Analysis of the Differential Expression Patterns of Sumoylation Enzymes E1, E2 and E3 in Ocular Cell Lines

Purpose: Protein sumoylation is a well established regulatory mechanism to control many cellular processes such as chromatin structure dynamics, transcriptional regulation of gene expression, cell proliferation and differentiation, cell transformation and carcinogenesis, autophagy and senescence. In the vertebrate vision system, we and others have revealed that sumoylation plays important roles in regulating differentiation of several ocular tissues during eye development. To further elucidate the functional mechanisms of sumoylation, in vitro assay systems are needed. Currently, the five major cell lines including alpha TN4-1, FHL124, HLE, N/N1003A and ARPE-19 have been extensively used to test the biochemical and molecular aspects of normal vision physiology and various disease processes. Thus, we conducted the study on the expression patterns of the three types of sumoylation enzymes, the activating enzymes SAE1 and UBA2, the conjugating enzyme UBC9, and the ligating enzymes such as RanBP2 and PIAS1 in these ocular cell lines. 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: we have obtained the following results: 1) For the mRNAs encoding E1 SAE1 and UBA2, E2 UBC9 and E3 PIAS1, the highest level of expression was observed in alpha TN4-1 cells; For the mRNA encoding RanBP2, the highest level of expression was detected in N/N1003A cells; 2) In contrast to the mRNA expression patterns, a similar level of the SAE1 protein was observed in the all five cell lines, and so is true with UBA2 protein in all cells except for N/N1003A where over fourfold of enrichment in UBA2 protein was observed compared with other cell lines; 3) A similar level of UBC9 protein was also detected in all cells except for N/N1003A where more than one-fold of decrease in UBC9 level was found compared with other cell lines; 4) For E3 ligases, we did not identify the regular PIAS1 band in N/N1003A cells, the remaining cells have a level of PIAS1 with difference of less than 0.6-fold; all cells except for FHL124 cells have a similar level of RanBP2, and a 70% drop in RanBP2 was observed in FHL124 cell. Conclusions: Our determination of the differential expression patterns of the three types of sumoylation enzymes in the 5 ocular cell lines help to understand sumoylation functions in vertebrate eye.