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M. F. Simpanya, L. L. David, L. J. Robertson, P. A. Wilmarth, F. J. Giblin; Age-Related Protein Modifications of Guinea Pig (Cavia porcellus) Lens Crystallins Analyzed by Two Dimensional Gel Electrophoresis and Mass Spectrometry. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4586.
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To identify age-related modifications (i.e. protein truncations, changes in isoelectric points and insolubilization) of guinea pig lens crystallins, and to produce two dimensional electrophoresis (2-DE) maps of cortical and nuclear water soluble (WS) and water insoluble (WI) proteins of young and old guinea pigs.
Lenses from 2.5-month and 24-month-old guinea pigs were dissected into nuclear and cortical regions, WS and WI fractions isolated by centrifugation, and proteins separated by 2-DE gels. Protein spots on 2-DE gels were excised, digested and tryptic peptides analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Spots not able to be identified by MALDI-MS were analyzed by LC-MS/MS.
Beyond the high abundance of ζ-crystallin, the crystallin composition of the guinea pig was unusual in that there was an abundance of nuclear βB3-crystallin. Similar to other mammals, βB3-crystallin was converted to partially truncated forms that were more abundant in the WI fraction in the nucleus of both young and old animals. Multiple truncated forms of βB1-crystallin with a wide range of pIs were also observed in all fractions and regions of lenses. However, βB1-crystallin was most extensively degraded in the WI fraction of both cortex and nucleus, which also exhibited decreases in intact βA3-crystallin. Little βB2-crystallin was found in the lens nucleus, but this protein was abundant in lens cortex. A truncated form of αA-crystallin was also apparent in the WS fraction of the young nucleus, which became the predominate form of αA-crystallin in the nuclear WI fraction of the old lens. This fraction also contained many additional truncated αA-forms that were less abundant in other lens fractions. The recently characterized γN-crystallin was readily observed in the cortex and nucleus of young lens, but was mainly a WI protein.
The results indicate aging of the guinea pig lens is associated with truncation and acidification of lens nuclear α- and β-crystallins. These changes may contribute to water-insolubilization since they were more pronounced in this fraction. This was especially true of αA-crystallin, whose truncation may contribute to crystallin insolubilization through loss of chaperone activity. The 2-DE maps created will prove useful for comparison with protein maps of guinea pig lens cataracts to elucidate cataract related changes and for comparison with other species.
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