In vitroproteolysis 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 A
Trang 1translocation into the cytosol of rat liver in vivo
Tatiana El Hage1,2, Paulette Decottignies3,4and Franc¸ois Authier1,2
1 INSERM, U756, Chaˆtenay-Malabry, France
2 Universite´ Paris-Sud, Faculte´ de Pharmacie, Chaˆtenay-Malabry, France
3 CNRS, UMR 8619, Orsay, France
4 Universite´ Paris-Sud, Orsay, France
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 Corynebacte-rium diphtheriaeand belongs to the A–B family of bac-terial exotoxins In the secreted form of DT, the A and
Keywords
cathepsin D; diphtheria toxin; endosome;
furin; translocation
Correspondence
F Authier, Inserm U756, Universite´
Paris-Sud, Faculte´ de Pharmacie, 5 rue
Jean-Baptiste Cle´ment, 92296 Chaˆtenay-Malabry,
France
Fax: +33 1 4683 5844
Tel: +33 1 4683 5528
E-mail: francois.authier@u-psud.fr
(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 adminis-tration 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 post-endosomal 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 vitroproteolysis 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 pro-teolyzed 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 previ-ously reported to participate in the export of DT from human endosomes
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.
Trang 2B 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
pro-cess 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
endocyto-sed using a clathrin-dependent pathway [5] Prior to,
during or after endocytosis of DT into early
endo-somes, 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
internal-ized 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
transmem-brane domain (tunnel model) [8,9]; (b) translocation of
the DT-A (low association) subunit through the
oligo-meric 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
thioredox-in 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
intox-ication 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
bind-ing 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
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 inter-action and degradation of internalized DT by unidenti-fied 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 meth-odology used in the present study, which is the first to use DT and an in vivo model, is similar to that devel-oped 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 physio-logical state Here, we report that rat hepatic endo-somes 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 addi-tion, 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 A with cytosolic fractions isolated from DT-injected rats, as well as an absence of in vitro translo-cation 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 lgÆ100 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 sub-unit) or 30–90 min (DT-B subsub-unit) 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
Trang 3Fig 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 con-tent 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 mobili-ties 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 gradi-ents (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 analy-sis 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 iso-lated 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 gÆmL)1were scored as truly endosomal and lysosomal, respectively.
Trang 4mDT, 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
pre-pared 15 min after mDT administration When LPS
fractions were incubated at 4C and analyzed by
reducing SDS⁄ PAGE followed by immunoblot analysis
using rabbit a-1275 antibody (upper blot 4C), most
of the intact mDT and mDT subunits appeared in a single broad region of density in the range 1.070– 1.100 gÆmL)1, which mainly coincided with the endo-somal marker early endosome antigen 1 (EEA1) or procathepsin D precursor (lower blots a-EEA1 and a-CD) When the LPS fraction was incubated at 37C for 60 min (Fig 1B, middle blot 37C ⁄ 60 min), a major shift of the mutant toxin to high density frac-tions 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
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 m M Hepes buffer (pH 7) or 25 m M 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 frag-ments (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 dith-iothreitol (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 l M [ 32 P]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 [ 32 P]-labeled EF-2 ( 105 kDa).
Trang 5membrane-bound distribution of endosomal
DT-degrading 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
DT-fragments 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-Ile-Pro-Gly-Lys sequence at the N-terminal of product
4c) Moreover, intermediate 4a displayed the
N-termi-nal sequence of DT (Gly-Ala-Asp-Asp-Val-Val), sug-gesting 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 neu-tral pH with cytosolic fraction in the presence of [32P]NAD A rapid and sustained ADP-ribosylation of cytosolic EF-2 was observed following endosomal pro-teolysis of DT at neutral pH (Fig 2B) However, [32P]ADP-ribose incorporation into EF-2 after endoso-mal 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 analy-sis The proteolytic activity directed against DT at
pH 7 was inhibited by hexa-d-arginine (HA), a com-petitive inhibitor of furin, and the metal-chelating
A B
Fig 3 Effect of protease inhibitors on the proteolysis of native DT by ENs ENs ( 1 lg) was incubated with 1 l M native DT at 37 C for
180 min in 25 m M Hepes buffer pH 7 (A) or 25 m M citrate-phosphate buffer (pH 4) (B) in the absence or presence of 3.5 lgÆmL)1PA, 1 l M
E64, 1 m M EDTA, 1 l M HA, 1 m M 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 Coo-massie 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).
Trang 6agent EDTA (Fig 3A, upper gels) Neutral
DT-degrading 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)7m), an inhibitor of aspartic acid
prote-ases, 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
charac-terized polyclonal antibodies to mature cathepsin D
and its proform [21,22] to deplete cathepsin D from
ENs (Fig 4A, left gel) Quantitative immunoprecipita-tion 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 pro-teolytic activity directed towards DT at pH 4, as assessed by reducing SDS⁄ PAGE analysis Immuno-depletion of ENs with antibodies to cathepsin B and its proform (a-CB 7183) [23] failed to remove the pro-teolytic activity
Hepatic endosomes are known to contain neutral peptidases such as insulin-degrading enzyme (IDE) [24] and furin [25] Endosomal neutral DT-degrading activ-ity 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 l M native DT at
37 C for 120 min in 25 m M citrate-phosphate buffer (pH 4) (gel on the left) or 25 m M 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 recombi-nant 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 poly-clonal (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).
