Proteolytic activation of internalized cholera toxin withinhepatic endosomes by cathepsin DClemence Merlen*, Domitille Fayol-Messaoudi*, Sylvie Fabrega, Tatiana El Hage, Alain Servinand Francois Authier

Institut National de la Sante et de la Recherche Medicale U510, Faculte de Pharmacie Paris XI, Chatenay-Malabry, France

Cholera toxin (CT) produced by Vibrio cholerae is

the major virulence factor responsible for the massive

secretory diarrhea of infected humans [1]. CT interacts

with intestinal epithelial cells and induces chloride

secretion due to toxin-mediated activation of adenylate

cyclase and elevation of intracellular cAMP. Activation

of adenylate cyclase results from ADP-ribosylation of

Arg201 of the a-subunit of the stimulatory GTP-binding

regulatory protein, Gsa, catalyzed by the toxin [2].

CT (� 84 kDa) is an oligomeric protein of the A-B

type composed of one activating A subunit (CT-A,

r.m.m. 27 400 Da) and five identical B subunits (CT-B,

r.m.m. 11 600 Da) arranged in a ring-like configur-

ation that bind ganglioside GM1 at the cell surface [3].

The CT-A subunit is comprised of two functional

domains termed the A1 and A2 peptides linked by

a single disulfide bond. The A1 peptide exhibits the

toxin’s ADP-ribosyltransferase activity which is neces-

sary for CT cytotoxic action. The A2 peptide contains

the endoplasmic reticulum-targeting motif KDEL at its

C-terminus. For full ADP-ribosylation of the stimula-

tory heterotrimeric GTPase Gsa, enzymatic production

of a degradative fragment generated from native CT

and structurally related to the A1 peptide must occur

followed by its targeting to the Gsa substrate. This

process, which takes 30–40 min in most cell types,

Keywords

cathepsin D; cholera toxin; endocytosis;

hepatocyte; proteolysis

Correspondence

F. Authier, INSERM U510, Faculte de

Pharmacie Paris XI, 5 rue Jean-Baptiste

Clement, 92296 Chatenay-Malabry, France

Fax: +33 1 46835844

Tel: +33 1 46835843

E-mail: francois.authier@cep.u-psud.fr

*These authors contributed equally to the

paper

(Received 29 April 2005, revised 1 July

2005, accepted 7 July 2005)

doi:10.1111/j.1742-4658.2005.04851.x

We have defined the in vivo and in vitro metabolic fate of internalized chol-

era toxin (CT) in the endosomal apparatus of rat liver. In vivo, CT was

internalized and accumulated in endosomes where it underwent degrada-

tion in a pH-dependent manner. In vitro proteolysis of CT using an endo-

somal lysate required an acidic pH and was sensitive to pepstatin A, an

inhibitor of aspartic acid proteases. By nondenaturating immunoprecipita-

tion, the acidic CT-degrading activity was attributed to the luminal form of

endosomal cathepsin D. The rate of toxin hydrolysis using an endosomal

lysate or pure cathepsin D was found to be high for native CT and free

CT-B subunit, and low for free CT-A subunit. On the basis of IC50 values,

competition studies revealed that CT-A and CT-B subunits share a com-

mon binding site on the cathepsin D enzyme, with native CT and free

CT-B subunit displaying the highest affinity for the protease. By immuno-

fluorescence, partial colocalization of internalized CT with cathepsin D was

confirmed at early times of endocytosis in both hepatoma HepG2 and

intestinal Caco-2 cells. Hydrolysates of CT generated at low pH by bovine

cathepsin D displayed ADP-ribosyltransferase activity towards exogenous

Gsa protein suggesting that CT cytotoxicity, at least in part, may be rela-

ted to proteolytic events within endocytic vesicles. Together, these data

identify the endocytic apparatus as a critical subcellular site for the accu-

mulation and proteolytic degradation of endocytosed CT, and define endo-

somal cathepsin D an enzyme potentially responsible for CT cytotoxic

activation.

Abbreviations

CT, cholera toxin; EN, endosomes; HI, human insulin; PA, pepstatin-A; PDI, protein disulfide isomerase.

FEBS Journal 272 (2005) 4385–4397 ª 2005 FEBS 4385

corresponds to the lag-phase required for CT to enter

the cell by endocytosis using clathrin-dependent and

-independent mechanisms [4,5], become activated and

move to its site of action.

Two major candidate compartments have been

proposed as being physiologically relevant to the

mechanism of activation of internalized CT. The first

activating pathway, identified in rat hepatocytes, has

been proposed to operate at an early stage of endo-

cytosis within endocytic vesicles [6,7]. Thus, using a

subcellular fractionation approach to address CT

compartmentalization and activation in vivo, it was

shown that activation of adenylate cyclase by CT in

intact liver requires the association and subsequent

processing of the toxin in an endosomal compart-

ment. However, the nature of the relevant enzymatic

activity (protease and ⁄or reductase) as well as the

mechanisms underlying CT-A subunit cytotoxic

action towards its substrate were not investigated.

The other well-characterized subcellular compart-

ment involved in CT activation is the endoplasmic reti-

culum (ER) where CT is transported in a retrograde

manner via the Golgi apparatus by a KDEL-dependent

mechanism [8]. After entry of CT into the ER, the

ER-chaperone protein disulfide isomerase (PDI) is

involved in facilitating the reduction of the internal

disulfide bond of CT [9] and preparation of the A1

peptide for retro-translocation to the cytosol by the

Sec61p channel [10].

In the present study, we have characterized the

endosomal processing of endocytosed CT in rat liver

and examined the sensitivity of toxin proteolysis to

pH, protease inhibitors and immunodepletion pro-

cedures using antibodies to well-defined endosomal

proteases. In addition, using a cell-free ADP-ribosyla-

tion assay, we have explored the potential physiologi-

cal significance of the endosomal proteolysis of CT in

its cytotoxic action. This has allowed us to characterize

the role of the aspartic acid protease cathepsin D in

the endosomal degradation of internalized CT. We

show that hydrolysates of CT generated in vitro by

pure cathepsin D displayed ADP-ribosyltransferase

activity towards exogenous Gsa protein.

