed by western blot analysis using the GT335 antibody and a human RPGR antibody raised against the N-terminus. To analyse the interaction between the WT and mutant RPGR constructs with TTLL5, a proximity ligation assay was performed using the Duolink® detection technology from Sigma-Aldrich. The level of fluorescence was quantified in both assays to determine the level of glutamylation and the interaction between both proteins.

Results : GT335 western blot showed that the full-length RPGR^ORF15 expressed with the wild type construct is strongly glutamylated. However, quantification of glutamylation levels by densitometry showed that all the mutations producing a truncated basic domain in RPGR^ORF15 cause a decrease in the level of glutamylation, between 50 and 90%, compared to the full-length protein. However, the binding between truncated forms of RPGR and TTLL5 appeared not to be affected, suggesting that there are other mediators involved in the glutamylation of RPGR^ORF15.

Conclusions : Mutations in the C-terminal of RPGR^ORF15 significantly reduce the level of glutamylation, despite apparently not impairing the binding to TTLL5. These results suggest that there are other proteins required for the glutamylation of RPGR^ORF15. Since glutamylation of RPGR^ORF15 influences its stabilization and folding, and its interaction with other proteins in the connecting cilia, it is critical to ensure that gene therapy vectors provide the fully glutamylated protein to the retina in order to be biologically active.

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