Analysis of Non-Sumoylated and Sumoylated Isoforms of Pax-6, the Master Regulator for Eye and Brain Development in Ocular Cell Lines

Title:Analysis of Non-Sumoylated and Sumoylated Isoforms of Pax-6, the Master Regulator for Eye and Brain Development in Ocular Cell Lines

Volume: 18 Issue: 8

Author(s): Fangyuan Liu, Ling Wang, Jia-Ling Fu, Yuan Xiao, Xiaodong Gong, Yunfei Liu, Qian Nie, Jia-Wen Xiang, Lan Yang, Zhigang Chen, Yizhi Liu*David Wan-Cheng Li*

Affiliation:

• The State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, #7 Jinsui Road, Guangzhou, Guangdong 510230,China

• The State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, #7 Jinsui Road, Guangzhou, Guangdong 510230,China

Keywords: Pax-6, Sumoylation, p68, p57, p48, p46, p43, p32 Pax-6.

Abstract: Purpose: Pax-6 is a master regulator for eye and brain development. Previous studies including ours have shown that Pax-6 exists in 4 major isoforms. According to their sizes, they are named p48, p46, p43 and p32 with the corresponding molecular weight of 48, 46, 43 and 32 kd, respectively. While p48 and p46 is derived from alternative splicing, p32 Pax-6 is generated through an internal translation initiation site. As for 43 kd Pax-6, two resources have been reported. In bird, it was found that an alternative splicing can generate a p43 Pax-6. In human and mouse, we reported that the p43 kd Pax-6 is derived from sumoylation: addition of a 11 kd polypeptide SUMO1 into the p32 Pax-6 at the K91 residue. Whether other Pax-6 isoforms can be sumoylated or not remains to be explored.

Methods: The 5 major ocular cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing fetal bovine serum (FBS) or rabbit serum (RBS) and 1% Penicillin- Streptomycin. The mRNA levels were analysed with qRT-PCR. The protein levels were determined with western blot analysis and quantitated with Image J.

Results: Both non-sumoylated and sumoylated isoforms of Pax-6 exist in 6 major types of ocular cells among which five are lens epithelial cells, and one is retinal pigment epithelial cell. Our results revealed that the most abundant isoforms of Pax-6 are the p32 and p46 Pax-6. These two major isoforms can be sumoylated to generate p43 (mono-sumoylated p32 Pax-6), p57 and p68 Pax-6 (mono- and di-sumoylated p46 Pax-6). In addition, the splicing-generated p48 Pax-6 is also readily detected.

Conclusion: Our results for the first time, have determined the relative isoform abundance and also the sumoylation patterns of pax-6 in 6 major ocular cell lines.

Cite this article as:

Liu Fangyuan , Wang Ling , Fu Jia-Ling , Xiao Yuan , Gong Xiaodong , Liu Yunfei , Nie Qian , Xiang Jia-Wen , Yang Lan , Chen Zhigang , Liu Yizhi *, Li Wan-Cheng David *, Analysis of Non-Sumoylated and Sumoylated Isoforms of Pax-6, the Master Regulator for Eye and Brain Development in Ocular Cell Lines, Current Molecular Medicine 2018; 18 (8) . https://dx.doi.org/10.2174/1566524019666190111153310