Aquaporin-0-protein interactions elucidated by crosslinking mass spectrometry.

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.

Deletion of beaded filament proteins or the C-terminal end of Aquaporin 0 causes analogous abnormal distortion aberrations in mouse lens.

Lens-specific beaded filament (BF) proteins CP49 and filensin interact with the C-terminus of the water channel protein Aquaporin 0 (AQP0). Previously we have reported that a C-terminally end-deleted AQP0-expressing transgenic mouse model AQP0ΔC/ΔC developed abnormal optical aberrations in the lens. This investigation was undertaken to find out whether the total loss of the BF structural proteins alter the optical properties of the lens and cause optical aberrations similar to those in AQP0ΔC/ΔC lenses; also, to map the changes in the optical quality as a function of age in the single or double BF protein knockouts as well as to assess whether there is any significant change in the water channel function of AQP0 in these knockouts. A double knockout mouse (2xKO) model for CP49 and filensin was developed by crossing CP49-KO and filensin-KO mice. Wild type, CP49-KO, filensin-KO, and 2xKO lenses at different ages, and AQP0ΔC/ΔC lenses at postnatal day-17 were imaged through the optical axis and compared for optical quality and focusing property. All three knockout models showed loss of transparency, and development of abnormal optical distortion aberration similar to that in AQP0ΔC/ΔC. Copper grid focusing by the lenses at 6, 9 and 12 months of age showed an increase in aberrations as age advanced. With progression in age, the grid images produced by the lenses of all KO models showed a transition from a positive barrel distortion aberration to a pincushion distortion aberration with the formation of three distinct aberration zones similar to those produced by AQP0ΔC/ΔC lenses. Water permeability of fiber cell membrane vesicles prepared from CP49-KO, filensin-KO and 2xKO models, measured using the osmotic shrinking method, remained similar to that of the wild type without any statistically significant alteration (P > 0.05). Western blotting and quantification revealed the expression of comparable quantities of AQP0 in all three BF protein KOs. Our study reveals that loss of single or both beaded filament proteins significantly affect lens refractive index gradient, transparency and focusing ability in an age-dependent manner and the interaction of BF proteins with AQP0 is critical for the proper functioning of the lens. The presence of BF proteins is necessary to prevent abnormal optical aberrations and maintain homeostasis in the aging lens.

Acceleration of age-induced proteolysis in the guinea pig lens nucleus by in vivo exposure to hyperbaric oxygen: A mass spectrometry analysis.

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.

Calmodulin Gates Aquaporin 0 Permeability through a Positively Charged Cytoplasmic Loop.

Aquaporin 0 (AQP0), the major intrinsic protein of the eye lens, plays a vital role in maintaining lens clarity by facilitating the transport of water across lens fiber cell membranes. AQP0 reduces its osmotic water permeability constant (Pf) in response to increases in the external calcium concentration, an effect that is mediated by an interaction with the calcium-binding messenger protein, calmodulin (CaM), and phosphorylation of the CaM-binding site abolishes calcium sensitivity. Despite recent structural characterization of the AQP0-CaM complex, the mechanism by which CaM modulates AQP0 remains poorly understood. By combining atomistic molecular dynamics simulations and oocyte permeability assays, we conclude that serine phosphorylation of AQP0 does not inhibit CaM binding to the whole AQP0 protein. Instead, AQP0 phosphorylation alters calcium sensitivity by modifying the AQP0-CaM interaction interface, particularly at an arginine-rich loop that connects the fourth and fifth transmembrane helices. This previously unexplored loop, which sits outside of the canonical CaM-binding site on the AQP0 cytosolic face, mechanically couples CaM to the pore-gating residues of the second constriction site. We show that this allosteric loop is vital for CaM regulation of the channels, facilitating cooperativity between adjacent subunits and regulating factors such as serine phosphorylation. Similar allosteric interactions may also mediate CaM modulation of the properties of other CaM-regulated proteins.

Lens transcriptome profile during cataract development in Mip-null mice.

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.

Spatial distributions of phosphorylated membrane proteins aquaporin 0 and MP20 across young and aged human lenses.

In the human ocular lens it is now realized that post-translational modifications can alter protein function and/or localization in fiber cells that no longer synthesize proteins. The specific sites of post-translational modification to the abundant ocular lens membrane proteins AQP0 and MP20 have been previously identified and their functional effects are emerging. To further understand how changes in protein function and/or localization induced by these modifications alter lens homeostasis, it is necessary to determine the spatial distributions of these modifications across the lens. In this study, a quantitative LC-MS approach was used to determine the spatial distributions of phosphorylated AQP0 and MP20 peptides from manually dissected, concentric layers of fiber cells from young and aged human lenses. The absolute amounts of phosphorylation were determined for AQP0 Ser235 and Ser229 and for MP20 Ser170 in fiber cells from the lens periphery to the lens center. Phosphorylation of AQP0 Ser229 represented a minor portion of the total phosphorylated AQP0. Changes in spatial distributions of phosphorylated APQ0 Ser235 and MP20 Ser170 correlated with regions of physiological interest in aged lenses, specifically, where barriers to water transport and extracellular diffusion form.

