Báo cáo khoa học: Endosomal proteolysis of diphtheria toxin without toxin translocation into the cytosol of rat liver in vivo pdf

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
¸
1
2
3
4

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, 5 rue Jeanˆ
´
Baptiste Clement, 92296 Chatenay-Malabry,
France
Fax: +33 1 4683 5844
Tel: +33 1 4683 5528

E-mail:
(Received 4 July 2007, revised 19 January
2008, accepted 8 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.

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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 min 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 min (DT-A subunit) or 30–90 min (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

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Fig. 1. 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 min after mDT administration, and immediately subfractionated on linear Nycodenz density gradients (panel 4 °C), or incubated with ATP and an ATP-regenerating system at 37 °C for 60 min 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 min 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.

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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 min after mDT administration. When LPS
fractions were incubated at 4 °C and analyzed by
reducing SDS ⁄ PAGE followed by immunoblot analysis

Activation and translocation of diphtheria toxin

using rabbit a-1275 antibody (upper blot 4 °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 1 (EEA1) or
procathepsin D precursor (lower blots a-EEA1 and
a-CD). When the LPS fraction was incubated at 37 °C
for 60 min (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. 2. 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;  1 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 4. The incubation mixtures were then analyzed by reducing SDS ⁄ PAGE
followed by Coomassie Brilliant Blue staining. The major degradation products generated at pH 7 (peptide 7a) or pH 4 (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 min at 37 °C with the ENs fraction in buffer pH 4 or 7 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 1 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 3 days. The arrow to the right indicates the mobility of [32P]-labeled EF-2 ( 105 kDa).

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membrane-bound distribution of endosomal DTdegrading activity was assessed by reducing SDS ⁄ PAGE
analysis of DT digestion performed at pH 7 and 4
(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 4. 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 4 or 7
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 min 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 7 was inhibited by hexa-d-arginine (HA), a competitive inhibitor of furin, and the metal-chelating
B

Fig. 3. Effect of protease inhibitors on the proteolysis of native DT by ENs. ENs ( 1 lg) was incubated with 1 lM native DT at 37 °C for

180 min in 25 mM Hepes buffer pH 7 (A) or 25 mM citrate-phosphate buffer (pH 4) (B) in the absence or presence of 3.5 lgỈmL)1 PA, 1 lM
E64, 1 mM EDTA, 1 lM HA, 1 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).

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agent EDTA (Fig. 3A, upper gels). Neutral DTdegrading activity was inhibited in a dose-dependent
manner by HA (IC50  1 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. 4. 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 1 lM native DT at
37 °C for 120 min 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).

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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 7.
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 min after DT injection. A parallel increase in the content of the 70-kDa fragment was
observed with anti-furin R2 serum, especially at 5 min
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 7 or 4 using reducing SDS ⁄ PAGE analysis (Fig. 5B). The neutral DT-degrading activity
(Fig. 5B, fraction 7 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 9 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 7
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(Fig. 5C, upper gel) or bovine cathepsin D at pH 4
(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 min 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 min at 37 °C in the presence or absence of ATP, followed by centrifugation at
100 000 g for 60 min (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

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Activation and translocation of diphtheria toxin

A

B

C

Fig. 5. 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 1 lM native DT for 90 min 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 6 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 do 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 min 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

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B

C

D

Fig. 6. 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 min after the administration
of native DT and then suspended in 0.15 M KCl containing 5 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 min 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

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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 min 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

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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

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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 do 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 5 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 5 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 min 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

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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 min post mDT injection.
The postmitochondrial supernatant (referred to as the LPS
fraction) was incubated for 60 min at 4 or 37 °C with
5 mm ATP, 1 mgỈmL)1 creatine kinase and 20 mm phosphocreatine. After cooling to 4 °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 min after the injection of native DT
(15 lgỈ100 g)1 body weight) were suspended at 1 mgỈmL)1
in 0.15 m KCl, 5 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 min 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 min at 380 mA in transfer buffer

1720

containing 25 mm Tris base and 192 mm glycine. The
membranes were blocked by 3 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 1 : 5000), mouse IgG against human furin R2
(diluted 1 : 200), rabbit IgG against either human furin
CT (diluted 1 : 200), rat TrxR1 (diluted 1 : 200), human
EF-2 (diluted 1 : 500) or human Hsp90 (diluted 1 : 3000),
rabbit polyclonal antisera against either mDT (diluted
1 : 30 000), mouse cathepsin D R291 (diluted 1 : 500), PE

(diluted 1 : 50 000) or CT (diluted 1 : 60 000)] in the
above buffer for 16 h at 4 °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 1 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 ( 1 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 6 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 6 mm CaCl2. After 1–90 min 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 ( 1 lg) at 37 °C for 90 min in 25 mm citratephosphate buffer (pH 4) or 25 mm Hepes buffer (pH 7.4)
containing 6 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 1 lm
[32P]NAD for 15–60 min at 30 °C. The reaction was
stopped by the addition of Laemmli sample buffer followed
by SDS ⁄ PAGE and autoradiography.

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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 4 °C in 800 lL of 20 mm sodium
phosphate buffer (pH 7). The fractions were then centrifuged
for 5 min 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 4 °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 4 with 0.5 m citrate-phosphate buffer or
pH 7 with 1 m Hepes buffer, and evaluated for DT-degrading activity by SDS ⁄ PAGE. In some experiments, the
eluted fractions that contained the highest DT-degrading
specific activity were subjected to SDS ⁄ PAGE followed by

western blotting to determine their content of cathepsin D
and furin.

Acknowledgements
We thank Pamela H. Cameron (McGill University,
Montreal, Canada) for reviewing the manuscript.

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FEBS Journal 275 (2008) 1708–1722 ª 2008 The Authors Journal compilation ª 2008 FEBS