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F.J. Giblin, J. Han, K.L. Schey; Identification of Sites of Truncation of AQP0 Induced by Hyperbaric Oxygen and UVA Light in vivo . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4088.
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We have shown previously using SDS–PAGE that both hyperbaric oxygen (HBO) and UVA light in vivo can cause truncation of the major lens membrane protein AQP0 in the guinea pig lens nucleus. Here we have used mass spectrometry to identify sites of the O2– and UVA–induced truncation.
Guinea pigs, initially 18 months–old, were treated with HBO 3x per week for 6 months or exposed to a low level of UVA light (0.5mW/cm2; 320–400nm; peak at 365nm) for 6 months. Both of these treatments induced an increased level of lens nuclear light scattering, as observed by slit–lamp examination. Lenses from treated and age–matched control animals were dissected into cortical and nuclear regions, and then homogenized, and the pellets washed with urea and NaOH to remove soluble and membrane–associated components. Membrane pellets were treated with trypsin in 10% acetonitrile/90% ammonium bicarbonate. Released tryptic peptides were desalted and subsequently identified and quantified by HPLC–tandem mass spectrometry.
Analysis of AQP0 C–terminal peptides revealed an increase in truncation at residues 250, 251, 253 and 257 in the nuclear fractions of lenses from HBO– and UVA–treated guinea pigs, compared to nuclear fractions from lenses of age–matched control animals. The overall truncation of AQP0 in both the O2– and UVA–treated lens nucleus was 60–80%, compared to 1–14% in the age–matched control nucleus. The major site of truncation in both models, accounting for about 50% of the total, was glycine 253. Only minimal truncation of AQP0 was detectable in the lens cortex of control and experimental animals.
The results indicate that both O2 and UVA light in vivo can increase truncation of the AQP0 C–terminus, specifically in the nuclear region of the lens. Although the mechanism of the truncation remains under investigation, it is noted that this modification of AQP0 occurs in the region of the observed pathology, i.e. in the nucleus where there is an increase in light scattering.
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