Peptidoglycan Recognition Protein Tag7 Forms a Cytotoxic Complex with Heat Shock Protein 70 in Solution and in Lymphocytes

The peptidoglycan recognition protein Tag7 is shown to form a stable 1:1 complex with the major stress protein Hsp70. Neither protein is cytotoxic by itself, but their complex induces apoptotic death in several tumor-derived cell lines even at subnanomolar concentrations. The minimal part of Hsp70 needed to evoke cytotoxicity is residues 450–463 of its peptide-binding domain, but full cytotoxicity requires its ATPase activity; remarkably, Tag7 liberated from the complex at high ATP is not cytotoxic. The Tag7-Hsp70 complex is produced by tag7-transfected cells and by lymphokine-activated killers, being assembled within the cell and released into the medium through the Golgi apparatus by a mechanism different from the commonly known granule exocytosis. Thus, we demonstrate how a heat shock protein may perform functions clearly distinct from chaperoning or cell rescue and how peptidoglycan recognition proteins may be involved in innate immunity and anti-cancer defense. The peptidoglycan recognition protein Tag7 is shown to form a stable 1:1 complex with the major stress protein Hsp70. Neither protein is cytotoxic by itself, but their complex induces apoptotic death in several tumor-derived cell lines even at subnanomolar concentrations. The minimal part of Hsp70 needed to evoke cytotoxicity is residues 450–463 of its peptide-binding domain, but full cytotoxicity requires its ATPase activity; remarkably, Tag7 liberated from the complex at high ATP is not cytotoxic. The Tag7-Hsp70 complex is produced by tag7-transfected cells and by lymphokine-activated killers, being assembled within the cell and released into the medium through the Golgi apparatus by a mechanism different from the commonly known granule exocytosis. Thus, we demonstrate how a heat shock protein may perform functions clearly distinct from chaperoning or cell rescue and how peptidoglycan recognition proteins may be involved in innate immunity and anti-cancer defense. The tag7/PGRP 1The abbreviations used are: PGRP, peptidoglycan recognition protein; AMP-PNP, 5-adenylyl-β,γ-imidodiphosphate; EGFP, enhanced green fluorescent protein; FasL, Fas ligand; HPLC, high performance liquid chromatography; Hsp, heat shock protein; rHsp70, recombinant Hsp70; LAK, lymphokine-activated killer; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight (mass spectrometry); Mts, metastasin; PBS, phosphate-buffered saline; tag, tumor-associated gene; rTag7, recombinant Tag7; TNF, tumor necrosis factor. gene, first found in mice (1.Kustikova O.S. Kiselev S.L. Borodulina O.R. Senin V.M. Afanas'eva A.V. Kabishev A.A. Genetika. 1996; 32: 621-628PubMed Google Scholar, 1.Kustikova O.S. Kiselev S.L. Borodulina O.R. Senin V.M. Afanas'eva A.V. Kabishev A.A. Genetika. 1996; 32: 621-628PubMed Google Scholar) and somewhat later in insects (2.Kiselev S.L. Kustikova O.S. Korobko E.V. Prokhortchouk E.B. Kabishev A.A. Lukanidin E.M. Georgiev G.P. J. Biol. Chem. 1998; 273: 18633-18639Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), belongs to a novel family of conserved genes (including four human members) that encode peptidoglycan recognition proteins (2.Kiselev S.L. Kustikova O.S. Korobko E.V. Prokhortchouk E.B. Kabishev A.A. Lukanidin E.M. Georgiev G.P. J. Biol. Chem. 1998; 273: 18633-18639Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 3.Kang D. Liu G. Lundstrom A. Gelius E. Steiner H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10078-10082Crossref PubMed Scopus (407) Google Scholar) with structural similarity to T-phage lysozyme in the C-proximal domain (2.Kiselev S.L. Kustikova O.S. Korobko E.V. Prokhortchouk E.B. Kabishev A.A. Lukanidin E.M. Georgiev G.P. J. Biol. Chem. 1998; 273: 18633-18639Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Recombinant PGRP and natural bovine PGRP have been reported to be bacteriostatic and bactericidal (2.Kiselev S.L. Kustikova O.S. Korobko E.V. Prokhortchouk E.B. Kabishev A.A. Lukanidin E.M. Georgiev G.P. J. Biol. Chem. 1998; 273: 18633-18639Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 4.Kibardin A.V. Mirkina I.I. Korneeva E.A. Gnuchev N.V. Georgiev G.P. Kiselev S.L. Dokl. Biochem. 2000; 372: 103-105PubMed Google Scholar). However, recombinant PGRP produced in a baculovirus system had no cytotoxic effect by itself (5.Mirkina I.I. Kibardin A.V. Korneeva E.A. Gnuchev N.V. Georgiev G.P. Kiselev S.L. Genetika. 2000; 36: 1492-1500PubMed Google Scholar), whereas we did observe Tag7-associated cytotoxicity in the conditioned medium of tag7-transfected cells (1.Kustikova O.S. Kiselev S.L. Borodulina O.R. Senin V.M. Afanas'eva A.V. Kabishev A.A. Genetika. 1996; 32: 621-628PubMed Google Scholar). Our previous studies have shown the following: (i) that lymphokine-activated killers exert their cytotoxicity through secreted as well as membrane-associated proteins (6.Liu C. Xu Z. Gupta D. Dziarski R. J. Biol. Chem. 2001; 276: 34686-34694Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 7.Tydell C.C. Yount N. Tran D. Yuan J. Selsted M.E. J. Biol. Chem. 2002; 277: 19658-19664Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar); (ii) that the secretory mechanism of cell lysis is not restricted to exocytosis of granzymes: e.g. cytotoxic proteins are released in the absence of calcium (8.Liu C. Gelius E. Liu G. Steiner H. Dziarski R. J. Biol. Chem. 2000; 275: 24490-24499Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar); and (iii) that the action of the latter can be largely blocked with anti-Tag7 antibodies (9.Sashchenko L.P. Gnuchev N.V. Lykyanova T.I. Redchenko I.V. Kabanova O.D. Lukanidin E.M. Blishchenko E.Yu. Satpaev D.K. Khaidukov S.V. Chertov O.Yu Immunol. Lett. 1993; 37: 153-157Crossref PubMed Scopus (28) Google Scholar). Hence, we make the suggestion that Tag7 initiates the cytotoxic events not by itself but in complex with some other protein(s). Searching for the Tag7 partner, here we show that in vitro Tag7 readily forms a complex with Hsp70. The latter is a multifunctional protein involved in protein translocation, folding, and multimer assembly (10.Sashchenko L.P. Gnuchev N.V. Redchenko I.V. Kabanova O.D. Lykyanova T.I. Blishchenko E.Yu. Satpaev D.K. Chertov O.Yu Immunol. Lett. 1994; 39: 243-247Crossref PubMed Scopus (6) Google Scholar) as an ATP-dependent molecular chaperone; again, Hsp70 has recently been reported to prevent apoptosis (11.Sashchenko L.P. Lykyanova T.I. Kabanova O.D. Mirkina I.I. Yatskin O.N. Pongor S. Gnuchev N.V. Immunol. Lett. 1996; 53: 25-29Crossref PubMed Scopus (8) Google Scholar, 12.Sashchenko L.P. Dukhanina E.A. Lukyanova T.I. Kabanova O.D. Romanova E.A. Shatalov Yu.V. Iashin D.V. Gnuchev N.V. Mol. Biol. (Mosc.). 2000; 34: 816-820PubMed Google Scholar) and to act outside as well as within the cell (13.Bukau B. Horwich A.L. Cell. 1998; 92: 351-366Abstract Full Text Full Text PDF PubMed Scopus (2429) Google Scholar). Further, we show that the complex of purified recombinant Tag7 and Hsp70 exerts marked cytotoxic activity at subnanomolar concentrations and that human lymphokine-activated killers enriched in CD3+ and CD8+ markers do use the Tag7-Hsp70 complex as a cytotoxic agent released through the Golgi apparatus. Cells—Mouse L929 fibroblasts, SV40-transformed African green monkey kidney COS-1, baby