Identification of crystallin modifications in the human lens cortex and nucleus using laser capture microdissection and CyDye labeling.
OBJECTIVE
With aging, lens crystallins undergo post-translational modifications (PTMs) and these modifications are believed to play a major role in age-related cataract development. The purpose of the present study was to determine the protein profiles of crystallins and their PTMs in the cortical and nuclear regions within an aging human lens to gain a better understanding about changes in crystallins as fiber cells migrate from cortical to nuclear region.


METHODS
Laser capture microdissection (LCM) was used to select and capture cells from cortical and nuclear regions of 12 mum, optimum cutting temperature (OCT) compound-embedded frozen lens sections from a 69-year-old human lens. Proteins were extracted and then analyzed by 2-D difference gel electrophoresis (2-D DIGE) with sulfonated indocyanine dye (CyDye) labeling. Crystallin identities and their PTMs were then determined by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) and Electrospray Ionization Quadripole Linear Ion-Trap Liquid Chromatography (ESI-QTRAP LC-MS/MS) mass spectrometry.


RESULTS
Crystallin fragments (M(r) <20 kDa) were present in both cortical and nuclear regions, while high molecular weight (HMW) aggregates (M(r) > 35 kDa) were mostly localized in the nuclear region. HMW complexes contained a relatively large number of truncated and modified beta-crystallins, compared to alpha- and gamma-crystallins, and two lens-specific intermediate filaments, CP49 (phakinin) and filensin. Modified alpha-crystallins were in low abundance in the nuclear region compared to the cortical region. Several PTMs, including deamidation, oxidation, phosphorylation, ethylation, methylation, acetylation, and carbamylation, were identified in virtually all crystallins and CP49. The data provide the first report of human lens crystallin profiling by a combination of LCM, 2D-DIGE, and mass spectrometric analysis.


CONCLUSIONS
The results suggested that as the fiber cells migrate from cortical region to the nuclear region, the crystallin degradation begins in the cortical region and continues in the nuclear region. However, a greater number of the HMW complexes exist mainly in the nuclear region.
Asomugha
C O
CO
Department of Vision Sciences, University of Alabama at Birmingham, Birmingham, AL 35294-4390, USA.
Gupta
R
R
Srivastava
O P
OP
D000368:Aged; D002467:Cell Nucleus / Q000378:metabolism*; D003459:Crystallins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D005453:Fluorescence; D005456:Fluorescent Dyes; D006801:Humans; D007834:Lasers; D007908:Lens, Crystalline / Q000166:cytology / Q000378:metabolism*; D042282:Microdissection / Q000379:methods*; D011499:Protein Processing, Post-Translational; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013194:Staining and Labeling
D003459:Crystallins; D005456:Fluorescent Dyes