Results

In vivo endocytosis of native CT and CT-B

subunit in rat liver

The kinetics of in vivo uptake of native CT and CT-B

subunit into endosomal fractions were first assessed

(Fig. 1). Rats were administered an intravenous injec-

tion of native CT or CT-B subunit (50 lg per 100 g

body weight) and killed 5–90 min postinjection.

Following preparation of hepatic endosomes the

amount of internalized CT-A and CT-B subunits was

determined by western blot analyses (Fig. 1A). A simi-

lar time-dependent increase in CT-A and CT-B

A

B

Fig. 1. Kinetics of appearance of CT-A and

CT-B subunits in hepatic endosomes after

ligand administration. (A) Endosomal frac-

tions were isolated at the indicated times

after the in vivo administration of native CT

or CT-B subunit, and evaluated for their con-

tent of both subunits by western blot analy-

sis using the polyclonal anti-CT Ig. Each lane

contained � 50 lg of endosomal protein.

Arrows to the right indicate the mobility of

CT-A (� 28 kDa) and CT-B subunit

(� 12 kDa). Molecular mass markers are

indicated to the left of each blot. (B) Assess-

ment of polyclonal antibody specificity

towards CT and individual A- and B-subunits

was performed using reducing and non-

reducing SDS ⁄ PAGE followed by western

blot analysis. Each lane contained 1 lg of

peptide. Arrows to the right indicate the

mobility of CT-A (� 28 kDa), CT-B (� 12 kDa),

and A1 peptide (� 21 kDa). A2 peptide was

not detected under these experimental

conditions. Molecular mass markers are

indicated to the left of each blot.

Endosomal proteolysis of cholera toxin by cathepsin D C. Merlen et al.

4386 FEBS Journal 272 (2005) 4385–4397 ª 2005 FEBS

subunits was observed in endosomal fractions 5–30 min

after native CT injection (Fig. 1A, upper blot), after

which the level of both subunits decreased. A slower rate

of B-subunit endocytosis was observed in response to

CT-B subunit administration and remained elevated up

to 90 min postinjection (Fig. 1A, lower blot).

Characterization of the polyclonal anti-CT antibody

C3062 by western blot analysis of pure CT or individ-

ual A- and B-subunits revealed a specificity for both

subunits under nonreducing conditions (Fig. 1B, upper

blot). Following chemical reduction of the A-subunit

interchain disulphide bond, the rabbit anti-CT anti-

body recognized the A1 peptide and B-subunit with no

immunoreactivity towards the A2 peptide (Fig. 1B,

lower blot).

Proteolysis of CT within hepatic endosomes

at acidic pH

We next examined the ability of hepatic endosomes

to degrade CT (Fig. 2). The luminal and membrane-

bound distribution of endosomal CT-degrading activity

was assessed by western blot analysis of CT digestions

performed at various pH (Fig. 2A). EN fractions

degraded native CT at pH 4 and 5, with degradation

decreasing markedly at pH 6–7. The CT-B subunit was

more efficiently degraded than the CT-A subunit

(Fig. 2A, EN, lane pH 4). A 25-kDa proteolytic frag-

ment of CT-A subunit was specifically generated at

pH 4–5. Another CT-A product of 22-kDa was

observed in hydrolysates at pH 6–7 and also in the

untreated toxin (lane -). Subfractionation of hepatic

endosomes (EN) into a soluble endosomal lysate (ENs)

revealed a similar pattern of proteolysis to that

observed for the EN fraction (Fig. 2A, ENs), suggest-

ing that the majority of CT-degrading activity in endo-

somes is soluble.

A soluble endosomal extract (ENs) was then assessed

by HPLC analysis for its ability to proteolyze native CT

in vitro at acidic (pH 4 and 5) and neutral pH (pH 7)

(Fig. 2B). Degradation of both A- and B-subunits was

pH-dependent with maximal degradation obtained at

A

B

Fig. 2. Effect of pH on the endosomal pro-

cessing of native CT. (A) Total (EN �10 lg)

and soluble endosomal fractions (ENs

�1 lg) were incubated with 7 lg native CT

at 37 �C for 60 min in 30 mM citrate-phos-

phate buffer at the indicated pH. The incu-

bation mixtures were then analyzed by

western blotting using the polyclonal anti-CT

antibody. The mobility of each CT subunit is

indicated on the right (CT-A �28 kDa; CT-B,

�12 kDa). (B) Representative RP-HPLC pro-

files obtained by incubating native CT (1 lM)

with ENs (� 1 lg) at 37 �C for 60 min in

175 mM citrate-phosphate buffer at the

indicated pH. Shown are absorbance profiles

at 214 nm. Untreated CT subunits had an

elution time of 60 min (CT-B) and 63 min

(CT-A) (HPLC profile CT). The endosomal

proteins alone did not give any detectable

peaks (results not shown).

C. Merlen et al. Endosomal proteolysis of cholera toxin by cathepsin D

FEBS Journal 272 (2005) 4385–4397 ª 2005 FEBS 4387

pH 4. Intermediate peptide peaks were only observed

at pH 4 in addition to the undegraded subunits which

had decreased in peak height (Fig. 2B).

Catalytic properties of endosomal CT-degrading

activity

We evaluated the ability of individual CT-A and CT-B

subunits to act as substrates for endosomal acidic pro-

teases (Fig. 3). The rate of hydrolysis of individual

subunits was determined using RP-HPLC analysis by

following the disappearance of the parent peptide after

a 2 h incubation with ENs at pH 4 (Fig. 3, lower pan-

els). The rate of peptide hydrolysis was found to be

very low with free CT-A subunit and high with free

CT-B subunit. However, when native CT was used as

a substrate the rates of proteolysis of both subunits pre-

sent in the AB5 pentamer were comparable to that of

the individual CT-B subunit (Fig. 3, lower panel, left).

