Negative charge of the glutamic acid 417 residue is crucial for isomerohydrolase activity of RPE65

“Mutations in the RPE65 gene cause severe congenital retinal dystrophies, such as juvenile severe retinitis pigmentosa and Leber congenital amaurosis (LCA), which lead to blindness [5-8].”

“RPE65, identified as an iron-dependent isomerohydrolase of the visual cycle, is a key enzyme for regeneration of 11-cis retinal, the chromophore of both rod and cone visual pigments [1-3].”

“Genetic analysis of LCA patients suggests that RPE65 gene mutations account for 3% [9] to 16% [10] of total cases of LCA.”

“RPE65, identified as an iron-dependent isomerohydrolase of the visual cycle, is a key enzyme for regeneration of 11-cis retinal, the chromophore of both rod and cone visual pigments [1-3].”

“Mutations in the RPE65 gene cause severe congenital retinal dystrophies, such as juvenile severe retinitis pigmentosa and Leber congenital amaurosis (LCA), which lead to blindness [5-8].”

“RPE65, identified as an iron-dependent isomerohydrolase of the visual cycle, is a key enzyme for regeneration of 11-cis retinal, the chromophore of both rod and cone visual pigments [1-3].”

“Mutations in the RPE65 gene cause severe congenital retinal dystrophies, such as juvenile severe retinitis pigmentosa and Leber congenital amaurosis (LCA), which lead to blindness [5-8].”

“Mutations in the RPE65 gene cause severe congenital retinal dystrophies, such as juvenile severe retinitis pigmentosa and Leber congenital amaurosis (LCA), which lead to blindness [5-8].”

“4A) has a seven-bladed propeller structure holding an iron ion coordinated by four highly conserved histidines (H180, H240, H313 and H527) [17].”

“However, the molecular mechanism by which RPE65-LCA mutations cause such severe visual deficiency remains elusive.”

“Previously, we have shown that the endogenous iron (II) ion [20] and the four conserved histidines are essential for the isomerase activity [16].”

“RPE65 is predominantly expressed in the retinal pigment epithelium (RPE) and associated with the endoplasmic reticulum membrane [4].”

“Alternatively, the negative charge of E417 could stabilize the carbocation transition state of the substrate developed in the course of the reaction [21,22].”

“Unlike previously reported RPE65 mutations [11,12,19], both E417D and E417Q demonstrated expression levels comparable with that of wtRPE65 in 293A-LRAT cells.”

“In this study, we investigated the E417Q variant of RPE65 which has been identified in LCA patients [14] and compared its activity with that of E417D.”

“QBI-293 A cells (Qbiogene, Carlsbad, CA) stably expressing LRAT (293A-LRAT) [16] were infected with adenoviruses expressing wtRPE65 and its mutants at MOI of 100 and the subcellular localization of recombinant RPE65 was elucidated using FractPrepTM kit (BioVision, Mountain View, CA).”

“To estimate the half-lives of wtRPE65 and its mutants, 293A-LRAT cells were infected with the adenoviruses at MOI of 20 and stability assays were performed as described previously [11].”

“Generation of recombinant adenoviruses (Ad-RPE65) was performed as described before [15].”

“Unlike previously reported RPE65 mutations [11,12,19], both E417D and E417Q demonstrated expression levels comparable with that of wtRPE65 in 293A-LRAT cells.”

“…from a complete lack of 11-cis retinoid production to very low but detectable levels (Bereta et al., 2008; Jin et al., 2005; Lorenz et al., 2008; Nikolaeva et al., 2010; Philp et al., 2009; Redmond et al., 2005; Takahashi et al., 2006) but available in vivo results from a knockin mouse appear…”

“This finding along with data from other biochemical studies on RPE65 (Nikolaeva et al., 2010; Takahashi et al., 2005), indicate that the negative charge of the second sphere Glu residues is critical for their role in supporting iron coordination by the primary sphere His residues for CCDs in…”

“Mutagenesis experiments combined with mutations observed in LCA patients have established that the second shell Glu residues are essential for RPE65 isomerase activity and even highly similar residues such as Asp and Gln are unable to functionally substitute for them (Hanein et al., 2004; Nikolaeva et al., 2010; Redmo…”

“Although the biochemical attribute of amino acid residues mutated is important in determining the enzyme activity of a mutant RPE65 (19), our result also suggests that the relative spatial distance between a mutation site and the catalytic site is a critical factor in determining whether the mutant RPE65 can be rescue…”

“3 The abbreviations used are: RAL, all-trans-retinal; CCD, carotenoid cleavage dioxygenase; ACO, apocarotenoid-cleaving oxygenase; BisTris, 2-[bis(2hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; BisTris propane, 1,3-bis[tris(hydroxymethyl)methylamino]propane; LCA Leber congenital amaurosis. crossmark THE JOURN…”

“In vitro and animal studies showed that not only R91W mutation but also many other mutations resulted in a drastic decrease of RPE65 (Chen et al., 2006; Takahashi et al., 2006; Bereta et al., 2008; Philp et al., 2009; Nikolaeva et al., 2010; Wright et al., 2013; S. Li et al., 2014, 2015).”

“Although the biochemical attributes of amino acid residue mutated are important in determining the enzyme activity of a mutant RPE65 (Nikolaeva et al., 2010), our results also suggest that the relative spatial distance between a mutation site and the catalytic site is a critical factor in determining whether the mutan…”

“26 More than 230 missense mutations lacking a clear pathogenicity classification or classified as VUS have been reported in public databases for RPE65 (ClinVar, https://www.ncbi.nlm.nih.gov/clinvar/ ?term=RPE65).”