Proteomic Analysis of Normal and Rhodopsin Pro347Leu Transgenic Pig Retina

Abstract

Purpose: : Proteomic analysis has the potential to reveal differences between normal and transgenic retina at the level of the effector molecules, proteins and peptides. This study commences comparative analysis of the normal porcine retina with that derived from pigs expressing pig ^Pro347^Leu rhodopsin which presents with a disease phenotype similar to that of human Retinitis Pigmentosa.

Methods: : Porcine eyes were resected, transported to the laboratory on ice and the retina carefully detached. Excised retinal specimens (5 mm²) were placed on polyvinylidene fluoride (PVDF) membrane photoreceptor layer uppermost. Retinal layer thicknesses was assessed using unfixed (10 µm) tissue sections and planar cryosections of each retinal layer were recovered, frozen in liquid nitrogen and stored at –80ºC. Excess tissues were frozen in liquid nitrogen and stored at –80ºC.

Results: : Removal of the appropriate layers was verified by transverse cryosectioning and histological staining of selected samples. Proteins extracted from the normal and transgenic (rho^Pro347^Leu) porcine retinae were resolved using IPG pH 4–7 in the first dimension and on gradient 12–14% polyacrylamide gels. Scanned gel images were subjected to Progensis image analysis; the mean number of protein spots in normal retina was 813±99 compared to a mean of 756±40 in transgenic retina. Analysis revealed 36 matching spots that exhibited a significant difference. ATP–synthase, creatine kinase B chain, and L–lactate dehydrogenase B chain were decreased 7.8–, 1.7– and 1.2–fold, respectively, in ^Pro347^Leu retina, while Stathmin 1 and cystatin B were 1.7 fold greater in the ^Pro347^Leu retina. Additional, as yet unidentified proteins demonstrated a16 fold greater spot density in ^Pro347^Leu retina, while another spot exhibited a 2 fold increase in density in the normal pig.

Conclusions: : Enriched separate retinal layer samples were generated by establishing section thickness parameters utilising rapid histology of unfixed tissue prior to planar cryosectioning. Planar cyrosectioning of frozen tissue (–25ºC) minimises autolysis providing a robust method for planar fractionation of retinal tissue for proteome analysis. The proteomes of normal and ^Pro347^Leu porcine retinae revealed significant differences in spot densities of a diverse range of proteins with the progression of the pathology.

Keywords: retinal degenerations: cell biology • proteomics • retina