hamster kidney BHK21, human erythroblastoid K562, human T-lymphoma Jurkat, mouse adenocarcinoma CSML-0, and human melanoma M3 cells were cultured in RPMI 1640 supplemented with 2 mm l-glutamine and 10% fetal calf serum (all from Invitrogen). The Tag7 cDNA was subcloned in pBK-CMV (Stratagene) and pEGFP (Clontech) vectors. For Tag7 or Tag7-EGFP expression, COS-1 or BHK21 cells were transiently transfected with the corresponding constructs using Maxifectin (Qiagen) reagents according to the manufacturer's recommendations, and the cells were used in 24 h. To prepare lymphokine-activated killers, human peripheral blood was obtained from healthy volunteers at the Cancer Research Center (Moscow); mononuclear cells were isolated by Ficoll-Hypaque gradient centrifugation and cultivated for 6 days with 1000 units/ml of recombinant interleukin-2 (Sigma); large granular lymphocytes were isolated by Percoll gradient centrifugation (6.Liu C. Xu Z. Gupta D. Dziarski R. J. Biol. Chem. 2001; 276: 34686-34694Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). Proteins and Antibodies—The cDNAs for recombinant mouse Tag7, mouse metastasin (Mts1)/S100A4, human 70-kDa heat shock protein 1A (Hsp70), and Hsp70 peptide-binding domain (385–539) were subcloned in pQE-31 and expressed in Escherichia coli M15[pREP4] (Qiagen); the proteins were purified on nickel nitrilotriacetic acid-agarose (Qiagen) as recommended by the manufacturer. Mouse rTag7 produced in yeast was kindly provided by Dr. S.V. Benevolensky. The p70 protein was isolated from L929 cell membranes as described elsewhere (17.Dukhanina E.A. Romanova E.A. Kiselev S.L. Sashchenko L.P. Dokl. Biol. Sci. 2000; 371: 224-225PubMed Google Scholar) and used to raise polyclonal antibodies in mice. Rabbit antibodies raised against recombinant Tag7 or Hsp70 were affinity-purified on CNBr-activated Sepharose 4B (Amersham Biosciences) coupled with rTag7 and rHsp70 in accordance with the manufacturer's manual. Rabbit antibodies against human TNFα were from Sigma, and 3A3 mouse monoclonal anti-Hsp70 was from Affinity Biore-agents, Inc. 3B5 mouse monoclonal anti-Hsp70 was a gift of Dr. I. V. Guzhova. Mouse monoclonal anti-GM130 was from Transduction Laboratories, Lexington, KY. 3C1 rat monoclonal anti-rTag7 was produced by a standard hybridoma protocol (14.Park H.S. Lee J.S. Huh S.H. Seo J.S. Choi E.J. EMBO J. 2001; 20: 446-456Crossref PubMed Scopus (266) Google Scholar), and clone 3C1B6 antibodies recognized both native and recombinant Tag7. Unless specified otherwise, the standard incubation time for protein complexing was 1 h at 37 °C in PBS pH 7. Cell membranes were isolated as described (15.Stuart J.K. Myszka D.G. Joss L. Mitchell R.S. McDonald S.M. Xie Z. Takayama S. Reed J.C. Ely K.R. J. Biol. Chem. 1998; 273: 22506-22514Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), and, after removing lipids with ether, membrane protein was suspended in PBS with a protease inhibitor mixture (Roche Diagnostics) to 3–5 mg/ml. To obtain secreted proteins, lymphocytes, after 6 days of activation with 1000 units/ml rIL-2, were incubated with either K562 cells (100:1) for 18 h in serum-free RPMI 1640 or with phorbol myristate acetate (20 ng/ml) and ionophore A23187 (200 nm); COS-1 cells 24 h after transfection were cultivated for 18 h in serum-free RPMI 1640. Cells were removed by centrifugation; the conditioned medium was tested for the presence of Tag7 or Tag7-EGFP by immunoblotting, and the supernatants were used in cytotoxic assays Affinity Chromatography, Immunoadsorption, and Immunoblotting—Tag7 produced in E. coli was coupled to CNBr-activated Sepharose 4B (Amersham Biosciences) according to the manufacturer's