Diphtheria toxin contains 535 residues (M
58,348) and comprises an A chain, which is equivalent to the
catalytic (C)
domain, and a B chain, which comprises
transmembrane (T) and receptor-binding (R) domains(1) . After
endocytosis, the acid environment of the endosome lumen triggers a
conformational change which allows the toxin to insert and then
translocate the C domain across the membrane bilayer(2) . The C
domain is then released into the cytoplasm. Hydrophobic helices in the
T domain are the most obvious sequences that may be involved in
translocation(1) . However, several studies have indicated that
the C domain may also participate actively in membrane interaction and
translocation(3, 4, 5, 6, 7, 8, 9) .
Furthermore, the R domain, which has been thought to act simply to bind
to the receptor for the toxin, becomes hydrophobic at low
pH(10) , raising the possibility it has an important role in
translocation.
In order to clarify the role each domain plays in
translocation, monoclonal antibodies to the C, T, and R domains and
polyclonal anti-peptide antibodies were used to examine the interaction
of all three toxin domains with the membrane bilayer. A system was used
in which the toxin is added externally to vesicles or entrapped within
the vesicle lumen. This latter system is analogous to endosomes,
allowing the detection of translocation(5, 11) . The
results show that all three domains traverse the bilayer and become
exposed to the trans side of the membrane after exposure to low pH.
This is a strong argument for the proposal that all three domains
participate directly in translocation of the C domain in
vitro. The observations that the unfolding of the C domain further
promotes translocation and that the toxin inserts in a mixture of
orientations are additional clues to the translocation mechanism.
EXPERIMENTAL PROCEDURES
Materials
ImmunoPlate MaxiSorp, flat-bottom
96-well microtiter plates were purchased from Nunc. Goat-anti rabbit Ig
labeled with alkaline phosphatase was obtained by Southern
Biotechnology Associates (Birmingham, AL). Goat anti-mouse Ig labeled
with alkaline phosphatase was obtained by Jackson Laboratories (West
Grove, PA) and Fisher Biotech. Sigma 104 phosphatase substrate (5-mg
tablets) and octyl glucoside (OG) were purchased from Sigma. DOPC and
DOPG were obtained from Avanti Polar Lipids (Pelham, AL).
Anti-fluorescein whole antibody and F
fragments (1-4
mg/ml in 0.1 M potassium phosphate, pH 8, with 2 mM sodium azide), rhodamine-DHPE, biotin-DHPE, biotin-X-DHPE, and
streptavidin were obtained from Molecular Probes (Eugene, OR). Phast
SDS-polyacrylamide gel electrophoresis gels and PhastSystem supplies
were obtained from Pharmacia Biotech Inc. Nitrocellulose membranes were
purchased from Schleicher & Schuell. Toxin was prepared as
described previously(4, 13) . Antibodies were prepared
as described in accompanying paper except that they were diluted with
Tris buffer (15 mM Tris-Cl, 150 mM NaCl, pH 7.2.)
prior to use. Antibody concentrations were identical to the antibody
concentration reported in the accompanying article(12) , except
where noted.
Lipid Preparation and Entrapping Toxin
LUV with
and without entrapped toxin were prepared by detergent dialysis as
described previously(5) . LUV contained (w/w) 79.5% DOPC, 20.2%
DOPG, and (as determined spectroscopically at 563 nm using an
= 73,000 M
cm
)
0.11-0.3% of rhodamine-DHPE. A small aliquot was removed from
each final preparation, and its rhodamine fluorescence (found to be
insensitive to detergent) relative to the initial (pre-dialysis)
mixture was measured to calculate lipid concentration. Toxin
concentration was measured by competition ELISA with native toxin added
externally to empty LUV used to prepare a standard curve (see below).
To detect entrapped toxin by ELISA, octyl glucoside (final
concentration, 15 mg/ml) was added to the entrapped (and standard)
samples to dissolve the vesicles. The percentage of initially added
toxin entrapped within the LUV was found to range between 5 and 10%.
