(Received for publication, January 31, 1997, and in revised form, March 24, 1997)
From the Cholera and Escherichia coli
heat-labile toxins (CT and LT) require proteolysis of a peptide loop
connecting two major domains of their enzymatic A subunits for maximal
activity (termed "nicking"). To test whether host intestinal
epithelial cells may supply the necessary protease, recombinant rCT and
rLT and a protease-resistant mutant CTR192H were prepared. Toxin action
was assessed as a Cl Over the last several years, we have used the human intestinal T84
cell line to examine the cell biology of Vibrio cholerae and
Escherichia coli heat-labile toxins on polarized epithelial cells. Both toxins (cholera toxin, CT1 , and labile toxin,
LT) are structurally, immunologically, and functionally
nearly identical (1-3). They account for the massive secretory
diarrhea seen in infections caused by these microorganisms (4-6).
CT and LT consist of five identical B subunits that bind ganglioside
GM1 at the cell surface and a single A subunit comprised of
two functional domains termed the A1 and A2
peptides (1, 2). The A1 peptide exhibits the toxin's
ADP-ribosyltransferase activity, which is necessary for signal
transduction. The A2 peptide tethers the A and B subunits
together and contains the endoplasmic reticulum-targeting motif K(R)DEL
at its COOH terminus. Enzymatic activity of the A subunit is latent.
For full ADP-ribosyltransferase activity, the peptide loop connecting
the A1 and A2 peptides must be proteolytically
cleaved at residue Arg-192 (7). After proteolytic cleavage (termed
"nicking"), the A1 and A2 peptides remain
covalently associated via a single disulfide bond. This bond is likely
to be reduced when the A1 peptide translocates across the
membrane to the cytosolic membrane surface (8). Translocation is
necessary for the A1 peptide to gain access to its
substrate the heterotrimeric GTPase Gs In nature, both toxins make initial contact with the intestinal cell
apical membrane but must gain access to adenylate cyclase on the
cytoplasmic surface of the basolateral membrane. This process takes
30-40 min. We have obtained evidence that this "lag phase" corresponds to the time required for CT to enter the cell via apical
endosomes and move to its site of action on the basolateral membrane by
transcytosis (11). The intracellular site where the A subunit
translocates across the membrane, however, remains undefined, but
available evidence indicates that this may occur as the
toxin-GM1 complex (or toxin alone) moves retrograde through Golgi cisternae or into the endoplasmic reticulum (12-15).
Several un-nicked bacterial toxins including anthrax protective
antigen, diphtheria toxin, Pseudomonas enterotoxin, and
possibly shiga toxin, require activation by endogenous proteases of the host cell (in these cases furin) to elicit a biologic response (16).
These toxins also require endocytosis for biologic activity and may
encounter furin within endosomes, trans-Golgi, or possibly at the membrane surface of the host cell. As many Vibrios
secrete their own proteases (17) and both CT and LT act within the
gastrointestinal tract, it has largely been assumed that V. cholerae activates its own toxin, and proteases in the gut lumen
activate LT. However, the very first events in the pathogenesis of
diarrhea due to both V. cholerae and E. coli
likely entail bacterial adhesion to the enterocyte surface, as
evidenced by the nature of identified invasion factors (type IV pilus,
surface glycoproteins, and inner membrane regulatory proteins such as
ToxR, (18, 19)). Thus, in vivo, both CT and LT may bind to
the intestinal cell apical membrane immediately after release from the
microbe.
Our aim in the present study was to examine whether the enterocyte
itself may proteolytically activate the nascent A subunits of cholera
or E. coli heat-labile toxins. As before, we utilized the
human intestinal cell line T84 to model the interaction between toxin
and intestinal epithelial cell. Our results show that a serine
protease(s) endogenous to the apical membrane or apical endocytic
compartment of T84 cells is sufficient to activate fully nascent CT or
LT. Proteolytic activation, however, is not apparent when toxin enters
the cell via the basolateral membrane, and this is rate-limiting for
signal transduction. These data provide evidence that in
vivo epithelial cells of the intestine (the physiologic target
cell of CT in nature) may activate the nascent A subunits of CT or LT
after toxin entry into apical endosomes or after binding apical
receptors.