The effect of various protease inhibitors on the aci-

dic CT-degrading activity contained in hepatic endo-

somes was next examined by western blot and HPLC

analyses (Fig. 4). The proteolytic activity directed

against both A- and B-subunits at pH 4 was inhibited

by pepstatin-A (PA), an inhibitor of aspartic acid pro-

teases (Fig. 4A, PA). No effect was observed with the

cysteine-protease inhibitor E-64 or the metal-chelating

agent EDTA. The 25-kDa proteolytic fragment of the

CT-A subunit, whose level was maximal after a 3 h

incubation, was not detected in the presence of pepsta-

tin-A. Two minor diffuse CT-A fragments of 22- and

18-kDa were also observed after 3 h of incubation,

however, their production was inhibited in the pres-

ence of pepstatin-A (for the 18-kDa species) or EDTA

(for the 22- and 18-kDa species). RP-HPLC analysis

of CT digestions performed at pH 4 using the ENs

fraction confirmed the inhibitory effect of pepstatin-A,

with no significant effect observed using E-64 and the

serine protease inhibitor PMSF (Fig. 4B,C).

Identification of endosomal CT-degrading

enzyme as cathepsin D

The inhibition of CT-degrading activity by pepstatin-

A, its low pH optimum and its presence in the endo-

somal lumen as a soluble form suggested cathepsin D

as a likely candidate for this activity. We therefore

used well characterized polyclonal antibodies to

mature cathepsin D and its proform [11,12] to deplete

cathepsin D from ENs (Fig. 5A). Quantitative immuno-

precipitation of cathepsin D using antibodies direc-

ted against the mouse (R291) and human enzyme

(M8147) removed greater than 88% of the endosomal

proteolytic activity directed towards both subunits

as assessed by RP-HPLC (Fig. 5B). Immunodeple-

tion of ENs with antibodies to cathepsin B and its

Fig. 3. Kinetics of proteolysis of native CT, CT-A subunit and CT-B subunit at pH 4 by a soluble endosomal lysate. Shown are representative RP-

HPLC profiles resulting from the incubation of native CT, CT-A or CT-B (1 lM) with ENs (� 1 lg) at 37 �C for 120 min in 175 mM citrate-phos-

phate buffer pH 4 (lower HPLC profiles). Shown are absorbance profiles at 214 nm. Elution profiles of native CT and individual CT-A and -B

subunits are shown for comparison (upper HPLC profiles).

Endosomal proteolysis of cholera toxin by cathepsin D C. Merlen et al.

4388 FEBS Journal 272 (2005) 4385–4397 ª 2005 FEBS

proform [13] failed to remove the proteolytic activity

(Fig. 5A).

To strengthen the physiological relevance of our

observations obtained in vitro with cell-free endosomes,

we studied the subcellular localization of internalized

CT or CTB-FITC and cathepsin D in hepatoma HepG2

and intestinal Caco-2 cells by immunofluorescence con-

focal microscopy (Fig. 6). Cells were incubated with

1 lm native CT for 30 min (HepG2 cells) or CTB-FITC

for 15 min (HepG2 and Caco-2 cells) when most of the

internalized ligands would be located in the endosomes

(Fig. 1A). CTB-FITC and monoclonal antibody D15-8

to the CT-B subunit (in green) demonstrated a highly

punctate staining pattern reminiscent of vesicular com-

partments. Costaining with antibody directed against

mature and precursor cathepsin D enzyme (in red)

revealed a partial colocalization (in yellow) of CT with

the intracellular aspartic acid protease.

Affinity-binding and degradation of native CT and

individual CT subunits by cathepsin D

Substrates of the same protease would be expected to

compete with each other for the enzyme binding site.

We therefore used a competition assay to evaluate

the ability of native CT and the individual A- and

B-subunits to inhibit degradation of the radiolabeled

substrate 125I-labelled TyrA14-HI by cathepsin D

(Fig. 7A). As reported previously [12,14], the well-

defined cathepsin D substrate HI was found to inhibit

A

C

B

Fig. 4. Effect of protease inhibitors on the proteolysis of native CT by hepatic endosomes. (A) EN fraction was incubated with 7 lg native

CT at 37 �C for 1 or 3 h in 30 mM citrate-phosphate buffer pH 4 without (buffer) or with 1% Me2SO (DMSO), 3.5 lgÆmL)1 pepstatin-A (PA),

0.1 mM E-64 or 1 mM EDTA. At the end of the incubation, the samples were analyzed by western blotting using the polyclonal anti-CT Ig.

Molecular mass markers are indicated to the left of each panel. Arrows indicate the mobility of the intact A (� 28 kDa) and B subunits

(� 12 kDa). (B) Representative RP-HPLC profiles obtained following the incubation of native CT with ENs in the presence or absence of

3.5 lgÆmL)1 pepstatin-A. Shown are absorbance profiles at 214 nm. Elution profile of native CT is shown for comparison (HPLC profile CT).

(C) ENs (� 1 lg) was incubated with 1 lM native CT at 37 �C for 60 min in 175 mM citrate-phosphate buffer pH 4 in the absence or pres-

ence of 3.5 lgÆmL)1 pepstatin-A (PA), 1% Me2SO (DMSO), 1 lM E-64, 1 mM PMSF or 1% MeOH. At the end of the incubation, the proteo-

lytic reaction was stopped with acetic acid (15%), and the incubation mixtures were analyzed by RP-HPLC. The rate of proteolysis of CT-A

and CT-B subunits was determined by following the disappearance of the peak area corresponding to the parent peptides.

C. Merlen et al. Endosomal proteolysis of cholera toxin by cathepsin D

FEBS Journal 272 (2005) 4385–4397 ª 2005 FEBS 4389

125I-labelled TyrA14-HI proteolysis by bovine cathepsin

D in a dose-dependent manner with an IC50 of 30 lm.Native CT was � 50 times more effective than HI at

competing for proteolysis of radiolabeled HI (IC50 of

0.6 lm). The CT-B subunit (IC50 of 0.9 lm) was able

to inhibit the degradation of radiolabeled HI compar-

ably to that of native CT, whereas the CT-A subunit

(IC50 of 9 lm) was found to be � 10–15 times less

potent than native CT.

