Loss of RPGR glutamylation underlies the pathogenic mechanism of retinal dystrophy caused by TTLL5 mutations.

Mutations in the X-linked retinitis pigmentosa GTPase regulator (RPGR) gene are a major cause of retinitis pigmentosa, a blinding retinal disease resulting from photoreceptor degeneration. A photoreceptor specific ORF15 variant of RPGR (RPGR(ORF15)), carrying multiple Glu-Gly tandem repeats and a C-terminal basic domain of unknown function, localizes to the connecting cilium where it is thought to regulate cargo trafficking. Here we show that tubulin tyrosine ligase like-5 (TTLL5) glutamylates RPGR(ORF15) in its Glu-Gly-rich repetitive region containing motifs homologous to the α-tubulin C-terminal tail. The RPGR(ORF15) C-terminal basic domain binds to the noncatalytic cofactor interaction domain unique to TTLL5 among TTLL family glutamylases and targets TTLL5 to glutamylate RPGR. Only TTLL5 and not other TTLL family glutamylases interacts with RPGR(ORF15) when expressed transiently in cells. Consistent with this, a Ttll5 mutant mouse displays a complete loss of RPGR glutamylation without marked changes in tubulin glutamylation levels. The Ttll5 mutant mouse develops slow photoreceptor degeneration with early mislocalization of cone opsins, features resembling those of Rpgr-null mice. Moreover TTLL5 disease mutants that cause human retinal dystrophy show impaired glutamylation of RPGR(ORF15) Thus, RPGR(ORF15) is a novel glutamylation substrate, and this posttranslational modification is critical for its function in photoreceptors. Our study uncovers the pathogenic mechanism whereby absence of RPGR(ORF15) glutamylation leads to retinal pathology in patients with TTLL5 gene mutations and connects these two genes into a common disease pathway.