Differential protein expressions in the emmetropization of chick retina by a proteomic approach

Abstract

Both in terms of the economic and social health aspects, the impact of myopia epidemic is high and far-reaching. It is believed that myopia development is a multifactorial disease but despite the intensity of myopia research in recent years, the molecular mechanism behind the myopia development is still not known. Global protein profilings and protein identifications have become possible with the new emerging proteomic technology using high resolution two-dimensional gel electrophoresis (2DE) and mass spectrometry (MS). Base on the previous ground works using chick as a myopia model, the present study explored if the chick retina is good tissue model for studying the molecular basis of myopia with proteomic technology. It is hypothesized that the study of retinal protein expressions may provide new insight on the downstream pathophysiological cascades in the myopic eye growth. In the first stage of the study, an animal model of compensated ametropia with chicks wearing goggles for different period was established. The protein extraction and the 2DE procedures from protein separation to protein staining were optimized experimentally. With an optimized proteomic workflow, the first chick retinal proteome database using the Matrix-Assisted Laser Desorption lonization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) was built in the second stage of the study. A total of 155 protein spots in the 2-D gels covering the 3-10 pH range were identified with manual in-gel digestion or using an automated robotic system. To allow for a global view of retinal proteins, the proteins were further classified according to their subcellular locations as well as their molecular functions. Most of the proteins (about 71%) were found to be presented in the cytoplasm. Others resided in the endoplasmic reticulum (7.2%), mitochondrion (7.2%), nucleus (5.6%), Golgi (3.2%), extracellular (1.6%) and plasma membrane (0.8%). There were remaining proteins (2.4%) returning with unknown subcellular location. Based on the gene ontology information, over 80% of the identified proteins fell into three major functional categories which were "catalytic activity" (39%), "binding" (33%) and "transporter activity" (10%). In the third stage, the differential protein expression of normal growing chick eyes at three time-points was studied and the capability of the established workflow in identifying candidate proteins in the early postnatal retinal growth was investigated. Four up-regulated and three down-regulated protein spots were found during the study period in which five of them could be successfully identified and their possible roles in the ocular growth were discussed. At the final stage, differential protein expressions in the emmetropization of chick retina were studied using both pooled and individual retinal samples. Using different experimental conditions with -10D, +10D and occluders for various deprivation periods, two candidate proteins were found to be differentially expressed in 2D gels in response to the treatments. In myopic eyes, Apolipoprotein AI (Apo-AI) was found to be down-regulated while destrin; actin depolymerising factor, ADF (Destrin) was found to be up-regulated in the defocused eye. Since these two proteins have yet to be related in the myopic growth, their functional roles in regulating eye growth through fibroblasts remodelling were explored and generally discussed. The information may provide an important link in the cascade of molecular activity during the myopia development. In addition, the feasibility of applying an emerging novel two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) technique in search for differential protein expressions in chick myopia was explored. Using a reverse fluorescent CyeDye TM experimental protocol, a number of differential expressed retinal proteins were detected and identified after the chicks were wearing -10D lens for 7 days. The 2D DIGE technology has the advantage of overcome some technical bottlenecks in the traditional 2DE and it offers a high level of confidence in comparing protein profiles across multiple gels.