The toxin concentration ranged from 7.4 to 20.0 µg/ml (average 12.3
µg/ml) and the lipid concentration ranged from 1.10 to 2.74 mg/ml
(average 1.72 mg/ml). Given the diameter of the vesicles (about 2500
Å; (5) ) and the toxin concentration used, about 65 toxin
molecules are entrapped within each vesicle.
Demonstrating Entrapped Toxin in the Native Conformation
Does Not React with Externally Added Antibodies
A competition
ELISA protocol was used as in the accompanying article(12) ,
with the exception that free dimer toxin was used as coating antigen.
For
-T1 (see accompanying article for antibody nomenclature), 115
µl of toxin-containing vesicles were diluted to 200 µl with
Tris buffer and then different aliquots were diluted with vesicles
without toxin to maintain 1 mg/ml lipid concentration. For
-C2 and
-R1, 88 µl of toxin-containing vesicles were diluted to 109
µl with Tris buffer and then different aliquots were diluted with
vesicles without toxin to maintain 1.7 mg/ml lipid concentration.
Tris-diluted antibodies were added and samples incubated for about 2 h
at room temperature. Samples were then diluted to 320 µl with Tris
buffer, and 100 µl aliquoted into the ELISA plate as described
previously(12) . Final antibody concentrations were 0.20
µg/ml for
-T1, 0.98 µg/ml for
-C2, and 0.56 µg/ml
for
-R1.
Demonstrating Entrapped Toxin Does React with Externally
Added Antibodies after the Addition of Detergent
For experiments
with
-T1, 9.4 µl of 240 mg/ml OG was added to a 115-µl
aliquot of LUV-entrapped toxin or toxin added externally to LUV having
the same protein and lipid concentrations to give an OG concentration
of 15 mg/ml (for
-C2 and
-R1, 6 µl of OG was added to
88-µl toxin-LUV aliquots). For
-T1, 35 µl of Tris buffer
was added and the samples incubated for 30 min at room temperature
(this step was skipped for the other antibodies). Then for
-T1, 40
µl of additional Tris buffer was added (15 µl for
-C2 and
-R1) and incubated for 15 min. Tris-diluted antibodies were added
and samples incubated for about 2 h at room temperature. Samples were
then diluted to 320 µl with Tris buffer, and 100 µl aliquoted
into the ELISA plate as described previously(12) . Final
antibody concentrations were as in the preceding section.
Assay for Antibody Permeation into the Lumen of Model
Membrane Vesicles
66-kDa FITC-dextran (predialyzed in 8-kDa
dialysis tubing to remove small dextrans) was entrapped in LUVs with or
without adenosine-3`-phosphate 5`-uridine phosphate-bound monomer
(79.7% DOPC, 20.2% DOPG, and 0.12% pyrene-DHPE) as described above.
Aliquots of coentrapped samples (lipid concentration of 1.8 mg/ml,
toxin concentration of 7.2 µg/ml, and a FITC-dextran concentration
of 0.39 µg/ml) were exposed to low pH by the addition of 200 mM sodium acetate, pH 4.4, for 30 min at room temperature (final pH
4.6). The samples were neutralized with 200 mM
Na
HPO
, diluted with Tris buffer to give a
volume of 800 µl, incubated for 15 min at room temperature, and
then fluorescein fluorescence was measured. 12 µl of an
anti-fluorescein isothiocyanate IgG (
-FITC) and, where desired,
aliquots of Tris-diluted
-R1 (10 µl),
-C1 (30 µl), or
-T2 (30 µl) were added. In the case of
-R1, the
-FITC was added just after antitoxin. In the other cases,
-FITC was added 15 min before antitoxin. Fluorescein fluorescence
was then monitored versus time. The final toxin and anti-toxin
antibody concentrations were 1.07 and 0.53 µg/ml for
-R1, 3.0
and 0.74 µg/ml for
-C1, and 2.6 and 0.62 µg/ml for
-T2. At the end of the incubation time, OG was added (50 µl of
200 mg/ml for
-R1 or 40 µl of 240 mg/ml for
-C1 and
-T2) and fluorescence was monitored for about 15 min.A similar
experiment using
-T2 was performed as for
-T2 above except
toxin was added externally to LUV with only FITC-dextran entrapped
(lipid concentration of 1.8 mg/ml and FITC-dextran concentration of
0.38 µg/ml) and at the end of the experiment 150 µl of 53 mg/ml
OG was added.
