In previous studies, we reported the presence of a large number of low-molecular-weight (LMW) peptides in aged and cataract human lens tissues. Among the LMW peptides, a peptide derived from αA-crystallin, αA66-80, was found in higher concentration in aged and cataract lenses. Additional characterization of the αA66-80 peptide showed beta sheet signature, and it formed well-defined unbranched fibrils. Further experimental data showed that αA66-80 peptide binds α-crystallin, impairs its chaperone function, and attracts additional crystallin proteins to the peptide α-crystallin complex, leading to the formation of larger light scattering aggregates. It is well established that Aβ peptide exhibits cell toxicity by the generation of hydrogen peroxide. The αA66-80 peptide shares the principal properties of Aβ peptide. Therefore, the present study was undertaken to determine whether the fibril-forming peptide αA66-80 has the ability to generate hydrogen peroxide. The results show that the αA66-80 peptide generates hydrogen peroxide, in the amount of 1.2 nM H2O2 per µg of αA66-80 peptide by incubation at 37°C for 4h. We also observed cytotoxicity and apoptotic cell death in αA66-80 peptide-transduced Cos7 cells. As evident, we found more TUNEL-positive cells in αA66-80 peptide transduced Cos7 cells than in control cells, suggesting peptide-mediated cell apoptosis. Additional immunohistochemistry analysis showed the active form of caspase-3, suggesting activation of the caspase-dependent pathway during peptide-induced cell apoptosis. These results confirm that the αA66-80 peptide generates hydrogen peroxide and promotes hydrogen peroxide-mediated cell apoptosis.
Figure 1. Hydrogen peroxide generation by crystallin derived peptides and β-amyloid peptide. A) Generation of H2O2 by αA66-80 peptide. αA66-80 peptide (1mg/mL) was incubated in 50mM phosphate buffer for different durations, up to 24 h. The sample was withdrawn every hour and H2O2 generation was monitored using Amplex red reagent. Closed circle, αA66-80 peptide; open circle, αA66-80pro. Inset shows the generation of H2O2 up to 24 h (avarage of three experiments). B) Relative amount of H2O2 generation by αA66-80 peptide, β-amyloid and α-crystallin. Assays were carried out in 50 mM phosphate buffer. H2O2 generation is presented in nano Molar scale. Data shown is the avarage of three independent experiments. H2O2 generation by αA66-80 peptide in the presence or absence of αA-crystallin or metal ion chelators or catalase is also shown in the figure. C) Comparision of H2O2 generation by LMW peptides isolated from 73-, 43- and 17-year-old human lenses. LMW peptides were isolated and assayed as described in methods section. The data obtained by Amplex red assay show a greater amount of H2O2 generation from LMW peptides of 73-year-old lenses. (The results shown are an avarage of three independent experiments.)
Proteins / peptides (100 µg)
H2O2 generation (nM)
Total lens extract (43-45yrs.)
5.7 + 0.9
11.3 + 1.2
6.1 + 0.2
7.5 + 0.2
6.7 + 0.3
0.5 + 0.0
0.2 + 0.0
LMW peptides from trypsin-digested α-fraction
208.1 + 6.4
LMW peptides from trypsin-digested βH -fraction
156.2 + 6.2
LMW peptides from trypsin-digested βL -fraction
184.4 + 7.9
LMW peptides from trypsin-digested γ -fraction
150.0 + 4.3
LMW peptides from trypsin-digested αA-crystallin (recombinant)
29.8 + 0.2
LMW peptides from trypsin-digested BSA
3.2 + 0.0
127 ± 12
4.5 + 0.3
Table 1 Hydrogen peroxide generation by lens crystallin fractions and crystallin-derived peptides*
Figure 2. A-D Nanosprary QTOF spectra of peptides with and without copper in aqueous solution. A) Peptide αA66-80, B) Peptide αA66-80 in presence of excess copper sulphate, C) Peptide αA66-80 (H79A), D) Peptide αA66-80 (H79A) in presence of excess copper sulphate. The αA66-80 peptide can bind upto 2 copper (peak at 1986.81 Da). The two-copper binding peak is suppressed in αA66-80 H79A peptide. The results suggest histidine role in copper binding and subsequent H2O2 generation by αA66-80 peptide. E) Generation of H2O2 by αA66-80 peptide in presence of different metal ions added to 50 mM Phoshpahte buffer, pH 7.2. To test whether addition of metal ions can increase the genration H2O2 by αA66-80 peptide, we incubated the peptide either alone or with Cu (11), Fe (III) or Zn(II), in 1 nM or 10 nM, for 4 hr in PO4 buffer. At the end of incubation, Amplex Red reagent was added and the mixtures were further incubated for 30 min in the dark at room temperature. The fluorescent intensities of the reaction mixtures were measured as mentioned in the method.
Figure 3. αA66-80 peptide--induced cell apoptosis and caspase-3. Peptide-induced cell apoptosis was assessed in the presence and absence of αA66-80 peptide or proline-substituted peptide (αA66-80 pro). A-C and G) TUNEL assay, in which the blue stain shows intact nucleus and the red stain indicates fragmented DNA inside the nucleus indicating cell apoptosis. D-F and H) Caspase-3 assay, in which the blue stain shows the nucleus and the green stain shows cleaved caspase-3 antibody reactivity. Note that most of the green stain is in the cytoplasmic region. I) Effect of catalase on Cos-7 cells treated with αA66-80 peptide. Cos-7 cells were cultured and treated as described under methods. Cells were stained with live/dead cell staining according to the EarlyTox live/dead cell potocol using Spectra Max i3 plate reader. αA66-80 (10µg); Catalase (500 units) and αA66-80(10µg) +Catalase (500 units) (The result shown are the avarage of three independent experiments.)
Figure 4. Entry of αA66-80 into cells. Pig primary LECs were treated with αA66-80-FITC for 24 h and the entry of fluorescent peptide (green) into the cells was visualized by observing the cells under fluorescent microscope after counter staining with DAPI (blue - nucleus) and Phalloidin (red - actin). Left image shows the composite of blue and green channels and the right image is a composite of all three channels.
Figure 5. Peptide αA66-80 forms amyloid-like fibrils. The peptide αA66-80 was incubated in phosphate buffer at 37°C. The samples were withdrawn at different time intervals (0 hr, 4 hr and 24 hr) and the fibril-like structure of αA66-80 peptides was visualized and recorded under TEM. A) 0 hr; B) 4 h; and C) 24 h incubations. The scale bar is 100 µm.
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