May 2004
Volume 45, Issue 13
ARVO Annual Meeting Abstract  |   May 2004
Protein–Protein Interactions of Lens Aquaporin 0 with Cytoskeletal Proteins
Author Affiliations & Notes
  • K.M. Lindsey
    Medical University of South Carolina, Charleston, SC
  • R.K. Crouch
    Medical University of South Carolina, Charleston, SC
  • K.L. Schey
    Medical University of South Carolina, Charleston, SC
  • Footnotes
    Commercial Relationships  K.M. Lindsey, None; R.K. Crouch, None; K.L. Schey, None.
  • Footnotes
    Support  NIH Grant 13462
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 3994. doi:
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      K.M. Lindsey, R.K. Crouch, K.L. Schey; Protein–Protein Interactions of Lens Aquaporin 0 with Cytoskeletal Proteins . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3994.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract: : Aquaporin 0 is the most abundant membrane protein in the lens and is a member of the aquaporin family of water channels. Mutations in the AQP0 gene underlie cataracts in mice and human, demonstrating the necessity of AQP0 for lens development and for maintaining transparency in the lens. The cytoplasmic C–terminal regions of the aquaporins have been proposed to play tissue–specific regulatory roles, and furthermore the C–terminus of AQP0 has been shown to bind calmodulin. Thus, regulation of AQP0 may occur through interaction of the C–terminus with other proteins in the lens fiber cells. Purpose: The goal of the present 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 N–terminal cysteine residues. An extracellular loop (residues 110–127) peptide column and a cysteine–blocked column were used as controls. Soluble human lens proteins were incubated on the peptide affinity columns and control columns. Proteins were eluted from the columns and treated with trypsin. In a second approach, anti–AQP0 antibodies crosslinked to Protein A beads were used to affinity–purify AQP0/protein complexes from detergent–solubilized human lens membrane proteins. Preimmune antibodies were used for the control. Eluted proteins were digested with trypsin. The resulting tryptic peptides from both the peptide affinity and antibody affinity experiments were analyzed by LC–MS/MS. Automated human database searching with the SEQUEST algorithm and manual interpretation were used to identify the proteins. Results: Results from the peptide affinity approach revealed binding of filensin and CP49 to the AQP0 C–terminal peptide affinity columns. Nonspecific binding was observed for lens crystallins. In addition, proteins affinity–purified via the AQP0 antibody column were identified as lens crystallins, cytoskeletal proteins including filensin and CP49, and a chloride channel protein. Conclusions: Protein–protein interactions of AQP0 have been revealed by affinity purification combined with mass spectrometry. Potentially important interacting proteins are the lens–specific cytoskeletal proteins, filensin and CP49. Identification of these protein–protein interactions could present novel cytoskeleton–membrane protein interactions in the lens and could provide insight on the role of AQP0 as a structural protein.

Keywords: protein structure/function • cell membrane/membrane specializations • cytoskeleton 

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