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J. Biol. Chem., Vol. 278, Issue 42, 41173-41181, October 17, 2003

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Cell Surface-bound Heat Shock Protein 70 (Hsp70) Mediates Perforin-independent Apoptosis by Specific Binding and Uptake of Granzyme B^*

Catharina Gross , Walter Koelch , Antonio DeMaio ¶, Nelson Arispe || and Gabriele Multhoff  **

From the University Hospital Regensburg, Department of Hematology, 93053 Regensburg, Germany, the University of Glasgow CRC Beatson Laboratories, Institute of Biomedical and Life Sciences, Glasgow G61 1BD, Scotland, United Kingdom, the ^¶Division of Pediatric Surgery and Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, and the ^||Department of Anatomy and Cell Biology, Uniformed Services of Health Sciences, Bethesda, Maryland 20814

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, aa^450–463) 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.

Received for publication, March 14, 2003 , and in revised form, June 19, 2003.

^* This work was supported by a grant of the Wilhelm-Sander Stiftung, European Union Grant TRANSEUROPE QLRT 2001 01936, and by multimmune GmbH. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: Prof. for Molecular Oncology, University Hospital Regensburg, Dept. of Hematology, Franz-Josef Strauss Allee 11, 93053 Regensburg, Germany. Tel.: 49-941-944-5575; Fax: 49-941-944-5572; E-mail: gabriele.multhoff{at}klinik.uni-regensburg.de.

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