Purchase this article with an account.
K. Varadaraj, S. S. Kumari, R. V. Patil, R. T. Mathias; Functional Characterization of a Novel Missense Mutation in the Putative Calmodulin Binding C-Terminal Domain of Human AQP0 That Results in Polymorphic Autosomal Dominant Lens Cataract. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1455.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To functionally characterize a missense mutation in the putative calmodulin binding domain of human AQP0 (hAQP0) at codon 233 (R233K; Lin et al., 2007, Mol. Vis., 13:1822) which caused polymorphic dominant lens cataract in a Chinese family for six generations.
Mutant R233K was created by site directed mutagenesis. Wild type and mutant were separately cloned into pcDNA3.1 containing a T7 promoter for cRNA expression in Xenopus oocytes, and a CMV promoter for MDCK cell expression. mCherry (from Dr. R. Y. Tsien, UC, San Diego) or EGFP was used as a tag in localization studies. Water permeability (Pw) was determined from the rate of volume change when each oocyte was subjected to hypotonic shock. Co-localization, trafficking and dominant negative effects were studied using fluorescent tags, immunostaining, fluorescence microscopy and FRET analysis. Necrosis- and apoptosis-specific staining and LDH assay were used to investigate cytotoxicity.
Membrane Pw of oocytes expressing R233K (12±4 µm/s) was significantly lower than those expressing wild type AQP0 (49±7 µm/s). Water injected oocytes showed a Pw value of 12±2 µm/s. When R233K was co-expressed with the WT AQP0, Pw was significantly reduced compared to the WT, reflecting dominant negative effect. hAQP0 protein localized in the plasma membrane whereas R233K protein was mostly in the ER. FRET studies revealed partial localization of WT with the co-expressed R233K mutant. Incubating the oocytes and cells expressing R233K with chemical chaperones did not correct the trafficking defect(s), as inferred by localization studies and lack of improvement in Pw. Necrosis and apoptosis were significantly higher in the R233K transfected cells than in the WT; however, necrosis was higher than apoptosis.
Heterologous expression of R233K showed the missense mutation in the C-terminal domain affects normal trafficking leading to reduced membrane Pw. Interactions of WT AQP0 with R233K mutant protein may account for the dominant negative effect. Since lysine substitution for arginine did not create a change in net charge, structure of the amino acid and interaction with other amino acid(s) appear more critical than charge for normal folding and trafficking. Loss of lens fiber cell Pw, and cytotoxicity due to mutant protein accumulation might have induced fiber cell disintegration causing polymorphic lens cataracts in this Chinese family. Correcting the R233K trafficking defect(s) by gene therapy using stem cells may be a feasible strategy to cure cataract due to this mutation.
This PDF is available to Subscribers Only