(Received for publication, August 15, 1995)
From the
The B-subunit pentamer of Escherichia coli heat-labile
enterotoxin (EtxB) is highly stable, maintaining its quaternary
structure in a range of conditions that would normally be expected to
cause protein denaturation. In this paper the structural stability of
EtxB has been studied as a function of pH by electrophoretic,
immunochemical, and spectroscopic techniques. Disassembly of the cyclic
pentameric structure of human EtxB occurs only below pH 2. As
determined by changes in intrinsic fluorescence this process follows
first-order kinetics, with the rate constant for disassembly being
proportional to the square of the H ion concentration,
and with an activation energy of 155 kJ mol
. A
C-terminal deletion mutant, hEtxB214, similarly shows first-order
kinetics for disassembly but with a higher pH threshold, resulting in
disassembly being seen at pH 3.4 and below. These findings are
consistent with the rate-limiting step for disassembly of human EtxB
being the simultaneous disruption of two interfaces by protonation of
two C-terminal carboxylates. By comparison, disassembly of the
B-subunit of cholera toxin (CtxB), a protein which shows 80% sequence
identity with EtxB, exhibits a much lower stability to acid conditions;
with disassembly of CtxB occurring below pH 3.9, with an activation
energy of 81 kJ mol
. Reasons for the observed
differences in acid stability are discussed, and the implications of
these findings to the development of oral vaccines using EtxB and CtxB
are considered.
Heat-labile enterotoxins from enterotoxinogenic Escherichia
coli of human (hEtx) ()or porcine (pEtx) origin are
oligomeric proteins comprising one A-subunit, M
27,000, and five B-subunits, M
11,700 (for
reviews, see (1, 2, 3) ). The A-subunits
possess ADP-ribosyltransferase activity while the B-subunits act as a
carrier system, binding to G
-ganglioside receptors found
ubiquitously on the surfaces of eukaryotic
cells(4, 5, 6) . The B-subunits of hEtx and
pEtx show 96% sequence identity to one another and approximately 80%
identity to the B-subunit of cholera toxin (CtxB)(7) . The
crystallographic structure of pEtx revealed that the B-subunits are
assembled into a pentameric toroid ring(8) . Each B-subunit
interacts extensively with its adjacent subunits via multiple
hydrophobic, hydrogen bond, and salt bridge interactions. Consequently,
the B-pentamers of both cholera toxin and heat-labile enterotoxins are
highly stable, maintaining their quaternary structure in a range of
conditions that would normally be expected to cause protein
denaturation. For example, harsh acidic conditions are required to
disassemble hEtxB(9) ; hEtxB is also stable in the presence of
1% of the ionic detergent sodium dodecyl
sulfate(9, 10) , in 8 M urea, in 7 M guanidinium chloride and when heated to temperatures
80
°C. (
)This stability, as well as their inherent potent
immunogenicity and capacity to bind to cell surface receptors, has
prompted considerable interest in the potential of CtxB and EtxB as
oral delivery vehicles for vaccinating the gut and other mucosa with
antigens or epitopes that have been chemically or genetically linked to
the B-subunit (for reviews, see (11) and (12) ).
The extreme stability of the quaternary structure of hEtxB and the importance of pH stability in withstanding the acidic environment of the stomach (required for effective immunization of the gut mucosal immune system) prompted a systematic study of the disassembly of these proteins at low pH.
Previous studies on pH-dependent stability of oligomeric enterotoxins, in which the change in steady state fluorescence anisotropy of CtxB labeled with dansyl chloride was monitored, have shown that the protein undergoes a progressive dissociation from the pentameric state below pH 4.0(13, 14) . Dissociation was reported to be sensitive to both pH and temperature. This lies within the expected range of stability for oligomeric proteins most of which are fully disassembled at pH 2.6(15, 16) .