instructions. The proteins of the membrane fractions from L929 cells were loaded onto a Tag7-Sepharose 4B column, washed with PBS and 0.5 m NaCl, eluted with 0.25 m triethylamine, concentrated, resolved by 12% PAGE with SDS, and stained with Coomassie Brilliant Blue R250 (Serva). Purified antibodies were also coupled to CNBr-activated Sepharose 4B. The Tag7-Hsp70 complex, the conditioned medium of COS-1-tag7 cells, and the conditioned medium of lymphokine-activated killers were loaded on antibody-conjugated Sepharose. Then the material was extensively washed with PBS plus 0.5 m NaCl followed by PBS, eluted with 0.25 m triethylamine, pH 12, or 50 mm ATP, concentrated, and resolved by 12% PAGE with SDS. After semidry transfer, polyvinylidene difluoride membranes (Amersham Biosciences) were routinely blocked and incubated with the 3C1 (1:500), 3A3 (1:2500), or 3B5 (1:500) antibody and horseradish peroxidase-conjugated secondary antibodies (Sigma anti-rat, 1:15,000 and Amersham rabbit anti-mouse, 1:40,000, respectively), and the Amersham ECL kit was used for visualization according to the manufacturer's instructions. Gel Filtration—Samples were equilibrated for 16 h at 25 °C in PBS and 5 μm ATP, loaded in a volume of 200 μl onto a pre-equilibrated TSK G2000 SW column (Amersham-Biosciences), and developed at a flow rate of 0.5 ml/min. The column was calibrated with ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), albumin (67 kDa), myoglobin (17.8 kDa), and cytochrome c (12.4 kDa). EGFP fluorescence was excited at 488 nm and measured at 507 nm. Cytotoxicity, ATPase, and Apoptosis—L929 cells were cultured in 96-well plates at a density of 3 × 104 cells/well; samples were added in 200 μl and incubated for 24 h at 37 °C. Cells were stained with trypan blue, coded samples were counted under the microscope, and at least 100 cells were scored for each group (16.Asea A. Rehli M. Kabingu E. Boch J.A. Bare O. Auron P.E. Stevenson M.A. Calderwood S.K. J. Biol. Chem. 2002; 277: 15028-15034Abstract Full Text Full Text PDF PubMed Scopus (1260) Google Scholar). Cytotoxicity was calculated as Cytotoxicity = (St - Sp)/(T - Sp) × 100%, where St is the number of stained cells, Sp represents spontaneously stained cells, and T is total cells. In some cases (Figs. 4A, 6A, and 7A), cell death was determined with the CytoTox 96 Assay kit (Promega); the discrepancy between the two assays never exceeded 7%.Fig. 6Confocal immunofluorescence analysis of BHK21 cells expressing pTag7-EGFP. A and D, cells transfected with pTag7-EGFP. Green indicates localization of fused protein in the cell. B, immunostaining with anti-GM130 mouse monoclonal antibodies for Golgi apparatus. E, immunostaining with anti-Hsp70 mouse monoclonal antibodies, followed by Cy3 anti-mouse secondary antibodies. C and F, overlays with yellow indicating co-localization. Both merged images pertain to the Golgi apparatus. Specimens were viewed with a Leica (Wetzlar, Germany) confocal microscope. Images were taken at ×100 magnification.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 7Tag7-Hsp70 complex is released by lymphocytes. Peripheral blood mononuclear cells of healthy volunteers were incubated for 6 days with 1000 units/ml rIL-2. On the sixth day, LAKs were co-incubated with K562 cells (100:1) for 18 h in serum-free RPMI; where specified, 5 μg/ml Brefeldin A or 10 nm Mts1 was added. Then samples of conditioned medium (Sn, about 100 ng total protein) (A) or membrane proteins (Mb, 75 ng) (B) were added to L929 cells. Cell death was determined after 24 h at 37 °C. Polyclonal antibodies to Tag7, TNFα, or Hsp70 were used at 1:500 dilution; ATP or AMP-PNP was at 5 