Similar experiments as above were also performed with
the Lr" conformation toxin. The coentrapped LUV had an initial lipid
concentration of 1.4 mg/ml, toxin concentration of 6.7 µg/ml, and a
FITC-dextran concentration of 0.27 µg/ml. For the experiments with
toxin added externally to LUV, the vesicles had an initial lipid
concentration of 1.3 mg/ml and FITC-dextran concentration of 0.27
µg/ml. The toxin concentrations were adjusted to equal those in the
Lr` samples before incubation at low pH for 30 min as above but at 37
°C. pH was neutralized as above at 37 °C, fluorescein
fluorescence was measured, and then
-FITC was added as above and
fluorescence remeasured. 15 min after pH neutralization, sample
temperature was decreased to 24 °C over 10 min, and then the
desired
-toxin was added as above, and the fluorescein
fluorescence monitored for 2 h.
Fluorescein fluorescence was
measured with a Spex 212 Fluorolog spectrofluorometer. The samples were
measured in quartz cuvettes having a 1-cm excitation and a 0.4-cm
emission path lengths. The excitation and emission wavelengths were 503
nm excitation and 523 nm emission.
Topography Assay-ELISA
An aliquot of
vesicle-entrapped toxin, or externally added toxin with the same
vesicle and toxin concentration, was brought to about pH 4.6 by the
addition of 200 mM sodium acetate pH 4.4 (about 1 volume of
acetate to 5 volumes of vesicles), and then incubated for 30 min at
either room temperature (to prepare Lr` conformation toxin) or 37
°C (to prepare Lr" conformation toxin). The sample was then
neutralized at the same temperature with 200 mM
Na
HPO
(1.4-2 volumes/volume of pH 4.6
sample) and enough Tris buffer to equalize toxin concentration between
preparations. Different aliquots were removed, and (to all but the
sample with the most toxin) empty LUV in Tris buffer was then added to
bring samples to the same volume without changing lipid concentration.
Tris buffer and Tris-diluted antibody was added to bring volume to 320
µl. The final antibody concentrations were 0.65 µg/ml for
-C1, 0.98 µg/ml for
-C2, 0.20 µg/ml for
-T1,
0.77 µg/ml for
-T2, 0.56 µg/ml for
-R1, 0.25
µg/ml for
-R2, 6.3 µg/ml for
-C
, and 4.7 µg/ml for
-T
. (These are the same concentrations as
used in the protocol for the competition ELISA in the absence of lipid;
see accompanying article(12) .) The ELISA results were
generally very reproducible when duplicates were compared. More
variability was observed when comparing experiments performed on
different days.To examine translocation at lower toxin to vesicle
ratios, a similar protocol was used except that the concentration of
toxin used for entrapment was decreased by 10-, 20-, or 40-fold.
