April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
Age-Related Protein Modifications of Guinea Pig (Cavia porcellus) Lens Crystallins Analyzed by Two Dimensional Gel Electrophoresis and Mass Spectrometry
Author Affiliations & Notes
  • M. F. Simpanya
    Eye Research Inst, Oakland University, Rochester, Michigan
  • L. L. David
    Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, Oregon
  • L. J. Robertson
    Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, Oregon
  • P. A. Wilmarth
    Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, Oregon
  • F. J. Giblin
    Eye Research Inst, Oakland University, Rochester, Michigan
  • Footnotes
    Commercial Relationships  M.F. Simpanya, None; L.L. David, None; L.J. Robertson, None; P.A. Wilmarth, None; F.J. Giblin, None.
  • Footnotes
    Support  NIH Grants EY02027 and EY014803 (FJG), EY007755 and EY010572 (LLD)
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 4586. doi:
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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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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : 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.

Methods: : 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.

Results: : 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.

Conclusions: : 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.

Keywords: aging • oxidation/oxidative or free radical damage • protein modifications-post translational 
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