Dissecting the disease mechanism of Leber congenital amaurosis type 5 (LCA5) using retinal affinity proteomics

Abstract

Purpose : Mutations in the LCA5 gene disrupt its cognate ciliary protein lebercilin, causing Leber congenital amaurosis (LCA), one of the most severe forms of inherited blindness. Previous proteomics studies performed in non-retinal cells (HEK293T) identified a lebercilin interactome that contained several submodules, including the ciliary intraflagellar transport (IFT) complex. This suggested that defective ciliary trafficking underlies the congenital blindness in LCA5 patients. In this study, we set out to identify the retina-specific interactome of lebercilin by applying two complementary retinal affinity proteomics approaches.

Methods : To optimize an ex vivo affinity purification of lebercilin interactors from bovine retinal extracts, we first purified correctly processed recombinant N-TAP tagged lebercilin bait-proteins, both wild-type (WT) and mutant (LCA5 p.P493TfsX1), from HEK293T cells employing an SDS ‘bait-purification’ procedure. These purified lebercilin baits were then used to pull-down specific interactors from bovine retinal lysates, followed by mass spectrometry (MS) analysis. For an in vivo approach, we used the AAV serotype AAV7m8 to intravitreally deliver a 3xFLAG-tagged human LCA5 cDNA in the eyes of Lca5^+/gt mouse pups (P2). After four weeks, retinas were isolated and subjected to affinity purification of lebercilin followed by MS analysis.

Results : Ex vivo, we found that all IFT-B proteins, two members of the multi subunit C-terminal to LisH (CTLH) complex, and the dynein light chain 1 complex are significantly enriched in purifications of lebercilin WT versus control. However, only the IFT-B and CTLH proteins were lost from the interactome due to the mutation.
In vivo, we found that two specific IFT-B proteins and four CTLH complex proteins were significantly enriched in AAV/3xFLAG-lebercilin purifications from mouse retinas compared to controls. Analysis of additional potential interactors is currently ongoing.

Conclusions : Our results indicate that the IFT complex and the CTLH complex, previously identified in HEK293T cells, are conserved in the retinal context. Furthermore, we identified that the interaction of all IFT-B proteins and several CTLH complex proteins is lost when lebercilin is mutated. Overall, these findings highlight the potential contribution of these protein complexes to the disease mechanism.

This is a 2021 ARVO Annual Meeting abstract.