Topography Assay-Western Blotting
To detect
antibody bound to vesicle-inserted toxin by Western blotting,
biotin-labeled LUV with entrapped or externally added toxin, were
prepared essentially as described above, with a composition of (w/w)
78.5% DOPC, 19.9% DOPG, 0.16% rhodamine-PE, 0.74% biotin-X-PE, and
0.74% biotin-PE. (Biotinylated lipid concentrations were based on the
manufacturer's specifications.) Vesicles with toxin were
incubated with 200 mM acetate, pH 4.4, and neutralized with
200 mM Na
HPO
as above to prepare toxin
in the Lr` and Lr" conformations. To prepare vesicles with native
toxin, premixed sodium acetate and 200 mM
Na
HPO
were added. 10 µl of Tris-diluted
antibodies were then added to bring volume to 120-124 µl and
incubated for 1 h at room temperature. The final toxin and antibody
concentrations were about 7.0 and 1.7 µg/ml, respectively, for
-C1 experiments, 6.5 and 0.54 µg/ml for
-T1 experiments,
and 6.5 and 0.67 µg/ml for
-R2 experiments.To pellet the
vesicles 10 µl of 1.3 mg/ml streptavidin (concentration determined
spectroscopically using 
= 40,542 M
cm
) was added and
samples incubated 30 min at room temperature(13) . 120 µl
of each sample was centrifuged using a Beckman airfuge at 26 p.s.i.
(95,000 rpm, 120,000
g) for 30 min using an A-100
rotor. 100 µl was removed and washed three times, using 100 µl
of Tris buffer followed by a 20-min spin for each wash. After the final
spin, 100 µl was removed and 20 µl of Tris buffer and 10 µl
of 110 mg/ml OG was added. After the pellet was completely dissolved by
vortexing, 15 µl of the sample was added to a 5-µl solution of
50 mM Tris-Cl, 1 mM Na
EDTA, 2.5% (w/v)
SDS, 0.01% (w/v) bromphenol blue, pH 8.0, and 7.5% (v/v)
2-mercaptoethanol) and boiled for 10 min. Then a 1-µl aliquot of
each sample was run on a 4-15% PhastGel using the PhastSystem
apparatus, transferred to a nitrocellulose membrane and detected as in
the accompanying paper(12) .
RESULTS
Preparations Used to Examine the Topography of
Membrane-inserted Toxin
Antibody binding was used to evaluate
the conformation of diphtheria toxin inserted in model membranes. Fig. 1schematically illustrates the preparation of the two types
of model membrane vesicles containing toxin that were examined. In one
preparation, the toxin was added externally to (outside of) model
membrane vesicles, and then inserted by exposure to low pH. In the
other preparation, the toxin was trapped in the aqueous lumen (inside)
of the vesicle and then inserted by exposure to low pH. In the latter
(entrapped) preparation, sites on the toxin exposed to external
solution after its insertion are those facing the trans surface of the
membrane (the side of the membrane opposite to that in which the toxin
inserts) whereas in the former (external toxin) preparation externally
exposed toxin sites face the cis side of the membrane (the side of the
membrane into which the toxin inserts). The ability to study toxin in
both of these preparations allows determination of the orientation of
specific sites on the toxin. Notice that, as shown in Fig. 1, if
toxin orients in a single direction, a monoclonal antibody will only
react with one preparation.
Figure 1:
Schematic
illustration of the preparation of membrane-inserted diphtheria toxin
and the assay for antibody binding to membrane-inserted toxin.
Entrapped toxin was prepared by detergent dialysis. Membrane-inserted
toxin was prepared by the low pH treatment of toxin added externally to
LUV and toxin entrapped within the lumen of an LUV. The side of
membrane insertion is considered the cis side, and the opposite side of
membrane insertion is considered the trans side. Notice that a given
site (illustrated as a black dot at the apex of toxin
molecule) is exposed on the external surface in only one preparation
when orientation is unidirectional.
Antibodies Do Not Bind to Entrapped Native
Toxin
Competition ELISA was used to demonstrate that toxin in
the entrapped toxin preparation was inaccessible to antibody prior to
membrane insertion. In this assay the amount of binding of antibody to
toxin is measured by the toxin's ability to inhibit antibody
binding to ELISA wells (see Fig. 1and ``Experimental
Procedures'').Antibody binding to entrapped native toxin was
measured with antibodies
-C2,
-T1, and
-R1. Fig. 2shows there is only very weak inhibition of antibody
binding assayed by an ELISA in the presence of entrapped toxin,
indicating that the entrapped toxin is inaccessible to antibody binding
and only small amounts of toxin may be outside of the vesicles. (
)(This was true even in samples tested after 4-6d
storage of the vesicles at 4 °C.) In contrast, when samples were
solubilized with the detergent OG, a strong toxin-induced inhibition of
antibody binding to ELISA plates, identical to that observed with toxin
not entrapped in vesicles, was observed (Fig. 2).