CT was
obtained from Calbiochem. Hanks' balanced salt solution (HBSS,
containing in g/liter 0.185 CaCl2, 0.098 MgSO4,
0.4 KCl, 0.06 KH2PO4, 8 NaCl, 0.048 Na2HPO4, 1 glucose, to which was added 10 mM HEPES, pH 7.4) was used for all assays unless otherwise stated. T84 cells obtained from ATCC were cultured and passaged as
described previously (20, 21). Cells from passages 69-88 were utilized
for these experiments.
For electrophysiological studies, confluent monolayers on Transwell
inserts were transferred to HBSS and preincubated with apical or
basolateral CT at 4 °C for 30 min prior to shifting to fresh HBSS at
37 °C. Measurements of short circuit current and resistance were
performed with 0.33-cm2 monolayers, and biochemical studies
were performed with 5-cm2 monolayers as described
previously (20-22).
E. coli XL1-Blue [recA1
lac A plasmid encoding CTR192H (pMGJ6705) was made by
oligonucleotide-directed mutation of a wt CT clone (pMGJ67) and will be described elsewhere.2 CT holotoxin made by
this clone contains A190G and R192H substitutions in CT A subunit.
rCTR192H was made by overnight induction of 500-ml mid-log phase
cultures (A600 nm = 0.6-0.8) of E. coli TX1 carrying the recombinant plasmid, grown in Luria broth at
30 °C using 200 µM
isopropyl-1-thio- To assay
toxin concentrations an enzyme-linked immunosorbent assay was used.
Periplasmic extracts containing recombinant toxins were applied to
96-well microtiter plates coated with ganglioside GM1 as
described previously (27). After 30 or 60 min at 37 °C, the plates
were washed with phosphate-buffered saline, pH 7.4, and the presence of
CT or LT bound to GM1 was assayed by routine techniques
using rabbit polyclonal antiserum raised against either CT A subunit or
CT B subunit (1:500 in phosphate-buffered saline) (11, 12) (both
cross-react with LT subunits), or mouse monoclonal against LT B subunit
(28). Apparent toxin concentrations were confirmed by SDS-PAGE and
Western blot using serial dilutions of CT or LT B subunit as
standards.
Periplasmic extracts (1 ml final volume) containing
recombinant CT or LT (15 nmol) were incubated with 0.2-2 mg/ml trypsin at 37 °C for 30 min as modified from methods described previously (7, 29, 30). The reaction was stopped by adding 200 mg/ml soybean
trypsin inhibitor at 4 °C. Nicking was assessed structurally by
SDS-PAGE and Western blot and functionally by increase in efficiency of
toxin-induced Cl Monolayers were incubated with apical or basolateral rCT or
CTR192H (20 nM) at 4 °C for 15 min before transferring
to fresh buffer at 37 °C or at 4 °C for the indicated times (up
to 180 min). Nicking of the CT A subunit was assessed as a 5-kDa shift in molecular mass after immunoprecipitation, SDS-PAGE, and Western blot
as described below.
To identify the "class" of protease likely responsible for nicking,
we utilized inhibitors of the proteolytic reactions catalyzed as
described in (31). Monolayers were preincubated with the specified
protease inhibitors or buffer alone (containing the carrier dimethyl
sulfoxide, Me2SO) at 4 °C for 30 min before adding 20 nM rCT or CTR192H. The following protease inhibitors were
used to determine "functionally" the class of protease involved
(based on reaction catalyzed). Serine-type peptidases were diisopropyl fluorophosphate (DFP, 2.5 µM), phenylmethylsulfonyl
fluoride (PMSF, 175 µM from 35 mM stock in
ethanol), and 3,4-dichloroisocoumarin (3,4-DCI, 1 mM from
100 mM stock in Me2SO). Cysteine-type
peptidases were leupeptin (1 µM, from 1 mM
stock in water) and
trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64, 10 µM from 100 mM stock in water).
Metallopeptidases were EDTA (5 mM), and 1,10-phenanthroline
(1 mM from 100 mM stock in Me2SO).
The aspartic-type peptidase was pepstatin (1 mM from 100 mM in Me2SO).