The kinetics of thermal stress induced denaturation of Aquaporin 0.

Aquaporin 0 (AQP0) is an integral membrane protein that facilitates water transport and cellular adhesion in the lens. Its dysfunction has been associated with cataractogenesis. Our earlier studies showed AQP0 undergoes aggregation when subjected to thermal stress and this aggregation seems to have been facilitated by mechanical agitation brought about by gentle stirring. The purpose of this study is to determine the secondary structural changes that precede aggregation and the role that α-crystallin plays in inhibiting those structural changes. Detergent solubilized calf lens AQP0 was subjected to thermal stress at 50°C for varying times. Secondary structural changes were measured by Circular Dichroism (CD) spectropolarimetry. Convex constraint analysis was used to deconvolute the CD spectra into pure component curves representing the secondary structural elements. Our results showed that under thermal stress, the α-helix content of AQP0 decreased from 50% to 7% with a concomitant increase from 0% to 52% in ß-sheet content. The time-dependent loss of α-helical structure and gain of ß-sheet structure appear to follow first-order kinetics with very similar values (∼30min) suggesting a single transition. In the presence of α-crystallin, this conversion to ß-sheet is minimized, suggesting that the protein structure that binds to the molecular chaperone is mostly the α-helical structure of AQP0.

Regional changes of AQP0-dependent square array junction and gap junction associated with cortical cataract formation in the Emory mutant mouse.

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.

Functional characterization of an AQP0 missense mutation, R33C, that causes dominant congenital lens cataract, reveals impaired cell-to-cell adhesion.

Aquaporin 0 (AQP0) performs dual functions in the lens fiber cells, as a water pore and as a cell-to-cell adhesion molecule. Mutations in AQP0 cause severe lens cataract in both humans and mice. An arginine to cysteine missense mutation at amino acid 33 (R33C) produced congenital autosomal dominant cataract in a Chinese family for five generations. We re-created this mutation in wild type human AQP0 (WT-AQP0) cDNA by site-directed mutagenesis, and cloned and expressed the mutant AQP0 (AQP0-R33C) in heterologous expression systems. Mutant AQP0-R33C showed proper trafficking and membrane localization like WT-AQP0. Functional studies conducted in Xenopus oocytes showed no significant difference (P > 0.05) in water permeability between AQP0-R33C and WT-AQP0. However, the cell-to-cell adhesion property of AQP0-R33C was significantly reduced (P < 0.001) compared to that of WT-AQP0, indicated by cell aggregation and cell-to-cell adhesion assays. Scrape-loading assay using Lucifer Yellow dye showed reduction in cell-to-cell adhesion affecting gap junction coupling (P < 0.001). The data provided suggest that this mutation might not have caused significant alterations in protein folding since there was no obstruction in protein trafficking or water permeation. Reduction in cell-to-cell adhesion and development of cataract suggest that the conserved positive charge of Extracellular Loop A may play an important role in bringing fiber cells closer. The proposed schematic models illustrate that cell-to-cell adhesion elicited by AQP0 is vital for lens transparency and homeostasis.

Differences in a Single Extracellular Residue Underlie Adhesive Functions of Two Zebrafish Aqp0s.

Aquaporin 0 (AQP0) is the most abundant lens membrane protein, and loss of function in human and animal models leads to cataract formation. AQP0 has several functions in the lens including water transport and adhesion. Since lens optics rely on strict tissue architecture achieved by compact cell-to-cell adhesion between lens fiber cells, understanding how AQP0 contributes to adhesion would shed light on normal lens physiology and pathophysiology. We show in an in vitro adhesion assay that one of two closely related zebrafish Aqp0s, Aqp0b, has strong auto-adhesive properties while Aqp0a does not. The difference appears to be largely due to a single amino acid difference at residue 110 in the extracellular C-loop, which is T in Aqp0a and N in Aqp0b. Similarly, P110 is the key residue required for adhesion in mammalian AQP0, highlighting the importance of residue 110 in AQP0 cell-to-cell adhesion in vertebrate lenses as well as the divergence of adhesive and water permeability functions in zebrafish duplicates.

AQP0 is a novel surface marker for deciphering abnormal erythropoiesis.