We then compared the rate of hydrolysis of the indi-

vidual CT-A and CT-B subunits by cathepsin D at

pH 4, followed by nonreducing SDS ⁄PAGE and Coo-

massie Blue staining (Fig. 7B). Pure cathepsin D was

found to degrade the CT subunits in a manner similar

to that observed for the hepatic endosomal fractions

(Fig. 2A), i.e. CT-B subunit (t1 ⁄ 2 < 15 min) > CT-A

subunit (t1 ⁄ 2 � 60 min).

Proteolytic activation of CT induced by

cathepsin D treatment

CT cleavage induced by cathepsin D may be an essen-

tial step for the cytotoxic activity of the toxin. Conse-

quently, we examined whether under conditions where

CT was partially processed by cathepsin D, we would

observe a corresponding change in the toxin cytotoxi-

city (Fig. 8). By western blot analysis, optimal degra-

dation of native CT was observed with a cathepsin D

concentration of 40 UÆmL)1Æmg)1 (Fig. 8A). After a

1 min incubation at this concentration, the 25-kDa

proteolytic fragment of the CT-A subunit previously

identified (Figs 2A and 4A) was detected. Another

22-kDa product, whose presence was observed in the

control condition (lane -), was prominently detected in

CT hydrolysates performed using 4 UÆmL)1Æmg)1 cath-

epsin D. Therefore, the 40 UÆmL)1Æmg)1 concentration

was used to follow the subsequent proteolytic activa-

tion of the toxin. CT was first partially processed by

cathepsin D at pH 4–7 using the above enzyme ⁄ sub-strate ratio and then incubated at neutral pH with

microsomal membranes in the presence of [32P]NAD

(Fig. 8B, upper panel). A rapid ADP-ribosylation of

microsomal Gsa was evident following proteolysis of

CT at acidic pH (pH 4–5), especially after a 15 min

digestion at pH 5. In vitro digestion of CT at pH 7, a

pH at which cathepsin D activity is nonexistent [12],

did not reveal any Gsa labeling even after 60 min of

proteolysis. Comparably, no detectable 32P-labeling of

Gsa was observed after treatment of CT under acidic

(pH 4.5 and 5.5) or neutral (pH 7.5) conditions

(Fig. 8B, middle panel). However, in vitro reduction of

native CT or the CT-A subunit at 37 �C and pH 7

Fig. 5. Effect of immunodepletion of cathep-

sins on endosomal CT-degrading activity. (A)

ENs fractions were immunodepleted of act-

ive cathepsin D (a-CD) or cathepsin B (a-CB)

using their respective polyclonal antibodies

which had been precoated onto protein G-

Sepharose beads. Following centrifugation,

the resultant supernatants were incubated

with 1 lM native CT in 175 mM citrate ⁄phosphate buffer pH 4 for 120 min at 37 �C,

and then analyzed by RP-HPLC. The rate

of proteolysis of CT-A and CT-B subunits

was determined by following the disappear-

ance of the peak area corresponding to the

parent peptides. (B) ENs were immunode-

pleted of active cathepsin D using polyclonal

antibodies directed against human (M8147)

or mouse (R291) cathepsin D, and the

resultant supernatants were tested for their

ability to degrade native CT at pH 4 as

described for panel A. The proteolytic reac-

tion was stopped with acetic acid (15%) and

the samples were analyzed by RP-HPLC.

Shown are absorbance profiles at 214 nm.

Endosomal proteolysis of cholera toxin by cathepsin D C. Merlen et al.

4390 FEBS Journal 272 (2005) 4385–4397 ª 2005 FEBS

with dithiothreitol (0.2 m final concentration) revealed

[32P]ADP-ribose incorporation into microsomal Gsa(Fig. 8B, lower panel), as reported previously [15]. The

possibility that the CT-A subunit was artefactually

reduced during the treatment with cathepsin D in vitro

was ruled out by the absence of production of the

A1-peptide when native CT or CT-A subunit was incu-

bated with cathepsin D in the presence of pepstatin-A

at both pH 4 and 7 (results not shown).

Discussion

As hepatic parenchyma accounts for a large fraction of

the total CT-binding sites in the body, subcellular frac-

tions from rat liver have been used to characterize the

initial step in the interaction of CT with cells [6,16,17].

The time-dependent internalization of CT in hepatic

cells was later assessed biochemically using isolated he-

patocytes [18] and morphologically using the rat liver

cell line KLTRYPV [19], a mouse hepatocyte cell line

[20] and primary culture hepatocytes [21]. In agreement

with these studies, we have found that in vivo both CT-A

and CT-B subunits undergo stoichiometric endocytosis

in rat liver and hepatoma HepG2 cells more rapidly and

to a greater extent than in intestinal Caco-2 cells. This

may reflect, in part, the greater binding capacity of

hepatocytes and hepatoma cells [6,16,17], the lower dis-

sociation constant of the hepatocyte–toxin interaction

[18] and ⁄or the higher content of ganglioside GM1 in

hepatocytes as compared to other cell types.

Previously, subcellular fractionation techniques used

to assess the in vivo localization of radiolabeled 125I-

labelled CT taken up by rat liver have shown that both

radioactive A- and B-subunits sequentially associate

with the plasma membrane, endosomes and lysosomes,

and that a very low level of A1 peptide occurred in the

endosomal compartment (< 5% of total endosomal

radioactivity recovered at 30 min postinjection) [6].

Unfortunately, in these studies, the nature of the endo-

somal alterations of the CT-A subunit was based on

(a) comparison of the electrophoretic mobility of the

endosomal radioactive CT-A subunit to that of the

chemically reduced 125I-labelled CT-A subunit; and (b)

TCA-precipitation which greatly underestimates CT-A

subunit degradation and reduction [6,18]. Moreover,

some of the intermediates might not have been radio-

labeled and therefore would have escaped detection.