Monolayers were incubated with apical or basolateral rCT
or CTR192H (20 nM) at 4 °C for 15 min before
transferring to 37 °C for the indicated times (to allow endocytosis
and entry of toxin into the cell). Control monolayers were kept at
4 °C for the duration of the experiment (90 or 120 min). Little or
no endocytosis occurs at 4 °C. After incubations at 37 °C were
complete, further cellular processing of internalized toxins was
quenched by returning the monolayers to 4 °C. Individual monolayers
were then removed intact on their filter supports, immersed in 0.6 ml
of 0.5% SDS, 20 mM Tris, 150 mM NaCl, 5 mM EDTA, 20 mM triethanolamine, 0.18 mM PMSF, and 20 µg/ml chymostatin, and heated at
100 °C for 3 min. The cell lysate was diluted with Triton X-100 to
form a mixed micelle buffer (0.25% SDS, 2% Triton X-100, final). DNA
was sheared by vortex at 4 °C for 30 min and removed by
centrifugation (12,000 × g for 10 min) in the presence
of Sepharose CL-4 (Pharmacia Biotech Inc.) until clear. Cell lysates
were precleared again by a 30-min incubation with protein A-Sepharose
(Pierce). This procedure solubilizes all cellular proteins and proteins
associated with the cell monolayer including the entire fraction of
toxin bound to the cell surface or internalized via endocytosis (11,
12, 25).
After cell lysis and solubilization of all cellular and cell-associated
proteins, CT A and B subunits were immunoprecipitated using polyclonal
antibodies raised against denatured toxin subunits eluted from SDS-PAGE
gels (11). Antisera against toxin subunits were first covalently
coupled to protein A beads using dimethyl pimelimidate (20 mM final). Immune complexes formed overnight at 4 °C
were recovered by centrifugation (3,000 × g, for 5 min) and washed five times with fresh mixed micelle buffer and finally once with buffer containing the same salts and 8% sucrose.
Immunoprecipitated proteins were released from the beads by heating in
100 µl of Laemmli sample buffer at 100 °C for 3 min and analyzed
by SDS-PAGE on 10-20% acrylamide gradient gels (reducing conditions),
transfer to 0.25-µm nitrocellulose, and Western blotting with
polyclonal antibody raised against CT B or A subunits (1:3,000 dilution
serum). Blots were developed using affinity-purified goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:2,500, Sigma)
and enhanced chemiluminescence (Amersham). Immunoblots were initially
blocked with 5% nonfat dry milk in 50 mM Tris, 200 mM NaCl, pH 7.4.
In recent studies utilizing recombinant CT and LT
(25), we noticed that the time courses of Cl
To test this idea, we proteolytically nicked recombinant rCT and rLT
in vitro by pretreatment with low dose trypsin. Pretreatment with trypsin resulted in nicking of >20% of total A subunit (as assessed by SDS-PAGE) without attenuating ADP-ribosyltransferase activity (assessed by the ability of nicked toxins to elicit a secretory response when applied to apical surfaces of T84 cells). Fig.
2 shows the time course of Cl
Table I.
Effect of trypsin treatment on kinetics of Cl Combined Program in Pediatric
Gastroenterology and Nutrition,
Department of Pathology
and Microbiology, School of Medical Sciences, University of Bristol,
Bristol BS8 1TD, United Kingdom
secretory response (Isc) elicited
from monolayers of polarized human epithelial T84 cells. When applied
to apical cell surfaces, wild type toxins elicited a brisk increase in
Isc (80 µA/cm2). Isc was reduced 2-fold, however, when
toxins were applied to basolateral membranes. Pretreatment of wild type
toxins with trypsin in vitro restored the "basolateral"
secretory responses to "apical" levels. Toxin entry into T84 cells
via apical but not basolateral membranes led to nicking of the A
subunit by a serine-type protease. T84 cells, however, did not nick
CTR192H, and the secretory response elicited by CTR192H remained
attenuated even when applied to apical membranes. Thus, T84 cells
express a serine-type protease(s) fully sufficient for activating the A
subunits of CT and LT. The protease, however, is only accessible for
activation when the toxin enters the cell via the apical membrane.
(9).
Toxin-induced ADP-ribosylation of Gs
activates adenylate
cyclase and raises intracellular cAMP levels, which in intestinal crypt
epithelial cells elicits a Cl
secretory response, the
primary transport event responsible for secretory diarrhea (10).
