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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) Ig–conjugated Sepharose, and hydroxylapatite. MALDI‐TOF‐MS fingerprinting revealed that this Ubi‐L adduct consists of an 8.5 kDa Ubi‐L and a Bcl2‐like protein, murine orthologue of a 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 post‐translational modification of Bcl‐G by Ubi‐L might be implicated in T‐cell activation.

The monoclonal nonspecific suppressor factor (MNSF) is a lymphokine product of a murine T-cell hybridoma that inhibits the generation of lipopolysaccharideinduced immunoglobulin-secreting cells in an antigennonspecific manner. A cDNA clone encoding MNSFj3 (an isoform of MNSF) was isolated and expressed in bacteria. The sequence obtained is virtually identical to the Fau protein, a product of the ubiquitously expressedfau gene with unknown function. Northern blot analysis demonstrated a single, 0.6-kb transcript. Specific polyclonal antibodies against synthetic peptides corresponding to the deduced amino acid sequences were elicited in rabbits. Immunoprecipitation experiments with these antibodies showed that MNSFj3 is released extracellularly in an aggregate form, albeit it lacks a signal peptide sequence. The anti-MNSFp affinity eluate from the MNSFproducing murine hybridoma (E17) and concanavalin A-activated splenocyte culture supernatants inhibited the immunoglobulin production by lipopolysaccharide-activated splenocytes. Recombinant MNSF.6 also showed a similar biologic activity. Thus, ubiquitin-like protein(s) may be involved in the regulation of the immune responses.

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