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 human NB cells.


RESULTS
In this study, we purified NB-p260 to homogeneity from human LA-N-1 NB cells by sequential ion exchange chromatography followed by preparative SDS gel electrophoresis. Purified NB-p260 exhibited rapid autodegradation despite the presence of various protease inhibitors. The autodegradation process precluded extensive N-terminal sequencing. However, 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 membrane 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 subunit c, NB-p260 also contained epitopes related to the human heat shock protein HSP90. In Western blots, a monoclonal anti-HSP90 antibody reacted with purified NB-p260 as well as with a predominant protein fragment of approximately 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-dependent manner.


CONCLUSIONS
Collectively, our data suggest that NB-p260, the apoptosis-mediating receptor for natural human anti-NB IgM, represents a novel surface protein of human NB cells containing polypeptide sequences related to the subunit c of the mitochondrial ATP synthase and the heat shock protein HSP90.
David
K
K
Department of Biochemistry and Molecular Biology, University of Hamburg, Germany. kdavid@crch.hawaii.edu
Heiligtag
S
S
Ollert
M W
MW
Teppke
M
M
Vogel
C W
CW
Bredehorst
R
R
D000818:Animals; D000912:Antibodies, Neoplasm / Q000276:immunology*; D000918:Antibody Specificity; D000951:Antigens, Neoplasm / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D017209:Apoptosis / Q000276:immunology*; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry; D006801:Humans; D007106:Immune Sera; D007113:Immunity, Innate; D007075:Immunoglobulin M / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009447:Neuroblastoma / Q000276:immunology / Q000473:pathology*; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D011480:Protease Inhibitors / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000737:chemistry; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured / Q000276:immunology
D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D018841:HSP90 Heat-Shock Proteins; D007106:Immune Sera; D007075:Immunoglobulin M; C120894:NB-p260 antigen; D010446:Peptide Fragments; D011480:Protease Inhibitors; D006180:Proton-Translocating ATPases