Endosomal proteolysis of diphtheria toxin without toxin translocation into the cytosol of rat liver in vivo Tatiana El Hage1,2, Paulette Decottignies3,4 and Francois Authier1,2 ¸ ˆ INSERM, U756, Chatenay-Malabry, France ˆ ´ ´ Universite Paris-Sud, Faculte de Pharmacie, Chatenay-Malabry, France CNRS, UMR 8619, Orsay, France ´ Universite Paris-Sud, Orsay, France Keywords cathepsin D; diphtheria toxin; endosome; furin; translocation Correspondence ´ F Authier, Inserm U756, Universite Paris´ Sud, Faculte de Pharmacie, rue Jeanˆ ´ Baptiste Clement, 92296 Chatenay-Malabry, France Fax: +33 4683 5844 Tel: +33 4683 5528 E-mail: francois.authier@u-psud.fr (Received July 2007, revised 19 January 2008, accepted February 2008) doi:10.1111/j.1742-4658.2008.06326.x A detailed proteolysis study of internalized diphtheria toxin (DT) within rat liver endosomes was undertaken to determine whether DT-resistant species exhibit defects in toxin endocytosis, toxin activation by cellular enzymes or toxin translocation to its cytosolic target Following administration of a saturating dose of wild-type DT or nontoxic mutant DT (mDT) to rats, rapid endocytosis of the intact 62-kDa toxin was observed coincident with the endosomal association of DT-A (low association) and DT-B (high association) subunits Assessment of the subsequent postendosomal fate of internalized mDT revealed a sustained endo-lysosomal transfer of the mDT-B subunit accompanied by a net decrease in intact mDT and mDT-A subunit throughout the endo-lysosomal apparatus In vitro proteolysis of DT, using an endosomal lysate, was observed at both neutral and acidic pH, with the subsequent generation of DT-A and DT-B subunits (pH 7) or DT fragments with low ADP-ribosyltransferase activity (pH 4) Biochemical characterization revealed that the neutral endosomal DT-degrading activity was due to a novel luminal 70-kDa furin enzyme, whereas the aspartic acid protease cathepsin D (EC 3.4.23.5) was identified as being responsible for toxin degradation at acidic pH Moreover, an absence of in vivo association of the DT-A subunit with cytosolic fractions was identified, as well as an absence of in vitro translocation of the DT-A subunit from cell-free endosomes into the external milieu Based on these findings, we propose that, in rat, resistance to DT may originate from two different mechanisms: the ability of free DT-A subunits to be rapidly proteolyzed by acidic cathepsin D within the endosomal lumen, and ⁄ or the absence of DT translocation across the endosomal membrane, which may arise from the absence of a functional cytosolic translocation factor previously reported to participate in the export of DT from human endosomes Diphtheria toxin (DT) is the causative agent of the acute disease diphtheriae and, in mammalian cells, mediates its cytotoxic effects by catalyzing the transfer of the ADP-ribosyl moiety of NAD+ to elongation factor-2 (EF-2), which arrests protein synthesis and kills the cells [1] DT is synthesized by Corynebacterium diphtheriae and belongs to the A–B family of bacterial exotoxins In the secreted form of DT, the A and Abbreviations CT, cholera toxin; CTF, cytosolic translocation factor; DT, diphtheria toxin; EEA1, early endosome antigen 1; EF-2, elongation factor-2; EN, endosomal fraction; ENs, soluble endosomal extract; HA, hexa-D-arginine; Hsp, heat shock protein; IDE, insulin-degrading enzyme; LPS, postmitochondrial supernatant; PA, pepstatin-A; PE, Pseudomonas exotoxin A; proHB-EGF, heparin-binding epidermal growth factor-like growth factor precursor; TrxR1, thioredoxin reductase-1 1708 FEBS Journal 275 (2008) 1708–1722 ª 2008 The Authors Journal compilation ª 2008 FEBS T El Hage et al B moieties are connected by both a peptide bond (Arg193–Ser194) and a disulfide bridge (Cys186– Cys201) Proteolytic nicking of the polypeptide and reduction of the disulfide are required for the A and B fragments to separate and for cytotoxicity to be expressed [2] To access its cytosolic target EF-2, DT must be transported across cellular membranes and into the cytoplasm [3,4] The first step in the intoxication process involves DT binding, via its B-subunit domain, to the heparin-binding epidermal growth factor-like growth factor precursor (proHB-EGF), and it is believed that DT-DT receptor complexes are endocytosed using a clathrin-dependent pathway [5] Prior to, during or after endocytosis of DT into early endosomes, furin mediates DT cleavage at the consensus motif within the 14-amino acid loop subtended by the disulfide bond connecting the A- and B-moieties [6] After a lag phase of 25 min, two-thirds of the internalized DT is proteolyzed and inactivated within the endosomal apparatus [7] Three models have been proposed as being physiologically relevant to the mechanism of translocation and ⁄ or cytosolic release of the DT catalytic domain: (a) translocation of DT across the endosomal membrane by its own transmembrane domain (tunnel model) [8,9]; (b) translocation of the DT-A (low association) subunit through the oligomeric DT-B (high association) subunit following its conformational change and insertion into the lipid bilayer (cleft model) [10]; and (c) the requirement of chaperonin heat shock protein (Hsp)90 and thioredoxin reductase-1 (TrxR1) as components of a cytosolic translocation factor (CTF) [11], as well as cytosolic factors (ATP, b-COP) [12] Another processing requirement for internalized DT to become fully active is reduction of the interchain disulfide bridge, but the intervening reductive steps, the nature of the relevant enzymatic activity and the subcellular site at which the disulfide bond is split (endosome and ⁄ or cytosol) are not understood Nevertheless, reduction of DT may represent the rate-limiting step in the diphtherial intoxication of eukaryotic cells [7] The existence of cells resistant to DT, such as rodent cells [13,14], suggests that the above requirements for DT activation and action may not be present in all mammalian cells The presence of functional DT binding sites in rat and mouse cells [15] suggests that the biochemical determinant(s) for resistance to DT in rodents must lie distal to the receptor binding step and involve some aspects of toxin internalization and ⁄ or endosomal degradation and ⁄ or translocation of the DT-A subunit Moreover, novel endosomal fragments of internalized DT that did not originate from furin Activation and translocation of diphtheria toxin activity have been identified in cells overexpressing DT receptor [16], suggesting that the endosomal proteolytic machinery may also degrade toxins and curtail their action This is in agreement with experiments using furin-deficient cells in which the intracellular interaction and degradation of internalized DT by unidentified protease(s) was reported [17] In the present study, we report a detailed processing study of internalized DT within