title: Functional characterization of an AQP0 missense mutation, R33C, that causes dominant congenital lens cataract, reveals impaired cell-to-cell adhesion.
abstract: 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.
authors.affiliation: Department of Physiology and Biophysics, Stony Brook University, Stony Brook, NY 11794-8661, USA.
authors.forename: Sindhu S
authors.initials: SS
authors.lastname: Kumari
authors.forename: Jason
authors.initials: J
authors.lastname: Gandhi
authors.forename: Mohammed H
authors.initials: MH
authors.lastname: Mustehsan
authors.forename: Semih
authors.initials: S
authors.lastname: Eren
authors.forename: Kulandaiappan
authors.initials: K
authors.lastname: Varadaraj
mesh_terms: D000818:Animals; D020346:Aquaporins / Q000235:genetics* / Q000378:metabolism; D002386:Cataract / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D002448:Cell Adhesion / Q000235:genetics; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism; D005260:Female; D006801:Humans; D007908:Lens, Crystalline / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D020125:Mutation, Missense; D012313:RNA / Q000235:genetics*; D014981:Xenopus
chemical_list: D020346:Aquaporins; D005136:Eye Proteins; C030328:aquaporin 0; D012313:RNA
keywords: AQP0; AQP0-R33C; AQP1; Aquaporin 0 R33C mutant; Aquaporin 1; FRET; Forster Resonance Energy Transfer; Pw; WT-AQP0; Water permeability; Wild type human aquaporin 0; cell-to-cell adhesion; congenital cataract; connexin 50; trafficking; water channel; water permeability