The ubiquitin-like protein monoclonal nonspecific suppressor factor β conjugates to endophilin II and regulates phagocytosis

Abstract

Monoclonal nonspecific suppressor factor β (MNSFβ) is a ubiquitously expressed member of the ubiquitin-like family that has been implicated in various biological functions. Previous studies have demonstrated that MNSFβ covalently binds to the intracellular proapoptotic protein Bcl-G in cells of the macrophage cell line Raw264.7, suggesting involvement of this ubiquitin-like protein in apoptosis. In this study, we purified a 62 kDa MNSFβ adduct from murine liver lysates by sequential chromatography on DEAE and anti-MNSFβ IgG-conjugated Sepharose. MALDI-TOF MS fingerprinting revealed that this MNSFβ adduct consists of an 8.5 kDa MNSFβ and endophilin II, a member of the endophilin A family. MNSFβ may conjugate to endophilin II with a linkage between the C-terminal Gly74 and Lys294. We confirmed this result by immunoprecipitation/western blot studies. Endophilin II was ubiquitously expressed in various tissues, although a truncated form was observed in liver. The 62 kDa MNSFβ-endophilin II was specifically expressed in liver and macrophages. Small interfering RNA-mediated knockdown of endophilin II and/or MNSFβ promoted phagocytosis of zymosan in Raw264.7 cells. Conversely, cotransfection of endophilin II and MNSFβ cDNAs inhibited the phagocytosis of zymosan. Such inhibition was not observed in cells expressing a mutant of endophilin II in which Lys294 was replaced by arginine. These results suggest that the post-translational modification of endophilin II by MNSFβ might be implicated in phagocytosis by macrophages. © 2009 FEBS.