BACKGROUND: Hematopoiesis occurs in the bone marrow, producing a complete spectrum of blood cells to maintain homeostasis. In addition to light microscopy, chromosome analysis, and polymerase chain reaction, flow cytometry is a feasible and fast method for quantitatively analyzing hematological diseases. However, because sufficient specific cell markers are scarce, dyserythropoietic diseases are challenging to identify through flow cytometry. METHODS: Bone marrow samples from C57BL/B6 mice and one healthy donor were analyzed using traditional two-marker (CD71 and glycophorin A) flow cytometry analysis. After cell sorting, the gene expressions of membrane proteins in early and late erythropoiesis precursors and in nonerythroid cells were characterized using microarray analysis. RESULTS: Among characterized gene candidates, aquaporin 0 (AQP0) expressed as a surface protein in early- and late-stage erythropoiesis precursors and was not expressed on nonerythroid cells. With the help of AQP0 staining, we could define up to five stages of erythropoiesis in both mouse and human bone marrow using flow cytometry. In addition, because patients with dyserythropoiesis generally exhibited a reduced population of APQ0high cells relative to healthy participants, the analysis results also suggested that the levels of APQ0high cells in early erythropoiesis serve as a novel biomarker that distinguishes normal from dysregulated erythropoiesis. CONCLUSIONS: AQP0 was successfully demonstrated to be a marker of erythroid differentiation. The expression levels of AQP0 are downregulated in patients with dyserythropoiesis, indicating a critical role of AQP0 in erythropoiesis. Accordingly, the level of AQP0high in early erythroid precursor cells may serve as a reference parameter for diagnosing diseases associated with dyserythropoiesis.

Localization and Ordering of Lipids Around Aquaporin-0: Protein and Lipid Mobility Effects.

Hydrophobic matching, lipid sorting, and protein oligomerization are key principles by which lipids and proteins organize in biological membranes. The Aquaporin-0 channel (AQP0), solved by electron crystallography (EC) at cryogenic temperatures, is one of the few protein-lipid complexes of which the structure is available in atomic detail. EC and room-temperature molecular dynamics (MD) of dimyristoylglycerophosphocholine (DMPC) annular lipids around AQP0 show similarities, however, crystal-packing and temperature might affect the protein surface or the lipids distribution. To understand the role of temperature, lipid phase, and protein mobility in the localization and ordering of AQP0-lipids, we used MD simulations of an AQP0-DMPC bilayer system. Simulations were performed at physiological and at DMPC gel-phase temperatures. To decouple the protein and lipid mobility effects, we induced gel-phase in the lipids or restrained the protein. We monitored the lipid ordering effects around the protein. Reducing the system temperature or inducing lipid gel-phase had a marginal effect on the annular lipid localization. However, restraining the protein mobility increased the annular lipid localization around the whole AQP0 surface, resembling EC. The distribution of the inter-phosphate and hydrophobic thicknesses showed that stretching of the DMPC annular layer around AQP0 surface is the mechanism that compensates the hydrophobic mismatch in this system. The distribution of the local area-per-lipid and the acyl-chain order parameters showed particular fluid- and gel-like areas that involved several lipid layers. These areas were in contact with the surfaces of higher and lower protein mobility, respectively. We conclude that the AQP0 surfaces induce specific fluid- and gel-phase prone areas. The presence of these areas might guide the AQP0 lipid sorting interactions with other membrane components, and is compatible with the squared array oligomerization of AQP0 tetramers separated by a layer of annular lipids.

Structural Properties of Protein-Detergent Complexes from SAXS and MD Simulations.

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-ß-maltoside molecules (ßDDM). 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 ßDDM numbers. Explicit-solvent methods predicted best agreement using 290-ßDDM 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 Claim of Anti-Cataract Potential of Heliotropium indicum: A Myth or Reality?

INTRODUCTION: Heliotropium indicum has several uses in traditional medicine attributable to its numerous bioactive compounds. It is used as a traditional remedy for cataracts in Ghana without any scientific verification. This study aimed at verifying the anti-cataract properties of an aqueous whole plant extract of H. indicum. METHODS: The effect (cataract score) of 30, 100, and 300 mg kg(-1) extract (bid for 21 days, per os) on the development of 30 µmol kg(-1) sodium selenite-induced cataract in 10-day-old rat pups was investigated. Soluble lens proteins alpha A and alpha B crystallins, total lens protein, total lens glutathione, and aquaporin 0 in enucleated lens homogenates were determined spectrophotometrically using commercially available kits. Histopathological studies on the lenses were also performed. The 2,2-diphenyl-1-picrylhydrazyl scavenging effect and linoleic acid autoxidation (antioxidant properties) of the extract (0.1-3.0 mg ml(-1)), compared to n-propyl gallate, were ascertained using standard procedures. RESULTS: Cataract scores showed that the extract, at all dose levels, significantly alleviated selenite-induced cataracts (P ≤ 0.001). Markers of lens transparency (aquaporin 0, alpha A and B crystallins), as well as total lens proteins and lens glutathione levels, were significantly preserved (P ≤ 0.01-0.001). The extract exhibited activity relevant for scavenging free radicals and inhibition of lipid peroxidation. Epithelial and lens fiber integrity in the histopathological assessment were maintained with HIE treatment. CONCLUSION: The aqueous whole plant extract of H. indicum significantly inhibited the development of cataracts in rats via multiple mechanisms.

Aquaporin-0 targets interlocking domains to control the integrity and transparency of the eye lens.

PURPOSE: 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.