Figure 2:
Demonstration of toxin entrapment in
vesicles by the inability of entrapped toxin to bind to externally
added antibodies. Binding of antibody to toxin was assayed by
competition ELISA, in which the percentage of absorbance is inversely
related to the amount of toxin binding (see text for details). Samples
contained: entrapped toxin before the detergent octyl glucoside was
added (
), after detergent added (
), or toxin added
externally to vesicles after detergent added (+). Left
panel, toxin binding to
-C2; middle panel,
-T1; right panel,
-R1. Points shown are the average of
duplicate samples. The absorbance values of duplicates were generally
within 10% of each other. OG has only a weak effect on antibody binding
(see Footnote 2).
Antibodies Do Not Permeate into the Vesicle Lumen Even
after Toxin Insertion into the Bilayer
Another requirement of
the antibody binding experiments is that antibodies do not permeate
into the vesicle lumen. This is demonstrated by the results above for
native toxin, but since the toxin forms pores upon low pH-induced
insertion (14, 15, 16) it is necessary to
demonstrate that antibodies do not permeate the vesicle after toxin
insertion. To do this the ability of an anti-fluorescein IgG
(
-FITC) to bind to entrapped FITC-dextran was monitored. The
interaction of
-FITC with fluorescein is easily measured by the
90% decrease in fluorescein fluorescence upon antibody binding (Table 1).
In these experiments fluorescein conjugated to a
66-kDa dextran was entrapped with or without toxin in the lumen of
model membrane vesicles. After exposure to low pH to insert toxin, and
the addition of anti-toxin antibodies where desired, the
-FITC was
added and the fluorescein fluorescence was monitored. Table 1shows samples with entrapped toxin and FITC-dextran do not
form pores large enough to release 66-kDa FITC-dextran or allow
-FITC to enter into the vesicle lumen even after exposure to low
pH.
This was true for toxin in both the Lr` and Lr"
conformations (see below). Binding of anti-toxin antibodies (either
-C1,
-T2, or
-R1) did not affect this result, showing
that anti-toxin interactions with toxin do not affect FITC-dextran and
IgG permeation. These results imply that antibody binding to toxin
inserted into vesicles will reflect the exposure of sites on the toxin
to the aqueous environment on the outside of the vesicles to antibody.
Antibody Binding to Membrane-inserted Toxin: All Domains
Penetrate to the Trans Face of the Membrane
In order to
determine what sites of toxin are exposed to the vesicle exterior upon
its insertion, antibody binding to membrane-inserted toxin was measured
using the competition ELISA (see above). Membrane-inserted toxin was
prepared by the low pH treatment of either toxin added externally to
vesicles or entrapped toxin. Experiments were performed both with low
pH-treated toxin in the Lr` (23 °C) conformation, (
)in
which the C domain is folded, and Lr" (37 °C) conformation, in
which the C domain is partially unfolded(4) .The relative
strength of binding for different antibodies is summarized in Table 2. One observation is that after membrane insertion of
toxin by exposure to low pH, antibodies can bind to the entrapped toxin
preparation. Since toxin remains membrane-bound(5) , this
reflects the exposure of sites in membrane-inserted toxin to the trans
face of the membrane. The IC
values obtained are generally
comparable to those obtained for toxin in solution (see accompanying
article(12) ), indicating that the translocation of a large
fraction of toxin molecules occurs. Furthermore, since interaction of
-C,
-T, and
-R antibodies with the toxin is observed,
there must be translocation of all three domains to the trans surface
of the vesicle.
