Evaluation of a Dominant–Negative Disease Mechanism in ELOVL4–Associated Macular Dystrophy

Abstract

Abstract: : Purpose: ELOVL4 is a member of the ELO family of proteins responsible for the elongation of very–long chain fatty acids. Protein truncating mutations in ELOVL4 have been identified in patients with two related forms of autosomal dominant macular degeneration; Stargardt–like macular dystrophy and autosomal dominant macular dystrophy. The purpose of this study was to determine if a dominant–negative mechanism may be responsible for the autosomal dominant nature of macular dystrophy linked to mutations in ELOVL4. Methods: Human ELOVL4 was cloned by RT–PCR from foveal RNA and disease–associated mutations were created by site–directed mutagenesis. Immunoaffinity tags were added to the ELOVL4 constructs and the heterologous expression of the proteins was examined in a cell culture model. The subcellular localization of the proteins was determined by immunofluoresence. The association of wild–type and mutant proteins was determined by co–immunoprecipitation followed by SDS–PAGE and Western blotting. Results: The affinity–tagged wild–type ELOVL4 protein localized correctly to the endoplasmic reticulum but the disease–associated mutant proteins demonstrated a significantly different localization pattern. When the wild–type and mutant proteins were co–expressed in a cultured cells, the cellular localization of the wild–type protein was altered. The wild–type and mutant proteins co–purified on an immunoaffinity column. Conclusions: Mutant ELOVL4 interacts with wild–type ELOVL4 leading to mislocalization of wild–type protein. These data suggest that the dominant–negative effect of mutant ELOVL4 on the localization and possible activity of ELOVL4 may contribute to the molecular mechanism underlying ELOVL4–mediated macular dystrophy.

Keywords: proteins encoded by disease genes • retinal degenerations: cell biology • retinal degenerations: hereditary