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

Abstract

RPE65 is the isomerohydrolase essential for regeneration of 11-cis retinal, the chromophore of visual pigments. Here we compared the impacts of two mutations in RPE65, E417Q identified in patients with Leber congenital amaurosis (LCA), and E417D on isomerohydrolase activity. Although both mutations decreased the stability of RPE65 and altered its subcellular localization, E417Q abolished isomerohydrolase activity whereas the E417D mutant retained partial enzymatic activity suggesting that the negative charge of E417 is important for RPE65 catalytic activity. Loss of charge at this position may represent a mechanism by which the E417Q mutation causes blindness in LCA patients.

Introduction

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]. RPE65 is predominantly expressed in the retinal pigment epithelium (RPE) and associated with the endoplasmic reticulum membrane [4]. 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]. Genetic analysis of LCA patients suggests that RPE65 gene mutations account for 3% [9] to 16% [10] of total cases of LCA. However, the molecular mechanism by which RPE65-LCA mutations cause such severe visual deficiency remains elusive. Recent studies have shown that a number of RPE65 mutants associated with LCA cause either partial or total loss of its isomerohydrolase activity [3, 11-13]. Here we focused on evaluation of the impacts of the mutation E417Q of RPE65 (associated with LCA [14]) on the stability, subcellular localization, and enzymatic activity of the protein. To investigate how the negative charge of residue E417 affects the enzymatic activity of RPE65, we constructed and studied the mutant E417D.

Materials and methods

Site-directed mutagenesis, expression and isomerohydrolase activity assay

Two point mutations E417Q and E417D in human RPE65 (hRPE65) were generated using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) using the wild-type hRPE65 (wtRPE65) cDNA as the template and confirmed by DNA sequencing from both strands as described before [11]. Generation of recombinant adenoviruses (Ad-RPE65) was performed as described before [15]. Measurements of the expression and isomerohydrolase activities of wtRPE65 and its mutants were performed as published previously [11, 15].

Sub-cellular fractionation and stability assay

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 sub-cellular localization of recombinant RPE65 was elucidated using FractPrep™ kit (BioVision, Mountain View, CA). The subcellular fractions were examined by Western blot analysis as described [11]. 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].

Modeling of the hRPE65 mutants structure

To analyze the 3-D structure of hRPE65 and its mutants, a sequence homology-based program Swiss Model (http://swissmodel.expasy.org/) was employed utilizing bovine RPE65 crystal structure (3FSN, http://www.pdb.org/pdb/explore/explore.do?structureId=3FSN) [17] as a template.

Results

RPE65 mutations E417Q and E417D significantly alter intracellular localization

To determine the sub-cellular localization of wtRPE65 and its E417Q and E417D mutants, we employed cell lysate fractionation. Western blot analysis of subcellular fractions revealed that wtRPE65 was predominantly present in the membrane fraction and at a lower level in the cytosolic fraction (Fig. 1A). In contrast, both E417D and E417Q mutants showed considerably decreased levels in the membrane fraction (Fig. 1B). Moreover, unlike wtRPE65, both mutant proteins were predominantly localized in the cytoskeletal fraction containing detergent-resistant inclusion bodies (Fig. 1B). Amounts of both of the mutants in the cytosolic fractions were found to be similar to that of wtRPE65.

Fig. 1.

Mutations in RPE65 alter its subcellular fractionation. The 293A-LRAT cells infected with Ad-wtRPE65, Ad-E417Q and Ad-E417D at MOI of 100 were harvested 18 h after infection, homogenized and fractionated. A, total cell lysate (32 μg), and equal amount of protein (8 μg) from the cytosolic, membrane, nuclear and cytoskeletal/including inclusion body fractions were analyzed by Western blot using the anti-RPE65 antibody. B, protein levels of wtRPE65 and the mutants were quantified by densitometry and presented as % of total protein levels of wtRPE65 or its mutants correspondingly (mean±S.D., n =3).