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) When necessary, antibodies were added simultaneously with samples. Immunoglobulin G from non-immune rabbits was used as a control. Dissociation constants for antibodies were estimated as described (17.Dukhanina E.A. Romanova E.A. Kiselev S.L. Sashchenko L.P. Dokl. Biol. Sci. 2000; 371: 224-225PubMed Google Scholar). ATP hydrolysis was measured as the increase in inorganic phosphate determined colorimetrically at 350 nm in a modified assay of Lin and Morales (18.Dean C.J. Manson M.M. Immunochemical Protocols. Humana Press, Totowa, NJ1992: 43-65Google Scholar). To assess DNA fragmentation, L929 cells (106) were fixed in ice-cold 70% ethanol, washed twice in PBS, and resuspended in 300 μl of PBS containing 12 μg of propidium iodide and 0.15 mg RNase. After 1 h at room temperature, samples were run in an EPICS Elite flow cytometer (Coulter) in the logarithmic channel of fluorescence. At least 104 cells per sample were analyzed, and the data were processed with EXPO32 software (Applied Cytometry Systems). Cells with fluorescence intensity less than the main peak were considered apoptotic. Immunofluorescence Microscopy—BHK21 cells were grown on 12-mm coverslips and transfected with pTag7-EGFP (tag7 cDNA subcloned in pEGFP vector, Clontech). The next day the cells were fixed with 3% paraformaldehyde in PBS. Background fluorescence was quenched with 50 mm ammonium chloride. Fixed cells were permeabilized with 0.2% saponin. Coverslips were then incubated for 1 h at room temperature with primary antibodies (3B5 and anti-GM130) diluted in PBS and 0.1% Tween 20 containing 5% dry milk and then with Cy3-labeled anti-mouse IgG secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove PA). Coverslips were mounted in Mowiol and examined with a Leica (Wetzlar, Germany) confocal microscope. Images were taken at ×100 magnification and captured at 1024 × 1024 pixels. Montages were prepared with Photoshop 4.0 (Adobe, Mountain View, CA). Mass Spectrometry—The p70 protein of the L929 cell membranes was purified by SDS-PAGE, the corresponding gel region was excised and extracted with acetonitrile, and the protein was digested with trypsin (Sigma) by a standard protocol (Ciphergen Biosystems, Inc.). The tryptic digest of p70 was analyzed by MALDI-TOF mass spectrometry (19.Liu C.C. Detmers P.A. Jiang S.B. Young J.D. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3286-3290Crossref PubMed Scopus (62) Google Scholar), and selected mass values from the spectra were used to search the Swiss-Prot protein database. Tag7 Binds with Hsp70 —To test the idea that Tag7 acquires cytotoxic activity upon forming a complex with another protein, solubilized membrane proteins from target L929 cells were subjected to affinity chromatography on Tag7-conjugated Sepharose. The specifically bound protein was eluted as a single peak of 70 kDa. A similar p70 was found in several other cell lines. The obvious next step was to establish the nature of this potential partner. By mass spectrometric analysis of tryptic peptides, the Tag7-binding p70 was found to be identical to the heat shock protein Hsp70. Recombinant Hsp70 was also shown to be bound specifically on Tag7-Sepharose. The identities of the isolated p70 and the rHsp70 were proven by Western blotting with mouse antibodies to p70 and rabbit antibodies to Hsp70, which were fully cross-reactive (Fig. 1A). To directly demonstrate the interaction, recombinant Tag7 and Hsp70 were mixed (10 nm each) and then immunoprecipitated with Sepharose-coupled antibodies against Tag7 or Hsp70. In either case, the immune complex contained both proteins (Fig. 1B), whereas control incubations with individual proteins showed that there was no cross-reactivity. Hence, one can state that Tag7 and Hsp70 form a specific complex in solution. Complexing in Vitro—To determine the stoichiometry of the Tag7-Hsp70 complex, the two proteins and their mixture were subjected to gel filtration. Preincubations and HPLC runs were carried out in the presence of 5 μM ATP to avoid interference from Hsp70 self-association (20.Gnuchev N.V. Sashchenko L.P. Iarovaia O.V. Dukhanina E. Lukianova T. Razin S.V. Biochem. Biophys. Res. Commun. 