Conjugation of ubiquitin-like polypeptide to intracellular acceptor proteins

Abstract Monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by a murine hybridoma, was originally found to inhibit the generation of LPS-induced immunoglobulin secreting cells. MNSF comprises of MNSFβ, an isoform of MNSF, and the other isoform, MNSFα. Ubiquitin-like segment (Ubi-L) of MNSFβ shows MNSF-like activity. Ubi-L (7.8 kDa) has 36% homology with 8.5 kDa ubiquitin. GST–Ubi-L was labeled with 125 I by the chloramine T method and tested for its conjugation to acceptor proteins in splenocyte lysates. 125 I –GST–Ubi-L conjugation on SDS–PAGE showed heterogeneous bands including 95 kDa GST–Ubi-L conjugation in the splenocyte, but not reticulocyte lysates. The Ubi-L adduct appeared to be MNSF-related molecule because anti-MNSF monoclonal antibody (mAb) recognized the 95 kDa band. The pattern of the conjugations was different from that seen in ubiquitination. Unlabeled GST–Ubi-L inhibited the conjugations, while ubiquitin did not. α-Lactalbumin, one of the target proteins for ubiquitination, failed to conjugate to GST–Ubi-L. In addition, covalent conjugation of ubiquitin to reticulocyte lysates was also interfered by GST–Ubi-L. These results suggest that Ubi-L may conjugate to acceptor proteins in a similar, but not in the same way as ubiquitination, and might play an important role in lymphoid cells.