An MIP/AQP0 mutation with impaired trafficking and function underlies an autosomal dominant congenital lamellar cataract

Abstract

Autosomal dominant congenital cataracts have been associated with mutations of genes encoding several soluble and membrane proteins. By candidate gene screening, we identified a novel mutation in MIP (c.494 G > A) that segregates with a congenital lamellar cataract within a south Indian family and causes the replacement of a highly conserved glycine by aspartate (G165D) within aquaporin0 (AQP0). Unlike wild type AQP0, expression of AQP0-G165D in Xenopus oocytes did not facilitate swelling in hypotonic medium. In transfected HeLa cells, wild type AQP0 localized at the plasma membrane while AQP0-G165D was retained within the secretory pathway, and localized mainly within the endoplasmic reticulum. These results suggest that mutation of this conserved glycine residue leads to improper trafficking of AQP0-G165D and loss of water channel function. They emphasize the importance of AQP0 for maintenance of lens transparency and identify a critical residue that is conserved among aquaporins, but has not previously been associated with disease-associated replacement.

Figure 1

A 494G>A nucleotide transition in the coding region of AQP0 segregates with the congenital lamellar cataract. A. Photographs show the congenital lamellar cataract in the proband’s right (top) and left (bottom) eyes. B. Pedigree of the family with autosomal dominant lamellar cataract. Black symbols indicate affected individuals and open symbols indicate unaffected individuals. The diagonal line indicates a deceased family member. Family members whose DNA was analyzed by sequencing and restriction enzyme digestion are indicated by asterisks. The proband is indicated with an arrow. C. Chromatogram of the DNA sequence from an unaffected individual (III.5) shows only the wild type AQP0 allele which encodes glycine (GGC) at codon 165. The sequence chromatogram from the proband (IV.1) shows both G and A at position 494; thus, the mutant allele contains a G to A transition resulting in the substitution of glycine for aspartate at amino acid residue 165. D. Diagram of the topology of AQP0 in the plasma membrane. Transmembrane helices are numbered and intervening loops are indicated by letters. Amino acid residue 165 is located within the 5^th transmembrane domain (red circle).

Figure 2

The AQP0-G165D mutant does not exhibit water channel function and does not traffic properly to the plasma membrane. A. Graph shows representative curves of the time course of swelling (measured as oocyte area) of individual oocytes injected with water (black triangles), or cRNA for wild type AQP0 (blue squares) or AQP0-G165D (red circles) after they were transferred to a 10% hypotonic solution. B. Graph shows the swelling rates for oocytes injected with water, wild type AQP0 cRNA (WT) or AQP0-G165D cRNA (G165D). Values are presented as mean ± standard deviation (n = 10 for water, n = 15 for WT AQP0 and n = 18 for AQP0-G165D). C. Immunoblots show the levels of immunoreactive AQP0 in cleared homogenates (H) or membrane-enriched pellets (P) prepared from oocytes injected with water or with cRNAs encoding wild type AQP0 or the mutant G165D. D. Photomicrographs show the distributions of immunoreactive AQP0 and an ER-resident protein (PDI) in HeLa cells that were transiently transfected with wild type AQP0 or AQP0-G165D. Twenty four hours later, cells were fixed and subjected to immunofluorescence using rabbit polyclonal anti-AQP0 and mouse monoclonal anti-PDI antibodies followed by Cy3-conjugated goat anti-rabbit and AlexaFluo®488-conjugated goat anti-mouse IgG antibodies. Arrows indicate immunoreactivity detected at the plasma membrane (only for wild type AQP0). Bar: 20 µm.