Mutations E417Q and E417D impair the protein stability of RPE65

To evaluate the impact of mutations of glutamic acid at position 417 on the protein stability, we measured the protein degradation rate after the blockade of translation by cycloheximide (CHX) in 293A-LRAT cells. The cells were separately infected with adenoviruses at MOI of 20 and incubated for 18 h, followed by the addition of 25 μg/ml of CHX. WtRPE65 and its mutant protein levels were measured by Western blot analysis at 0, 2, 6, and 10 h after the CHX addition (Fig. 2A) and semi-quantified by densitometry (Fig. 2B). WtRPE65 was found to have high stability, with an apparent half-life longer than 10 h. In contrast, both mutants showed dramatically accelerated rates of protein degradation with half-lives of less than 2 h (Fig.2B).

Fig. 2.

RPE65 mutations E417Q and E417D cause deterioration of the protein stability. A, The adenoviruses infected 293A-LRAT cells were harvested at consecutive time intervals after blockage by CHX and RPE65 levels were determined by Western blot. B, RPE65 protein levels were semi-quantified by densitometry and expressed as % of that before the addition of CHX (mean±S.D., n=4).

The enzymatic activity of RPE65 is abolished in the E417Q mutant, but partially retained in the E417D mutant

Both E417Q and E417D mutants were expressed in 293A-LRAT cells using adenovirus with MOI of 100. Both mutants had protein expression levels of approximately 70% of wtRPE65 (Fig. 3A, B). Enzymatic activities of the mutants were compared with that of wtRPE65 expressed in 293A-LRAT cells at the same MOI of 100. At the comparable protein levels, wtRPE65 generated significant amounts of 11-cis retinol (Fig. 3C); the E417D mutant produced a decreased but detectable amount of 11-cis retinol, while the E417Q mutant did not show any detectable isomerohydrolase activity under the same assay conditions (Fig. 3D and E). Further, to determine the effect of RPE65 abundance in the cell lysate on isomerohydrolase activity, the cells were infected separately with Ad-wtRPE65 and Ad-E417D at different MOIs (from 10 to 100). The 11-cis retinol generated in these reactions increased proportionally with increasing MOI for both wtRPE65 and E417D (Fig. 3F). Nevertheless, at all tested MOIs, the amount of 11-cis retinol generated by E417D was approximately 5-fold lower than the amount generated by wtRPE65 (Fig. 3F).

Fig. 3.

Expression level and isomerohydrolase activities of E417O and E417D. The 293A-LRAT cells were infected with Ad-wtRPE65, Ad-E417D and Ad-E417Q at MOI of 100. A, the cells were harvested 24 hr after the infection, and 25 μg of total proteins from each sample along with positive and negative controls (1 μg of bovine microsomes (BMF) and 25 μg of uninfected cells) were immunoblotted with the antibodies for RPE65 (top) and β-actin (bottom). B, protein levels of wtRPE65 and its mutants from the cells infected with MOI of 100 (mean±S.D., n=3). C–E, Cell lysates from (A) (250 μg of protein) were used for isomerohydrolase activity assays. C, cells infected with Ad-wtRPE65; D and E, cells infected with Ad-E417D and Ad-E417Q, respectively. Peak 1, retinyl esters; peak 2, all-trans retinal; peak 3, 11-cis retinol. F, Dependence of isomerohydrolase activities of wtRPE65 protein and E417D mutant on MOIs.

Discussion

RPE65 protein is the only known enzyme in the visual cycle with the ability to catalyze the conversion of all-trans retinyl esters to 11-cis retinol [1-3]. Mutations in the RPE65 gene are known to cause inherited retinal dystrophies such as LCA [14, 18]. However, the mechanism by which RPE65 mutations leading to the disease is still unknown. 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. The results showed that E417Q completely lost enzymatic activity, while E417D retained partial activity. This result suggests that the negative charge at residue E417 is crucial for the enzymatic activity of RPE65 and the visual defect in patients with E417Q may be ascribed to the loss of the negative charge at E417.

