title: Cell surface-bound heat shock protein 70 (Hsp70) mediates perforin-independent apoptosis by specific binding and uptake of granzyme B.
abstract: Cell surface-bound heat shock protein 70 (Hsp70) renders tumor cells more sensitive to the cytolytic attack mediated by natural killer (NK) cells. A 14-amino acid Hsp70 sequence, termed TKD (TKDNNLLGRFELSG, aa450-463) could be identified as the extracellular localized recognition site for NK cells. Here, we show by affinity chromatography that both, full-length Hsp70-protein and Hsp70-peptide TKD, specifically bind a 32-kDa protein derived from NK cell lysates. The serine protease granzyme B was uncovered as the 32-kDa Hsp70-interacting protein using matrix-assisted laser desorption ionization time-of-flight mass peptide fingerprinting. Incubation of tumor cells with increasing concentrations of perforin-free, isolated granzyme B shows specific binding and uptake in a dose-dependent manner and results in initiation of apoptosis selectively in tumor cells presenting Hsp70 on the cell surface. Remarkably, Hsp70 cation channel activity was also determined selectively in purified phospholipid membranes of Hsp70 membrane-positive but not in membrane-negative tumor cells. The physiological role of our findings was demonstrated in primary NK cells showing elevated cytoplasmic granzyme B levels following contact with TKD. Furthermore, an increased lytic activity of Hsp70 membrane-positive tumor cells could be associated with granzyme B release by NK cells. Taken together we propose a novel perforin-independent, granzyme B-mediated apoptosis pathway for Hsp70 membrane-positive tumor cells.
authors.affiliation: University Hospital Regensburg, Department of Hematology, 93053 Regensburg, Germany.
authors.forename: Catharina
authors.initials: C
authors.lastname: Gross
authors.forename: Walter
authors.initials: W
authors.lastname: Koelch
authors.forename: Antonio
authors.initials: A
authors.lastname: DeMaio
authors.forename: Nelson
authors.initials: N
authors.lastname: Arispe
authors.forename: Gabriele
authors.initials: G
authors.lastname: Multhoff
mesh_terms: D017209:Apoptosis; D015153:Blotting, Western; D002412:Cations; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002845:Chromatography; D002857:Chromium / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D053804:Granzymes; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D007477:Ions; D007694:Killer Cells, Natural / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D054353:Perforin; D010743:Phospholipids / Q000378:metabolism; D052899:Pore Forming Cytotoxic Proteins; D011233:Precipitin Tests; D011485:Protein Binding; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D002412:Cations; D018840:HSP70 Heat-Shock Proteins; D007477:Ions; D008562:Membrane Glycoproteins; D010455:Peptides; D010743:Phospholipids; D052899:Pore Forming Cytotoxic Proteins; D002857:Chromium; D054353:Perforin; D045304:Cytochromes c; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases