Aquaporin-0 (AQP0) constitutes 50 % of the lens membrane proteome and plays important roles in lens fiber cell adhesion, water permeability, and lens transparency. Previous work has shown that specific proteins, such as calmodulin (CaM), interact with AQP0 to modulate its water permeability; however, these studies often used AQP0 peptides, rather than full-length protein, to probe these interactions. Furthermore, the specific regions of interaction of several known AQP0 interacting partners, i.e. αA and αB-crystallins, and phakinin (CP49) remain unknown. The purpose of this study was to use crosslinking mass spectrometry (XL-MS) to identify interacting proteins with full-length AQP0 in crude lens cortical membrane fractions and to determine the specific protein regions of interaction. Our results demonstrate, for the first time, that the AQP0 N-terminus can engage in protein interactions. Specific regions of interaction are elucidated for several AQP0 interacting partners including phakinin, α-crystallin, connexin-46, and connexin-50. In addition, two new interacting partners, vimentin and connexin-46, were identified.

Hyperbaric oxygen (HBO) treatment of animals or ocular lenses in culture recapitulates many molecular changes observed in human age-related nuclear cataract. The guinea pig HBO model has been one of the best examples of such treatment leading to dose-dependent development of lens nuclear opacities. In this study, complimentary mass spectrometry methods were employed to examine protein truncation after HBO treatment of aged guinea pigs. Quantitative liquid chromatography-mass spectrometry (LC-MS) analysis of the membrane fraction of guinea pig lenses showed statistically significant increases in aquaporin-0 (AQP0) C-terminal truncation, consistent with previous reports of accelerated loss of membrane and cytoskeletal proteins. In addition, imaging mass spectrometry (IMS) analysis spatially mapped the acceleration of age-related αA-crystallin truncation in the lens nucleus. The truncation sites in αA-crystallin closely match those observed in human lenses with age. Taken together, our results suggest that HBO accelerates the normal lens aging process and leads to nuclear cataract.

Major intrinsic protein or aquaporin-0 (MIP/AQP0) functions as a water channel and a cell-junction molecule in the vertebrate eye lens. Loss of MIP function in the lens leads to degraded optical quality and cataract formation by pathogenic mechanisms that are unclear. Here we have used microarray-hybridization analysis to detect lens transcriptome changes during cataract formation in mice that are functionally null for MIP (Mip-/-). In newborn Mip-/- lenses (P1) 11 genes were up-regulated and 18 were down-regulated (>2-fold, p=<0.05) and a similar number of genes was differentially regulated at P7. The most up-regulated genes (>6-fold) in the Mip-/- lens at P1 included those coding for a mitochondrial translocase (Timmdc1), a matrix metallopeptidase (Mmp2), a Rho GTPase-interacting protein (Ubxn11) and a transcription factor (Twist2). Apart from Mip, the most down-regulated genes (>4-fold) in the Mip-/- lens at P1 included those coding for a proteasome sub-unit (Psmd8), a ribonuclease (Pop4), and a heat-shock protein (Hspb1). Lens fiber cell degeneration in the Mip-/- lens was associated with increased numbers of TUNEL-positive cell nuclei and dramatically elevated levels of calpain-mediated proteolysis of αII-spectrin. However red-ox status, measured by glutathione and free-radical levels, was similar to that of wild-type. These data suggest that while relatively few genes (∼1.5% of the transcriptome) were differentially regulated >2-fold in the Mip-/- lens, calpain hyper-activation acts as a terminal pathogenic event during lens fiber cell death and cataract formation.