Materials, Cell Culture, and Electrophysiology
endA1
gyrA96 thi hsdR17 supE44
relA1 {F
proAB lacIq lacZ
M15 Tn10}] (23)
or TX1 harboring plasmids encoding wild type (wt) CT or LT were
inoculated from stocks into 200 ml of Luria-Bertani medium (L-broth)
(24) supplemented with 200 µg/ml ampicillin and 10 µg/ml
tetracycline. The cells were grown at 37 °C on a rotary shaker. When
the culture had reached an optical density of 0.2 (600 nm),
isopropyl-1-thio-
-D-galactopyranoside (0.5 mM final) was added, and the cells were cultured for an
additional 2 h. The cells were then collected by centrifugation
(6,000 rpm, 4 °C for 15 min) and washed in 4 ml of ice-cold
phosphate-buffered saline (140 mM NaCl, 10 mM
Na2HPO3/NaH2PO3, pH
7.2) and then resuspended in 8 ml of ice-cold 0.3 M
sucrose, 0.1 M
Na2HPO3/NaH2PO3, pH
7.6. EDTA (5 mM) and lysozyme (10 µg/ml) were added and
the cells kept on ice for an additional 15 min with occasional
agitation. The treated cells were centrifuged (12,000 rpm at 4 °C
for 5 min), and the supernatant containing the periplasmic extract was
removed, dialyzed overnight against two changes of HBSS (1:1,000 v/v), separated into 20-50-µl aliquots, flash frozen, and stored at
80 °C until required. Periplasmic extracts prepared from control E. coli strain XL1-Blue not harboring toxin clones or
harboring clones containing wt B subunits alone were electrically
silent when applied to T84 cell monolayers (i.e. they did
not elicit a secretory response or affect monolayer resistance).
Periplasmic extracts of wt rCT and rLT were characterized in a previous
publication (25).
-D-galactopyranoside. Cells were harvested by centrifugation (6,000 × g, 10 min at
4 °C), washed in 10 ml of TEN (50 mM Tris, pH 7.4, 1 mM EDTA, 0.2 M NaCl), resuspended in 25 ml of
TEN containing 1 mg/ml polymyxin B, and incubated at 37 °C for 10 min. This extract was applied to a galactose affinity resin (Pierce) at
room temperature, washed with 20 ml of TEN, and eluted with 1 M galactose in TEN (26). Pooled fractions were dialyzed
extensively against HBSS and stored at
80 °C. Analysis of protein
content in these pooled fractions by SDS-PAGE (reducing conditions)
showed single protein bands at
28 and 11 kDa (corresponding to
A and B subunits) when stained by Coomassie Blue.
secretion after applying the nicked
toxin to basolateral reservoirs of T84 cell monolayers. Proteolytic
damage to the recombinant toxin was assessed functionally as a
reduction in potency of toxin-induced Cl
secretion after
applying the nicked toxin to apical reservoirs of T84 cell
monolayers.
Proteolytic Activation of rLT and rCT by Host Epithelia: Role of
Cell Polarity
secretion
elicited by basolaterally applied toxins (20 nM) were slower and significantly less robust than we had previously observed with toxin preparations purified from V. cholerae
supernatants. In contrast, when these same recombinant toxins were
applied to apical membranes, there was little or no delay in the
anticipated time course of the secretory response. In all past
experiments (11, 12, 21, 25, 32), we consistently found that toxin preparations purified from V. cholerae supernatants elicited
Cl
secretion with a shorter lag phase and more rapid rate
of onset when applied to basolateral rather than apical surfaces of the same cells (Fig. 1, panel A). Unmistakably,
the faster rate of signal transduction elicited by recombinant toxins
(rCT or rLT) entering the cell through the apical endosome was
altogether opposite to that observed previously in earlier studies
(Fig. 1, panels B and C). As preparations of
native CT purified from V. cholerae supernatants are
proteolytically nicked and preparations of recombinant toxins expressed
in E. coli XL1-Blue are not, we hypothesized that when
applied to basolateral surfaces of T84 cell monolayers the attenuated
time courses of signal transduction elicited by recombinant rCT or rLT
were due to the absence of or a delay in A subunit activation. In
vitro, the enzymatic activities of both CT and LT are enhanced
10-fold by proteolytic cleavage (activation) of the A subunit (7).
Fig. 1.
Time course of CT (20 nM) and LT
(40 nM)-induced Cl secretion when applied to
apical or basolateral surfaces of T84 cell monolayers. Panel
A, CT prepared from V. cholerae supernatants (proteolytically nicked). Panel B, recombinant (not nicked)
CT prepared from E. coli. Panel C, recombinant
(not nicked) LT prepared from E. coli.