Finally, the nature of the relevant enzymatic activity

(protease and ⁄or reductase) was not investigated. In

addition, the fraction of hepatocyte-associated 125I-

labelled CT that was converted into A1 peptide

was < 4% after 60 min, suggesting that the endo-

somal reductive pathway may represent a minor meta-

bolic fate for internalized CT within the endosomal

apparatus [18]. Alternatively, the radioactive iodine on

the CT-A subunit might have had a detrimental effect

A

B

C

Fig. 6. Partial colocalization of internalized

CT and cathepsin D in hepatoma HepG2

and intestinal Caco-2 cells.HepG2 (A and B)

and Caco-2 cells (C) were treated with 1 lM

native CT for 30 min (B) or 1 lM CTB-FITC

for 15 min (A and C), fixed with paraformal-

dehyde and permeabilized with Triton X-100

prior to staining. In panels A and C, HepG2

and Caco-2 cells were treated with poly-

clonal anti-(cathepsin D) Ig R291. In Panel B,

HepG2 cells were treated with both poly-

clonal anti-(cathepsin D) Ig R291 and anti-

CTB monoclonal antibody D15-8. The mono-

clonal antibody D15-8 directed against the

B-subunit was concluded to be highly speci-

fic as it recognized only the B-subunit by

western blot analysis (results not shown).

CT is shown in green and cathepsin D is

shown in red. Merged images on the right

indicate the extent of colocalization (yellow).

Scale bar, 7.6 lm (A and B) or 8.5 lm (C).

Fluorescent images were captured at two

emission wavelengths (488 and 543 nm).

C. Merlen et al. Endosomal proteolysis of cholera toxin by cathepsin D

FEBS Journal 272 (2005) 4385–4397 ª 2005 FEBS 4391

on the endosomal proteolytic and reductional systems

by affecting the degradation and ⁄or reduction rates.

Although unable to document CT reduction in endo-

cytic structures, our in vivo and in vitro endosome

studies clearly show that, under conditions ensuring

acidification of these structures, internalized unlabeled

CT-A subunit was progressively processed into degra-

dative fragments. However, as a possible consequence

of diffusion of degradative products out of the endo-

somal apparatus and ⁄or transfer of CT metabolites to

lysosomes, a net detection of CT proteolytic fragments

was only observed in our in vitro assays.

Exploiting the fact that two substrates competing

for the same enzyme will inhibit each other [14], we

have defined the affinity of native CT and its individ-

ual subunits for the cathepsin D protease. On the basis

of IC50 values, the CT-A and CT-B subunits and HI

share a common binding site on the cathepsin D

enzyme. Competition studies revealed that native CT

and CT-B subunit displayed nearly equivalent affinity

for cathepsin D (IC50 of 0.6–0.9 lm). However, the

CT-A subunit and the cathepsin D substrate HI [12]

were found to be 10–15 times less potent. These

competition studies correlated with results obtained

from degradation studies using endosomal fractions or

pure bovine cathepsin D in which the rate of peptide

hydrolysis was found to be low with the A-subunit

and high with the B-subunit. Interestingly, insulin

elicits inhibition of CT-stimulated adenylate cyclase

activity in both hepatocytes and the P9 immortalized

hepatocyte cell line [22]. Attenuating effects on CT

action may well originate from the ability of both pep-

tides to accumulate into hepatic endocytic vesicles and

to interact with endosomal cathepsin D (this study and

[12]). Comparably, it has been reported that the CT-B

subunit alters the progression of exogenous antigens

along the endocytic processing pathway, and prevents

or delays efficient epitope presentation and T-cell sti-

mulation [23]. Studies have suggested the potential role

of cathepsin D in conjunction with cathepsins S and L

in degrading endocytosed antigens and the invariant

chain within antigen-presenting cells [24]. CT may have

the ability to alter the immune response by reducing

cathepsin D activity and its subsequent processing of

antigen in antigen-presenting cells [23]. In either case,

it is clear that the endosomal cathepsin D activity

exhibits a high specificity that limits further proteolysis

of the bioactive CT-degrading fragment.

The present studies indicate that both CT-A and

CT-B subunits are high-affinity substrates for the

endosomal protease cathepsin D. We confirmed our

earlier biochemical and morphological studies that had

localized active cathepsin D to endosomal subcellular

fractions of rat liver [11,12] and early endocytic EEA1-

positive vesicles of rat hepatocytes [12]. Thus, we have

previously reported that internalized glucagon [11] and

insulin [12] are partially processed within hepatic endo-

somes by the endopeptidase activity of cathepsin D.

The participation of cathepsin D in the endosomal

proteolysis of mannose-BSA and parathyroid hor-

mone-(1)84), in macrophages [25,26], invariant chain

and endocytosed antigens in antigen-presenting cells

[24] and b-amyloid precursor protein in astrocytoma

cells [27] has been comparably demonstrated. Import-

antly, the role of cathepsin D in conjunction with cath-

epsin B in the endosomal processing, membrane

translocation and cytotoxicity of ricin A chain has also

been established [28].

CT-A– 5 15 30 60 90 – 5 15 30 60 (min of incubation)

CT-A(28-kDa)

CT-B(12-kDa)

CT-B

Fig. 7. Affinity-binding and degradation of native CT, CT-A subunit

and CT-B subunit by cathepsin D. (A) Competition of native CT, CT-

A subunit, CT-B subunit and HI for the degradation of 125I-labelled

TyrA14-HI by cathepsin D. Bovine cathepsin D (0.025 UÆmL)1) was

incubated with 125I-labelled TyrA14-HI (75 fmol) for 20 min at 37 �Cin 0.1 M citrate-phosphate buffer pH 4 with the indicated concentra-

tions of unlabeled peptides. The amount of degraded radiolabeled

insulin was determined by precipitation with TCA. Results are the

mean of three separate experiments and are expressed as a per-

centage of degradation observed in the absence of added unlabeled

peptides. (B) CT-A or -B subunit (7 lg) was incubated with cathep-

sin D (40 UÆmL)1Æmg)1) at 37 �C in citrate-phosphate buffer pH 4

for the indicated times. The incubation mixtures were then ana-

lyzed by nonreducing SDS ⁄ PAGE followed by Coomassie Brilliant

Blue staining. Arrows indicate the mobility of intact CT-A (28 kDa)

and CT-B (12 kDa) subunits.

