Molecular changes in selected epithelial proteins in humankeratoconus corneas compared to normal corneas

[摘要] Purpose: The purpose of the study was to determine molecular changesin selected epithelial proteins in human keratoconus (KC) corneascompared to normal corneas.Methods: Two-dimensional (2-D) gel electrophoretic profiles ofepithelial cell proteins from normal and keratoconus corneas werecompared, and the selected protein spots that showed either up- ordownregulation were identified. The desired spots were identified aftertrypsin digestion and mass spectrometric analysis. Based on the results,two proteins, α-enolase and β-actin, were further analyzed byimmunohistochemical and western blot methods, using respectiveantibodies. To determine the presence of mRNA of the two proteins in theepithelial cells, RT-PCR studies were performed.Results: On comparison of the 2-D gel electrophoretic proteinprofiles, two protein spots were identified in normal corneas that wereeither absent or present at lower levels in keratoconus corneas. The twospots were determined to be α-enolase (48 kDa) and β-actin (42kDa) by matrix-assisted laser desorption ionization-time of flight(MALDI-TOF), and ES-MS/MS mass spectrometric methods.Immunohistochemical analysis revealed that α-enolase andβ-actin were present at extremely low levels in the epithelialsuperficial and wing cells of the keratoconus corneas compared to thesecells of normal corneas. 2-D gel electrophoresis followed by westernblot analysis revealed relatively greater degradation of the twoproteins in the keratoconus corneas compared to normal corneas. RT-PCRanalysis showed the mRNA expression of the two proteins in theepithelial cells of both normal and keratoconus corneas.Conclusions: The results showed relatively low or negligible levelsof α-enolase and β-actin in the wing and superficial epithelialcells of keratoconus corneas compared to normal corneas. This wasattributed to relatively greater degradation of the two proteins inkeratoconus corneas compared to normal corneas.

[学科分类] 生物化学/生物物理