The carboxyl terminal mutational hotspot of the ciliary disease protein RPGRORF15 (retinitis pigmentosa GTPase regulator) is glutamylated in vivo

Mutations in RPGR(ORF15) (retinitis pigmentosa GTPase regulator) are a major cause of inherited retinal degenerative diseases. RPGRORF15 (1152 residues) is a ciliary protein involved in regulating the composition and function of photoreceptor cilia. The mutational hotspot in RPGR(ORF15) is an unusual C-terminal domain encoded by exon ORF15, which is rich in polyglutamates and glycine residues (Glu-Gly domain) followed by a short stretch of basic amino acid residues (RPGR(C2) domain; residues 1072-1152). However, the properties of the ORF15-encoded domain and its involvement in the pathogenesis of the disease are unclear. Here we show that RPGR(ORF15) is glutamylated at the C-terminus, as determined by binding to GT335, which recognizes glutamylated substrates. This reactivity is lost in two mouse mutants of Rpgr, which do not express RPGR(ORF15) due to disease-causing mutations in exon ORF15. Our results indicate that RPGR(ORF15) is posttranslationally glutamylated in the Glu-Gly domain and that the GT335 antibody predominantly recognizes RPGR(ORF15) in photoreceptor cilia.