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KM Lindsey, RK Crouch, KL Schey; Protein-Protein Interactions of the C-terminus of Lens Aquaporin 0 . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4641.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:To identify lens cytosolic proteins that interact with the C-terminus of human AQP0. Methods:Peptides were synthesized to mimic the C-terminal region of AQP0 and were conjugated to the photoactive crosslinker benzophenone-4-maleimide (MBP) via an additional N-terminal cysteine residue. The peptide sequences corresponded to residues 251-263 and 239-263 of AQP0. The conjugated peptides were incubated with human lens cytosolic proteins and UV-irradiated for varying lengths of time to activate the benzophenone moiety. To test the specificity of the conjugated peptides, irradiation was also performed in the presence of unconjugated peptides. The crosslinked mixtures were analyzed by mass spectrometry and immunoblotting with an anti-AQP0 (residues 256-263) antibody. Results:Upon increasing irradiation time, the 251-263 MBP-peptide conjugate crosslinked to cytosolic proteins in a dose-dependent manner. According to immunoblot analysis, six bands were immunoreactive following crosslinking, with the most intense signal at 22kDa. After crosslinking, a new signal appeared at approximately 23kDa as determined by MALDI mass spectrometry. The presence of unconjugated peptide decreased the signal of the crosslinked products. In the absence of the conjugated peptide, no immunoreactivity was observed, indicating the specificity of the antibody. Conclusion:The C-terminus of AQP0 interacts with cytosolic proteins in the lens fiber cells, and this interaction may be involved in the function of AQP0. Identification of the crosslinked proteins is underway by mass spectrometric analysis.
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