(Received for publication, March 27, 1995)
From the
Cholera toxin and the related Escherichia coli heat-labile enterotoxin are hexameric proteins comprising one
A-subunit and five B-subunits. In this paper we report the generation
and characterization of a monoclonal antibody, designated LDS47, that
recognizes and precipitates in vivo assembly intermediates of
the B-subunit (EtxB) of E. coli heat-labile enterotoxin. The
monoclonal antibody is unable to precipitate native B-subunit
pentamers, thus making LDS47 a useful probe for studying the early
stages of enterotoxin biogenesis. The use of LDS47 to monitor the in vivo turnover of newly synthesized B-subunits in the
periplasm of E. coli demonstrated that (i) the turnover of
unassembled B-subunits followed an apparent first order process and
(ii) it occurred concomitantly with the assembly of native B-pentamers (k = 0.317 ± 0.170 min; t = 2.2 min). No other proteins were co-precipitated with the
newly synthesized B-subunits; a finding that implies that unassembled
B-subunits do not stably associate with other periplasmic proteins
prior to their assembly into a macromolecular complex.
The use of overlapping synthetic peptides corresponding to the entire EtxB polypeptide demonstrated that the epitope recognized by LDS47 is located within the amino-terminal decapeptide of the B-subunit. From the x-ray structural analysis of the toxin (Sixma, T., Kalk, K., van Zanten, B., Dauter, Z., Kingma, J., Witholt, B., and Hol, W. G. J. (1993) J. Mol. Biol. 230, 890-918), this region appears to resemble a curved finger that clasps the adjacent B-subunit. Thus, this region might be expected to be exposed in the unfolded or unassembled subunit, but to become partially buried upon assembly and thus inaccessible to recognition by the monoclonal antibody.
The folding and assembly of polypeptide chains into compact,
stable, and functional three-dimensional structures represents a
remarkable biological phenomenon (Kim and Baldwin, 1990; Jaenicke,
1991). An intrinsic feature of such folding and assembly processes is
the sequestration from the aqueous solvent of segments of the
polypeptide. This arises as a consequence of the burial of secondary
structural elements to form the hydrophobic core of the protein and,
for certain proteins, the formation of complementary interfaces
involved in interdomain or intersubunit interactions (Jaenicke, 1991).
One powerful tool capable of probing protein folding and assembly
events in vivo, as well as in vitro, are antibodies
that specifically recognize conformational or assembly intermediates in
proteins (Goldberg, 1991). Although early work employed polyclonal
antisera as investigative tools (Hamlin and Zabin, 1972, Creighton et al., 1978), it is now recognized that monoclonal antibodies
(mAbs) ()embody the necessary features of homogeneity and
specificity that make them exquisite probes of protein conformation
(Goldberg, 1991). mAbs have been used, for example, to study domain
assembly in the
subunit of Escherichia coli tryptophan
synthase (Friguet et al., 1986, Djavadi-Ohaniance et
al., 1986), to investigate the structure of ribosome-bound nascent
polypeptides (Fedorov et al., 1992), to monitor the assembly
of trimeric proteins such as bacterial porins (Fourel et al., 1992) and the tailspike protein of bacteriophage P22 (Friguet et al., 1990, 1994), and to assess conformational changes in
proteins when they interact with macromolecules such as that which
occurs when the
-subunit of E. coli RNA polymerase
interacts with promoter regions in DNA (Sharif et al., 1994).
Our studies have focussed on the in vivo folding and
assembly pathways of a family of enterotoxins that are responsible for
causing diarrheal disease in humans and farm animals (Levine et
al., 1983; Black, 1986; Glass, 1986; Albert and Ansaruzzaman,
1993). These include cholera toxin (Ctx) produced by Vibrio
cholerae and heat-labile enterotoxin (Etx) produced by certain
enterotoxinogenic strains of E. coli, which exhibits
approximately 80% sequence identity to Ctx (Yamamoto et al.,
1987). The toxins are comprised of six noncovalently associated
subunits; a single toxic A-subunit (M =
28,000) which has ADP-ribosyltransferase activity and five identical
B-subunits (M
= 12,000 each) arranged as a
planar ring structure which binds to G
receptors on
intestinal cells (Holmgren et al., 1973; Moss and Richardson,
1978; Sixma et al., 1991). Both the A- and B-subunits are
synthesized in the cytoplasm as precursor proteins with typical
amino-terminal signal sequences that target the precursors across the
bacterial cytoplasmic membrane (for a review, see Hirst(1995)). The
signal sequences are cleaved off, and the mature toxin subunits are
released into the periplasm, where they fold and assemble to form the
native AB
holotoxin complex (Hirst et al., 1984a,
1984b; Hofstra and Witholt, 1984, 1985; Hirst and Holmgren 1987a,
1987b; Hardy et al., 1988). The various intra- and
intermolecular interactions that occur during folding and assembly of
the toxin subunits have yet to be fully explored. It has been
established that a periplasmic thiol-disulfide oxidoreductase (DsbA),
which catalyzes intrachain disulfide bond formation, is essential for
toxin subunit folding (Yu et al., 1992), and an 8-kDa
polypeptide, of unknown identity, has also been implicated to
transiently associate with newly synthesized B-subunits (Hofstra and
Witholt, 1985). When Ctx or Etx are expressed in E. coli, the
holotoxin remains entrapped in the periplasm, whereas when they are
expressed in V. cholerae they are efficiently secreted to the
extracellular milieu (Neill et al., 1983; Hirst et
al., 1984b). Recombinant strains that express the B-subunit alone
produce stable B-pentamers that are devoid of enterotoxic activity.
