Sequence confirmation and characterization of the mouse Ssxa gene: Ssxa protein is cleaved and the N-terminal cleaved fragment translocates into the nucleus.

This is the first demonstration of a stop codon in the sequence of mouse Ssxa and characterization of the biological behavior of Ssxa protein. Cancer testis antigen (CTA) is known as a target of immunotherapy against cancer, and Ssxa is one of the CTAs. Although a CTA would be useful to establish a mouse cancer vaccine model using endogenous antigen, the stop codon was not identified in the sequence of Ssxa cDNA that was previously reported. In this study, the gene sequence of Ssxa was different from the previous report in which several mouse CTAs were analyzed. Initially, we identified the correct cDNA sequence of mouse Ssxa by 3'-rapid amplification of cDNA ends and found a new exon containing the stop codon (Exon X). Ssxa mRNA expression was determined by reverse transcription-PCR (RT-PCR) in four mouse cancer cell lines and the testis but not in other normal organs. We found that the molecular weight of recombinant Ssxa protein is 12 kDa, and we generated an anti-Ssxa antibody which recognizes the C-terminus of Ssxa. Two vectors expressing fusion proteins (pSsxa-GFP and pGFP-Ssxa) were generated and fluorescence in the nucleus was observed only in the pGFP-Ssxa transfected cells. Therefore, we conclude that the N-terminal cleaved fragment of Ssxa, which has a KRAB domain (nuclear localization signal), translocates into the nucleus after cleavage of the C-terminus.