Genetic and Biochemical Analyses of the Ush2A Protein (Usherin)

Abstract

Abstract: : Purpose:To determine the subcellular localization and in vivo function of the Ush2A protein. Methods: The murine Ush2A coding sequence was determined by RT–PCR of retinal mRNA followed by DNA sequencing. Polyclonal antibodies were generated against recombinant fusion proteins corresponding to the N– and C–terminal regions of murine Ush2A. The murine Ush2a gene was disrupted by gene–targeting in embryonic stem cells. Targeted ES cells were microinjected into blastocysts and male chimeras were crossed with WT mice. WT and Ush2a–/– mice were analyzed by a variety of assays including light and electron microscopy, immunofluorescence, and immunoblotting. Results: The murine Ush2A gene encodes a large polypeptide of 5193 amino acids, which shares 82% sequence similarity with its human ortholog. Immunoblotting of retinal extracts confirmed the exceptional large size of the Ush2A protein (with an apparent M.W. of 600 kDa). Immunoblotting did not find an N–terminal variant of Ush2A (170 KDa isoform) as previously reported, indicating that this is not a major isoform. Ush2A is expressed primarily in photoreceptors, and was not detected in the RPE or choroid. In photoreceptors, Ush2A was enriched in the vicinity of the connecting cilia. Retinal phenotype of mice lacking Ush2A is still under investigation. Conclusions: Ush2A is a large matrix protein expressed in photoreceptor cells. It is not a generalized basement membrane component as previously reported. Our data suggest that the Ush2A protein may have a role in the long–term maintenance of the photoreceptor ciliary structure.

Keywords: transgenics/knock-outs • photoreceptors • genetics