[View Larger Version of this Image (15K GIF file)]
secretion
elicited by recombinant rCT (20 nM)and rLT (40 nM), nicked or not nicked by pretreatment with trypsin
in vitro. When applied apically, toxins not pretreated with
trypsin elicited a brisk secretory response from T84 cells (upper
panels). When applied to basolateral membranes, however, the
secretory responses elicited by these recombinant toxins were clearly
attenuated (lower panels). Signal transduction by toxin
entering the cell through the basolateral membrane displayed a longer
lag phase and slower rate of onset compared with the time courses of
Cl
secretion elicited by the same toxin preparations
applied apically. Pretreatment of rCT or rLT with trypsin in
vitro, however, resulted in a faster time course of signal
transduction elicited by the basolaterally applied toxins, presumably
due to nicking of the rA subunits (lower panels,
+Trypsin). Trypsin had no effect on the time course of
apically applied toxins (upper panels, +Trypsin). These data (summarized in Table I) show that proteolytic
activation of CT or LT is rate-limiting for signal transduction when
toxin enters the cell via the basolateral (but not apical) cell
surface. As such, these results suggest that entry of nascent CT or LT into the apical endosome leads to cleavage of the A subunit at position
Arg-192, but this does not occur when toxin enters the cell via the
basolateral endocytic pathway.
Fig. 2.
Effect of trypsin treatment on the time
course of rCT- and rLT-induced Cl secretion.
[View Larger Version of this Image (30K GIF file)]
secretion
elicited by recombinant LT or CT entering the cell via apical or
basolateral cell surfaces
secretory response was assessed as a short circuit
current (Isc). In vitro activation of either recombinant
toxin by trypsin treatment resulted in a significant acceleration of
signal transduction when CT or LT entered the cell from the basolateral but not apical membrane. Mean ± S.E. N represents the
number of independent experiments with each experiment comprised of two independent samples/group. For wt rLT and nicked rLT applied apically, calculated means were based on N = 3 and
N = 2, respectively. For wt rLT and nicked rLT applied
basolaterally, calculated means were based on N = 5 and
N = 3, respectively.
Toxin
Trypsin
+Trypsin
Lag
phase
dIsc/dt
Peak Isc
Lag phase
dIsc/dt
Peak Isc
min
µA/cm2/min
µA/cm2
min
µA/cm2/min
µA/cm2
wt LT (40 nM)
Apical
N = 2-3
46 ± 2
0.5 ± 0.1
39
± 3
51 ± 3
0.33 ± 0.08
38 ± 4
Basolateral
N = 3-5
66 ± 3
0.23 ± 0.07
26
± 6
42 ± 3a
0.75 ± 0.05a
55
± 6a
wt CT (20 nM)
Apical
N = 3
45 ± 2
1.5 ± 0.2
78
± 0.7
45 ± 2
1.6 ± 0.5
82 ± 8
Basolateral
N = 3
61 ± 6
0.8 ± 0.3
49
± 16
37 ± 7a
2.6 ± 0.9a
83
± 12a
a
Statistically significant differences between
parameters describing the time course of CI secretion induced by
recombinant toxins nicked or not nicked in vitro with
trypsin as assessed by ANOVA (p = 0.0001).
To examine whether T84 cells
could activate nascent CT by proteolytic nicking of the A subunit, we
again utilized recombinant toxins. rCT was applied to apical (Fig.
3, upper panel) or basolateral (Fig. 3,
lower panel) reservoirs of T84 monolayers at 4 °C and then shifted to 37 °C or kept at 4 °C for the indicated times. The entire fraction of toxin associated with the monolayers was then
solubilized by lysing the monolayers in 0.5% SDS at 100 °C as
described under "Experimental Procedures." Toxin subunits were concentrated by immunoprecipitation, and nicking was assessed as a
shift in apparent molecular mass of the A subunit from 28 to 22 kDa by
SDS-PAGE and Western blot. Fig. 3 shows the results of these studies.
The first lane from the left in the upper
panel shows that when toxin was applied apically, incubations at
4 °C led to proteolytic nicking of the A subunit. At 37 °C
(third through seventh lanes in the upper
panel), nicking was apparent by 15 min and progressed steadily
over time. When applied to basolateral cell surfaces, however, nicking
of the A subunit was not apparent even after a 2-h incubation
(lower panel). Proteolysis of as little as 0.5% of total
toxin bound to T84 cell monolayers can be detected by Western blot
(assessed by serial dilution's of toxin bound at 4 °C). Thus, toxin
entry into the cell via apical endosomes resulted in cleavage of the A
subunit into A1 and A2 peptides. Nicking was
time-dependent and appeared able to occur at the cell surface as evidenced by proteolysis of toxin bound to apical cell surface receptors at 4 °C.