hepatic endosomes obtained from rat, a toxin-resistant species The methodology used in the present study, which is the first to use DT and an in vivo model, is similar to that developed to investigate the metabolic fate of internalized cholera toxin (CT) in the endo-lysosomal apparatus [18,19] This methodology has proven useful in relating endosomal processes to toxin cytotoxicity in a physiological state Here, we report that rat hepatic endosomes contain a luminal truncated furin that cleaves the Arg193–Ser194 peptide bond in the connecting A-B region of internalized DT at neutral pH In addition, cathepsin D (EC 3.4.23.5) generates DT cleavages at the Met14–Glu15 and Leu434–Pro435 peptide bonds of DT at acidic pH and releases DT fragments with low ADP-ribosyltransferase activity These events coincided with an absence of in vivo association of DT or DT-A with cytosolic fractions isolated from DTinjected rats, as well as an absence of in vitro translocation of DT or DT-A from cell-free endosomes into the external milieu Results In vivo endocytosis of DT and nontoxic mutant DT (mDT) within the endo-lysosomal apparatus of rat liver The kinetics of in vivo uptake of native DT or mDT into the endosomal fraction (EN) were first assessed (Fig 1A) Rats were administered an intravenous injection of either toxin (50 lgỈ100 g)1 body weight) and killed 5–90 post-injection Hepatic endosomes were isolated and the amount of internalized DT (Fig 1A, upper blots) or mDT (Fig 1A, lower blots) was determined by reducing SDS ⁄ PAGE followed by western blotting using rabbit (a-1275) antibody or horse (a-PV) anti-DT serum Following injection of native DT, a short association of the 62-kDa DT form was observed in EN at 5–15 min, whereas the level of individual subunits was maximal at 30 (DT-A subunit) or 30–90 (DT-B subunit) In response to the in vivo administration of mDT, which displays a single substitution of Glu52 for Gly in the A-chain, the kinetics of association with hepatic endosomes of FEBS Journal 275 (2008) 1708–1722 ª 2008 The Authors Journal compilation ª 2008 FEBS 1709 Activation and translocation of diphtheria toxin T El Hage et al Fig Kinetics of the appearance of DT and mDT in the endo-lysosomal apparatus after toxin administration (A) EN were isolated at the indicated times after the in vivo administration of native DT (upper blots) or nontoxic mutant mDT (lower blots), and evaluated for their content of internalized toxins by reducing SDS ⁄ PAGE followed by western blot analysis using polyclonal anti-DT 1275 (a-1275; blots on the left) or PV (a-PV; blots on the right) sera Each lane contained approximately 50 lg of endosomal protein Arrows to the right indicate the mobilities of intact toxins ( 62 kDa), B-subunits ( 37 kDa) and A-subunits ( 25 kDa) Molecular mass markers are indicated to the left of each blot (B) The LPS fraction was isolated 15 after mDT administration, and immediately subfractionated on linear Nycodenz density gradients (panel °C), or incubated with ATP and an ATP-regenerating system at 37 °C for 60 prior to subfractionation on linear Nycodenz density gradients (panel 37 °C ⁄ 60 min) mDT was evaluated for each subfraction using reducing SDS ⁄ PAGE followed by western blot analysis using polyclonal anti-DT 1275 Thirty microliter of each subfraction were loaded onto each lane Arrows to the left indicate the mobilities of immunodetected mDT ( 62 kDa), mDT-B subunit ( 37 kDa) and mDT-A subunit ( 25 kDa) The LPS fraction was also isolated from control rats and incubated with ATP and an ATP-regenerating system at 37 °C for 60 prior to subfractionation on linear Nycodenz density gradients (lower blots a-EEA1 and a-CD) The content of EEA1 and cathepsin D was evaluated by immunoblotting for each subfraction isolated from the Nycodenz gradients Arrows to the right indicate the mobilities of EEA1 (180 kDa), procathepsin D precursor (64 kDa) and mature cathepsin D (45 and 31 kDa) Components appearing at densities in the ranges 1.075–1.105 and 1.11–1.14 gỈmL)1 were scored as truly endosomal and lysosomal, respectively 1710 FEBS Journal 275 (2008) 1708–1722 ª 2008 The Authors Journal compilation ª 2008 FEBS T El Hage et al mDT, as well as mDT-A or mDT-B subunits, were similar to that of wild-type DT The levels of both subunits decreased after 30 min, especially when using anti-DT 1275 serum, suggesting a degradation state of DT-A and DT-B subunits at this locus However, a stronger immunoreactivity of endosome-associated mDT was observed compared to the nonmutant toxin, which could result from a higher binding property of mDT with membrane lipids and proteins [20] and ⁄ or a higher level of mDT endocytosis in rat hepatocytes Next, we used the in situ liver model system for analysis of endosome–lysosome transfer to determine the post-endosomal fate of internalized mDT (Fig 1B) Transfer of mDT, mDT-A and mDT-B subunits from the endosomal to the lysosomal compartment was examined by Nycodenz density gradient analysis of postmitochondrial supernatant (LPS) fractions prepared 15 after mDT administration When LPS fractions were incubated at °C and analyzed by reducing SDS ⁄ PAGE followed by immunoblot analysis Activation and translocation of diphtheria toxin using rabbit a-1275 antibody (upper blot °C), most of the intact mDT and mDT subunits appeared in a single broad region of density in the range 1.070– 1.100 gỈmL)1, which mainly coincided with the endosomal marker early endosome antigen (EEA1) or procathepsin D precursor (lower blots a-EEA1 and a-CD) When the LPS fraction was incubated at 37 °C for 60 (Fig 1B, middle blot 37 °C ⁄ 60 min), a major shift of the mutant toxin to high density fractions was observed, which partially coincided with the lysosomal marker mature cathepsin D enzyme (lower blot a-CD) This was accompanied by a corresponding loss of intact mDT and mDT-A and mDT-B subunits from the endosomal position Proteolytic activation of DT within hepatic endosomes We examined the ability of hepatic endosomes to degrade native DT (Fig 2A) The luminal and A B Fig Effect of pH and assessment of the degradation products generated from native DT by endosomal DT-degrading activity (A) Total (EN;  10 lg) and soluble (ENs;  lg) endosomal fractions were incubated with 10 lg native DT at 37 °C for the indicated times in 25 mM Hepes buffer (pH 7) or 25 mM citrate-phosphate buffer pH The incubation mixtures were then analyzed by reducing SDS ⁄ PAGE followed by Coomassie Brilliant Blue staining The major degradation products generated at pH (peptide 7a) or pH (peptides 4a, 4b and 4c) were subjected to N-terminal sequence analysis Arrows to the left indicate the mobilities of intact DT ( 62 kDa), DT-B subunit ( 37 kDa) and DT-A subunit ( 25 kDa) Arrows to the right indicate the mobilities of intact DT ( 62 kDa) and uncharacterized DT fragments (60, 35 and 12 kDa) (B) Native DT was