Trang 7against 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
con-tent of furin by immunoblotting with antibodies directed
against either the catalytic or carboxyl-terminal domain
of furin (Fig 4B) The EN showed intense
immuno-reactivity for the 90-kDa furin enzyme using either
anti-furin serum By contrast, anti-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
anti-genicity 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
time-dependent decrease in the content of the 90-kDa furin
was observed at 5–60 min after DT injection A
paral-lel 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
proteo-lytic activity associated with soluble endosomal
pro-teins 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
cathep-sin D and furin by western blotting ucathep-sing
anti-cathep-sin 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
anal-ysis (Fig 5B) The neutral DT-degrading activity
(Fig 5B, fraction 7 of upper gel) coincided with
elu-tion 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
(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 trea-ted with Pseudomonas exotoxin A (PE) (Fig 6A, blot a-PE) or CT (Fig 6A, blot a-CT) at 5–90 min post-injection, 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 iso-lated after injection of DT were incubated in buffered isotonic medium for 30–60 min at 37C in the pres-ence or abspres-ence 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 process-ing of DT in liver endosomes (Fig 6B, left blot) By contrast, immunoprecipitation of DT from superna-tants 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 iso-lated from control rats (Fig 6C, lane –) revealed a con-centration of TrxR1 equivalent to that observed in the cytosolic fraction (Fig 6C, blots a-TrxR1, lane –)
A weak immunoreactivity for Hsp90 was observed in
Trang 8control 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
endo-somes may represent a physiological site of TrxR1
localization and action, they do not demonstrate
physi-cal interaction between TrxR1 and internalized DT
Therefore, coimmunoprecipitation studies were
under-taken (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 DT PV or
anti-DT 1275 sera As expected, anti-DT, as well as free anti-DT-A and DT-B subunits, were detected in immunoprecipi-tates using anti-DT antibodies However, no DT immunoreactivity was found in TrxR1 immunoprecipi-tates (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
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
trun-cated furin ( 70 kDa) (B) Eluted fractions (5, 7, 9, 10 and 12) were tested for their ability to degrade 1 l M native DT for 90 min at 37 C in
25 m M Hepes buffer (pH 7) (upper gel) or 25 m M 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 UÆmL)1Æmg)1) in 25 m M Hepes buffer (pH 7) containing 6 m M CaCl 2 (upper gel) or cathepsin D (40 UÆmL)1Æmg)1) in
25 m M 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).
Trang 9are 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
exhi-bit a similar range in DT sensitivity [3] However, the
rat has surface membrane receptors for DT that have
properties indistinguishable from the receptors on
sen-sitive cells [4,13] Moreover, rat EF-2 is equally as
good a substrate for in vitro DT-catalyzed
ADP-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 extra-endosomal milieu (or cytosol), is responsible for part
C D
A B
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 m M MgCl2and, when indicated, 10 m M ATP After the indicated time of incuba-tion at 37 C, endosomes were sedimented by ultracentrifugation and the pellets (endosome-associated material) and supernatants (extra-endosomal 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 iso-lated 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 immuno-precipitated 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).
Trang 10(if not all) of the DT insensitivity in rats Another
pos-sibility 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 lgÆ100 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 vivocompartmentalization and processing activation
of DT in rat liver parenchyma, clearly demonstrates
that the toxin is internalized into endosomes as a
sin-gle nonproteolyzed and nonreduced monomeric chain
polypeptide where, at later stages, it undergoes
proteo-lytic processing (major pathway) and reduction (minor
pathway) by the endo-lysosomal apparatus The
kinet-ics of in vivo DT internalization observed in our
bio-chemical studies are comparable to that previously
reported morphologically using in vitro cellular
sys-tems, 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
bac-terial toxins such as CT [18,19] and plant toxins such
as ricin [27] are taken up by the rat liver and
subse-quently accumulate in a low-density endosomal
com-partment 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
endo-cytosis involves only the clathrin-dependent pathway
[5] Moreover, in the present study, the rate of
endoso-mal 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
en-dosomal proteolysis of CT involves only the cathepsin
D enzyme and requires endosomal acidification [18,19]
Using the in situ rat liver model system for
endo-some–lysosome fusion [29], we show here, as
previ-ously 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
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 internal-ized DT in DT sensitive cells [12] and extends this physiological location of DT to the lysosomal appara-tus of insensitive cells
Comparable to the endosomal degradation of inter-nalized ricin A-chain in macrophages [30], the endo-somal 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 prote-olytic activity in several respects: (a) the neutral DT-degrading 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 deple-tion of the neutral degrading activity; and (c) the neu-tral 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 cellu-lar and acellucellu-lar in vitro systems [6,17] However, the previously reported transmembrane property of furin [31] would argue against a role for furin in the proteo-lysis of DT by a soluble endosomal lysate Interest-ingly, 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 responsi-ble 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 nondenatu-rating and denatunondenatu-rating conditions, confirming that the 70-kDa truncated soluble furin represents a proteo-lytically active species
In agreement with our demonstration of a role for a novel soluble 70-kDa furin in the endosomal process-ing 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 protec-tive antigen, PE, Shiga and Shiga-like toxins, and bot-ulinum toxin [32]
Immunoblotting in the present study confirmed the detection of furin in rat liver endosomes [25] and rat