The C terminus of lens aquaporin 0 interacts with the cytoskeletal proteins filensin and CP49.
OBJECTIVE
Aquaporin 0 (AQP0), the most abundant membrane protein in the lens, is a water-permeable channel, has a role in fiber cell adhesion, and is essential for fiber cell structure and organization. The purpose of this study was to identify proteins that interact with the C terminus of AQP0, by using a proteomics approach, and thus further elucidate the role of AQP0 in the human lens.


METHODS
AQP0 C-terminal peptides and AQP0 antibody affinity chromatography were used for affinity purification of interacting human lens proteins. Purified proteins were digested with trypsin, analyzed by liquid chromatography (LC)-tandem mass spectrometry and identified after database searching and manual examination of the mass spectral data. Colocalization of AQP0 with filensin and CP49, two proteins identified after mass spectrometric analysis, were examined by immunoconfocal and immunoelectron microscopy of lens sections.


RESULTS
The proteomics approach used to identify affinity-purified proteins revealed the lens-specific intermediate filament proteins filensin and CP49. With immunoconfocal microscopy, regions of colocalization of AQP0 with filensin and CP49 at the fiber cell plasma membrane in the lens cortex were defined. Immunoelectron microscopy confirmed that filensin and AQP0 were present in the same membrane compartments.


CONCLUSIONS
These studies suggest a novel interaction between an aquaporin water channel and intermediate filaments, an interaction through which AQP0 may maintain lens fiber cell shape and organization.
Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston 29403, USA.
Kristie M
KM
Lindsey Rose
Robert G
RG
Gourdie
Alan R
AR
Prescott
Roy A
RA
Quinlan
Rosalie K
RK
Crouch
Kevin L
KL
Schey
D000818:Animals; D020346:Aquaporins / Q000378:metabolism*; D015153:Blotting, Western; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D005136:Eye Proteins / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007381:Intermediate Filament Proteins / Q000378:metabolism*; D007908:Lens, Crystalline / Q000378:metabolism* / Q000648:ultrastructure; D008562:Membrane Glycoproteins / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D040901:Proteomics; D051381:Rats
D020346:Aquaporins; D005136:Eye Proteins; D007381:Intermediate Filament Proteins; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; C030328:aquaporin 0; C079098:filensin; C084808:phakinin