Kumari SS et al. (2013), Functional characterization of an AQP0 missense...

XB-ART-47854

Exp Eye Res 2013 Nov 01;116:371-85. doi: 10.1016/j.exer.2013.09.019.

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

Kumari SS , Gandhi J , Mustehsan MH , Eren S , Varadaraj K .

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Species referenced: Xenopus
Genes referenced: aqp1 gja8 mip
GO keywords: cellular water homeostasis [+]

???displayArticle.disOnts??? cataract 15 multiple types
???displayArticle.omims??? CATARACT 15, MULTIPLE TYPES; CTRCT15

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	Graphical abstract
	Fig. 1. (A). Schematic representation of human AQP0. Monomeric structure shows folds, helix assignment, and location in the membrane. Membrane-spanning helices are denoted as H1–H6 and loops as LA–LE. The two pore lining helices are shown as HB and HE. Highly conserved NPA motifs in loops B and E (shaded green) that line the water pore of aquaporin are shown. NH2, amino terminus; COOH, carboxy terminus. ‘+’ and ‘−’ represent amino acid charges in extracellular and cytoplasmic domains. (B). AQP0 mutations in humans and mice. Schematic illustration of the locations of eleven mutations in humans (red) and four mutations in mice (black) that cause inherited lens cataracts. AQP0 secondary structure domain designations are as given in (Fig. 1A). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
	Fig. 2. Expression of WT-AQP0 and AQP0-R33C in the Xenopus laevis oocytes. (a–c) Immunostaining of cryosections of oocytes injected with distilled water or AQP0 cRNAs. Expression of WT-AQP0 or mutant AQP0-R33C was visualized by immunostaining using anti-AQP0 antibody: (a) Water injected oocytes, (b) WT-AQP0 cRNA injected and (c) mutant AQP0-R33C cRNA injected oocytes. (d) Western blot analysis of oocytes injected with cRNA of WT-AQP0 (lanes 1) or mutant AQP0-R33C (lane 2).
	Fig. 3. Localization and colocalization of WT-AQP0-mCherry and AQP0-R33C-EGFP. (a, b) Epifluorescent images of MDCK cells transfected with the WT-AQP0-mCherry and AQP0-R33C-EGFP, respectively. c. Western blot analysis of MDCK cells expressing WT-AQP0-mCherry (lane 1) and AQP0-R33C-EGFP (lane 2). (d–f) Cells cotransfected with WT-AQP0-mCherry and AQP0-R33C-EGFP constructs; (d) cotransfected cell viewed under mCherry fluorescent filter; (e) the same cells under an EGFP fluorescent filter; (f) overlaid image of (d) and (e).
	Fig. 4. Protein colocalization. (a–f) Forster Resonance Energy Transfer in MDCK cells cotransfected with WT-AQP0-mCherry and mutant AQP0-R33C-EGFP or WT-AQP0-EGFP and WT-AQP0-mCherry. Cells were cotransfected with: WT-AQP0-mCherry and AQP0-R33C-EGFP (a-c), WT-AQP0-mCherry and WT-AQP0-EGFP (d–f); (a, d) cells excited at 587 nm and emission recorded at 610 nm; (b, e) cells excited at 488 nm and emission recorded at 507 nm; (c, f) cells excited at 470 nm and emission recorded at 640 nm fluorescence due to FRET; (c, f) fluorescence indicating colocalization of mutant AQP0-R33C-EGFP and WT-AQP0-mCherry or WT-AQP0-EGFP and WT-AQP0-mCherry proteins in the same oligomer or within 100Å. (g–i) Localization of WT-AQP0-EGFP, AQP0-R33C-EGFP in the ER. MDCK cells were coexpressed with WT-AQP0-EGFP or WT-AQP0-mCherry and organelle light ER-RFP. (g, j) Images taken under EGFP filter; (h, k) images taken under Texas Red filter; (i, l) merged images of (g, h) and (j, k), respectively showing yellow color due to colocalization of the respective protein (green) in the ER along with the organelle light (red). The green fluorescence at the periphery of the images (i) and (l) indicates plasma membrane localization of WT-AQP0-EGFP and AQP0-R33C-EGFP, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
	Fig. 5. Water permeability of WT-AQP0 and AQP0-R33C. (A) Membrane water permeability of Xenopus laevis oocytes injected with distilled water, 5 ng/oocyte cRNA of WT-AQP1, 25 ng/oocyte cRNA of WT-AQP0 or 25 ng/oocyte cRNA of AQP0-R33C. An oocyte was placed in a hypotonic solution and initial rate of swelling was estimated. A simple curve fit to the data was obtained to calculate the oocyte membrane water permeability as described in the “Materials and Methods' section. Relative volume due to water uptake of oocytes injected with distilled water (control), human AQP1-cRNA, human WT-AQP0-cRNA or human mutant AQP0-R33C-cRNA is plotted. (B) Oocyte membrane water permeability of nine swelling assays (each assay with 15 oocytes; (mean ± SD)). Compared to control all assays showed significant increase in water permeability, P < 0.0001. There was no significant difference in water permeability between fluorescent protein tagged and untagged WT-AQP0, AQP0-R33C or WT-AQP0 + AQP0-R33C, P > 0.05.