digested in vitro for 90 at 37 °C with the ENs fraction in buffer pH or containing dithiothreitol (0.2 M) The treated DT (10 lg) was then incubated for the indicated times at 30 °C with the cytosolic fraction ( 200 lg) in 0.1 M Hepes buffer (pH 7.4) in the presence of lM [32P]NAD Samples were then subjected to SDS ⁄ PAGE and analyzed by autoradiography The dried gels were exposed to X-ray film at )80 °C for days The arrow to the right indicates the mobility of [32P]-labeled EF-2 ( 105 kDa) FEBS Journal 275 (2008) 1708–1722 ª 2008 The Authors Journal compilation ª 2008 FEBS 1711 Activation and translocation of diphtheria toxin T El Hage et al membrane-bound distribution of endosomal DTdegrading activity was assessed by reducing SDS ⁄ PAGE analysis of DT digestion performed at pH and (Fig 2A) EN degraded native DT efficiently both at neutral and acidic pH (Fig 2A, upper gels) DT-A and DT-B subunits were specifically generated at pH 7, whereas three major DT fragments of 60, 37 and 12 kDa were observed at pH Subfractionation of hepatic EN into a soluble endosomal lysate (ENs) revealed a similar pattern of proteolysis (Fig 2A, lower gels), suggesting that the majority of the neutral and acidic DT-degrading activity in endosomes is soluble The cleavage sites in the major metabolites of DT were determined by N-terminal sequence analysis Edman degradation of intermediate 7a generated the Ser-Val-Gly-Ser-Ser-Leu peptide, suggesting that the major cleavage produced at neutral pH affected the peptide bond between Arg193–Ser194 At acidic pH, N-terminal sequence analysis of the major DTfragments revealed cleavages between Met14–Glu15 (as demonstrated by the Glu-Asn-Phe-Ser-Ser-Tyr sequence at the N-terminal of product 4b) and Leu434-Pro435 (as demonstrated by the Pro-Thr-IlePro-Gly-Lys sequence at the N-terminal of product 4c) Moreover, intermediate 4a displayed the N-termiA nal sequence of DT (Gly-Ala-Asp-Asp-Val-Val), suggesting that the cleavage is located within the carboxyl-terminal region of the toxin We next examined whether, under conditions where DT was processed by ENs, a corresponding change in the toxin cytotoxicity would be observed (Fig 2B) DT was first partially processed by ENs at pH or under reducing conditions, and then incubated at neutral pH with cytosolic fraction in the presence of [32P]NAD A rapid and sustained ADP-ribosylation of cytosolic EF-2 was observed following endosomal proteolysis of DT at neutral pH (Fig 2B) However, [32P]ADP-ribose incorporation into EF-2 after endosomal digestion of DT under acidic conditions was very low, even after 60 of incubation (Fig 2B) Catalytic properties of endosomal neutral and acidic DT-degrading activity We next evaluated the effects of various protease inhibitors on the neutral (Fig 3A) and acidic (Fig 3B) DT-degrading activity by reducing SDS ⁄ PAGE analysis The proteolytic activity directed against DT at pH was inhibited by hexa-d-arginine (HA), a competitive inhibitor of furin, and the metal-chelating B Fig Effect of protease inhibitors on the proteolysis of native DT by ENs ENs ( lg) was incubated with lM native DT at 37 °C for 180 in 25 mM Hepes buffer pH (A) or 25 mM citrate-phosphate buffer (pH 4) (B) in the absence or presence of 3.5 lgỈmL)1 PA, lM E64, mM EDTA, lM HA, mM phenylmethanesulfonyl fluoride (PMSF), 1% MeOH or 1% Me2SO (upper gels), or in the presence of HA and PA at the indicated concentrations (lower gels) The incubation mixtures were then analyzed by reducing SDS ⁄ PAGE followed by Coomassie Brilliant Blue staining Arrows to the right in (A) indicate the mobilities of intact DT ( 62 kDa), DT-B subunit ( 37 kDa) and DT-A subunit ( 25 kDa) Arrows in (B) indicate the mobilities of intact DT ( 62 kDa) and uncharacterized DT fragments (60, 35 and 12 kDa) 1712 FEBS Journal 275 (2008) 1708–1722 ª 2008 The Authors Journal compilation ª 2008 FEBS T El Hage et al agent EDTA (Fig 3A, upper gels) Neutral DTdegrading activity was inhibited in a dose-dependent manner by HA (IC50  lm) (Fig 3A, lower gel) At acidic pH, endosomal DT-degrading activity was strongly inhibited by pepstatin-A (PA) (IC50 < 10)7 m), an inhibitor of aspartic acid proteases, and EDTA (Fig 3B) Inhibition of acidic DT-degrading activity by PA and its presence in the endosomal lumen as a soluble form suggested cathepsin D as a likely candidate for the degrading activity We therefore used well characterized polyclonal antibodies to mature cathepsin D and its proform [21,22] to deplete cathepsin D from Activation and translocation of diphtheria toxin ENs (Fig 4A, left gel) Quantitative immunoprecipitation of cathepsin D using antibodies directed against the mouse (a-CD R291) or human enzyme (a-CD M8147) removed a major part of the endosomal proteolytic activity directed towards DT at pH 4, as assessed by reducing SDS ⁄ PAGE analysis Immunodepletion of ENs with antibodies to cathepsin B and its proform (a-CB 7183) [23] failed to remove the proteolytic activity Hepatic endosomes are known to contain neutral peptidases such as insulin-degrading enzyme (IDE) [24] and furin [25] Endosomal neutral DT-degrading activity was depleted by anti-furin R2 serum directed A B C Fig Effect of immunodepletion of cathepsins and furin on endosomal DT-degrading activity (A) ENs fractions were immunodepleted of active cathepsin D (a-CD), cathepsin B (a-CB), furin (a-furin) or insulin-degrading enzyme (a-IDE) using their respective antibodies, which had been precoated onto protein G-Sepharose beads Following centrifugation, the resultant supernatants were incubated with lM native DT at 37 °C for 120 in 25 mM citrate-phosphate buffer (pH 4) (gel on the left) or 25 mM Hepes buffer (pH 7) (gel on the right) The incubation mixtures were then analyzed by reducing SDS ⁄ PAGE followed by Coomassie Brilliant Blue staining Arrows indicate the mobilities of intact DT ( 62 kDa) and uncharacterized DT fragments ( 60, 35 and 12 kDa) (B) A total (EN) and soluble (ENs) endosomal fraction, and recombinant furin were evaluated by immunoblotting for their immunoreactivity with monoclonal (a-furin R2) or polyclonal (a-furin CT) antibody to furin Arrows indicate the mobilities of intact (90 kDa) or truncated furin (70 and 57 kDa) (C) Rat liver EN were isolated at the indicated times after the in vivo administration of native DT and evaluated by western blotting for their content of furin using monoclonal (a-furin R2) or polyclonal (a-furin CT) antibody to furin Molecular mass markers are indicated to the left of each blot Arrows to the right indicate the mobilities of intact (90 kDa) or truncated furin (70 kDa) FEBS Journal 275 (2008) 1708–1722 ª 2008 The Authors Journal compilation ª 2008 FEBS 1713 Activation and translocation of diphtheria toxin T El Hage et al against the catalytic domain of furin (Fig 4A, right gel) However, immunoprecipitation using antibody to the carboxyl-terminal domain