To determine the class of protease responsible for this cleavage (as
defined by reaction catalyzed; 31), we preincubated T84 cell monolayers
with serine, cysteine, aspartic, and metalloprotease inhibitors before
applying recombinant CT. As shown in Fig. 4, A and B, cleavage of rCT A subunits was inhibited
completely by the serine protease inhibitors DFP (2.5 µM), and 3,4-DCI (1 mM). PMSF (175 µM) blocked cleavage of the rA subunit strongly but incompletely. Nicking was not inhibited by the metalloprotease inhibitors EDTA (5 mM) and 1,10-phenanthroline (1 mM) or by the cysteine peptidase inhibitor leupeptin (1 µM). The aspartic-type protease inhibitor pepstatin (1 µg/ml) and the cysteine peptidase inhibitor E-64 (1 mM)
displayed incomplete inhibition. Both pepstatin and E-64 may interfere
with reactions catalyzed by proteases of other classes, notably those
that may exhibit significant thiol dependence or those maximally active
at low pH and dependent on carboxyl groups (31).
When taken together, these data provide evidence that the nascent A subunits of CT and LT are likely activated by a serine protease(s) endogenous to the apical membrane or apical endosome, or both, of human intestinal T84 cells. In contrast, entry into T84 cells via the basolateral endocytic pathway did not result in detectable nicking of the A subunit (Fig. 3, lower panel). Nevertheless, recombinant toxins applied to basolateral cell surfaces elicited an attenuated but clearly detectable secretory response (Figs. 1 and 2).
Protease-resistant CT VariantTo confirm our interpretation
of these results, we prepared recombinant rCT replacing Arg-192 with
histidine (CTR192H). This mutation inactivates the trypsin nicking site
connecting the A1 and A2 peptides of the A
subunit. Fig. 5 shows that CTR192H was not nicked by T84
cells, even after 2-h incubation at 37 °C. CTR192H, however, was
still able to elicit a secretory response (Fig. 6). When
applied basolaterally, the secretory response elicited by CTR192H was
similar to that of wt but not nicked rCT (Fig. 6, lower
panel, and Table II). In contrast, when applied to
apical cell surfaces, the time course of Cl secretion
elicited by wt rCT was accelerated (presumably due to nicking), but the
response elicited by mutant CTR192H remained dramatically attenuated
(Fig. 6, upper panel, and Table II). These data confirm our
findings that intestinal T84 epithelial cells proteolytically activate
nascent rCT when the toxin enters the cell through the physiologically
relevant apical membrane.
|
The results of these studies show that nascent rCT (and presumably rLT) can be proteolytically activated by a serine protease(s) endogenous to the apical membrane or apical endosome (or both) of human intestinal T84 cells. In contrast, toxin entry into the cell via basolateral endosomes did not result in nicking of either rCT or rLT A subunit, and this appears to be rate-limiting for signal transduction. These data provide evidence that in vivo, after colonization of the intestine by V. cholerae or E. coli, the process of toxin binding and entry into the host intestinal cell via the physiologically relevant apical membrane may be fully sufficient for activation of the nascent enterotoxins secreted by these microbes.
Although full activity of both CT and LT requires proteolytic cleavage
of the exposed loop connecting the A1 and A2
fragments of their respective A subunits (residues 187-199), the amino
acid sequences of these functionally conserved regions lack any real sequence homology, with the exception of Arg at position 192 (1, 33).
The importance of this residue in toxin action is confirmed by
replacing Arg-192 with His in recombinant toxin CTR192H. Neither trypsin treatment in vitro nor proteases endogenous to T84
cells in situ were able to cleave the A subunit of CTR192H
into A1 and A2 fragments, and this was
correlated with a clear decrease in the ability of mutant CTR192H to
induce Cl secretion. Similar results confirming the
importance of Arg-192 in LT were obtained by replacing Arg-192 with Gly
as described recently by Grant et al. (34) and Dickinson and
Clements (35).
