眼细胞系中眼和脑发育的主调控因子 Pax-6 的非 sumoylated 和 sumoylated 异构体分析。

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

机构信息

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

出版信息

Curr Mol Med. 2018;18(8):566-573. doi: 10.2174/1566524019666190111153310.

DOI:10.2174/1566524019666190111153310

PMID:30636604

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.

摘要

目的

Pax-6 是眼睛和大脑发育的主要调节因子。包括我们在内的先前研究表明，Pax-6 存在 4 种主要的异构体。根据它们的大小，分别命名为 p48、p46、p43 和 p32，相应的分子量为 48、46、43 和 32 kd。p48 和 p46 是通过选择性剪接产生的，而 p32 Pax-6 则是通过内部翻译起始位点产生的。至于 43 kd Pax-6，已经有两种来源的报道。在鸟类中，发现一种选择性剪接可以产生 p43 Pax-6。在人和小鼠中，我们报道 p43 kd Pax-6 是通过 SUMO 化产生的：在 K91 残基处将一个 11 kd 的多肽 SUMO1 添加到 p32 Pax-6 中。其他 Pax-6 异构体是否可以 SUMO 化还有待探索。

方法

在含有胎牛血清（FBS）或兔血清（RBS）和 1%青霉素-链霉素的 Dulbecco 修改的 Eagle 培养基（DMEM）中培养 5 种主要的眼部细胞系。通过 qRT-PCR 分析 mRNA 水平。通过 Western blot 分析测定蛋白质水平，并使用 Image J 定量。

结果

非 SUMO 化和 SUMO 化的 Pax-6 异构体存在于 6 种主要的眼部细胞类型中，其中 5 种是晶状体上皮细胞，1 种是视网膜色素上皮细胞。我们的结果表明，Pax-6 最丰富的异构体是 p32 和 p46 Pax-6。这两种主要的异构体可以 SUMO 化生成 p43（单 SUMO 化的 p32 Pax-6）、p57 和 p68 Pax-6（单 SUMO 化和双 SUMO 化的 p46 Pax-6）。此外，还容易检测到由剪接产生的 p48 Pax-6。