Purpose: It is now well established that protein sumoylation acts as an important
regulatory mechanism modulating functions over three thousand proteins. In the vision system,
protein conjugation with SUMO peptides can regulate differentiation of multiple ocular tissues. Such
regulation is often explored through analysis of biochemical and physiological changes with various
cell lines in vitro. We have recently analyzed the expression levels of both mRNAs and proteins for
seven de-sumoylation enzymes (SENPs) in five major ocular cell lines. In continuing the previous
study, here we have determined their cellular localization of the seven de-sumoylation enzymes
(SENP1, 2, 3, 5, 6, 7 and 8) in the above 5 major ocular cell lines using immunocytochemistry.
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 localization of the 7 major de-sumoylation enzymes (SENPs) in the 5 major
ocular cell lines were determined with immunohistochemistry. The images were captured with a
Zeiss LSM 880 confocal microscope.
Results: 1) The SENP1 was localized in both cytoplasm and nucleus of 3 human ocular cell lines,
FHL124, HLE and ARPE-19; In N/N1003A and αTN4-1, SENP 1 was more concentrated in the
cytoplasm. SENP1 appears in patches; 2) SENP2 was distributed in both cytoplasm and nucleus
of all ocular cell lines in patches. In HLE and ARPE-19 cells, SENP2 level was higher in nucleus
than in cytoplasm; 3) SENP3 was almost exclusively concentrated in the nuclei in all ocular cells
except for N/N1003A cells. In the later cells, a substantial amount of SENP3 was also detected in
the cytoplasm although nuclear SENP3 level was higher than the cytoplasmic SENP3 level.
SENP3 appeared in obvious patches in the nuclei; 4) SENP5 was dominantly localized in the
cytoplasm (cellular organelles) near nuclear membrane or cytoplasmic membrane ; 5) SENP6 was
largely concentrated in the nuclei of all cell lines except for αTN4-1 cells. In the later cells, a
substantial amount of SENP6 was also detected in the cytoplasm although nuclear SENP6 level
was higher than the cytoplasmic SENP6 level. 6) SENP7 has an opposite localization pattern
between human and animal cell lines. In human cell lines, a majority of SENP7 was localized in
nuclei whereas in mouse and rabbit lens epithelial cells, most SENP7 was distributed in the
cytoplasm. SENP8 was found present in human cell lines. The 3 human ocular cell lines had
relatively similar distribution pattern. In FHL124 and ARPE-19 cells, SENP8 was detected only in
the cytoplasm, but in HLE cells, patches of SENP8 in small amount was also detected in the nuclei.
Conclusions: Our results for the first time defined the differential distribution patterns of seven desumoylation
enzymes (SENPs) in 5 major ocular cell lines. These results help to understand the
different functions of various SENPs in maintaining the homeostasis of protein sumoylation patterns
during their functioning processes.