Molecular signature for senile and complicated cataracts derived from analysis of sumoylation enzymes and their substrates in human cataract lenses

Abstract Sumoylation is one of the key regulatory mechanisms in eukaryotes. Our previous studies reveal that sumoylation plays indispensable roles during lens differentiation (Yan et al. 2010. Proc Natl Acad Sci USA. 107:21034–21039; Gong et al. 2014. Proc Natl Acad Sci USA. 111:5574–5579). Whether sumoylation is implicated in cataractogenesis, a disease largely derived from aging, remains elusive. In the present study, we have examined the changing patterns of the sumoylation ligases and de‐sumoylation enzymes (SENPs) and their substrates including Pax6 and other proteins in cataractous lenses of different age groups from 50 to 90 years old. It is found that compared with normal lenses, sumoylation ligases 1 and 3, de‐sumoylation enzymes SENP3/7/8, and p46 Pax6 are clearly increased. In contrast, Ubc9 is significantly decreased. Among different cataract patients from 50s to 70s, male patients express more sumoylation enzymes and p46 Pax6. Ubc9 and SENP6 display age‐dependent increase. The p46 Pax6 displays age‐dependent decrease in normal lens, remains relatively stable in senile cataracts but becomes di‐sumoylated in complicated cataracts. In contrast, sumoylation of p32 Pax6 is observed in senile cataracts and increases its stability. Treatment of rat lenses with oxidative stress increases Pax6 expression without sumoylation but promotes apoptosis. Thus, our results show that the changing patterns in Ubc9, SENP6, and Pax6 levels can act as molecular markers for senile cataract and the di‐sumoylated p46 Pax6 for complicated cataract. Together, our results reveal the presence of molecular signature for both senile and complicated cataracts. Moreover, our study indicates that sumoylation is implicated in control of aging and cataractogenesis.


Animals
The study was performed using 4 weeks old C57BL/6J mice, 4 weeks old Sprague-Dawley (SD) rats, and 3 months old pigs. Mice were housed in standard cages in a specific pathogen-free facility of Sun Yat-sen University. The room was maintained on a 12h light-dark cycle at a constant temperature of 25 o C and 50% humidity and the animals were fed with commercial laboratory food and sterilized water. SD rats were purchased from Zhongshan Medical School of Sun Yat-sen University. The 3 months old pigs were raised in the animal facility of Hunan Normal University. In all the cases, animal protocols to use the above species were approved by the IACUC of Zhongshan Ophthalmic Center of Sun Yat-sen University.

Collection of lens capsular epithelial samples
Collection of human capsular epithelia from cataract lenses of different age groups was approved by the Institutional Review Board of the Zhongshan Ophthalmic Center (ZOC).
Informed consent was obtained from each of the cataract patients. For senile cataractous samples, the lens capsules from cataract patients were collected at surgery by the physicians in Zhongshan Ophthalmic Center of Sun Yat-sen University. According to the patient age, capsular samples from 50 to 59 years old were pooled together and labeled as 50s (Table S1); those from 60 to 69 years old were pooled together and labeled as 60s (Table S2); those from 70 to 79 years old were pooled together and labeled as 70s (Table S3), and those from 80 to 89 years old were pooled together and labeled as 80s (Table S4). For complicated cataractous samples, patients with cataract and other complications including diabetes, glaucoma or other syndromes (Table S5) were pooled together for the study described in figure 5. As control, the capsular samples from the lenses of human donors (two female individuals with age of 45 and 56, and one male individual with age of 65), adult mice, rats and pigs as described in the above section were dissected out in the laboratory.

Lens organ culture
The 4 weeks old C57BL/6J mice were sacrificed by CO2 inhalation. The eyeballs were removed and the lenses were carefully dissected by a posterior approach (Li et al., 1995, Li andSpector, 1996). Dissected lenses were placed in a 10-cm dish containing 20 ml meidium199, then incubated at 37 o C with a 5% CO2 gas phase for 12h. The medium 199 was prepared with ion-exchange double-distilled water and supplemented with 26mM NaHCO3, with a pH adjusted to 7.2, then sterilized by filtration through 0.22um filter.
After cultured for 12h, transparent lenses were selected for further experimentation.

Glucose oxidase (GO) treatment
For each sample, three transparent lenses were transferred into a 6-cm petri dish containing 7 ml of medium 199 supplemented with 10 mU glucose oxidase (GO) (Li et al., 1995;Gong et al., 2018), which continuously generates cytotoxic H2O2 in an average of 100 M in a 24 h period.

Apoptosis assays
The percentage of apoptosis in GO-treated mouse lenses were determined by cellTiter-Glo ® luminescent cell viability assay kit (G7573, Promega) (Crouch et al., 1993) and verified with live/dead viability/cytotoxicity kit (L3224,Thermofisher Scientific) according to the company instruction.

Total protein extraction and western blot analysis
Total proteins were extracted from cultured animal lenes with RIPA buffer (50 mM Tris·HCl (pH7.4), 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.1% SDS, 1% sodium deoxyholate) and homogenized as described below (Li et al., 1995;Li and Spector, 1996;Li et al, 2005;Yan et al., 200 and 2010;Gong et al., 2014 and2018). For various capsular samples of lens epithelium, each pooled capsular samples from 23 to 41 patients (see Table S1 to S5 for details), or from 20 animal lenses were transferred to an Eppendorf tube containing 200 l RIPA buffer and homogenized on ice with an Eppendorf tube micropestle (Brinkmann Instruments Inc.). For each sample, the protein concentration was determined as previously described Xiao et al., 2010).

Automated western immunoblotting
The simple western immunoblots were performed on a PeggySue (ProteinSimple) as previously described (Dahl et al. 2016). Briefly, each sample was loaded with 0.9 g total protein and then analyzed with the Size Separation Master Kit and Split Buffer (12-230 kDa) according to the manufacturer's standard instruction using the antibodies described above. The dilution factors are 1:100 for Pax6, Ubc9, Uba2, PIAS1, SENP3, SUMO2/3, and 1:50 for SENP1, SENP2, and SUMO1, as well as 1:20 for AOS1, RanBP2, SENP6, SENP7 and SENP8. The Campass software (Protein Simple, version 4.1.5) was used to program the PeggSue-robot and for presentation (and quantification) of the western blots. Output western blot style data were displayed with exposure time indicated, and the quantification data were displayed from the software-calculated average of seven exposures (1-512 s).

Protein solubility analysis
To compare protein solubility of human capsular epithelium samples from different age groups, total protein from each sample was first extracted as described above. The supernatant was considered as soluble protein. The remaining pellet was dissolved in modified protein extraction buffer with 5% CHAPs to replace 1% NP-40, and the suspended protein solutions were further sonicated on ice for 3 min, immersed in ice for 3 min. This cycle was repeated 5 times to help dissolving the pellet, the protein solution was then used for determination of protein concentration as described (Marshak et al. 1998). The protein solubility (Table S6) was calculated by dividing the initial soluble proteins with the total proteins from combination of two extractions. Samples displayed in red were used for automated western blot analysis.  Samples displayed in red were used for automated western blot analysis. Samples displayed in red were used for automated western blot analysis. Samples displayed in red were used for automated western blot analysis.   according to the manufacturer's standard instruction using anti-SENP7 antibody (for antibody information, see Experimental Procedures) with a dilution factor of 1:20. The