Endosomal proteolysis of cholera toxin by cathepsin D C. Merlen et al.

4392 FEBS Journal 272 (2005) 4385–4397 ª 2005 FEBS

The endosomal CT-degrading activity described here

differs from the neutral Ca2+-dependent furin enzyme

which participates in the proteolytic activation of other

bacterial toxins [29] as follows: (a) furin recognition

sequences (-Arg-X-Lys ⁄Arg-Arg- or -Lys ⁄Arg-X-X-X-

Lys ⁄Arg-Arg-) are not present in the CT molecule [30];

(b) no degradation products were observed when pure

active furin was incubated with CT (results not

shown); and (c) the acidic CT-degrading activity was

not inhibited by the metal-chelating agent EDTA,

whereas Ca2+ and a neutral pH were strictly required

for furin activity [31].

Correlations between CT action, endocytosis and

processing were first made in cell fractionation studies

using rat hepatocytes intoxicated with CT in vivo [6,7].

In these experiments, the endosomal processing of the

A-subunit was closely associated with activation of

adenylate cyclase and CT-induced activation of adeny-

late cyclase appeared to depend on endosome acidifica-

tion [7,18]. Thus, the acidotropic drug chloroquine

reduced adenylate cyclase activation by CT and length-

ened the lag phase both in vivo [7] and in vitro [18].

In addition intracellular CT, after a 30–60 min lag

phase, was localized within the ER in murine hepatocyte

BNL CL.2 cells [20] and other various cell types [32]

using electron microscopy, fluorescence assay and sub-

cellular fractionation. That CT followed a retrograde

pathway into the Golgi and then the ER, with subse-

quent ER processing participating in CT cytotoxic

action, was provided by four independent lines of

evidence: (a) inactivating mutations or removing the

KDEL motif attenuated the efficiency of toxin action

greater than 10-fold in polarized intestinal T84 cells [33];

(b) inhibition of CT action correlated with brefeldin A-

induced disruption of Golgi structure and function [34];

(c) reduction of the A-subunit was shown within the ER

lumen in human intestinal Caco-2 cells and depended

on catalysis by PDI [35]; (d) interaction between the

A1-peptide and the protein translocation channel of the

ER was shown, followed by transport of the A1-peptide

through the Sec 61p translocon into the cytosol [36]. In

cell types other than hepatocytes, CT-induced cellular

responses were brefeldin A-sensitive but independent

of organelle acidification (chloroquine-insensitive) [37],

suggesting that the dual-compartmental activation of

intracellular CT may be cell-type specific. The discrep-

ancies between these data sets may also be due to the

experimental approaches: an in vivo model used for the

A

B

Fig. 8. Effect of cathepsin D treatment on CT-catalyzed ADP-ribosylation of microsomal Gsa. (A) Native CT (7 lg) was incubated in vitro with

cathepsin D (40 or 4 UÆmL)1Æmg)1) at 37 �C in citrate-phosphate buffer pH 4 for the indicated times. The incubation mixtures were then ana-

lyzed by western blotting using the polyclonal anti-CT Ig. Molecular mass markers are indicated to the left. Arrows indicate the mobility of

the intact A (� 28 kDa) and B subunits (� 12 kDa). (B) Native CT and ⁄ or CT-A were digested in vitro with cathepsin D (40 UÆmL)1Æmg)1) at

37 �C in citrate-phosphate buffer pH 4–7 for the indicated times or incubated at 37 �C in citrate-phosphate buffer pH 4.5–7.5 with or without

dithiothreitol (0.2 M) for 30 min. The treated CT (5 lg) was then incubated for 45 min at 30 �C with microsomal proteins (� 50 lg) in sodium

phosphate buffer pH 7.2 in the presence of 0.52 lM [32P]NAD. Samples were then subjected to SDS ⁄ PAGE and analyzed by autoradio-

graphy. Dithiothreitol and cathepsin D samples were exposed to X-ray films at )80 �C for 1 or 5 days, respectively. The arrow indicates the

mobility of 32P-labeled Gsa (� 45 kDa).

C. Merlen et al. Endosomal proteolysis of cholera toxin by cathepsin D

FEBS Journal 272 (2005) 4385–4397 ª 2005 FEBS 4393

studies on hepatocytes [6,7] and an in vitro model for

the studies on intestinal and other cell types [34–37]. On

the other hand, CT is internalized into cells by both

clathrin-dependent and -independent endocytosis and

these pathways may be differentially affected by acido-

tropic agents and carboxylic ionophores.

Previous studies have shown that CT administration

to rats markedly and significantly increased rat liver

endosome acidification [38]. The more acidic pH of

these endocytic vesicles might facilitate (a) the inter-

action of internalized CT with endosomal cathepsin D

which displays an optimum activity at pH 4; and (b)

the translocation of the A-subunit (or A-subunit frag-

ment) across the endosomal membrane, which requires

a low pH [18].

The subcellular site where endosomal activated CT

ADP-ribosylates Gsa protein and activates adenylate

cyclase remains to be clarified. Several lines of evidence

now indicate that trimeric G proteins and adenylate

cyclase are located in the endosomal compartment of

various cells [39–41]. Previous studies report the pres-

ence of the a-subunit of Gs protein [40], a fluoride-sen-

sitive adenylate cyclase activity [7,42,43] and adenylate

cyclase VI [40] in rat liver endosomal fractions. These

observations are also in agreement with related studies

on endosomal fusion where Gsa appears to be implica-

ted as fusion between endosomes was abolished using

CT in the presence of NAD, an antibody against the

C-terminus of Gsa or synthetic peptides that preferen-

tially activate Gsa [44]. Finally, activation of hepatic

adenylate cyclase after CT injection into rats occurs

first in endosomal fractions and, later, in plasma mem-

brane fractions, suggestive of sequential activation of

adenylate cyclase in these compartments [7].

