Identification of crystallin modifications in the human lens cortex and nucleus using laser capture microdissection and CyDye labeling

Abstract

Purpose: 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.

Figure 1

Tissue section of a human lens. A: Whole tissue section (12 μm) of 69-year-old lens stained with hematoxylin and eosin, as seen under a stereomicroscope with 4× magnification. All three major regions are present in this section, however the nucleus stained more faintly because of possible increased hydrophobicity of the proteins in this region. B: Unstained section showing three major lenticular regions: Nucleus (N), Inner Cortex (IC), and Outer Cortex (OC), as seen during LCM with the Zeiss/PALM Microbeam microscope at its lowest magnification of 2.5×.

Figure 2

Separation of 65-year-old human lens proteins using 15% polyacrylamide gel by SDS–PAGE analysis and identification of excised bands by MALDI-TOF mass spectrometry. A: SDS–PAGE image of proteins in three lenticular regions with Coomassie Blue R250 staining showing mostly LMW species. Samples from the outer and inner cortices were repeated in the last two lanes on the right side of the gel. B: Expanded image seen in A. Boxes outline the bands excised for mass spectrometric analysis and crystallin identifications of excised bands were based on MALDI-TOF data.

Figure 3

Typhoon-scanned fluorescence images from a 2D-DIGE of a 69-year-old human lens. Equal concentrations of protein (20 μg) from the cortex and nucleus were labeled with different fluorescent dyes and analyzed by 2D-DIGE. A: Internal standard labeled with Cy2 (Ex 488 nm, Em 520 nm). B: Cortical proteins (outer and inner cortex pooled) labeled with Cy3 (Ex 532 nm, Em 580 nm). C: Nuclear proteins labeled with Cy5 (Ex 633 nm, Em 670 nm). D: Overlay of A, B, and C. Spots fluorescing white are protein species present in both cortex and nucleus, while those fluorescing blue and red are localized to the corresponding regions as shown in B and C, respectively.

Figure 4

Coomassie blue-stained gel from 2D-DIGE of 69-year-old human lens. The gel was stained with Coomassie blue R250 for the purpose of manually picking the individually labeled spots (circled and numbered in the figure) for identification by ESI-QTRAP LC/MS/MS. The gels in this figure are technical replicates, so corresponding spots were picked and pooled for identification.

Figure 5

Standard 2D gel of a 69-year-old human lens. IEF was done in the first dimension using an 11 cm IPG strip, pH 5–8, followed by SDS–PAGE using a 15% polyacrylamide gel in the second dimension. The gel was stained with Coomassie blue R250, and shows a profile similar to 2D-DIGE gels of the same tissue. However, HMW (>35 kDa) aggregates are not distinguished as individual spots, rather they appeared as non-descript bands.