To see if the toxin concentration affects
this translocation, toxin was entrapped at lower toxin-to-vesicle
ratios than in the protocol used above (65 toxin molecules/vesicle),
and the binding of antibodies
-C2,
-T2, and
-R2 was
measured. After low pH treatment of toxin entrapped at 6.5, 3.2, or 1.6
toxin molecules/vesicle, binding of all three antibodies was observed
(data not shown).)
In at least the experiments with
-T2 and
-R2, the efficiency of binding appeared to be
independent of toxin/vesicle ratio. Since there are so few toxins
trapped per vesicle, these results suggest that translocation to the
trans side of membrane does not involve the formation of large toxin
oligomers.
Antibody Binding to Membrane-inserted Toxin: Toxin
Inserts in a Mixture of Orientations
Another observation evident
from the antibody binding data is that all of the anti-toxin antibodies
are able to bind to both membrane-inserted toxin in both the externally
added and entrapped toxin preparations. The only way this could occur
is if the toxin inserts in a mixture of orientations, such that some
toxin molecules have sites facing one side of the membrane, and others
in the same sample have the same sites facing the opposite side of the
membrane (see schematic figures). The observation that the IC
for antibody binding to entrapped and externally added toxin
preparations are generally similar implies that there is a large
fraction of toxin molecules facing in each orientation. This result
could be interpreted in terms of a mixture of transmembraneous toxin
molecules facing in different directions or a mixture of
transmembraneous toxin and toxin that only penetrates the membrane
incompletely.The IC
is on the average somewhat lower
for externally added toxin than for entrapped toxin in the Lr`
conformation, and averaging the difference in IC
values
for externally added and entrapped toxin suggests that about 70% of the
molecules have sites oriented facing the cis side of the membrane and
30% are oriented facing the trans side in this conformation. However,
the difference in IC
could potentially be influenced by a
difference in reactivity as well as the number of sites facing cis and
trans sides.
Antibody Binding to Membrane-inserted Toxin: Effect of
Toxin Conformation
Monoclonal antibody binding to
membrane-inserted toxin in the Lr` and Lr" conformations was examined.
In general, binding is slightly tighter when the toxin is in the Lr`
conformation, as judged by a lower IC
.
-C2 behavior
is somewhat different (see Table 2), showing closer to equal
binding to Lr` and Lr" toxin. Binding of the anti-peptide antibodies
-C
and
-T
to
toxin differs from that of the monoclonals in that binding to the Lr"
toxin is tighter than binding to the Lr` toxin. It is possible that the
increased degree of unfolding in the Lr" toxin promotes the binding of
antipeptide antibodies by increasing the exposure of peptide epitopes,
whereas the non-contiguous surface epitopes seen for monoclonal
antibodies(17, 18) , are somewhat disrupted by the
increased unfolding in the Lr" toxin.
Antibody Binding to Membrane-inserted Toxin: Evidence
That Increased Translocation of C and T, but Not R Domain, Occurs upon
C Chain Unfolding
The IC
values in Table 2show that for externally added toxin most
antibodies bind the Lr` conformation toxin more tightly than the Lr"
conformation toxin. The weaker binding of an antibody to its epitope on
externally added toxin in the Lr" conformation may be explained in one
of two ways; 1) there is a structural change of an epitope to a weaker
binding form in the Lr" conformation, or 2) in the Lr" conformation the
epitope has translocated to the trans side of the membrane bilayer (and
so faces the antibody-inaccessible vesicle lumen for most of the toxin
molecules). It is possible to distinguish these possibilities by
comparing antibody binding to entrapped toxin in the Lr` and
Lr" conformations. If the decrease in antibody binding to toxin in Lr"
conformation were due to a change in epitope structure, antibody
binding would show a decrease in binding to Lr" toxin relative to Lr`
toxin similar to that seen with externally added toxin. However, if the
weaker reactivity of the Lr" toxin is due to translocation of the
epitope across the bilayer, the ratio of reactivity of the entrapped
Lr" toxin relative to Lr` toxin should be higher than for externally
added toxin.The second explanation is seen to be correct for
several epitopes when IC
values are examined. Table 3shows the IC
Lr"/IC
Lr` ratio is
lower for entrapped toxin than for externally added toxin in several
cases, i.e. the reactivity of several antibodies with Lr"
toxin normalized to the reactivity of the Lr` toxin is higher in the
entrapped preparation than in the externally added preparation. We have
defined the change in the Lr"/Lr` IC
ratio as the relative
translocation index. This is a measure of how much a site moves to the
trans face upon unfolding of the C domain, the major change in the
toxin conformation on going from Lr` to Lr" conformation (5) .
