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Marie Vestergaard Lukassen, Ebbe Toftgaard Poulsen, Jan J. Enghild; Identification of redox-sensitive protein interactions in the human cornea. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2264. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The architecture of the stroma is of key importance in maintaining one of the unique features of the cornea, namely its transparency. Furthermore the cornea absorbs the majority of the UV-radiation, which can induce photochemical redox reactions leading to breakage of disulfide bonds. The insoluble collagen meshwork forms a scaffold for supporting corneal homeostasis that interacts with the soluble proteins expressed within the cornea. Understanding the redox-sensitivity of the cornea is important, as UV-radiation is the most common cause of radiation injury to the eye. This study aims to map the redox-sensitive protein interactions in the cornea. These interactions might be important for both the architecture of the cornea and it’s ability to withstand UV-radiation.
Corneal proteins were analyzed by alternative 2D gel electrophoresis, separating the proteins by unreduced SDS-PAGE in the first dimension. The first-dimensional gel lanes were excised and incubated under conditions that facilitate redox reactions before separation in the second dimension by SDS-PAGE. Redox-sensitive proteins would deviate from the diagonal on the 2D gels and could subsequently be identified by mass spectrometry.
The 2D gels of the cornea showed several spots deviating from the diagonal due to redox-sensitive protein interactions. Spots aligned in the vertical direction indicate redox sensitive-bonded partners. The most intense proteins identified by mass spectrometry were type VI and XII collagen, transforming growth factor β-induced protein (TGFBIp), complement C3 and IgG kappa. Some of the proteins migrated below the diagonal due to inter-chain disulfide bond breakage and others as a result of reducible protein-protein bonds. The location of TGFBIp indicates a redox-sensitive bond to collagen type VI and XII.
In this study, we have identified the redox-sensitive protein interactions in the human cornea. Some of the spots below the diagonal on the 2D gels indicated reducible interactions between different proteins. TGFBIp have previously been shown to be disulfide bonded to collagen type XII in the cornea in agreement with our results. Our results also indicated a disulfide bond between TGFBIp and collagen type VI. Further characterization of the redox-sensitive protein interactions in the cornea will improve our understanding of the corneal architecture and it’s resistance toward UV-induced redox-damage.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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