RESUMO
PURPOSE: The purpose of the study was to determine molecular changes in selected epithelial proteins in human keratoconus (KC) corneas compared to normal corneas. METHODS: Two-dimensional (2-D) gel electrophoretic profiles of epithelial cell proteins from normal and keratoconus corneas were compared, and the selected protein spots that showed either up- or downregulation were identified. The desired spots were identified after trypsin digestion and mass spectrometric analysis. Based on the results, two proteins, alpha-enolase and beta-actin, were further analyzed by immunohistochemical and western blot methods, using respective antibodies. To determine the presence of mRNA of the two proteins in the epithelial cells, RT-PCR studies were performed. RESULTS: On comparison of the 2-D gel electrophoretic protein profiles, two protein spots were identified in normal corneas that were either absent or present at lower levels in keratoconus corneas. The two spots were determined to be alpha-enolase (48 kDa) and beta-actin (42 kDa) by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF), and ES-MS/MS mass spectrometric methods. Immunohistochemical analysis revealed that alpha-enolase and beta-actin were present at extremely low levels in the epithelial superficial and wing cells of the keratoconus corneas compared to these cells of normal corneas. 2-D gel electrophoresis followed by western blot analysis revealed relatively greater degradation of the two proteins in the keratoconus corneas compared to normal corneas. RT-PCR analysis showed the mRNA expression of the two proteins in the epithelial cells of both normal and keratoconus corneas. CONCLUSIONS: The results showed relatively low or negligible levels of alpha-enolase and beta-actin in the wing and superficial epithelial cells of keratoconus corneas compared to normal corneas. This was attributed to relatively greater degradation of the two proteins in keratoconus corneas compared to normal corneas.