of furin (a-furin CT) or to IDE (a-IDE) failed to remove the DT-degrading activity observed at pH Hepatic endosomes were then evaluated for their content of furin by immunoblotting with antibodies directed against either the catalytic or carboxyl-terminal domain of furin (Fig 4B) The EN showed intense immunoreactivity for the 90-kDa furin enzyme using either antifurin serum By contrast, anti-furin R2 serum, which is directed against the catalytic domain of furin, showed immunoreactivity for the soluble 70-kDa form of furin in both the total EN and the luminal content (ENs) Comparably, recombinant truncated furin was revealed by immunoblotting as a 57-kDa form using anti-furin R2 serum However, anti-furin CT serum failed to detect either the 70-kDa soluble form or the 57-kDa recombinant truncated furin To determine whether DT alters the degradation state of furin in the endosomal apparatus, we prepared endosomes after DT injection and assessed furin antigenicity towards both anti-furin sera (Fig 4C) Using anti-furin R2 serum (left blot), the 90-kDa furin (major species) was found in endosomes from untreated rats, as well as the truncated 70-kDa furin (minor species) Using both anti-furin sera, a timedependent decrease in the content of the 90-kDa furin was observed at 5–60 after DT injection A parallel increase in the content of the 70-kDa fragment was observed with anti-furin R2 serum, especially at post-injection (Fig 4C, left blot) To verify the involvement of two enzymes in the endosomal processing of internalized DT, the proteolytic activity associated with soluble endosomal proteins was further purified on a TSK-GEL G3000 HPLC column (Fig 5) Eluted fractions 5–12 (Fig 5A, upper panel) were assayed for their content of cathepsin D and furin by western blotting using anti-cathepsin D R291 or anti-furin R2 sera, respectively (Fig 5A, blots), as well as for their DT-degrading activity at pH or using reducing SDS ⁄ PAGE analysis (Fig 5B) The neutral DT-degrading activity (Fig 5B, fraction of upper gel) coincided with elution of the 70-kDa immunoreactive furin (Fig 5A, lower blot) The fraction with the highest acidic DT-degrading activity (Fig 5B, fraction of lower gel) revealed a strong immunoreactivity towards the mature 45-kDa cathepsin D enzyme (Fig 5A, upper blot) To determine whether furin and cathepsin D are capable of generating the same degradation products as those observed with endosomes, DT was subjected to in vitro digestion with human furin at pH 1714 (Fig 5C, upper gel) or bovine cathepsin D at pH (Fig 5C, lower gel) Reducing SDS ⁄ PAGE analysis revealed DT proteolytic products with molecular masses identical to those observed with EN or ENs fractions at both neutral and acidic pH (Fig 2A) Assessment of a functional translocation complex for DT in hepatic endosomes To evaluate a possible defect in DT-A translocation to its cytosolic target, we measured the presence of the DT-A subunit in cytosolic fractions prepared from DT-injected rats using western blot analysis (Fig 6A) No detectable immunoreactivity for the DT-A subunit was observed, even at late stages of DT endocytosis (Fig 6A, blot a-DT1275), whereas the cytosolic target of DT-A, EF-2, was easily detected (Fig 6A, blot a-EF-2) However, a cytosolic translocation of the cytotoxic A-subunit was clearly observed in rats treated with Pseudomonas exotoxin A (PE) (Fig 6A, blot a-PE) or CT (Fig 6A, blot a-CT) at 5–90 postinjection, consistent with a selective retention of DT-A within rat liver endosomes To directly assess the translocation of internalized DT into the extra-endosomal milieu, endosomes isolated after injection of DT were incubated in buffered isotonic medium for 30–60 at 37 °C in the presence or absence of ATP, followed by centrifugation at 100 000 g for 60 (Fig 6B) Western blot analysis of DT associated with sedimentable endosomes showed a progressive decrease in immunoreactive DT, as well as DT-A and DT-B subunits, confirming the processing of DT in liver endosomes (Fig 6B, left blot) By contrast, immunoprecipitation of DT from supernatants followed by western blot analysis using anti-DT PV serum did not reveal any immunoreactivity towards DT or the individual DT-subunits (Fig 6B, right blot), suggesting that DT and DT degradation products remained strictly associated with endosomes Recently, TrxR1 and the chaperonin Hsp90 have been proposed to be components of a CTF complex that participates in the translocation and release of cytosolic DT-A subunit from early endosomes of human T cells [11] Consequently, we attempted to evaluate the contribution of these enzymes to the endosomal disulfide-reducing activity (TrxR1) and membrane translocation of toxin peptides (TrxR1 and Hsp90) by assessing hepatic endosomes and cytosol for their content of TrxR1 and Hsp90 (Fig 6C) EN isolated from control rats (Fig 6C, lane –) revealed a concentration of TrxR1 equivalent to that observed in the cytosolic fraction (Fig 6C, blots a-TrxR1, lane –) A weak immunoreactivity for Hsp90 was observed in FEBS Journal 275 (2008) 1708–1722 ª 2008 The Authors Journal compilation ª 2008 FEBS T El Hage et al Activation and translocation of diphtheria toxin A B C Fig Characterization of endosomal DT-degrading activity by gel-filtration HPLC (A) ENs ( 260 lg) was applied to a TSK-GEL G3000 HPLC column The HPLC profile shows the absorbance at 214 nm The eluted fractions (5–12) were evaluated for their content of cathepsin D and soluble truncated furin by immunoblotting with their respective polyclonal (a-CD R291) or monoclonal (a-furin R2) antibody Arrows to the right indicate the mobilities of the immunoreactive proform ( 64 kDa) and mature form ( 45 kDa) of cathepsin D, and soluble truncated furin ( 70 kDa) (B) Eluted fractions (5, 7, 9, 10 and 12) were tested for their ability to degrade lM native DT for 90 at 37 °C in 25 mM Hepes buffer (pH 7) (upper gel) or 25 mM citrate-phosphate buffer (pH 4) (lower gel) The incubation mixtures were then analyzed by reducing SDS ⁄ PAGE followed by Coomassie Brilliant Blue staining Arrows to the right indicate the mobilities of intact DT ( 62 kDa), DT-B subunit ( 37 kDa), DT-A subunit (25 kDa) and uncharacterized DT fragments (60, 35 and 12 kDa) (C) Native DT (10 lg) was incubated at 37 °C with furin (50 mL)1Ỉmg)1) in 25 mM Hepes buffer (pH 7) containing mM CaCl2 (upper gel) or cathepsin D (40 mL)1Ỉmg)1) in 25 mM citrate-phosphate buffer (pH 4) (lower gel) for the indicated times The incubation mixtures were then analyzed by reducing SDS ⁄ PAGE followed by Coomassie Brilliant Blue staining Arrows to the right indicate the mobilities of intact DT ( 62 kDa), DT-B subunit ( 37 kDa), DT-A subunit ( 25 kDa) and uncharacterized DT fragments (60, 35 and 12 kDa) control endosomes (Fig 6C, upper blot a-Hsp90, lane –), whereas a strong immunoreactivity was detected in control cytosol (Fig 6C, lower blot