1997; 234: 655-659Crossref PubMed Scopus (11) Google Scholar). (Indeed, 200–4000 kDa aggregates were formed by Hsp70 alone and in mixture with Tag7 in preliminary experiments without ATP.) Fig. 2A shows that, under such conditions, Hsp70 alone behaved as a 70-kDa entity, and Tag7 alone acted as a 20-kDa entity, whereas in their mixture most of the protein was eluted in the 90-kDa peak. The latter contained both Tag7 and Hsp70, as evidenced by Western blotting (Fig. 2B). These data strongly suggest that the two proteins form a 1:1 complex. Further, similar analysis was performed with a fusion Tag7-EGFP (50 kDa) constructed for subsequent in situ fluorescence analysis; HPLC demonstrated formation of a 120-kDa fluorescent complex with Hsp70 (Fig. 2C), thus confirming the 1:1 stoichiometry as well as proving the adequacy of the labeled construct. EGFP itself did not interact with Hsp70 (not shown). The association process was found to be markedly ATP-dependent. Very little of the 90-kDa complex was formed by purified proteins in the absence of ATP; a moderate molar excess (5 μM ATP at 1 μM each protein) ensured fast complexing, whereas 50 mm caused the complex to dissociate. If the complex was adsorbed on anti-Tag7 or anti-Hsp70-Sepharose and then treated with 50 mm ATP, Hsp70 was eluted in the former case and Tag7 in the latter (lane 1 in Fig. 3). The Tag7-Hsp70 Complex Induces Cell Death in Culture—Next, we checked whether this newly found complex could indeed be cytotoxic. As shown in Fig. 4A, Tag7-Hsp70 at 1 nm caused the death of about one-third of L929 cells in 24 h, although either protein added separately had no appreciable effect. The cytotoxic action of the complex was prevented by the simultaneous addition of purified polyclonal antibodies against either rTag7 or rHsp70 (but not by immunoglobulins from nonimmunized animals). As above, all incubation media included 5 μm ATP; without it, the cytotoxic effect was considerably lower and less reproducible. The concentration dependence of cytotoxicity assessed with equimolar mixtures of Tag7 and Hsp70 (Fig. 4B) shows that the maximal effect was already reached at 0.1 nm, corroborating the specificity of the phenomenon. The L929 cells exposed to Tag7-Hsp70 died by an apoptotic pathway as evidenced by increased DNA fragmentation (Fig. 4C); the profiles obtained with either protein alone did not differ from the control. We also checked the effect of Tag7-Hsp70 on some other cell lines: tumor-derived Jurkat, M3, and CSML-0 were susceptible, whereas human lymphocytes and fibroblasts from primary cell culture were not. Role of Hsp70 Domains in Functional Interaction with Tag7—Further, we tried to determine some structural requirements for the cytotoxic activity. Hsp70 has two domains of known function, ATP binding and peptide binding (20.Gnuchev N.V. Sashchenko L.P. Iarovaia O.V. Dukhanina E. Lukianova T. Razin S.V. Biochem. Biophys. Res. Commun. 1997; 234: 655-659Crossref PubMed Scopus (11) Google Scholar). Cytotoxicity was evoked by the incubation of Tag7 with the isolated peptide-binding domain (Hsp70 amino acids 385–539) and its tetradecapeptide 450–463 (TKDNNLLGRFELSG), considered crucial for target protein binding (21.Kim B.B. Dikova E.B. Sheller V. Dikov M.M. Gavrilova E.M. Egorov A.M. J. Immunol. Methods. 