This paper shows
that nonlabeled, i.e. native, hEtxB pentamers exhibit
remarkable acid stability, only undergoing disassembly, to a monomeric
state, at pH values below 2.0. Since hEtxB is stable up to pH 11 ()this represents, to the authors knowledge, the most
pH-stable oligomeric complex yet reported. We demonstrate that the
stability of hEtxB pentamers in buffers at pH values of 2.0 or above is
due to the presence of an intersubunit salt bridge between the
C-terminal carboxylate of the B-subunit, Asn-103, and an adjacent
subunit in the pentamer. The extreme acid stability of hEtxB pentamers
contrasts with CtxB which is shown to disassemble at pH values below
3.8. The implications of these findings for the use of EtxB or CtxB as
potential carriers for oral delivery of antigens are discussed.
When the purified preparations of hEtxB and hEtxB214 were analyzed
by SDS-polyacrylamide gel electrophoresis and silver stained, only
bands corresponding to the B-subunits were detected. The yield of
purified protein was typically around 10-15 mg/liter of Vibrio sp. 60 culture. Protein concentration was determined
using the Bio-Rad protein microassay according to the instructions
recommended by the manufacturer. The concentrations of hEtxB and
hEtxB214 pentamers were determined using a G ELISA as
described previously(20) .
Figure 1: Acid-mediated disassembly of hEtxB. A, equal amounts of hEtxB were applied to each lane of a SDS-polyacrylamide gel after subjection to buffers of differing pH and stained with Coomassie Blue. Lanes 1 and 13, molecular weight markers; lane 2, pH 7.0; lanes 2-11, pH 6.0 to 1.5 in 0.5 pH unit decremental steps. Mass of markers, in kDa, are indicated. B, far-uv CD spectra for hEtxB after incubation for 15 min at room temperature at pH 2.6 (solid line) and pH 1.5 (dashed line).
Spectroscopic parameters were observed to change in parallel with
the disassembly process. Circular dichroism spectroscopy showed a large
concomitant change in the secondary structure of the protein,
consistent with a significant reduction in sheet (Fig. 1B). At 20 °C and pH values of
2.00-7.50 no time dependent changes were seen in the intensity of
the fluorescence maxima (excitation 280 nm, emission 347 ± 1 nm)
of hEtxB over 1 h (excluding photobleaching). However, at 20 °C and
pH values of 1.90 or lower, a time dependent decrease in the intensity
of the fluorescence maxima was observed. This decrease followed a
first-order process (r
> 0.993) with the rate
constant for the observed process increasing with decreasing pH (Fig. 2). Over the pH range 1.90 to 1.30 the initial
fluorescence intensity (t = 0) was equal to 100
± 5% of the fluorescence intensity of hEtxB at pH 2.00 and the
ratio of the final fluorescence intensity (t =
)
to the initial fluorescence intensity was 54 ± 2%.
Figure 2:
Kinetics of acid-mediated disassembly of
hEtxB monitored by changes in the intrinsic fluorescence (excitation
280 nm, emission 347 nm) at pH 2.0 (), pH 1.6 (
), and pH 1.5
(
). For the kinetics of disassembly at pH 1.6 and 1.5 the line of
best fit plotted is to a first-order process (r
= 0.999 and 0.999).
At 20
°C, the first-order rate constants for disassembly, as determined
by fluorescence and G ELISA, showed a linear dependence on
the square of the H
ion concentration, with an
apparent first-order rate constant for disassembly of zero at pH
= 1.90 ± 0.03 (r
= 0.997, Fig. 3). The identity of the rate constants determined by ELISA
and fluorescence confirms that the time dependent fluorescence
intensity change is monitoring the disassembly process. At 27 °C
the first-order rate constants for disassembly, as determined by
fluorescence, showed a linear dependence on the square of the
H
ion concentration (Fig. 3) with an apparent
first-order rate constant for disassembly of zero at pH = 1.92
± 0.06 (r
= 0.992). An Arrhenius
plot for pH-dependent hEtxB disassembly, at a pH of 1.68, gave a value
for the activation energy for disassembly of 155.2 ± 4.1 kJ
mol
(r
= 0.998, Fig. 4).
Figure 3:
Linear dependence of the rate constant for
acid-mediated disassembly of hEtxB on the square of the
H ion concentration (r
=
0.997). The rate constants were determined by intrinsic fluorescence
(
) and G
ELISA (
) at 20 °C and intrinsic
fluorescence (
) at 27 °C. Values shown are the mean ±
standard deviation (n = 3).
