Exploring the roles of the connecting cilium in photoreceptor health

Abstract

Defects in proteins functioning at the photoreceptor connecting cilium/transition zone (CC/TZ) have been linked to retinal degenerative disorders such as retinitis pigmentosa (RP) and cone-rod dystrophy (CRD). Mutations in eyes shut homolog (EYS, RP25), a secreted ciliary protein with laminin globular (LG) domains, have been linked to RP and CRD. Previously, some LG domains have been shown to interact with O-mannosyl glycans of α-dystroglycan (α-DG). Additionally, mutations in pomgnt1, an enzyme that plays a critical role in the synthesis of these glycans, have also been linked to RP (RP76). At the CC/TZ, the tectonic protein complex functions to maintain the unique biochemical environments of the inner segments (IS) and outer segments (OS) of photoreceptors. Mutations in tectonic complex proteins have been linked to ciliopathies that often include ocular abnormalities. The pathogenic mechanism underlying these mutations are poorly understood; thus, we hypothesized that EYS is an extracellular ciliary protein that interacts with α-DG and the tectonic complex. This project investigated the role of EYS, TMEM216, a member of the tectonic complex, and O-mannosyl glycans of α-DG in photoreceptor health. We determined that the C-terminal LG domains of EYS interacted with the O-mannosyl glycan epitope of α-DG. In pomgnt1 zebrafish mutants, EYS-glycan binding was reduced, and the secretion of EYS to the CC/TZ was significantly disrupted. Furthermore, in the pomgnt1 mutant retina, a substantial accumulation of EYS protein was observed in the soma of photoreceptors. Interestingly, deletion of pomgnt1 resulted in a pattern of photoreceptor degeneration similar to that previously observed in eys zebrafish mutants. By contrast, deletion of TMEM216 did not disrupt localization of EYS or of other tectonic complex proteins, yet photoreceptor degeneration was still observed in these animals. Our study has identified a previously unknown interaction between the LG domain-containing EYS and O-mannosyl glycans. These findings provide novel insight into the functional role of EYS around the CC/TZ and suggest the importance of O-mannosyl glycosylation in the regulation of protein secretion. Furthermore, our results suggest a mechanistic link between the disruption of glycosylation and photoreceptor degeneration, providing a new perspective on the underlying mechanisms behind RP25 and RP76.