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K.M. Lindsey, R.K. Crouch, K.L. Schey; Protein-Protein Interactions of the C-Terminus of Lens Aquaporin 0 . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4479.
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© ARVO (1962-2015); The Authors (2016-present)
The cytoplasmic C-terminus of the major lens membrane protein AQP0, a putative regulatory domain, has been shown to bind calmodulin and is the site of cataractogenic mutations in CatFr mice. Purpose: The purpose of this study is to identify lens proteins that interact with the C-terminus of AQP0. Methods: Synthetic peptides identical to the AQP0 C-terminal sequence (residues 251-263 and 240-263) were immobilized on sulfhydryl reactive affinity columns via additional N-terminal cysteine residues. An extracellular loop (residues 110-127) peptide column and a cysteine-blocked column were used as controls. Soluble lens proteins were incubated on the peptide affinity columns and control columns. Eluted proteins were analyzed by MALDI-MS followed by proteolytic digestion and LC-MS/MS analysis. MS/MS data were analyzed by SEQUEST and manual interpretation to identify eluted proteins. In a second approach, an anti-AQP0 antibody/Protein A column was used to affinity purify AQP0/protein complexes from detergent-solubilized lens membranes, and the eluted proteins were analyzed by gel electrophoresis and immunoblotting with an anti-AQP0 antibody. Following trypsin treatment of the eluted fractions, the resulting peptides were analyzed by LC-MS/MS, and the SEQUEST algorithm was used to identify proteins. Results: Initial results revealed specific binding of filensin to both peptide affinity columns. Nonspecific binding was observed for lens crystallins. In addition, proteins affinity-purified via the antibody column were identified as lens crystallins, cytoskeletal proteins including filensin, and a chloride channel protein. Conclusions: Protein-protein interactions of AQP0 have been revealed by affinity purification combined with mass spectrometry. A potentially important interaction is with the cytoskeletal protein filensin.
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