	Fig. 6. Cell aggregation assay using rotary gyratory shaker. (A). Cell aggregation exhibited by adhesion-deficient L-cells expressing empty vector, AQP1, E-cadherin, WT-AQP0, AQP0-R33C or WT-AQP0 + AQP0-R33C in relation to incubation time. (Nt – total number of particles at time ‘t’ of incubation; N0 - initial number of particles). (B). Cell-to-cell adhesion assay using a microplate reader. Over a monolayer of L-cells expressing empty vector (negative, control), AQP1 (negative, control), E-cadherin (positive control), WT-AQP0, AQP0-R33C or WT-AQP0 + AQP0-R33C corresponding cells loaded with CellTracker Red were plated. A microplate reader was used to obtain data as described in the ‘Materials and Methods’ section. Stars (, ) indicate statistically significant reduction in cell-to-cell adhesion in AQP0-R33C or WT-AQP0 + AQP0-R33C compared to the WT-AQP0. (C). Cell-to-cell adhesion assay using a fluorescence microscope. Over a monolayer of L-cells expressing empty vector, AQP1, E-cadherin, WT-AQP0 or AQP0-R33C corresponding cells loaded with CellTracker Red were plated. At the end of the procedure described in the ‘Materials and Methods’ section, cells were imaged under an epifluorescent microscope (Zeiss). Cells/aggregates were counted and values were plotted. (D). Histogram showing the extent of cell-to-cell adhesion exhibited after 45 min of incubation by samples tested using the fluorescence assay. Compared to E-cadherin, WT-AQP0 exhibited significantly low (P < 0.001) cell-to-cell adhesion. **Compared to WT-AQP0, mutant AQP0-R33C exhibited significantly low cell-to-cell adhesion.
	Fig. 7. Scrape-loading dye transfer assay showing AQP0-facilitated gap junction coupling. (A). Western blotting of (a) Cx50, (b) WT-AQP0 and AQP0-R33C proteins to show expression of the respective proteins on L-cells. (B). Lucifer yellow (LY) dye transfer in L-cells stably expressing vector, WT-AQP0, AQP0-R33C, Cx50, WT-AQP0 + Cx50 or AQP0-R33C + Cx50. The extent of dye transfer was quantified by measuring distance from the scrape line to the dye front of LY. (C). Bar graph representing the data collected, presented as mean ± SD. (n = 5). *Increase in gap junction coupling in WT-AQP0 + Cx50 transfected cells was statistically significant (P < 0.001) compared to Cx50 transfected cells; #Reduction in gap junction coupling in AQP0-R33C + Cx50 transfected cells was statistically significant (P < 0.001) compared to WT-AQP0 + Cx50 transfected cells.
	Fig. 8. Schematic models illustrating the possible cause for autosomal dominant congenital lens cataract due to R33C missense mutation. WT-AQP0: It has been postulated that positive charges in the extracellular loops of AQP0 play a significant role in cell-to-cell adhesion. WT-AQP0 monomers form tetramers. Each tetramer carries a total of 20 positive charges (5/monomer) in Extracellular Loops, A, C and E. These positive charges electrostatically attract negatively charged lipids of the opposing fiber cell plasma membrane to come closer, thus reducing the extracellular space between the fibers and enabling firm and tight cell-to-cell adhesion. The proximity of fiber cells facilitates gap junction hemichannels to couple and form channels for intercellular communication, which aids in microcirculation and homeostasis of the avascular lens. R33C-AQP0: Mutation at codon 33 in the Extracellular Loop A of AQP0 resulted in replacement of positively charged amino acid arginine (R) to a neutral amino acid cysteine (C). Mutant AQP0-R33C tetramer has only 16 positive charges in extracellular loops due to loss of an arginine to cysteine in each monomer. This reduction might have decreased the pulling and holding forces between opposing fiber cells due to the fewer number of positive charges interacting with negative charges of the plasma membrane lipids, leading to wider intercellular space between adjacent fibers compared to that in the WT-AQP0. Reduced cell-to-cell adhesion of AQP0-R33C might have eventually caused loss of gap junction coupling, compromising intercellular communication, microcirculation and homeostasis of the lens.

References [+] :

Aerts, Hydrodynamic characterization of the major intrinsic protein from the bovine lens fiber membranes. Extraction in n-octyl-beta-D-glucopyranoside and evidence for a tetrameric structure. 1990, Pubmed