Hsp90, lane –) DT administration did not alter the distribution of TrxR1 or Hsp90 in the endosomal and cytosolic pools (Fig 6C) Although these data demonstrate that hepatic endosomes may represent a physiological site of TrxR1 localization and action, they not demonstrate physical interaction between TrxR1 and internalized DT Therefore, coimmunoprecipitation studies were undertaken (Fig 6D) Immunoprecipitation of internalized DT or TrxR1 from endosomes isolated 15 after DT injection was effected with the indicated antibodies followed by immunoblotting with anti-DT PV or antiDT 1275 sera As expected, DT, as well as free DT-A and DT-B subunits, were detected in immunoprecipitates using anti-DT antibodies However, no DT immunoreactivity was found in TrxR1 immunoprecipitates (Fig 6D) Discussion The present study aimed to explore the biochemical determinant(s) that confer DT insensitivity to rats Whereas man, monkey, rabbit, guinea pig and chicken FEBS Journal 275 (2008) 1708–1722 ª 2008 The Authors Journal compilation ª 2008 FEBS 1715 Activation and translocation of diphtheria toxin A T El Hage et al B C D Fig Assessment of a functional cytosolic translocation complex for DT associated with hepatic endosomes (A) Cytosolic fractions were isolated at the indicated times after the in vivo administration of native DT (blots a-DT1275 and a-EF-2), native PE (blot a-PE) or native CT (blot a-CT), and evaluated for their content of the cytotoxic A-subunit of DT (DT-A), PE (PE-A) or CT (CT-A) using their respective polyclonal antibody, and for their content of EF-2 using polyclonal anti-EF-2 serum Arrows to the left indicate the mobilities of DT-A ( 25 kDa), PE-A ( 37 kDa), CT-A ( 28 kDa) and EF-2 ( 105 kDa) No immunoreactivity for DT-A was observed with cytosolic fractions (B) Membrane translocation of toxin peptides in cell-free endosomes containing in vivo internalized DT The EN was isolated 15 after the administration of native DT and then suspended in 0.15 M KCl containing mM MgCl2 and, when indicated, 10 mM ATP After the indicated time of incubation at 37 °C, endosomes were sedimented by ultracentrifugation and the pellets (endosome-associated material) and supernatants (extraendosomal material) were evaluated for their content of DT peptides using polyclonal anti-DT 1275 antibody, either directly (pellets) or after DT immunoprecipitation (supernatants) Arrows to the right and left indicate the mobilities of intact DT ( 62 kDa), DT-B subunit ( 37 kDa) and DT-A subunit ( 25 kDa) No immunoreactivity of DT was observed with supernatants (C) Endosomal and cytosolic fractions were isolated at the indicated times after the in vivo administration of native DT, and evaluated by western blotting for their content of TrxR1 and Hsp90 using their respective polyclonal antibody Arrows to the left indicate the mobilities of immunodetected TrxR1 ( 58 kDa) or Hsp90 ( 90 kDa) (D) The EN was isolated 15 after the in vivo administration of native DT and lysed The endosomal lysate was then immunoprecipitated using anti-DT PV, anti-DT 1275 or anti-TrxR1 sera, and immunoprecipitates were then immunoblotted with antibodies to DT as indicated Arrows to the left indicate the mobilities of DT ( 62 kDa), DT-B subunit ( 37 kDa) and DT-A subunit ( 25 kDa) are examples of sensitive species, rats and mice are examples of insensitive species, requiring a 10 000-fold higher dose before toxic symptoms are noted, and cell lines derived from sensitive and insensitive species exhibit a similar range in DT sensitivity [3] However, the rat has surface membrane receptors for DT that have properties indistinguishable from the receptors on sensitive cells [4,13] Moreover, rat EF-2 is equally as good a substrate for in vitro DT-catalyzed ADP1716 ribosylation Therefore, the biochemical determinant(s) for DT resistance in rats must lie distal to the receptor binding step and presumably involve some aspects of the endosomal process The present study is consistent with this view Our in vivo and in vitro study supports the contention that the marked degradation of DT-A subunit within the endosomal lumen, as well as the absence of translocation of DT-A subunit to the extraendosomal milieu (or cytosol), is responsible for part FEBS Journal 275 (2008) 1708–1722 ª 2008 The Authors Journal compilation ª 2008 FEBS T El Hage et al (if not all) of the DT insensitivity in rats Another possibility is that the rat DT receptor, despite its ability to bind to and internalize DT, might not be operational within the endosomal apparatus [14] The DT receptor concentration of purified rodent liver plasma membranes is comparable to that found for the insulin receptor, which is highly expressed on these same membranes [13] Accordingly, we have found a marked uptake of DT into rat liver at an early time of sacrifice (5 post-injection) after an acute injection of toxin (50 lgỈ100 g)1 body weight giving a DT-concentration of approximately 0.25 lm within the plasma matrix) The present study, which is the first subcellular fractionation approach to address the in vivo compartmentalization and processing activation of DT in rat liver parenchyma, clearly demonstrates that the toxin is internalized into endosomes as a single nonproteolyzed and nonreduced monomeric chain polypeptide where, at later stages, it undergoes proteolytic processing (major pathway) and reduction (minor pathway) by the endo-lysosomal apparatus The kinetics of in vivo DT internalization observed in our biochemical studies are comparable to that previously reported morphologically using in vitro cellular systems, such as monkey Vero cells [7], human WI-38 cells and mouse 3T3 fibroblasts [26] Indeed, using a similar in vivo experimental approach to that used in the present study, we and others have shown that bacterial toxins such as CT [18,19] and plant toxins such as ricin [27] are taken up by the rat liver and subsequently accumulate in a low-density endosomal compartment where toxin processing begins Our results extend these observations to DT and show that the rate of internalization into hepatic endosomes is slower for CT and ricin (30–40 min) than for DT (5 min) This may well originate from the ability of CT and ricin to be endocytosed using both clathrin-dependent and -independent mechanisms [28], whereas DT endocytosis involves only the clathrin-dependent pathway [5] Moreover, in the present study, the rate of endosomal processing of internalized DT was much faster than that of internalized CT [18] This may in part reflect the two sequential steps in DT processing (before and after endosomal acidification), whereas endosomal proteolysis of CT involves only the cathepsin D enzyme and requires endosomal acidification [18,19] Using the in situ rat liver model system for endosome–lysosome fusion [29], we show here, as previously was the case for native CT [19], a progressive lysosomal