Characterization of ubiquitin-like polypeptide acceptor protein, a novel pro-apoptotic member of the Bcl-2 family

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Monoclonal nonspecific suppressor factor(MNSF) is a cytokine with antigen nonspecific suppressive activity. MNSFβ (a subunit of MNSF) is a 14.5-kDa fusion protein consisting of a protein with 36% identity with ubiquitin and ribosomal protein S30. The ubiquitin-like segment (Ubi-L) may be cleaved from MNSFβ in the cytosol. Recently, we have observed that Ubi-L covalently binds to intracellular proteins in mitogen-activated murine T helper type 2 clone, D.10 cells. In this study, we purified a 33.5-kDa Ubi-L adduct from D.10 cell lysates by sequential chromatography on DEAE, anti-Ubi-L antibody conjugated Sepharose, and hydroxylapatite. MALDI-TOF-Mass fingerprinting revealed that this Ubi-L adduct consists of 8.5-kDa Ubi-L and Bcl-2-like protein, murine orthologue of previously cloned human BCL-G gene product with pro-apoptotic function. Murine Bcl-G mRNA was highly expressed in testis and significantly in spleen. In addition, the level of Bcl-G mRNA expression was increased in concanavalin A- and interferon γ-activated D.10 cells. The 33.5-kDa-Ubi-L adduct was expressed in spleen but not in testis, even though Bcl-G protein was highly expressed in this tissue. The antisense oligonucleotide to Bcl-G significantly decreased the level of the Ubi-L-adduct formation in concanavalin A-activated D.10 cells and the proliferative response of the D.10 cells. These results suggest that the posttranslational modification of Bcl-G by Ubi-L might be implicated in T cell activation.