In summary, we have characterized in vivo and

in vitro the metabolic fate of internalized CT within

the endosomal apparatus of rat liver. We have found

that proteolytic degradation of CT was mediated by

endosomal cathepsin D which assisted in the release of

CT-A fragment(s) that are active towards the Gsaprotein. Studies are currently underway to elucidate

the sites of cleavage of CT by cathepsin D and to

determine the endosomal degradative CT-A frag-

ment(s) responsible for the endosomal ADP-ribosyla-

tion of the Gsa substrate.

Experimental procedures

Peptides, ligand radioiodination, antibodies,

protein determination and materials

CT-A and -B subunits, native CT, CT-B subunit-FITC,

HI and bovine cathepsin D (EC 3.4.23.5), 15 UÆmg)1,

were purchased from Sigma (St Louis, MO, USA). HI

was radioiodinated by the lactoperoxidase method and

purified by RP-HPLC to specific activities of 150–

300 lCiÆlg)1 as previously described [45]. Rabbit polyclo-

nal anti-(CT C3062) was from Sigma. Mouse monoclonal

antibody directed against CT-B subunit (anti-CTB D15-8)

was a gift from F. Nato (Institut Pasteur, Paris, France).

Rabbit anti-(mouse cathepsin D R291) Ig [11,12], sheep

anti-(human cathepsin D M8147) [12] and rabbit anti-(rat

cathepsin B 7183) Ig [13] were obtained from J.S. Mort

(Shriners Hospital for Crippled Children, Montreal,

Quebec) and used to immune deplete samples of native

mature enzymes as described previously [11–13]. HRP-

conjugated goat anti-(rabbit IgG) Ig was from Bio-Rad

(Hercules, CA, USA). The protein content of isolated

fractions was determined by the method of Lowry et al.

[46]. Nitrocellulose membranes and Enhanced Chemi-

Luminescence (ECL) detection kit were from Amersham.

Protein G-Sepharose was from Pharmacia (Peapack, NJ,

USA). Pepstatin-A, E-64 and PMSF were from Sigma.

HPLC grade acetonitrile and trifluoroacetic acid (TFA)

were obtained from Baker Chemical Co. (Phillipsburg,

NJ, USA). All other chemicals were obtained from com-

mercial sources and were of reagent grade.

Animals and injections

In vivo procedures were approved by the INSERM

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 were fasted for 18 h prior to being

killed. Native CT or CT-B subunit (50 lg per 100 g

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.

Isolation of subcellular fractions from rat liver

Subcellular fractionation was performed using established

procedures [11–13,45]. Following injection of native CT or

CT-B subunit, rats were killed and livers rapidly removed

and minced in isotonic ice-cold homogenization buffer as

previously described [11–13,45].

Microsomal (P) fraction was isolated by differential cen-

trifugation as previously described [11–13,45]. The endo-

somal (EN) fraction was isolated by discontinuous sucrose

gradient centrifugation and collected at the 0.25–1.0 m

sucrose interface [11–13,45]. The soluble endosomal extract

(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 homo-

genizer (15 strokes with Type A pestle) followed by cen-

trifugation at 150 000 g for 60 min as previously described

[11–13,45].

Endosomal proteolysis of cholera toxin by cathepsin D C. Merlen et al.

4394 FEBS Journal 272 (2005) 4385–4397 ª 2005 FEBS

Immunoblot analysis

Electrophoresed samples were transferred onto nitrocellu-

lose membranes for 60 min at 380 mA in transfer buffer

containing 25 mm Tris base and 192 mm glycine. The mem-

branes were blocked by a 3 h incubation with 5% (w ⁄ v)skimmed milk in 10 mm Tris ⁄HCl pH 7.5, 300 mm NaCl

and 0.05% (v ⁄ v) Tween-20. The membranes were then

incubated with primary antibody [rabbit polyclonal anti-

serum against native CT C3062 (diluted 1 : 60000)] in the

above buffer for 16 h at 4 �C. The blots were then washed

3 times with 0.5% (w ⁄ v) skimmed milk in 10 mm Tris ⁄HCl

pH 7.5, 300 mm NaCl and 0.05% (v ⁄ v) Tween-20 over a

period of 1 h at room temperature. The bound Ig was

detected using HRP-conjugated goat anti-(rabbit IgG) Ig.

In vitro proteolysis of CT peptides by hepatic

endosomes and cathepsin D

ENs (� 1 lg) or EN (1–15 lg) were incubated for varying

lengths of time at 37 �C with 30 lg native CT, CT-A sub-

unit or CT-B subunit in 90 lL of 175 mm citrate-phosphate

buffer (pH 4–7) containing 50 mm MgCl2, in the presence

or absence of protease inhibitors. To determine the inte-

grity of the CT-A and -B subunits, the ENs samples were

acidified with acetic acid (15%) and immediately loaded

onto a RP-HPLC column. ENs or EN was also incubated

with nonreducing SDS ⁄PAGE sample buffer (62.5 mm

Tris ⁄HCl, pH 6.8, 2% SDS, 10% glycerol) for 15 min at

65 �C, followed by SDS ⁄PAGE and western blot analysis.

For some experiments, native CT, CT-A subunit or

CT-B subunit was digested in vitro with bovine cathepsin D.

CT peptides (7 lg) were incubated with cathepsin D

(4–40 UÆmL)1Æmg)1) in 90 lL of 175 mm citrate-phosphate

buffer, pH 4–7, containing 50 mm MgCl2 for 1–90 min at

37 �C. The proteolytic reaction was stopped by addition of

nonreducing SDS ⁄PAGE buffer (for western blot analysis

and Coomassie Brilliant Blue staining) or ADP-ribosylation

buffer (for ADP-ribosylation analysis).