transfer of internalized DT, which was accompanied by a net decrease in its immunoreactivity throughout the gradient This result suggests that DT and DT subunits are proteolyzed within the endosomal Activation and translocation of diphtheria toxin apparatus [16], as well as within lysosomal vesicles The cotransfer of DT subunits to lysosomes in response to DT injection confirms previous studies that have documented a lysosomal association of internalized DT in DT sensitive cells [12] and extends this physiological location of DT to the lysosomal apparatus of insensitive cells Comparable to the endosomal degradation of internalized ricin A-chain in macrophages [30], the endosomal processing of internalized DT begins prior to ATP-dependent acidification of the endosomal lumen The endosomal neutral DT-degrading enzyme described in the present study is similar to furin proteolytic activity in several respects: (a) the neutral DTdegrading activity was inhibited by metal-chelating reagents and hexa-arginine, an inhibitor profile similar to that of furin [31]; (b) immunoprecipitation of furin from a soluble endosomal extract led to major depletion of the neutral degrading activity; and (c) the neutral degrading activity produced a cleavage pattern for the DT substrate at the Arg193–Ser194 peptide bond that was similar to that generated using pure furin This is in agreement with previous studies using cellular and acellular in vitro systems [6,17] However, the previously reported transmembrane property of furin [31] would argue against a role for furin in the proteolysis of DT by a soluble endosomal lysate Interestingly, in the present study, we also provide the first evidence for endosomal compartmentalization of a 70-kDa soluble form of furin in rat liver Hence, the 70-kDa furin protein identified is most likely responsible for the endosomal neutral DT-degrading activity due to: (a) its association with soluble endosomal proteins; (b) the similar elution profile of the neutral DT-degrading activity and the immunoreactive 70-kDa furin polypeptide by gel-filtration HPLC; and (c) its binding to monoclonal antibody R2 under nondenaturating and denaturating conditions, confirming that the 70-kDa truncated soluble furin represents a proteolytically active species In agreement with our demonstration of a role for a novel soluble 70-kDa furin in the endosomal processing of endocytosed DT, the endoprotease furin has been also proposed to catalyze these reactions towards various bacterial toxins both at the cell surface and ⁄ or within endocytic compartments [32] Bacterial toxins that require proteolytic cleavage mediated by furin to express full toxic activity include Clostridium septicum alpha-toxin [33], Aerolysin [34], anthrax toxin protective antigen, PE, Shiga and Shiga-like toxins, and botulinum toxin [32] Immunoblotting in the present study confirmed the detection of furin in rat liver endosomes [25] and rat FEBS Journal 275 (2008) 1708–1722 ª 2008 The Authors Journal compilation ª 2008 FEBS 1717 Activation and translocation of diphtheria toxin T El Hage et al liver subcellular fractionation studies have shown that endogenous furin is partially co-distributed with early endosome markers [35] These data are consistent with previous morphological studies that demonstrated a minor localization of furin in early endosomes and suggest that, following the endocytosis of cell surface furin to early endosomes [36], proteolytic maturation occurs in the endosomal apparatus and leads to the generation of a 70-kDa soluble active form of furin It still remains to be determined whether endosomal processing of furin observed after DT-treatment is a direct effect of toxin and whether maturation of the transmembrane furin occurs by an unimolecular autoprocessing event or intermolecular processing by endosomal cathepsins or other endosomal endopeptidases Interestingly, it has been shown that, upon reaching the cell surface or during cycling between the transGolgi network and cell surface, furin may undergo further processing because a shortened but still active form of the endoprotease is detected in the media of cells overexpressing native furin [37] PACE4, another furin-related protease, was shown to cleave and activate DT and anthrax toxin protective antigen at neutral pH in a Ca2+ dependent manner [38], although one report suggested that PACE4 processes anthrax toxin protective antigen, but not DT [39] Consistent with our previous studies [18,19], we conclude from the present study that the endosomal acidic DT-degrading activity was likely the aspartic acid protease cathepsin D, as indicated by the following observations: (a) the inhibitor profile and the pH optimum of the acidic proteolytic activity were similar to those of cathepsin D; (b) immunodepletion of cathepsin D from ENs led to a major loss of the acidic proteolytic activity; (c) the elution profile of the acidic DT-degrading activity from the gel-filtration HPLC column was identical to that of immunoreactive cathepsin D; and (d) pure cathepsin D produced a cleavage pattern for DT that was similar to that generated using the EN Two major degradation products of DT that were generated using endosomal lysate at acidic pH result from proteolytic cleavages occurring at residues Met14–Glu15 and Leu434–Pro435, two peptide bonds that fit the specificity pattern for nonpolar sites cleaved by cathepsin D [22] This is in agreement with the endopeptidase activity of cathepsin D in addition to its specificity for hydrophobic sites [22], which previously have been shown to induce various internal cleavages within polypeptide hormones [21,22], as well as in plant [30] and bacterial toxins within endosomes [18,19] Depending on the type of toxin, endosomal proteolysis by cathepsin D is either a prerequisite for toxicity (CT [18,19], ricin A-chain 1718 [30]) or attenuates cytotoxicity (DT; present study) Interestingly, endosomal proteolysis of DT resembles that of ricin A-chain for which two proteases, cathepsins B and D, are implicated in its processing, with the first reducing cytotoxicity and the second enhancing the catalytic effect Due to the low molecular mass degradation products observed at pH 4, it is clear that the endosomal acidic DT-degrading activity favors internal cleavages within both DT subunits, leading to DT fragments with low (if any) ADP-ribosyltransferase activity On the other hand, endosomal proteolysis of DT at neutral pH exhibits high specificity towards furin recognition sequences, limiting further proteolysis of the catalytic DT-A subunit that displayed efficient ADP-ribosylation action towards cytosolic EF-2 Using cell-free endosomes isolated from human T cells (HUT 1026 TG cells), characterization of a CTF complex implicated in the translocation of DT across endosomal membranes has identified the presence of TrxR1 and Hsp90 [11] A functional role for TrxR1 and Hsp90 in the translocation and ⁄ or cytosolic release of cytotoxic DT-A subunit was established through immunoprecipitation and ⁄ or the use of inhibitors However, it was also shown that TrxR1 alone was not sufficient for in vitro translocation of the DT-A catalytic domain and a potential role for TrxR1 in the reduction of the interchain disulfide bond of DT was not demonstrated [11] The endosomal DT-reducing activity described in the present study differs from TrxR1 in several respects: (a) an acidic pH was required for maximal reducing activity (results not shown), whereas the catalytic activity of TrxR1 is optimal at pH 7; (b) immunoprecipitation of endosomal TrxR1 failed to co-immunoprecipitate endocytosed DT or DT-subunits, suggesting that TrxR1 and DT might not be physically associated; and (c) the acidic DT-reducing activity was mainly recovered within the endosomal lumen (results not shown), whereas the endosomal pool of TrxR1 would be associated with the cytosolic side of the endosomal membrane However, c-interferon-inducible lysosomal thiol reductase, a luminal endo-lysosomal reductase that is capable of reducing protein disulfides at acidic pH, may well represent the enzymatic activity responsible for reduction of internalized DT [40] Alternatively, the redox potential of the endosomal environment may represent the mechanism involved in the reduction of DT Physiological reductants, which have been proposed to contribute to the reducing activity in lysosomes, include cysteine and cysteinyl glycine for which specific lysosomal transporters have been identified [41] However, their presence in endosomes has not been FEBS Journal 275 (2008) 1708–1722 ª 2008 The Authors Journal compilation ª 2008 FEBS T El Hage et al demonstrated, making their involvement in the endosomal reduction of DT still questionable [41] With respect to Hsp90, we did not detect its association with endocytic vesicles isolated from control rats, nor its endosomal recruitment upon DT-treatment Thus, although other experiments using in vitro cell systems and ⁄ or RNA interference are required to confirm our in vivo data, our report is consistent with the hypothesis that, in the rat species, TrxR1 and ⁄ or Hsp90 not participate in a chaperone complex facilitating the transport of endosomal DT to the cytosolic compartment In summary, we have found that internalized DT was rapidly proteolyzed within rat hepatic endosomes both before (by a soluble form of furin) and after (by cathepsin D) ATP-dependent acidification without subsequent translocation of endosomal fragments to the cytosol Although it is suggested that proHB-EGF is responsible for DT binding and internalization in rat hepatocytes, we provide no direct information on the nature and fate of the rat liver DT receptor(s) Studies are currently underway to elucidate whether proHBEGF is responsible for the high level of DT binding in hepatocytes and to determine the fate of the DT binding activity upon endo-lysosomal translocation of DT Experimental procedures Peptides, enzymes, antibodies, protein determination, N-terminal sequencing and materials Native DT, a CRM197 mDT that has a Gly to Glu mutation at amino acid 52 in the A-chain and native PE were purchased from Calbiochem (San Diego, CA, USA) Native CT, acridine orange, bovine cathepsin D, 15 mg)1, and recombinant truncated human furin, 2000 mL)1, were purchased from Sigma (St Louis, MO, USA) Horse antiDT IgG (a-PV) was obtained from Pasteur Vaccin (Ville d’Avray, France) Rabbit anti-mDT (a-1275) and rabbit ´ anti-DT168-220 (a-D4) [42] were obtained from M Leonetti (CEA, Gif-sur-Yvette, France) Western blot analysis revealed a strong affinity for all anti-DT antibodies (horse a-PV and rabbit a-D4 or a-1275) and similar specificity towards the A- and B-subunits Rabbit polyclonal anti-CT or anti-PE were from Sigma Rabbit anti-mouse cathepsin D R291 [21,22], sheep anti-human cathepsin D M8147 [22] and rabbit anti-rat cathepsin B 7183 [23] were obtained from J S Mort (Shriners Hospital for Crippled Children, Montreal, Canada) Mouse monoclonal antibody 9B12 directed against human IDE [22] was obtained from R A Roth (Stanford University, Stanford, CA, USA) Rabbit polyclonal antibody against the carboxy terminus of human furin (a-furin CT) and polyclonal antibody against Activation and translocation of diphtheria toxin human EF-2 were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA) Mouse antihuman IgG recognizing the catalytic domain of furin (a-furin R2) was purchased from Alexis Biochemicals (San Diego, CA, USA) Rabbit polyclonal antibody against rat TrxR1 was purchased from Upstate Rabbit polyclonal antibody against human Hsp90 was obtained from J.-M Renoir (UMR8612, Chatenay-Malabry, France) ˆ Horseradish peroxidase-conjugated goat anti-rabbit IgG and rabbit anti-horse IgG were from Sigma The protein content of isolated fractions was determined by the method of Lowry et al [43] N-terminal sequence data were obtained by automated Edman degradation using a Procise sequencer (Applied Biosystems, Inc., Foster City, CA, USA) equipped with an online phenylthiohydantoin amino acid analysis system Nitrocellulose membranes and the enhanced chemiluminescence detection kit were from Amersham (Orsay, France) Protein G-Sepharose was from Pharmacia (Orsay, France) Pepstatin-A, E-64 and phenylmethanesulfonyl fluoride were from Sigma HA was from Calbiochem All other chemicals were obtained from commercial sources and were of reagent grade Animals and injections In vivo procedures were approved by the institutional committee for use and care of experimental animals Male Sprague–Dawley rats, body weight 180–200 g, were obtained from Charles River France (St Aubin Les Elbeufs, France) and fasted for 18 h prior to sacrifice Native DT, mDT, PE or CT (15–50 lgỈ100 g)1 body weight) in 0.4 mL of 0.15 m NaCl was injected within s into the penile vein under light anaesthesia with ether Rat liver subcellular fractionation Subcellular fractionation was performed using established procedures [18,19] Following injection of toxins, rats were sacrificed and livers rapidly removed and minced in ice-cold isotonic homogenization buffer as previously described [18,19] The EN was isolated by discontinuous sucrose gradient centrifugation and collected at the 0.25–1.0 m sucrose interface [18,19,21–23,44] The ENs was isolated from the EN fraction by freeze ⁄ thawing in mm Na-phosphate (pH 7.4) and disrupted in the same hypotonic medium using a small Dounce homogenizer (15 strokes with type A pestle) followed by centrifugation at 150 000 g for 60 as previously described [18,19,21–23,44] Rat liver cytosolic (S) fractions was isolated by differential centrifugation as previously described [25,44] EN fractions revealed no significant enrichment of lysosomal enzyme markers (N-acetyl-b-dglucosaminidase, relative