Through literature research, we found that glutathione (GSH) and 25-hydroxycholesterol (25HC) should prevent and treat cataracts, respectively. We wanted to first see if we could simulate cataract formation and reproduce the effects of GSH and 25HC seen by other researchers. If successful, then we can later test our own constructs using the same cataract model.
We set up the model by extracting soluble proteins from the lens of Priacanthus macracanthus, a common freshwater fish that we purchased from a market (Figure 01 left). There are two distinct parts to the lens: an outer soft layer called the cortex and an inner layer called the nucleus (Figure 01 right). We focused on the lens nucleus, because that part contains older cells and is more prone to cataract formation. The lens nucleus was placed into Tris Buffer and gently shaken overnight. After centrifuging, the supernatant contains dissolved protein from the lens nucleus. We then incubated the lens solution with hydrogen peroxide (H2O2), since H2O2 is the main reactive oxygen species that oxidizes lens proteins and induces cataracts.
Figure 1 (left). Priacanthus macracanthus purchased from the market. (right) The two spheres on the left are lens with cortex and nucleus and the two smaller spheres on the right are nucleus.
To quantify the severity of cataracts in our model, we used spectrophotometer to measure absorbance of the protein solution (Figure 03). As lens proteins get oxidized, absorbance should increase because protein clumps that form will scatter the emitted light. To find a wavelength of light for data collection, we compared the absorbance of an untreated proteins, H2O2-treated proteins, and heat-denatured proteins as a positive control. We observed an absorbance peak at 397.5 nm for insoluble lens proteins, and chose to collect all future absorbance values at that wavelength.
Figure 3. CT-2400 Spectrophotometer used to measure absorbance value
We tested different concentrations of H2O2 on this protein solution to see if cataracts form. Our results show that increasing concentrations of H2O2 lead to more severe cataracts (Figure 04). We also ran a protein gel to compare the sizes of untreated and H2O2-treated proteins. After treatment with H2O2, there was an increase in higher bands, which is consistent with the idea that proteins are clumping and aggregating (Figure 05). Together, the protein gel and our lens cataract model suggest that our model accurately represents cataract development.
Figure 4. Percent change in absorbance of lens solutions treated with H2O2 Figure 5. SDS Page. The left lane is lens protein solution seated in room temperature for two days. The middle lane is lens protein solution treated with 10mM of H2O2 for two days. The right lane is the molecular ladder.
Testing Prevention of Cataracts with GSH
GSH is the main antioxidant in the lens and prevents H2O2 from oxidizing crystallin proteins. After GSH converts H2O2 into water, it becomes GSSG, which will be recycled back to GSH with the help of glutathione reductase (GSR) (figure 06). Older cells in the nucleus are unable to produce GSR efficiently, so over time GSSG builds up. Our project is to deliver the GSR into the lens in order to facilitate the conversion of GSSG to GSH. We purchased GSH from Sigma Aldrich. 8 mg of GSH was added to the protein solution prior to the addition of H2O2 .
Figure 6. Glutathione and Reduced Glutathione recycling pathway
Figure ???. Caption not provided!!!
Protein solutions with GSH and H2O2 have an absorbance value lesser than those with just protein solutions with H2O2 . The smaller absorbance value indicates smaller protein aggregation and shows GSH has preventative effect. (figure #)
Testing Treatment of Cataracts with CH25H
We select 25 HC (25 hydroxycholesterol) to be our treatment because it reverses aggregation and restores solubility of the lens crystallin protein by stabilizing the natural state of the proteins.
In order to verify our research, we use commercially bought 25 HC from Sigma Aldrich to treat the cataracts model. Commercial 25HC came in powder form, and was dissolved in 95% ethanol; thus, for our negative control, we added both H2O2 and ethanol into the protein solution. We also bought vet eyedrops for cataracts from OcluVet to be the positive control (Figure 1). Figure 2 shows the setup of the experiment.
Figure 1. The Ocluvet vet eyedrop contains antioxidants that can treat cataracts. We used this eyedrop as our positive control for cataracts treatment.
Figure 2. Lens Treatment Experiment after 48hrs. (left) Control, Vet eyedrop treated cataracts solution, 50uM 25HC treated cataracts solution, 20uM 25HC treated cataracts solution. Each treatment is added after adding H2O2 for 24 hours.
Two sets of experiments were conducted to prove that 25 HC treats cataracts. For the first set, we added H2O2 into fresh fish lens protein solution, and waited for 24 hours before adding the treatment. After adding 25HC into the cataracts model, we measured the absorbance of the treated protein solutions with a UV spectrophotometer in 24 hours increments (F3). We used Tris buffer to blank the UV spectrophotometer before measuring the absorbances for the 25HC treated tubes, and since the vet eyedrop is yellow, we blanked the spectrophotometer with Tris buffer containing vet eyedrop before measuring vet eyedrop treated cataracts solution. Absorbance increases due to increase of opaqueness of the protein solution. Thus, we assume that, the higher the absorbance, the more cataracts is formed due to protein aggregation.
Figure 4. Percent change in absorbance of the treated cataracts lens protein solution after 48 hours
As shown in Figure 4, absorbance values increased when lens proteins were incubated with H2O2 for 48 hours. Adding vet eyedrops did not lower the absorbance as much as treatment with 25HC, suggesting that 25HC is more effective at reducing cataracts. In addition, a higher concentration of 25HC lowered the absorbance even further (50 μM compared to 20 μM). Two trials were conducted and the error bars show that the difference between untreated and 25HC-treated proteins is significant.
Construct Design
Prevention: GSR-HIS
We want to express glutathione reductase (GSR) to catalyze the reduction of GSSG to GSH, the main antioxidant in the lens. This will prevent crystallin proteins from being oxidized by H2O2 . We also want to extract the proteins produced by E. coli so that no bacteria is used in our final delivery prototype. Thus, we include a histidine tag behind the gene we want to express. Proteins with a histidine tag can be detected and purified.
Figure X. Full Construct.
Prevention: GSR-HIS
The purchased GSR cDNA has two internal PstI and three EcoRI cutting sites. After making silent mutations to the sequence, we sent the cDNA to MissionBiotech for mutagenesis to remove these cutting sites. Once we had the correct sequence of GSR (with 5 point mutations), we designed primers to add the BioBrick prefix and suffix in order to clone GSR into a BioBrick backbone. The primer designs were sent to Tri-I Biotech for oligo synthesis, and polymerase chain reaction (PCR) was set up. We first cloned GSR behind BBa_K880005 (strong promoter + strong ribosome binding site; Figure AA), then front-inserted this before a new plasmid (Figure BB) containing 10x Histidine tag (BBa_K844000) and double terminator (BBa_B0015). The sequence of the full construct was confirmed by sequencing (Tri-I Biotech).
Figure BB. His + term cloning
Figure AA. 1kb ladder, GSR alone, samples after are K880005 + GSR PCR checks. Boxed bands are correct (slightly higher than GSR alone)
Treatment
We also found a molecule that can restore solubility of protein clumps and lens transparency. It is called 25-hydroxycholesterol (25HC) (Makley et al., 2015). 25HC can be produced from cholesterol, which is abundant in the lens, by the enzyme cholesterol 25-hydroxylase (CH25H). We produced CH25H for delivery into the lens and treatment of cataracts.
Figure ???. Caption not provided!!!
Citations
Prevention
GSR Eyedrop
Treatment
25HC Eyedrop
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Eyedrops
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