Cleavage of RXRalpha by a lysosomal enzyme, cathepsin L-type protease

In this study, we characterized a protease responsible for the cleavage of RXRalpha in two human derived cell lines, HepG2 and JEG-3 cells. Electrophoretic mobility shift assay (EMSA) combined with antibody supershift analysis suggested that contamination of cytoplasmic components during nuclear extract preparation could result in complete cleavage of RXRalpha at its N-terminus in JEG-3 cells, while such proteolytic activity was much less evident in HepG2. When the nuclei were purified in the presence of leupeptin, only full-length RXRalpha was found in the extracts prepared from both JEG-3 and HepG2 cells, suggesting a member of cysteine protease family is responsible for the cleavage. The presence of the protease in the cytoplasm, but not in the nucleus, was confirmed by incubating full-length 35S-labeled RXRalpha with each fraction. The cytoplasmic fraction from JEG-3 and HepG2 cells cleaved RXRalpha into smaller sizes with molecular mass of 45, 43, and 31 kD. Immunoprecipitation with antibodies recognizing distinct epitopes indicated that the cleaved RXRalpha with the size of 45 and 43 kD were truncated at N-terminus in which most of the A/B domain was absent. Using a series of protease inhibitors, the enzyme cleaving RXRalpha was characterized as cathepsin L-type protease. The enzyme activity in JEG-3 cells was much higher than that in HepG2 cells. This is the first demonstration that RXRalpha is cleaved by a lysosomal enzyme, cathepsin L-type protease.