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Stephen Barnes, Supraja R Sridhar, Mason W Harris, Landon Wilson, Kelly Walters, Ali Arabshahi, Janusz Kabarowski; Membrane association of alpha-crystallins with cataract formation in the ICR/f rat. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2503.
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© 2017 Association for Research in Vision and Ophthalmology.
The Ihara hereditary cataractous rat (ICR/) develops bilateral lens cataracts spontaneously in 10-12 weeks. It is considered an important model of senile cataract disease. We have shown in this model using imaging mass spectrometry that αA-crystallin undergoes an accelerated rate of proteolysis into at least 16 peptide species compared to non-cataractous rats. In the present study we have examined whether αA-crystallin and other lens protein undergo changes in cellular compartmentation and whether this is associated with corresponding changes in lens lipid distribution.
Using the ICR/f rat colony at UAB, groups of rat were sacrificed at different ages during the stages of appearance of lens cataracts (30, 45, 60, 80, and 100 days of age) as well as at later ages (120, 140 and 180 days of age). Lens were recovered and quickly frozen in liquid N2. Lens were ground to a powder in liquid N2 using a glass pestle and mortar and proteins extracted with 50 mM Tris-HCl, pH 7.4 containing Roche protease inhibitor cocktail for water-soluble fraction, 6 M urea-50 mM Tris-HCl, pH 7.4, to recover membrane-associated fraction and 1% SDS-glycine buffer, pH 8.8 for a lipid-soluble fraction. Each fraction was analyzed by 15%-SDS-PAGE and proteins visualized using Coomassie Blue. Lens lipids extracted with chloroform-methanol were subject to MSMS(ALL) and differential ion mobility mass spectrometry.
At day 30, >80% of the lens proteins were in the water-soluble fraction. The lens was optically clear. At each time point after this, the amount of water-soluble protein declined. Initially, there was an increase in the water-insoluble, urea-soluble fraction, but as the rats got older, the proportion in the urea-insoluble, SDS-soluble fraction had an even greater increase, so much so that at 180 days >80% of the proteins were in this fraction. Imaging mass spectrometry of rodent lens lipds revealed that the outer cortical region of the lens was enriched with diacyl lipids (predominantly phosphatidylcholines and sphingomyelins). However, with aging there was a large decrease in the diacyl species in the nuclear region which were replaced by the corresponding monacyl species.
Aging in the ICR/f rat is accompanied by first increasing extrinsic membrane association and then intrinsic membrane association. This is accompanied by altered membrane composition, a potential driving force of cataract formation.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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