Autosomal dominant retinitis pigmentosa mutations in inosine 5′-monophosphate dehydrogenase type I disrupt nucleic acid binding

Abstract

Two mutations of IMPDH1 (inosine 5'-monophosphate dehydrogenase type I), R224P and D226N, have recently been found to cause adRP (autosomal dominant retinitis pigmentosa). IMPDH1 catalyses the rate-limiting step in guanine nucleotide biosynthesis and also binds single-stranded nucleic acids. In the present paper, we report the biochemical characterization of the adRP-linked mutations, R224P and D226N, and a potentially pathogenic mutation, V268I. The adRP-linked mutations have no effect on enzyme activity, protein stability or protein aggregation. These results suggest strongly that the mutations do not affect enzyme activity in vivo and thus do not perturb the guanine nucleotide pool. The R224P mutation changes the distribution of enzyme between the nucleus and cytoplasm. This effect was not observed with the D226N mutation, so the relevance of this observation to disease is unclear. In contrast, both mutations decrease the affinity of nucleic acid binding and both fail to co-immunoprecipitate RNA. These observations suggest that nucleic acid binding provides a functional assay for adRP pathogenicity. The putative adRP-linked mutation V268I also disrupts nucleic acid binding, which suggests that this mutation is indeed pathogenic.

Retinal isoforms of inosine 5′-monophosphate dehydrogenase type 1 are poor nucleic acid binding proteins

“…The canonical IMPDH1-GFP was cloned into pCI using the Xho I/Not I fragment from pH1GFP [12] and was expressed under the control of the CMV promoter. The wild-type gene was also mutated to the D226N form in the cloned plasmid.…”

“…Binding of IMPDH1 to single-stranded DNA (ssDNA) was performed as previously described [12,16]. The sequence of the DNA pool used was 5′-GGGAATGGATCCACATCTACGAATTC-N 30 -TTCACTGCAGACTTGACGAAGCTT-3′, where N 30 denotes a 30 bases-long random sequence.…”

“…First, the adRPlinked mutations of IMPDH1 have no effect on enzymatic activity [7,12]. Moreover, most of the mutations are located in a subdomain that is not required for enzymatic function [13,14].…”

“…We have discovered that IMPDH binds nucleic acids, and that this interaction is mediated by the subdomain [16]. The adRP-linked mutations perturb this nucleic binding function, decreasing both affinity and specificity of this interaction [7,12].…”

“…Both IMPDH1(546) and IMPDH1(595) contain a 32 residue C-terminal extension while IMPDH1(595) has an additional 49 residues on the N-terminus. Since polyHis and GFP tags do not perturb either the enzymatic or nucleic acid binding actitvities of IMPDH [12,15], these N-and C-terminal extensions need not perturb function. The retinalspecific expression of IMPDH1 isoforms and the compensating presence of IMPDH2 in other tissues may account for the photoreceptor-specific effects of the adRP/LCA-causing mutations, but do not explain the pathological mechanism.…”