Interestingly, a translocation index close to 2, indicating increased
translocation for toxin in the Lr" conformation, is seen for the anti-C
and some anti-T antibodies, but not for the anti-R antibodies, where
the index is close to 1. This suggests that additional C and T domain
sites move to the trans face in the Lr" toxin but the location of at
least some R domain sites do not change location. The increased
movement of sites to the trans face in the Lr" toxin confirms and
amplifies previous observations of increased toxin movement to the
trans surface of the membrane in the Lr" toxin (5) and studies
showing a role of C chain unfolding in translocation(5, 19) (see ``Discussion'').
Direct Demonstration of Antibody Binding to
Membrane-inserted Toxin
Monoclonal antibody binding to
membrane-inserted diphtheria toxin was more directly detected than in
the ELISA by using centrifugation to isolate anti-toxin-bound vesicles
containing membrane-inserted toxin. After solubilization, pelleted
vesicles were analyzed for bound antibodies by probing with anti-mouse
antibodies, and for toxin by probing with polyclonal anti-toxin. Fig. 3shows the results of such experiments using
-C1,
-T1, and
-R2. When the toxin preparations were not exposed to low pH (native toxin), none of the antibodies bound to
the vesicles (lanes 1 and 4 in each panel, upper
half). This rules out nonspecific antibody binding to the
vesicles. As expected in a sample with vesicle entrapped toxin,
pelleted toxin is observed (lane 4 in each panel, lower
half), but with externally added toxin, even the toxin does not
pellet (lane 1 in each panel, lower half) because it
does not bind to the vesicles.
Figure 3:
Translocation of toxin domains to the
trans surface of the bilayer after low pH treatment assayed by western
blotting. Vesicles with antibody bound to membrane-inserted toxin were
isolated by centrifugation. Western blotting followed by reaction with
alkaline phosphate conjugated to anti-mouse antibodies was used to
directly detect the amount of antibody bound to the vesicles (top
half). Western blotting followed by reaction with horse polyclonal
anti-toxin antibodies and rabbit anti-horse antibodies conjugated to
alkaline phosphate was used to detect the amount of membrane-inserted
toxin in each sample (bottom half).
-C1,
-T1, and
-R2 bound to vesicles with membrane-inserted toxin were run on a
non-reducing 4-15% Phast System gel. Lanes 1-3 show antibody bound to toxin added externally to vesicles. Lanes 4-6 show antibody bound to toxin originally
entrapped within vesicles. Samples in lanes 1 and 4 contained toxin not treated at low pH. Samples in lanes 2 and 5 contained toxin treated at low pH and 23 °C.
(Lr` conformation). Samples in lanes 3 and 6 contained toxin treated at low pH and 37 °C (Lr"
conformation).
In contrast, when toxin was inserted
by exposure to low pH, the antibodies bound to the vesicles both with
externally added (lanes 2 and 3 in each panel) and
entrapped toxin (lanes 5 and 6 in each panel), in
agreement with the ELISA results. This confirms the conclusion that
there is translocation of sites on all three domains of the toxin to
the trans face upon insertion. (
)
DISCUSSION
Structural Model For Membrane-inserted Diphtheria
Toxin
This study shows that antibodies can be used to examine
the structure of membrane-inserted diphtheria toxin. The observation
that all three domains of the toxin undergo translocation to the trans
face of the membrane requires a model of translocation in which all
three domains participate in the insertion and translocation process in vitro (but perhaps not in vivo, see below).