Figure 4:
Arrhenius plot for disassembly of hEtxB
() at pH 1.68 (r
= 0.998) and hEtxB214
(
) at pH 2.60 (r
= 0.998). The rate
constants were determined by intrinsic fluorescence. Values shown are
the mean (n = 3).
Thus both direct and indirect methods for
determining the quaternary structure of hEtxB have shown that
disassembly from the pentameric state occurs at pH values below 2.0.
Measurements of the changes in two independent parameters showed that
there was a linear dependence of the first-order rate constant for
disassembly on the square of the H ion concentration
below pH 1.9, with an activation energy of 155 kJ
mol
.
A decrease in protein stability at acidic pH
values arises from protonation of amino acids in the protein either
giving rise to a structure destabilizing interaction, e.g. protonation of a buried side chain, or by the removal of a
stabilizing salt bridge. The only groups typically found in proteins
with pK values around the range at which hEtxB
disassembles are C-terminal carboxylates(21) . The C-terminal
carboxylate has been implicated in an inter-subunit salt bridge with
Lys-23 of an adjacent subunit(6) . We therefore postulated that
the pH-dependent disassembly of hEtxB was directly related to
protonation of the C-terminal carboxylate with the resultant loss of
the the inter-subunit salt bridge. To test this hypothesis, the pH
dependence of disassembly was examined in hEtxB214, a mutant protein in
which the C-terminal amino acid Asn-103 had been deleted.
SDS-gel electrophoresis showed a sharp pH dependence for disassembly below pH 3.5, as seen by a transition from the protein running in its pentameric form to its monomeric form (data not shown).
At 25 °C and pH
values of 3.50-7.50 no time dependent changes were seen in the
intensity of the fluorescence maxima (excitation 280 nm, emission 347
nm) of hEtxB214 over 1 h. However, at 25 °C a time dependent
decrease in the fluorescence intensity maxima was observed at pH values
of 3.40 or lower. This decrease followed a first-order process with the
rate of the process increasing with decreasing pH. Between pH 3.40 and
2.60 the initial fluorescence intensity (t = 0) was
equal to 100 ± 3% of the fluorescence intensity of native
hEtxB214 at pH 3.50; but the ratio of the final fluorescence intensity (t = ) to the initial fluorescence intensity
varied from 68% at pH values of 2.60 to 2.90 to 32% at pH 3.30.
At
25 °C the first-order rate constants for disassembly of hEtxB214
showed a linear dependence on the square of the H ion
concentration, with an apparent first-order rate constant for
disassembly of zero at pH = 3.29 ± 0.05 (r
= 0.998, Fig. 5).
Figure 5:
Linear dependence of the rate constant for
acid-mediated disassembly of hEtxB214 on the square of the H ion concentration (r
= 0.998). The
rate constants were determined by intrinsic fluorescence. Values shown
are the mean ± standard deviation (n =
2).
An Arrhenius plot for the
pH-dependent disassembly of hEtxB214 at a pH of 2.60, chosen to yield
similar rate constants for disassembly over the same temperature range
as hEtxB, gave a value for the activation energy for disassembly of
127.9 ± 2.2 kJ mol (r
= 0.998, Fig. 4).
Thus, as for wild-type hEtxB,
the mutant hEtxB214 pentamer disassembles at acidic pH, with the rate
constant for disassembly showing a linear dependence on the square of
the H ion concentration below a threshold pH. However,
the mutant pentamer is clearly destabilized relative to the wild-type.
The pH threshold for disassembly was at pH 3.3, some 1.4 pH units
higher than that for wild-type, while the activation energy for
disassembly was 27 kJ mol
less than for the
wild-type. We conclude that the presence of terminal residue of
wild-type hEtxB is necessary for enhanced acid stability.
The decrease in
fluorescence intensity maxima followed a first-order process with the
rate constant increasing with decreasing pH. At 25 °C the
first-order rate constants for disassembly of CtxB showed a linear
dependence on the square of the H ion concentration,
with an apparent first-order rate constant for disassembly of zero at
pH = 3.70 ± 0.09 (r
= 0.999, Fig. 6).