For the in vitro degradation of 125I-labelled TyrA14-HI by

bovine cathepsin D, the radiolabeled HI (70 fmol) was

incubated with 0.01 UÆmL)1 cathepsin D and various con-

centrations of unlabeled native CT, CT-A subunit or CT-B

subunit in 0.1 m citrate-phosphate buffer, pH 4, for 20 min

at 37 �C. The amount of radiolabeled HI-degraded was

assayed by precipitation with 2 mL of ice-cold 10% (v ⁄v)TCA for 2 h at 4 �C. The samples were then centrifuged at

10 000 g for 20 min at 4 �C, and the supernatants and pel-

lets evaluated for their radioactive content using a Packard

c-counter (Perkin Elmer, Wellesley, MA, USA).

CT-catalyzed ADP-ribosylation

Native CT was first digested by incubation at 37 �C for

1–60 min in 175 mm citrate-phosphate buffer pH 4–7 and

50 mm MgCl2 as described above. Native CT and CT-A

were also treated with 0.2 m dithiothreitol at 37 �C for

30 min in 175 mm citrate-phosphate buffer pH 7 and

50 mm MgCl2. The samples were then neutralized with

0.5 m sodium-phosphate buffer pH 7.2. Microsomal mem-

branes (� 50 lg) were then incubated with the pretreated

CT (5 lg) in an ADP-ribosylation buffer containing

0.54 lm [32P]NAD, 50 mm sodium phosphate buffer

pH 7.2, 0.5 mm GTP, 1 mm ATP, 5 mm MgCl2 and 10 mm

thymidine for 45 min at 30 �C. The reaction was stopped

by the addition of Laemmli sample buffer [47] followed by

SDS ⁄PAGE and autoradiography.

Immunodepletion studies

ENs was immunodepleted of active cathepsin B or cathep-

sin D prior to the digestion step by incubating ENs

(0.15 mgÆmL)1) with antibodies coated onto protein G-

Sepharose 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 immu-

nodepleted supernatants were used in the toxin degradation

assay. The reaction was terminated by the addition of 15%

(v ⁄ v) acetic acid and immediately analyzed by RP-HPLC.

HPLC separation of CT peptides

RP-HPLC was performed on a Beckman Coulter System

Gold model 127 liquid chromatograph equipped with a

Rheodyne sample injector fitted with a 0.5 mL loop and a

lBondapak C18 column (Waters, Milford, MA, USA;

0.39 · 30 cm, 10 lm particle size). Samples were chroma-

tographed using a mixture of 0.1% (v ⁄ v) TFA in water

(solvent A) and 0.1% (v ⁄ v) TFA in acetonitrile (solvent

B) with a flow rate of 1 mLÆmin)1. Elution was carried

out using three sequential linear gradients of 0–5% solvent

B (10 min), 5–15% solvent B (5 min) and 15–39% solvent

B (32 min), followed by an isocratic elution of 39% sol-

vent B (20 min). Eluates were monitored on-line for

absorbance at 214 nm with a LC-166 spectrophotometer

(Beckman Coulter, Fullerton, CA, USA).

Cell culture and immunofluorescence

Human hepatoma (HepG2) cells were grown in Dulbecco’s

modified Eagle’s medium (DMEM) supplemented with

10% (v ⁄ v) fetal bovine serum, 1% (w ⁄ v) penicillin ⁄ strepto-mycin, and 1% (w ⁄ v) glutamine in an atmosphere of 95%

air ⁄ 5% CO2 (v ⁄ v). Human intestinal Caco-2 cells were

grown in DMEM supplemented with 15% (v ⁄ v) fetal

bovine serum and 1% (w ⁄ v) glutamine in an atmosphere of

90% air ⁄ 10% CO2 (v ⁄ v).Cells grown on glass coverslips were washed once with

phosphate buffered saline (NaCl ⁄Pi) before incubation with

C. Merlen et al. Endosomal proteolysis of cholera toxin by cathepsin D

FEBS Journal 272 (2005) 4385–4397 ª 2005 FEBS 4395

serum-free DMEM containing 1 lm native CT or CT-B sub-

unit-FITC. After 15–40 min at 37 �C, cells were washed three

times with NaCl ⁄Pi and fixed with 3% (v ⁄ v) paraformalde-

hyde in NaCl ⁄Pi for 20 min. Fixed cells were treated with

100 mm NH4Cl for 15 min, washed with NaCl ⁄Pi, permeabi-

lized with 0.1% (v ⁄ v) Triton X-100 in NaCl ⁄Pi for 4 min

following by 0.5% (w ⁄ v) saponine in NaCl ⁄Pi for 10 min.

Permeabilized cells were then blocked for 20 min with 10%

(v ⁄ v) horse serum in NaCl ⁄Pi. Mouse monoclonal antibody

to CT-B subunit D15-8 (diluted 1 : 50) or rabbit polyclonal

antibody to mouse cathepsin D R291 (diluted 1 : 50) were

used as primary antibodies, and detected with either Alexa

Fluor 488-conjugated goat anti-mouse (diluted 1 : 100)

(Molecular probes) or Texas Red-conjugated goat anti-rabbit

(diluted 1 : 400) (Jackson Immunoresearch). Laser-scanning

confocal microscopy was performed using a Zeiss LSM 510

confocal (Axiovert 100 m) inverted microscope equiped

with a Zeiss X63 ⁄ 1.4 NA oil immersion objective lens

(plan-Apochromat). Fluorescence images were acquired with

argon (wavelength 488 nm) and helium neon (wavelength

543 nm) lasers. Simultaneous images corresponding to Alexa

Fluor 488 and Texas Red fluorescence were obtained using

the multitracking function of the microscope.

Acknowledgements

We thank Pamela H. Cameron (McGill University,

Montreal, Quebec, Canada) for reviewing the manu-

script. We thank Dr F. Nato (Institut Pasteur, Paris,

France) for the kind gifts of monoclonal anticholera

toxin antibodies. We thank Dr V. Nicolas (IFR 75

INSERM, Faculte de Pharmacie, Chatenay-Malabry,

France) for assistance in confocal microscopy.

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