The two codon 863 variants, G863A and delG863, show a decrease in both basal and retinal-stimulated ATPase, with G863A showing the greater decrease (Fig. 4d).

Variants S206R and L541P have reduced basal ATPase and S206R shows little, and L541P shows no, stimulation by retinal (Fig. 4a).

Patient 4 with an early frameshift mutation in one allele and a downstream p.Val552Ile mutation in the second allele has a mild form of STGD1. Because the frameshift mutation is likely to result in a null allele, any residual functional activity of ABCA4 would arise from ABCA4 harboring the p.Val552Ile missense mutation. Our in vitro studies showing that the p.Val552Ile variant expresses at close to WT levels, exhibits normal N-Ret-PE binding properties, and has only a modest reduction in ATPase activity (Table 2) are consistent with the mild disease phenotype of patient 4. Another study has also reported that the p.Val552Ile mutation is associated with a STGD1 disease phenotype.50 However, in silico predictions on the pathological relevance of this mutation have been variable.20,13 On the basis of allele frequencies in controls versus patients, it has been argued that the p.Val552Ile is most likely benign.20 At a protein level, a hydrophobic amino acid residue valine is replaced with another hydrophobic residue isoleucine. Accordingly, this substitution would be predicted to have only a marginal impact on ABCA4 protein structure and function. However, valine at position 552 of ABCA4 is invariable among vertebrate species, including other mammals, chicken, Xenopus, and Japanese puffer fish (Takifugu rubripes), attesting to the likely importance of valine at this position. Collectively, these studies suggest that the p.Val552Ile is a mild mutation in which the pathogenicity may only be displayed in selected cases. More specifically, the p.Val522Ile would display a mild STGD1 phenotype when combined with a null allele as in the case of patient 4 or a missense mutation with little or no activity such as the p.Asn965Ser mutation,37,51 but would not display a disease phenotype in a patient homozygous for this mutation or patients in which this mutation is combined with a mutation that shows significant ABCA4 function because under these circumstances sufficient ABCA4 activity would be realized to prevent the accumulation of toxic retinoids.

Next, we measured the effect of disease-associated mutations on the ATPase activity of ABCA4. WT and ABCA4 variants were solubilized in CHAPS, purified by immunoaffinity chromatography, and subsequently reconstituted into PE-containing liposomes at similar protein concentrations. The ATPase activity of the mutants in the presence and absence of N-Ret-PE substrate is shown in Figure 6A, ​A,6B.6B. As previously reported,25,47 addition of 40 μM all-trans retinal to WT ABCA4 resulted in a 1.8- to 2.5-fold increase in ATPase activity (Fig. 6). The ATPase activity of the mutants was measured at the same protein concentration as WT ABCA4 to determine the effect of the mutation on the functional activity of ABCA4. Five mutants (p.Val552Ile, p.Ala1038Val, p.Ala1357Thr, p.Ala1794Pro, and p.Leu2027Phe) showed reduced basal ATPase activity relative to WT ABCA4 (∼40%–85%), but this activity was stimulated 1.6- to 3.0-fold by the addition of all-trans retinal. On the other hand, p.Gly72Arg, p.Met448Lys, p.Leu541Pro, p.Gly1091Glu, p.Gly1961Glu, and p.Arg2077Trp variants showed drastically reduced basal activity with little or no substrate stimulation.Open in a separate windowFigure 6ATPase activity of ABCA4 variants. The ATPase activity of immunopurified and reconstituted ABCA4 variants was measured in the presence or absence of all-trans retinal. (A) Quantification of the basal and retinal-stimulated ATPase activity of ABCA4 variants normalized to WT basal ATPase activity. ATPase assays were carried out using similar concentrations of purified ABCA4. Data expressed as an average ± SD for n ≥ 3 independent experiments. (B) Representative curves of specific ATPase activity as a function of all-trans retinal concentration for WT and ABCA4 variants. (C) Relative basal ATPase activity of WT and A1794P using equal amounts of transfected HEK293T cells. Data expressed as an average ± SD. Measurements were done in triplicate.To more directly evaluate the expression and function of the p.Ala1794Pro variant, we transfected HEK293T cells separately with WT ABCA4 and the p.Ala1794Pro mutant cDNAs at similar levels. After solubilization in CHAPS buffer, the samples were subjected to high-speed centrifugation and the supernatant was reconstituted into liposomes for analysis of its basal and substrate activated ATPase activity. As shown in Figure 6C, the p.Ala1794Pro had a significantly reduced activity due largely to the low expression of this variant. These studies indicate that only a small fraction of the p.Ala1794Pro mutant folds into a functionally active protein and correlates well with the phenotype of patient 5.

The measurement of ATPase activity has been the only assay available to study the effects of mutations on ABCA4 function. We employed this assay to examine the effects of [L541P; A1038V], R602W and C1490Y mutations on in vitro ATP hydrolysis. Constructs containing wild-type and mutated ABCA4 cDNAs, tagged with the eight amino acid bovine opsin C-terminal epitope (1D4), were expressed in COS7 cells and proteins were purified on a 1D4 affinity column. CHAPS-solubilized ABCA4 was incubated subsequently with ATP, and the hydrolysis rate was estimated with the charcoal method (31).The rate of ATP hydrolysis of the complex allele [L541P; A1038V] was decreased to 68.1% of wild-type ABCA4 (Fig. 3).

G818EER51 ± 1363 ± 5111 ± 825 ± 312 ± 32Moderate/Severe

Analysis of the rates of hydrolysis of ATP and CTP in the mutant protein demonstrated that they were significantly reduced (Fig. 3). The results presented in Fig. 3A indicated that the ATPase function of G863A mutant protein was reduced ∼3-fold as compared with NBD1wt, indicating ∼70% of inhibition of the ATPase activity. TheVmax (ATPase) for G863A mutant was 128 pmol/min/mg and that of the wild-type NBD1 was 584 pmol/min/mg (Table II). A time-course analysis of ATP hydrolysis using 2.5 μg of protein (Fig. 3C) suggested the actual rates of ATP hydrolysis were attenuated 3-fold as a consequence of the mutation. We have earlier reported that the NBD1wt has significantly higher CTPase than ATPase activity (1717.Biswas, E.E.Biochemistry. 2001; 40:8181-8187CrossrefScopus (25)PubMedGoogle Scholar). However, in the mutant G863A protein the CTPase activity was reduced (Fig.3B). In this case, the CTP hydrolysis of G863A was reduced to ∼30% of the activity of NDB1wt. The Vmaxfor G863A mutant was 104 pmol/min/mg and that of the wild-type NBD1 was 376 pmol/min/mg (Table II).