In experimental studies of solubilized membrane proteins, the detergent corona influences the protein behavior and the resulting measurement. Thus, combinations of experimental techniques with atomistic modeling have been used to resolve corona structural parameters and distributions. Here, we used small-angle X-ray scattering (SAXS) data and molecular dynamics simulations to study a model protein-detergent complex (PDC) consisting of aquaporin-0 and dodecyl-β-maltoside molecules (βDDM). The corona morphology of single snapshots was found to be rough, but it is smooth and compacted in 100-ns-scale ensemble averages. Individual snapshots therefore were unable to accurately represent the ensemble information as captured by experimental SAXS. Mimicking of annular lipids by detergent was also observed. SAXS prediction using different published methods was used to identify optimal βDDM numbers. Explicit-solvent methods predicted best agreement using 290-βDDM PDCs, but implicit-solvent methods gave unclear predictions due to overcompensation by free solvation-layer density parameters. Thus, ensemble-based approaches and physically motivated constraints will help to extract structural information from SAXS data.

The Emory mutant mouse has been widely used as an animal model for human senile cataract since it develops late-onset hereditary cataract. Here, we focus on the regional changes of aquaporin-0 (AQP0) and connexins that are associated with the cortical cataract formation in the Emory mutant mice. Emory mutant and CFW wild-type mice at age 1-16 months were used in this study. By using an established photography system with dissecting microscopy, the opacities were first detected at the anterior or posterior lens center surface in Emory mice at age 7 months, and gradually extended toward the equator during the 16 months examined. Scanning EM verified that disorganized and fragmented fiber cells were associated with the areas of opacities within approximately 200 μm from the lens surface, indicating that Emory mouse cataracts belong to the cortical cataracts. Freeze-fracture TEM further confirmed that cortical cataracts exhibited extensive wavy square array junctions, small gap junctions and globules. Immunofluorescence analysis showed that in contrast to the high labeling intensity of AQP0-loop antibody, the labeling of AQP0 C-terminus antibody was decreased considerably in superficial fibers in Emory cataracts. Similarly, a significant decrease in the labeling of the antibody against Cx50 C-terminus, but not Cx46 C-terminus, occurred in superficial and outer cortical fibers in Emory cataracts. Western blotting further revealed that the C-termini of both AQP0 and Cx50 in Emory cataracts were decreased to over 50% to that of the wild-type. Thus, this systematic study concludes that the Emory mouse cataract belongs to the cortical cataract which is due to regional breakdown of superficial fibers associated with formation of AQP0-dependent wavy square array junctions, small gap junctions and globules. The marked decreases of the C-termini of both AQP0 and Cx50 in the superficial fibers may disturb the needed interaction between these two proteins during fiber cell differentiation and thus play a role in the cortical cataract formation in Emory mutant mice.

OBJECTIVE: Lens fiber cell membranes contain aquaporin-0 (AQP0), which constitutes approximately 50% of the total fiber cell membrane proteins and has a dual function as a water channel protein and an adhesion molecule. Fiber cell membranes also develop an elaborate interlocking system that is required for maintaining structural order, stability, and lens transparency. Herein, we used an AQP0-deficient mouse model to investigate an unconventional adhesion role of AQP0 in maintaining a normal structure of lens interlocking protrusions.
METHODS: The loss of AQP0 in AQP0(-/-) lens fibers was verified by Western blot and immunofluorescence analyses. Changes in membrane surface structures of wild-type and AQP0(-/-) lenses at age 3 to 12 weeks were examined with scanning electron microscopy. Preferential distribution of AQP0 in wild-type fiber cell membranes was analyzed with immunofluorescence and immunogold labeling using freeze-fracturing transmission electron microscopy.
RESULTS: Interlocking protrusions in young differentiating fiber cells developed normally but showed minor abnormalities at approximately 50 μm deep in the absence of AQP0 in all ages studied. Strikingly, protrusions in maturing fiber cells specifically underwent uncontrolled elongation, deformation, and fragmentation, while cells still retained their overall shape. Later in the process, these changes eventually resulted in fiber cell separation, breakdown, and cataract formation in the lens core. Immunolabeling at the light microscopy and transmission electron microscopy levels demonstrated that AQP0 was particularly enriched in interlocking protrusions in wild-type lenses.
CONCLUSIONS: This study suggests that AQP0 exerts its primary adhesion or suppression role specifically to maintain the normal structure of interlocking protrusions that is critical to the integrity and transparency of the lens.