Structural and functional roles of deamidation of N146 and/or truncation of NH2- or COOH-termini in human αB-crystallin.

PURPOSE: The purpose of the study was to determine the relative effects of deamidation and/or truncation on the structural and functional properties of αB-crystallin. METHODS: Using wild-type (WT) αB-crystallin and the αB deamidated mutant (i.e., αB N146D), we generated NH(2)-terminal domain deleted (residues no. 1-66; αB-NT), deamidated plus NH(2)-terminal domain deleted (αB N146D-NT), COOH-terminal extension deleted (residues no. 151-175; αB-CT), and deamidated plus COOH-terminal extension deleted (αB N146D-CT) mutants. All of the proteins were purified and their structural and functional (chaperone activity with insulin as target protein) properties were determined and compared to WT αB-crystallin. RESULTS: The desired deletions in the αB-crystallin mutants were confirmed by DNA sequencing and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric analysis. The homomers of αB-CT and its deamidated form (αB N146D-CT) became water insoluble, whereas the αB N146D, αB-NT, and αB N146D-NT species remained water-soluble. CD spectroscopic studies revealed that the mutants with deletion of NH(2)- or COOH-termini or deamidation showed increased ß-sheet and decreased α-helical contents with the exception of αB N146D-CT, which showed a substantial increase in α-helix and decrease in ß-sheet content. Results of intrinsic Trp fluorescence suggested little change in Trp microenvironment of αB N146D relative to WT αB, but substantial alterations on deletion of COOH-terminal extension or a combination of this deletion plus deamidation. Hydrophobic binding studies using the hydrophobic probe 8-anilino-1-naphthalene sulfonate (ANS) showed that, relative to WT αB structure, the N146 deamidation, COOH-terminal extension deletion or a combination of this deamidation and deletion resulted in a relatively compact structure whereas the NH(2)-terminal domain deletion and a combination of this deletion plus deamidation resulted in a relaxed structure. All the αB mutants showed higher molecular mass ranging between 1.2×10(6) to 5.4×10(6) Da, relative to WT αB which had a molecular mass of 5.8×10(5) Da. Chaperone activity across all αB species decreased in the following order: WTαB > αB N146D-CT > αB N146D-NT > αB-NT > αB-CT > αB N146D. Specifically, substantial losses in chaperone activity (only 10% to 20% protection) were seen in αB N146D, αB-NT, and αB-CT. However, in the species with the combination of deamidation plus NH(2)- or COOH-terminal deletion, the percent protection was about 24% in αB N146D-NT and about 40% in αB N146D-CT. CONCLUSIONS: Although all mutants formed oligomers even after deamidation, on deletion of either NH(2)-terminal domain or COOH-terminal extension or a combination of these deletions and deamidation, their structural properties were substantially altered. The results suggested that the NH(2)-terminal domain is relatively more important than the COOH-terminal extension for the chaperone function of αB. The non-deamidated N146 residue, NH(2)-terminal domain and COOH-terminal extension are also of critical importance to the maintenance of αB-crystallin chaperone activity.

Structural and functional properties of NH(2)-terminal domain, core domain, and COOH-terminal extension of αA- and αB-crystallins.

PURPOSE: The purpose of the present study was to determine the biophysical and chaperone properties of the NH(2)-terminal domain, core domain and COOH-terminal extension of human αA- and αB-crystallins and correlate these properties to those of wild type (WT) αA- and αB-crystallins. METHODS: WT αA- and αB-crystallins cloned into pET 100D TOPO vector, were used as templates to generate different constructs encoding specific regions (NH(2)-terminal domain [NTD], core domain [CD], and COOH-terminal extension, [CTE]). The specific regions amplified by PCR using plasmid DNA from WT αA and WT αB were: αA NTD (residues 1-63), αA CD (residues 64-142), αA CTE (residues 143-173), αB NTD (residues 1-66), αB CD (residues 67-146), and αB CTE (residues 147-175). Resultant blunt-end PCR products were ligated to a pET 100 Directional TOPO vector. DNA sequencing results confirmed the desired constructs. Positive clones were transformed into the BL21 Star (DE3) expression cell line. Protein expression and solubility were confirmed by SDS-PAGE and western blot analysis using a monoclonal antibody against a 6× His-tag epitope. Proteins were purified using Ni(2+)-affinity column chromatography, under native or denaturing conditions, and used for biophysical and chaperone function analyses. RESULTS: A total of five constructs were successfully generated: αA NTD, αA CD, αB NTD, αB CD, and αB CTE. SDS-PAGE and western blot analyses showed that αA CD and αB CD were present in both the soluble and insoluble fractions, whereas mutant preparations with NTD alone became insoluble and the mutant with CTE alone became soluble. All purified constructs showed alterations in biophysical properties and chaperone function compared to WT α-crystallins. αA NTD and αB CTE exhibited the most notable changes in secondary structural content. Also, αA NTD and all αB-crystallin constructs showed altered surface hydrophobicity compared to their respective WT α-crystallins. CONCLUSIONS: Although the individual α-crystallin regions (i.e., NH(2)-terminal domain, core domain, and COOH-terminal extension) exhibited varied biophysical properties, each region alone retained some level of chaperone function. The NH(2)-terminal domains of αA and αB each showed the maximum chaperone activity of the three regions with respect to their WT crystallins.

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

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.