The functionally defined T84 cell protease(s) responsible for cleavage of the A subunit displayed complete sensitivity to both DFP and 3,4-DCI and likely represents a membrane-associated serine protease (31). E-64 (1 mM) also inhibited nicking, though not completely. The related cysteine peptidase inhibitor leupeptin, however, did not. The aspartic-type protease inhibitor pepstatin appeared to have an effect on nicking. As such, these data provide some evidence that the apically located serine-type protease(s) responsible for toxin activation may exhibit significant thiol or pH dependence, or both (such as that found for the carboxypeptidase C family, prolyl oligopeptidase, and a subset of the subtilisin family) (31).
The molecular identity of the protease responsible for nicking the CT A subunit remains unknown. Although intestinal epithelia in vivo express at least two endoproteases and numerous peptidases on their lumenal membranes (36), the activities of cell surface or endosomal proteases in human T84 cells have not been systematically examined. T84 cells do express the serine protease tissue-kallikrein (37, 38), together with a novel serpin protease inhibitor kalistatin (39). These data raise the possibility that T84 monolayers may activate nascent CT and LT by utilizing tissue-kallikrein. T84 cells also express furin, as evidenced by the ability of T84 cells to activate, process, and respond to edema factor/protective antigen of nascent anthrax toxin.3 Nascent anthrax toxin must be cleaved by furin to elicit a response from intact cells. However, the expression of furin by T84 cells cannot explain our results as neither CT nor LT contains the RXXR amino acid motif required for furin cleavage (16). Whatever the molecular identity, this functionally defined protease must display a remarkable degree of apical polarization as little or no activity can be detected when CT or LT enters the cell from basolateral membranes.
The results of these studies also show that proteolytic nicking of the
A subunit may not be necessary for toxin action, as both CTR192H and wt
but un-nicked rCT (i.e. basolaterally applied) elicit a
Cl secretory response from T84 monolayers. Similar
findings were reported by Grant and co-workers for a nearly identical
mutation in LT tested on Chinese hamster ovary and nonpolarized Caco-2 cells (34). It has also been well described that the intact A subunit
displays clear (though attenuated) enzymatic activity in
vitro (7). Nevertheless, it remains possible that after entry into
basolateral or apical endosomes, both wt and mutant CTR192H may be
nicked at position 192 or at an alternative site(s), in very small
amounts not detectable by Western blot, by the same or another
protease. When taken together, however, these data provide evidence
that in vivo protease-resistant variants such as CTR192H may
continue to display the ability to elicit secretory diarrhea though
with attenuated potency.
Since the A subunits of both CT and LT maintain extensive and stable
noncovalent interactions with the B pentamer (2, 7), the ability of
un-nicked CTR192H to elicit a Cl secretory response
raises the distinct possibility that domain A1 of CTR192H
may translocate across the membrane and exhibit enzymatic activity in
the cytosol while tethered via the A2 domain to the B
subunit on the contralateral (lumenal) membrane surface. If this is
correct, it is also possible that the A1 peptide of nicked
CT or LT may not fully dissociate from the A2 peptide and B
subunit after entry into the cell. If so, this would fit nicely with
our previous observations that in polarized cells signal transduction
by CT is not diffusion-limited even after translocation of the A
subunit (21) and that both the pentameric B subunit and translocated A
subunit appear to travel together across the cell en route to the
basolateral membrane (11). Alternatively, it remains possible that the
A1 peptide of both mutant and wt toxin may dissociate from
the A2 peptide/B subunit-GM1 complex after a
small (but not measurable) amount of nicking by the same or another
protease as discussed above or that proteolytic cleavage of the A
subunit is not required for complete dissociation of A subunit from the
B pentamer. In support of this possibility, the entire CT A subunit
including the A2 peptide appears to separate from the
pentameric B subunit after toxin entry into Vero cells (13, 14).
In summary, these studies show that in nature the host intestinal cell may engage V. cholerae or E. coli in a form of molecular interaction by supplying the necessary protease to activate the nascent A subunits of cholera or E. coli heat-labile toxins. As modeled by the T84 cell system, a protease(s) endogenous to the enterocyte apical membrane, or endosome, or both is fully sufficient to nick the A subunits of CT and LT after they are released from the microbe and bind to the cell surface. Soluble proteases produced by V. cholerae or endogenous to the gut lumen may have little or no effect on the activation of these enterotoxins in vivo.