In the present study, the majority of both E417Q and E417D mutant proteins were localized in the cytoskeletal/inclusion body fraction in contrast with the predominantly membrane-associated wtRPE65 protein. This mislocalization may contribute to the accelerated degradation rate of the mutant proteins. The half-life of wtRPE65 was longer than 10 h, which is in agreement with that of a previously published study [11, 12], whereas the half-life of each mutant was found to be shorter than 2 h. The lower stability of both E417Q and E417D mutants of RPE65 indicates that their protein folding is disturbed. Unlike previously reported RPE65 mutations [11, 12, 19], both E417D and E417Q demonstrated expression levels comparable with that of wtRPE65 in 293A-LRAT cells. Therefore, the lack of enzymatic activity in the E417Q mutant is not caused by decreased protein levels, but rather it is a clear result of the amino acid substitution, indicating that E417 is a critical residue for RPE65 activity.

To elucidate the role of E417 in the catalytic mechanism of RPE65, we performed structural analysis using molecular modeling of RPE65 and mutants. The molecular model of wtRPE65 (Fig.4A) has a seven-bladed propeller structure holding an iron ion coordinated by four highly conserved histidines (H180, H240, H313 and H527) [17]. Previously, we have shown that the endogenous iron (II) ion [20] and the four conserved histidines are essential for the isomerase activity [16]. The conserved E417 is located at a distance which allows it to form a hydrogen bond with H313 (<3 Å) (Fig. 4B) in the iron binding domain. Interestingly, substitution of Q for E417 does not significantly alter the distance between the Q417 and H313, and molecular modeling predicts that hydrogen bonding could occur between these residues (Fig. 4D). Nevertheless, E417Q mutation leads to a complete loss of RPE65 enzymatic activity. This is in agreement with the previously published data where replacement of E417 by uncharged A or Q caused a complete loss of RPE65 isomerase activity [3].

Fig. 4.

RPE65 molecular model showing the vicinity of Fe²⁺ binding site, A, Predicted structure of RPE65, where the conserved Fe²⁺–binding site marked with red dashed line. B, a close-up view of predicted structure of wt RPE65 in proximity of E417 residue and the iron binding site; C-D, close-up views of predicted structures of RPE65 with a mutation E417D (C) and the patient mutant E417Q (D). The colors are coded as a carbon (white), a nitrogen (blue), an oxygen (red); and the Fe²⁺ ion is presented as a gray sphere.

To distinguish the role of the conserved negatively charged acidic residue at the position 417 (E) and the impact of its mutation to non-charged residue (Q), E417 was replaced by another negatively charged residue having a shorter side chain (D). The replacement of E417 by D retained more than 20% of wtRPE65 enzymatic activity even though this residue is located at a longer distance (5.77 Å) and cannot form a hydrogen bond to H313 (Fig. 4C). In fact, the difference in the enzymatic activity between E417D and wtRPE65 might be even less if we take into account that the amount of the protein found in the membrane fraction for E417D is three times less than that for the wtRPE65 (Fig.1B) and the membrane association is essential for RPE65 activity. Thus, we conclude that a negative charge of glutamic acid (E) residue 417 rather than the hydrogen bond between the E417 and H313 is essential for the isomerase activity of RPE65. An increase in the distance between D417 and H313 appears to result in a decrease in the electrostatic effect of D417 and a concomitant 20% decrease in enzymatic activity of E417D mutant. The negative charge of E417 is likely to affect the pK_a of H313 and in turn may modulate its metal ion binding capacity. Alternatively, the negative charge of E417 could stabilize the carbocation transition state of the substrate developed in the course of the reaction [21, 22].

In summary, the present study shows that the negative charge of E417 is essential for the isomerohydrolase activity of RPE65. Mutation of E417Q decreases the protein stability, alters subcellular localization and abolishes isomerohydrolase activity of RPE65 which results in the development of retinal dystrophy and vision loss.

Abbreviations

Ad-RPE65

adenovirus expressing RPE65

LCA

Leber congenital amaurosis

MOI

multiplicity of infection

RPE

retinal pigment epithelium

CHX

cycloheximide