Despite the prejudice that the T domain, with its potentially
transmembrane
-helices, should be the main part of the toxin
undergoing insertion, the observation that isolated C (3, 6) and R domains ( (10) and data not
shown) become hydrophobic at low pH, isolated C domain can membrane
insert (3) and proteolytic evidence for R domain insertion (23) support a role for these domains in the insertion and
translocation process. Since the C and R domains lack long hydrophobic
stretches, this brings up the question of the structure of
membrane-inserted C and R domains. We have proposed that transmembrane
-sheets, similar to those formed by porins(20) , may be
formed by these domains(21) .
-Strand 4 in the R domain is
rich in hydrophobic residues, while
-strands 8 and 10 in the R
domain and
-strands 4, 5, 6, and 8 in the C domain have the
striking regions of alternating hydrophilic and hydrophobic residues
found in transmembraneous
-strands. An alternate possibility is
that these strands insert in the membrane in a non-transmembraneous
fashion.It has been observed that isolated catalytic,
transmembrane, and receptor binding domains of Pseudomonas exotoxin A become hydrophobic at low pH(22) . This may
mean that the translocation mechanism for diphtheria toxin and exotoxin
A is similar. This is interesting in terms of the similar secondary
structure, but relative lack of sequence similarity, between the
transmembrane and receptor binding domains of diphtheria toxin and Pseudomonas exotoxin A.
Unfolding of the C Domain and Translocation of Sites to
the Trans Surface
There is an increasing body of evidence that
the partial unfolding of the C domain promotes its
translocation(5, 23, 24) . Jiang et al.(5) showed that upon the unfolding of the C domain at 37
°C, additional regions of the A and B chain become exposed to the
trans side of membrane-inserted toxin, suggesting translocation of both
chains across a membrane bilayer. The observations in this report
showing increased movement to the trans surface for toxin in the Lr"
conformation support this proposal, and indicate that the transmembrane
insertion of some part of the T domain is also promoted by C domain
unfolding. A model of toxin translocation is shown in Fig. 4. It
shows that the C domain and certain regions of the T domain translocate
across the bilayer upon unfolding of the C domain by heating to 37
°C, while the R domain does not translocate across the bilayer.
Since our anti-T antibodies are against the amino-terminal end of the T
domain, it suggests that some of the amino-terminal end of the T domain
translocates across the bilayer upon C domain unfolding. This proposal
is supported by protease and mutagenesis studies, which show that the
amino-terminal end of the T domain is important for translocation of
the toxin across a membrane(25, 26, 27) . The
observation that this change does not occur for the R domain suggests
the R domain may not change its degree of insertion during the
translocation process. In future studies it will be interesting to see
how the exposure of T and R domains to the trans surface changes upon
the release of the C domain from the membrane.
Figure 4:
Schematic illustration of toxin
translocating across a model membrane bilayer. The toxin inserts into
the bilayer with a mixed orientation, and upon exposure to 37 °C,
additional sites on the C domain and certain regions of the T domain
translocate to the opposite side of the bilayer (shown for the
predominant form only).
Orientation of Membrane-inserted Toxin in
vivo
The observation that diphtheria toxin inserts in a mixed
orientation brings up the question of whether this can occur in
vivo. This cannot be answered at present, but it should be noted
that several studies suggest this possibility. Two studies of toxin
interaction with cells have been interpreted as indicating the presence
of two populations of toxin, only one of which
translocates(28, 29) . In entry via endosomes it has
been reported that only a third of the toxins
translocated(29) , which agrees with the percent toxin
penetration to the trans surface found in this study. Another study has
shown that not all toxin molecules insert such that they are accessible
to a specific chemical modification(30) . This could be
interpreted in terms of toxin oligomers with mixed orientations.
Nevertheless, it should be remembered the mixture of orientations
(specifically the minor (30%) orientation) may not occur in vivo.