1990; 131: 213-222Crossref PubMed Scopus (23) Google Scholar), whereas the corresponding Hsc70 tetradecapeptide, which has two changes, R458K and S462T, was totally ineffective (Fig. 4B). However, with Hsp fragments, an appreciable cytotoxic effect was attained only at concentrations three orders of magnitude higher than that of the full-sized protein. Together with the ATP requirement, this means that the ATP-binding moiety is also quite important. ATP Hydrolysis Is Necessary for Tag7-Hsp70 Cytotoxicity—In the conventional chaperone cycle, ATP hydrolysis in the active center of Hsp70 is needed for the bound target protein to assume the proper conformation, whereas the reaction product, ADP, stabilizes the complex; further replacement of ADP with ATP leads to its dissociation (22.Lin T.I. Morales M.F. Anal. Biochem. 1977; 77: 10-17Crossref PubMed Scopus (195) Google Scholar, 23.Geromanos S. Freckleton G. Tempst P. Anal. Chem. 2000; 72: 777-790Crossref PubMed Scopus (76) Google Scholar, 24.Benaroudj N. Fouchaq B. Ladjimi M.M. J. Biol. Chem. 1997; 272: 8744-8751Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). We found (Fig. 4D) that the ATPase activity of Hsp70 increased significantly upon complexing with Tag7, which itself was inactive in this respect. Cytochrome c, used as a control, was without effect. The positive control was metastasin (Mts1 or S100A4), which, as we have shown previously, interacts with the plasma membrane calcium-ATPase (29.Dukhanina E.A. Dukhanin A.S. Georgiev G.P. Dokl. Akad. Nauk. 1999; 365: 826-828PubMed Google Scholar); its effect on Hsp70 was close to that of Tag7. The Hsp70-Mts1 complex was not cytotoxic. Interestingly, the simultaneous addition of Mts1 and Tag7 to Hsp70 hindered the formation of a cytotoxic Tag7-Hsp70 complex, as Mts1 successfully competed with Tag7 for Hsp70. However, a 10-fold excess of Mts1 added after Tag7 could not interfere with Tag7-Hsp70 cytotoxicity (Fig. 4E; see also the subsection "Tag7-HSP70 Is Released by Lymphokine-activated Killers"). In this context, we checked whether ATP cleavage in Tag7-Hsp70 was essential to its cytotoxicity (Fig. 4E). Indeed, the complex failed to kill target cells in the presence of 5 μM AMP-PNP, a nonhydrolyzable ATP analog. Notably, Tag7 liberated from the complex by ATP elution again had no cytotoxic effect (rightmost bar in Fig. 4E); i.e. stable complexing with Hsp70 rather than mere correct folding was required to induce cell death. Detection of Tag7-Hsp70 in tag7-Transfected Cells—Proceeding to the in vivo level, we first had to check whether such a complex could indeed be produced by a live cell, and we thus started with transfecting standard laboratory lines to express tag7. In line with earlier studies (1.Kustikova O.S. Kiselev S.L. Borodulina O.R. Senin V.M. Afanas'eva A.V. Kabishev A.A. Genetika. 1996; 32: 621-628PubMed Google Scholar), the conditioned medium of COS-1-tag7 cells exhibited cytotoxicity that could be suppressed with antibodies to Tag7 as well as to Hsp70 (Fig. 5A). Just as in the in vitro experiments described above, the cytotoxic component was immunoadsorbed by anti-Tag7 and anti-Hsp70-Sepharose; elution with 50 mm ATP liberated Hsp70 and Tag7, respectively (Fig. 3, lane 2), confirming the presence of a Tag7-Hsp70 complex. To check whether the complex was formed already within the cell, similar assays were run on the membrane fraction from the transfected COS-1 cells, yielding the same results (Fig. 5A). Further, nonreducing SDS-PAGE of membrane proteins bound to