Initial characterization of the apoptosis-inducing receptor for natural human anti-neuroblastoma IgM

Background Human neuroblastoma (NB) cells contain a 260 kDa surface antigen (NB-p260), which serves as receptor for natural human IgM antibodies (anti -NB IgM). Upon binding to NB-p260, these antibodies induce apoptosis in hum an NE cells. Procedure and Results, In this study, we purified NB-p260 to h omogeneity from human LA-N-1 NE cells by sequential ion exchange chromatogr aphy followed by preparative SDS gel electrophoresis. Purified NB-p260 exhi bited rapid autodegradation despite the presence of various protease inhibi tors. The autodegradation process precluded extensive N-terminal sequencing . How ever, from repeat N-terminal sequence analysis, a consensus sequence of seven amino acid residues emerged that exhibited significant homology to the subunit c of the human mitochondrial ATP synthase, a hydrophobic membr ane protein of 7.6 kDa. Western blot analyses demonstrated that purified NB -p260 was recognized by polyclonal antibodies raised against both subunit c -containing storage bodies and a synthetic peptide consisting of amino acid residues 32-45 of subunit c. In addition to peptide sequences related to s ubunit c, NB-p260 also contained epitopes related to the human heat shock p rotein HSP90. In Western blots, a monoclonal anti-HSP90 antibody reacted wi th purified NB-p260 as well as with a predominant protein fragment of appro ximately 90 kDa that appeared during the process of NB-p260 autodegradation . The anti-HSP90 antibody was also capable of binding to the surface of LA- N-1 cells and inhibiting the binding of human anti-NB IgM in a dose-depende nt manner. Conclusions. Collectively, our data suggest that NB-p260, the ap optosis-mediating receptor for natural human anti-NE IgM, represents a nove l surface protein of human NE cells containing polypeptide sequences relate d to the subunit c of the mitochondrial ATP synthase and the heat shock pro tein HSP90. Med. Pediatr. Oncol. 36: 251-257, 2001. (C) 2001 Wiiey-Liss, In c.