Figure 3: Protein yield and ATP-binding capacity of 37 naturally occurring ABCR variants produced in transiently transfected 293 cells.Membranes were analysed by immunoblotting with affinity-purified anti-ABCR antibodies (top) and photoaffinity labelling with α-32P azido-ATP (bottom). We loaded 1 μg (immunoblotting) or 2.5 μg (azido-ATP labelling) of total membrane protein, as determined by Bradford assay, per track. The mutations that reside in NBD-1 and NBD-2 are indicated above the corresponding lanes. The relative levels of the different variant proteins and the extent of azido-ATP labelling were observed to be highly reproducible in multiple independent experiments. ABCR (large arrowhead); an endogenous 55-kD protein (small arrowhead) serves as an internal control for azido-ATP labelling. Molecular mass standards are shown on the left in kD.Full size imageThe combination of immunoblotting and azido-ATP labelling revealed defects in more than 75% of the variants tested. Among the variants with reduced yield and/or ATP binding are G863A and delG863, the two protein products of a guanosine2588→cytosine mutation that both generates a glycine-to-alanine substitution at codon 863 and activates a cryptic splice acceptor site in exon 17 that results in the removal of codon 863 from approximately 50% of the transcripts10. This is the most common allele among STGD patients in Northern Europe, representing roughly 20% of disease-associated alleles. It is also present at a frequency of approximately 3% in the general population in Northern Europe and approximately 1% in the United States population7,9,10. Genotype-phenotype correlations suggest that it is a mild allele and that it leads to STGD only when paired with a more severe allele10. Relative to wild type, the G863A variant is subtantially impaired and the delG863 variant is mildly impaired (Fig. 3).

Functional studies of the arRP-associated complex allele [L541P; A1038V] also showed abnormal localization to rod IS although the IF studies revealed a different staining pattern than R602W, because the mutant protein forms fine aggregates in the IS (Fig. 2E and F). The aggregate formation suggests a mechanism distinct from that observed for R602W may be responsible for the retention of [L541P; A1038V] in the IS. To determine which mutation prevents translocation of [L541P; A1038V] to ROS, we examined functionally the L541P and A1038V mutants independently. We observed retention of L541P in the IS (Fig. 2G and H) and correct localization of A1038V to ROS (Fig. 2I and J). These results suggest that L541P is a disease-causing mutation that affects ABCA4 processing.

AR197197–057CF 3 feet OD; CF 2 feet OSRP[L541P; A1038V][L541P; A1038V]197–069CF 5 feet OD; HM OSRP[L541P; A1038V][L541P; A1038V]

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).

Patient 8, a 13-year-old individual carrying two mutations, the p.Leu541Pro and p.Ala1038Val, most likely as a complex allele, had an early disease onset with 20/200 VA and stage 4 FC (Table 1). Previous reports have documented the severity and early age of onset associated with the p.[Leu541Pro;Ala1038Val] complex variant.36,38 Our screening studies failed to detect an additional mutation in ABCA4 in patients 6, 8, or 12.

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.

In contrast, we found that the expression levels of the WT and all three mutant proteins were equivalent as determined by western blotting (Fig. 3B).

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

both the P and PV ABCA4 variants exhibited a dramatically reduced basal ATPase activity (∼30% of WT ABCA4), which did not increase upon the addition of all-trans-retinal. Thus, ABCA4 variants carrying the P mutation were functionally impaired.

Amounts of A2E in the eyes of Abca4PV/PV, Abca4−/− and WT mice at the ages of 1, 3, 6, 12 and 15 months were quantified by reverse-phase high-performance liquid chromatography (HPLC) (Fig. 9B). Mice were raised under a regular 12-h light (∼10 lux)/12-h dark cycle. Age-dependent A2E accumulation was noted in all genotypes, with Abca4PV/PV and Abca4−/− mice accumulating about 5-fold more A2E than WT mice. No statistically significant differences in A2E accumulation were found between Abca4PV/PV and Abca4−/− animals.