Figure 6:
Linear
dependence of the rate constant for acid-mediated disassembly of CtxB
on the square of the H ion concentration (r
= 0.999). The rate constants were
determined by intrinsic fluorescence. Values shown are the mean
± standard deviation (n =
2).
An Arrhenius plot for the pH-dependent disassembly,
at a pH of 3.00, gave a value for the activation energy for disassembly
of 81.3 ± 1.0 kJ mol (r
= 0.999, data not shown).
Time dependent changes in
intrinsic fluorescence showed that disassembly of CtxB pentamers occurs
at acidic pH. As for hEtxB and the C terminally truncated hEtxB,
disassembly occurred below a pH threshold value, with the rate constant
for disassembly being linearly dependent on the square of the
H ion concentration. However, the CtxB pentamer was
significantly less stable than the hEtxB pentamer, since the threshold
for disassembly was at pH 3.8, 1.9 pH units higher than that for hEtxB.
The activation energy for acid-mediated disassembly of CtxB was 81 kJ
mol
, approximately half the value found for hEtxB
disassembly. We conclude that the B-subunit pentamer of E. coli heat-labile enterotoxin exhibits a much higher pH stability than
the closely related CtxB pentamer.
The measurement of time dependent changes in intrinsic
fluorescence provides a convenient means of monitoring the kinetics of
changes in protein conformation. This method relies upon the occurrence
of significant changes in the environment of at least one of the
proteins aromatic amino acids. Over the pH ranges that hEtxB, hEtxB214,
and CtxB remain stable pentamers, their emission maxima were around 347
nm when excited at 280 nm. At these wavelengths the intrinsic
fluorescence of these proteins is predominantly due to the single
conserved tryptophan at position 88. The crystal structure of pEtxB
revealed that this residue is located at the basal plane of the
pentamer, forming part of the G binding site (6, 8) . Disassembly of the pentameric state was found
to result in a time, and pH, dependent loss of both G
binding and a decrease in the intensity of the intrinsic
fluorescence of the protein.
The crystal structure of pEtx revealed that the C-terminal carboxylate forms an inter-subunit salt bridge with Lys-23 of the adjacent subunit (6) . From the pH dependence for disassembly we postulated that acid-mediated disassembly of hEtxB below pH 2.0 was a direct result of protonation of the C-terminal carboxylate with the resultant loss of the inter-subunit salt bridge. At such low pH values it is probable that the side chain carboxylates of the aspartate and glutamate residues would be protonated leaving the salt bridge involving the C-terminal carboxylate as the only inter-subunit salt bridge. Hence acid-mediated disassembly of hEtxB is proposed to occur with the loss of the last, structure stabilizing, inter-subunit salt bridge between the C-terminal carboxylate and Lys-23.
Consistent with this hypothesis was the finding that the C-terminal
deletion mutant hEtxB214 disassembles at much higher pH values.
Previous work (18) has shown that there were no apparent
differences between hEtxB and hEtxB214 with regard to SDS
susceptibility, in vivo assembly, or G binding,
hence it is likely that both proteins have nearly identical quaternary
structures. From the crystal structure of pEtx it is unlikely that a
salt bridge could be formed between the C-terminal carboxylate in
hEtxB214 and Lys-23 of an adjacent subunit, however, the
-carboxylate of the C-terminal Glu-102 may be able to form this
inter-subunit salt bridge. Whether or not this salt bridge is formed,
all of the inter-subunit salt bridges would involve side chain
carboxylates and it is probable that these would be protonated in the
range pH 4.5 to 3.5. Since hEtxB214 disassembles at pH values below 3.3
it is likely that, as with hEtxB, disassembly occurs with the loss of
the last inter-subunit salt bridge.