specific activity = 1.5; acid phosphatase, relative specific activity = 2.2) with the yield of FEBS Journal 275 (2008) 1708–1722 ª 2008 The Authors Journal compilation ª 2008 FEBS 1719 Activation and translocation of diphtheria toxin T El Hage et al enzymes accounting for < 0.2% that of homogenate Comparably, EN fractions accounted for only 1% of the alkaline phosphodiesterase, confirming minimal contamination by plasma membrane These data are comparable with previously published studies [22,45] The recovery of organelle enzyme markers in the nonsedimentable cytosolic S fraction was very low, conforming well with our previously published biochemical characterizations [22,45] In vitro endosome–lysosome transfer assay The cell-free endosome–lysosome transfer of in vivo internalized mDT was performed as described previously [19,29,46] Rats were sacrificed 15 post mDT injection The postmitochondrial supernatant (referred to as the LPS fraction) was incubated for 60 at or 37 °C with mm ATP, mgỈmL)1 creatine kinase and 20 mm phosphocreatine After cooling to °C, incubation mixtures were subjected to centrifugation on linear Nycodenz gradients, as described previously [19,29,46] The distribution of mDT, mDT-A subunit, mDT-B subunit and enzyme activities were analyzed using reducing SDS ⁄ PAGE followed by western blot analysis and components appearing at densities in the ranges 1.065–1.11 and 1.11–1.145 gỈmL)1 were scored as endosomal and lysosomal, respectively The specificity of the endo-lysosomal transfer of internalized proteins and that the density shift involves a direct interaction between endosome and lysosome organelles have both previously been demonstrated [29] Cell-free proteolysis and translocation of endosome-associated DT EN isolated 15 after the injection of native DT (15 lgỈ100 g)1 body weight) were suspended at mgỈmL)1 in 0.15 m KCl, mm MgCl2 and 25 mm Hepes (pH 7) in the presence or absence of 5–10 mm ATP Samples were incubated at 37 °C for various periods and subjected to reducing SDS ⁄ PAGE followed by western blotting to determine the endosomal content and integrity of DT and DT-A and DT-B subunits To specifically assess the membrane translocation of degradative DT products, incubation mixtures were centrifuged for 60 at 100 000 g Pelleted endosomes were then subjected to reducing SDS ⁄ PAGE followed by western blot analysis using anti-DT 1275 serum Supernatants were first immunoprecipitated using anti-DT 1275 serum and then subjected to reducing SDS ⁄ PAGE followed by western blot analysis using anti-DT PV serum Immunoblot analysis Electrophoresed samples were transferred onto nitrocellulose membranes for 60 at 380 mA in transfer buffer 1720 containing 25 mm Tris base and 192 mm glycine The membranes were blocked by h of incubation with 5% skimmed milk in 10 mm Tris-HCl (pH 7.5), 300 mm NaCl and 0.05% Tween-20 The membranes were then incubated with primary antibody [horse IgG against native DT (diluted : 5000), mouse IgG against human furin R2 (diluted : 200), rabbit IgG against either human furin CT (diluted : 200), rat TrxR1 (diluted : 200), human EF-2 (diluted : 500) or human Hsp90 (diluted : 3000), rabbit polyclonal antisera against either mDT (diluted : 30 000), mouse cathepsin D R291 (diluted : 500), PE (diluted : 50 000) or CT (diluted : 60 000)] in the above buffer for 16 h at °C The blots were then washed three times with 0.5% skimmed milk in 10 mm Tris–HCl (pH 7.5), 300 mm NaCl and 0.05% Tween-20 over a period of h at room temperature The bound immunoglobulin was detected using horseradish peroxidase-conjugated goat anti-rabbit IgG, goat anti-mouse IgG or rabbit antihorse IgG In vitro proteolysis of DT peptides by hepatic endosomes, cathepsin D and furin ENs ( lg) or EN (1–15 lg) were incubated for varying lengths of time at 37 °C with 10 lg of native DT in 19 lL of 25 mM citrate-phosphate buffer (pH 4) or 25 mm Hepes buffer (pH 7) containing mm CaCl2, in the presence or absence of protease inhibitors To determine the integrity of DT, the proteolytic reaction was stopped by the addition of reducing or nonreducing SDS ⁄ PAGE sample buffer (62.5 mm Tris–HCl, pH 6.8, 2% SDS, 10% glycerol) followed by SDS ⁄ PAGE and either Coomassie Brilliant Blue staining or western blot analysis For some experiments, DT (10 lg) was digested in vitro with bovine cathepsin D (40 mL)1Ỉmg)1) in 50 mm citrate-phosphate buffer (pH 4), containing 50 mm MgCl2 or human furin (50 mL)1Ỉmg)1) in 50 mm Hepes buffer (pH 7), containing mm CaCl2 After 1–90 at 37 °C, the proteolytic reaction was stopped by the addition of reducing SDS ⁄ PAGE buffer followed by SDS ⁄ PAGE and Coomassie Brilliant Blue staining DT-catalyzed ADP-ribosylation of cytosolic EF-2 Native DT (30 lg) was first digested by incubation with ENs ( lg) at 37 °C for 90 in 25 mm citratephosphate buffer (pH 4) or 25 mm Hepes buffer (pH 7.4) containing mm CaCl2 and 0.2 m dithiothreitol The pretreated DT (10 lg) was then neutralized with 0.5 m Hepes (pH 7.4) and incubated with the cytosolic fraction ( 200 lg) in 0.1 m Hepes buffer containing lm [32P]NAD for 15–60 at 30 °C The reaction was stopped by the addition of Laemmli sample buffer followed by SDS ⁄ PAGE and autoradiography FEBS Journal 275 (2008) 1708–1722 ª 2008 The Authors Journal compilation ª 2008 FEBS T El Hage et al Immunodepletion studies ENs was immunodepleted of active cathepsin B, cathepsin D, IDE or furin prior to the digestion step by incubating ENs (0.15 mgỈmL)1) with antibodies coated onto protein GSepharose beads for 16 h at °C in 800 lL of 20 mm sodium phosphate buffer (pH 7) The fractions were then centrifuged for at 10 000 g, and the resultant immunodepleted supernatants were used in the toxin degradation assay Characterization of endosomal DT-degrading activity using gel-filtration HPLC ENs was loaded onto a TSK-GEL G3000 SWXL HPLC column [0.78 · 30 cm; Tosoh Corporation (Tessenderlo, Belgium)] equilibrated at °C with 50 mm Na-phosphate buffer (pH 6) The column was washed with 30 mL of Naphosphate buffer (pH 6) using a flow rate of 0.5 mLỈmin)1 Eluates were monitored on-line for absorbance at 214 nm with a LC-166 spectrophotometer (Beckman Coulter, Roissy, France) Each fraction (0.5 mL) was immediately adjusted to pH with 0.5 m citrate-phosphate buffer or pH with m Hepes buffer, and evaluated for DT-degrading activity by SDS ⁄ PAGE In some experiments, the eluted fractions that 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into. .. penetration of diphtheria toxin Reduction of the interchain disulfide bridge is the rate-limiting step of translocation in the cytosol J Biol Chem 268, 1567– 1574 Oh KJ, Senzel L, Collier RJ & Finkelstein... alpha -toxin [33], Aerolysin [34], anthrax toxin protective antigen, PE, Shiga and Shiga-like toxins, and botulinum toxin [32] Immunoblotting in the present study confirmed the detection of furin in