anti-Tag7-Sepharose revealed a 90-kDa band reacting with both antibodies (Fig. 5B). To locate the Tag7-Hsp70 complex in the cell, we used confocal microscopy of BHK cells transfected with the tag7-EGFP construct and stained with anti-Hsp70 and anti-Golgi antibodies (Fig. 6). Though the distribution of Hsp70 was expectedly rather diffuse, its co-localization with the bulk of Tag7 fluorescent fusion in the Golgi apparatus is quite obvious. Tag7-Hsp70 Is Released by Lymphokine-activated Killers—Having shown that a cytotoxic Tag7-Hsp70 complex can arise in solution and in transfected cells, we had to demonstrate this phenomenon in a natural system to speak about its biological relevance. To this end, human lymphocytes activated with interleukin-2 for 6 days and enriched in cytotoxic T-lymphocyte markers CD3+ and CD8+ (further referred to as LAKs) were co-incubated with K562 cells (100:1). Their conditioned medium killed some 28% of L929 cells in our standard assay (it should be noted that the latter proved quite selective for the events under study: indeed, L929 cells are devoid of the Fas receptor, and granzymes have little effect without perforin, which is apparently not produced by these LAKs; Refs. 8.Liu C. Gelius E. Liu G. Steiner H. Dziarski R. J. Biol. Chem. 2000; 275: 24490-24499Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 9.Sashchenko L.P. Gnuchev N.V. Lykyanova T.I. Redchenko I.V. Kabanova O.D. Lukanidin E.M. Blishchenko E.Yu. Satpaev D.K. Khaidukov S.V. Chertov O.Yu Immunol. Lett. 1993; 37: 153-157Crossref PubMed Scopus (28) Google Scholar). Cell death could be prevented with polyclonal antibodies against Tag7 or Hsp70, but not those against TNFα (Fig. 7A); the antibodies specifically recognized the agents released by LAKs, as evident from the calculated dissociation constants (5.3 × 10-11m for anti-Tag7 and 2.5 × 10-11m for anti-Hsp70). The addition of 5 μM ATP to the assay medium raised the cytotoxicity to 35%, whereas 5 μM AMP-PNP completely blocked it (Fig. 7A), just as with Tag7-Hsp70 in vitro. The LAK-conditioned medium was passed through anti-Tag7 or anti-Hsp70-Sepharose; elution with 50 mm ATP yielded Hsp70 in the former case and Tag7 in the latter (Fig. 3, lane 3). All these data indicated that CD3+ and CD8+ lymphocytes released the Tag7-Hsp70 complex in response to contact with target cells. To completely rule out the possibility that the cytotoxic proteins were secreted by the K562 cells (even if unlikely in view of their minority), LAKs were instead induced with phorbol myristate acetate and ionophore A23187 (26.Greene L.E. Zinner R. Naficy S. Eisenberg E. J. Biol. Chem. 1995; 270: 2967-2973Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), with quite the same results (not shown). To ascertain whether the complex was formed within the lymphocytes, the membrane fraction was isolated therefrom and found to be similar to the conditioned medium in L929 cytotoxicity and antibody sensitivity (Fig. 7B). As with transfected COS-1 cells, electrophoresis of the membrane material under mild conditions yielded a 90-kDa band reacting with antibodies to Tag7 and to Hsp70 (Fig. 5B). To ascertain the pathway of the cytotoxic agent, we used Brefeldin A, which is known to irreversibly block protein translocation from endoplasmic reticulum to Golgi (25.Multhoff G. Pfister K. Germann M. Hantschel M. Gross C. Hafner M. Hiddemann W. Cell Stress Chaperones. 2001; 6: 337-344Crossref PubMed Scopus (157) Google Scholar). Its addition to LAKs during their contact with K562 cells abolished the cytotoxicity of the conditioned medium (Fig. 7A), suggesting that Tag7-Hsp70 is exported through the Golgi machinery. In parallel, we