CtxB was found to disassemble at
higher pH values than hEtxB and to have a significantly lower
activation energy for disassembly (81 kJ mol, c.f. 151 kJ mol
). Since CtxB and hEtxB
share 81% sequence identity (over 90% similarity) and x-ray
crystallographic analyses of pEtx and CtxB have revealed that the
B-subunits possess the same structural fold, it is reasonable to
presume that the B-pentamers of the enterotoxin family have near
identical tertiary and quaternary structures(3) . Indeed, all
of the amino acids implicated in side chain inter- or intra-subunit
salt bridges and those implicated in side chain inter-subunit hydrogen
bonding are fully conserved. Thus, the differences in pH-dependent
stability of CtxB and hEtxB are likely to arise from differences in
residues that are protonatable. The two changes in such residues are
Tyr-18 and Asn-94 in hEtxB, which are both histidines in CtxB. Neither
of these residues are particularly deeply buried in the crystal
structure of pEtx nor are they in regions of high charge density.
However, the side chains of the corresponding residues in pEtx are
within 3 Å of each other. Hence in CtxB, at pH values below pH 4,
where histidines are likely to be protonated, there will be
electrostatic repulsion between these two residues. This repulsion may
be the cause of the much lower stability of CtxB compared with hEtxB.
hEtxB, hEtxB214, and CtxB all show a linear dependence of the
first-order rate constant for disassembly on the square of the
H ion concentration. This dependence implies a
requirement for two protonation events in the rate-limiting step. This
is consistent with protonation of the C-terminal carboxylate being the
only requirement for acid-mediated disassembly, if two subunits require
this group to be protonated simultaneously for disassembly to occur.
This requirement for two groups to be protonated, i.e. for two
inter subunit interfaces to be disrupted, can be rationalized due to
the cyclic nature of the quaternary structure of hEtxB (see Fig. 7). If a single interface is broken in a cyclic pentamer
the structure is still pentameric whereas if two interfaces are broken
simultaneously smaller structures, e.g. a trimer and a dimer,
are formed. These smaller oligomeric structures are probably very
unstable in the conditions in which they are formed, since only a
single interface need be disrupted for their further disassembly, and
are likely to be rapidly broken down into monomeric units.
Figure 7: Schematic representation of the requirement for two protonation events in the rate-limiting step for disassembly.
The
demonstration that hEtxB is more acid-stable than CtxB has important
implications for the use and development of these proteins as oral
vaccine components and vaccine delivery vehicles. Disassembly of
B-subunit pentamers significantly reduces their capacity to induce an
anti-toxin immune response because of the loss of their
G-binding property, which normally aids uptake and
presentation to the immune system. (
)The effectiveness of
regimes to neutralize stomach acid when CtxB or EtxB are given
perorally as components of oral vaccines (22, 23) is
not known and may vary from individual to individual. The findings
reported here suggest that the EtxB pentamer should be better at
withstanding the acidic environment of the stomach and thus be a more
effective oral immunogen.
CtxB and EtxB are also being evaluated as
oral delivery vehicles for vaccinating the gut with antigens or
epitopes that have been genetically or chemically fused to the
B-subunit(12, 24, 25, 26, 27, 28, 29) .
Fusions to EtxB have been exclusively designed such that epitope or
antigen extensions are present at the C terminus of the
B-subunit(12) . This reflects the availability of convenient
restriction sites, the initial demonstration that the C terminus of
EtxB may be extended without interfering with B-subunit assembly or
G binding, and the choice of whether to target fusion
proteins to the cytoplasm or periplasm. The finding reported here that
the acid stability of EtxB is dependent on an intersubunit salt bridge
involving the C-terminal carboxylate would suggest that the recombinant
EtxB-fusion proteins (which have C-terminal extensions) should
disassemble at higher pH values than the wild-type EtxB pentamer.
Indeed, we have found that an (Asn-Ala-Asn-Pro)
extension
at the C terminus of hEtxB (30) results in a pentameric protein
which disassembles below pH 3.52, with an activation energy for
disassembly of 140 kJ mol
. (
)Thus, our
findings on the molecular basis of the acid stability of EtxB should
guide the design of new conjugates which ensure retention of the native
C-terminal carboxylate.
The work detailed here on acid-mediated
disassembly not only allows insight into the molecular basis of the
extreme stability of the quaternary structure of hEtxB and the
rationalization of chimeric vaccine design, but also forms the basis
for further work on the reassembly pathway of this remarkable
protein.