To further analyze the in vivo pathway of enterotoxin folding and assembly, we have sought to generate a monoclonal antibody that specifically precipitates unassembled B-subunits and lacks the capacity to precipitate the native assembled B-subunit pentamers. Other laboratories have previously obtained a wide range of specific and cross-reactive mAbs to EtxB and CtxB that either exclusively recognize the pentameric B-subunit or which recognize both the pentameric B-subunit and denatured B-subunit monomers (Remmers et al., 1982; Robb et al., 1982; Holmes and Twiddy, 1983; Lindholm et al., 1983; Belisle et al., 1984a, 1984b; Svennerholm et al., 1986; Finkelstein et al., 1987; Kazemi and Finkelstein, 1990). However, none of those mAbs exhibited an exclusive specificity for monomeric B-subunits.
In this paper, we report the generation and characterization of a new mAb, designated LDS47, that recognizes and precipitates in vivo assembly intermediates of the B-subunit of E. coli heat-labile enterotoxin, but fails to precipitate the native B-subunit pentamer or holotoxin. We show that the epitope recognized by LDS47 is located in the amino-terminal 10 amino acids of the B-subunit polypeptide. We also demonstrate that the epitope, which is not recognized in the native B-pentamer, becomes accessible to the antibody once the pentamer binds to plastic or nitrocellulose surfaces. The use of LDS47 in monitoring the turnover of newly synthesized B-subunits in the periplasm of E. coli is described, and the implications of our findings for understanding toxin subunit assembly are discussed.
BALB/c female mice (8 to 10 weeks old) were immunized
intraperitoneally with 10 µg of EtxB/nitrocellulose suspension in
0.5 ml of PBS, followed by a second 10-µg intraperitoneal dose 7
weeks later. A final 10-µg intraperitoneal dose was administered
after 4 weeks, and the mice were sacrificed 3 days later. Spleen cells
were isolated and mixed with NS0 myeloma cells at a ratio of 4:1 and
fused using a modification of the method described by
Köhler and Milstein (1975) at 37 °C using 0.8
ml of 50% (w/v) polyethylene glycol (Boehringer Mannheim). The cell
suspension was added in 1-ml aliquots to each well of 2 24 well
plates in RPMI/Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) containing 15% (v/v) fetal calf serum (Seralab
batch 001010) and incubated at 37 °C in 5% CO
(=
day 0). Hypoxanthine and azaserine were added on day 1. Medium in the
wells was replaced twice weekly with 1 ml of RPMI/Dulbecco's
modified Eagle's medium + 15% fetal calf serum +
hypoxanthine and azaserine. Supernatants were screened by an
enzyme-linked immunosorbent assay (ELISA) on day 11, and positive
supernatants containing antibodies of the desired specificity cloned to
a density of 1 cell per well by limiting dilution without feeder cells
(Harlow and Lane, 1988). Hybridomas were cultured and then stored
frozen in liquid nitrogen.
ELISAs were also performed using purified cholera toxin B-subunit from List Biologicals (Campbell, CA).
mAb LDS47 was purified from ascitic fluid on a
Protein G-agarose column. The ascites were clarified by centrifugation
at 12,000 rpm for 10 min at 20 °C in a Sigma 4K10 centrifuge,
filtered through a 0.22-µm filter, and then applied to a 2-ml
Protein G-agarose column equilibrated in 20 mM phosphate
buffer, pH 7.0. The adsorbed IgG was eluted from the column using 0.1 M glycine-HCl buffer, pH 2.7, with 1.0-ml fractions collected
into tubes containing 0.06 ml of 1 M Tris-HCl, pH 9.0. The
presence of IgG in fractions was monitored by measuring absorbance at
280 nm. Peak fractions were pooled and dialyzed against PBS and then
lyophilized. The purified mAb was dissolved in water at a concentration
of approximately 5 mg/ml, as determined by A.
SDS-PAGE was used to assess antibody purity, and an ELISA was used to
confirm its activity.
Western
blotting was performed as described above (cf. Hybridoma
Production) except that proteins were electroblotted onto Hybond C
membranes (Amersham), and, when the SDS gels contained pentameric EtxB,
the electroblotting was performed for 45 min to permit efficient
transfer of the pentamer. Nonspecific binding sites were blocked by
overnight incubation of the membrane in PBS containing 1% (w/v) BSA.
Blots were washed in PBS containing 0.05% v/v Tween 20 (PBS/T) for 3
10-min incubations with agitation. Primary antibodies were
diluted in 0.1% (w/v) BSA in PBS/T and allowed to react with blots for
1 to 2 h at room temperature with agitation. Blots were washed as
previously and incubated with goat anti-mouse IgG conjugated to
horseradish peroxidase diluted 10,000-fold for 1-2 h. The blots
were then washed and developed with an enhanced chemiluminescence
system (ECL, Amersham) using the conditions recommended by the
manufacturer.
For peptides not containing either arginine or tryptophan residues,
cleavage was carried out over 4 h. For peptides containing either one
or both of those residues, cleavage was performed overnight. Cleaved
peptides were then precipitated in ice-cold diethyl ether (3 15
ml washes at 2592 rpm for 7 min at 4 °C in a Sigma 3K10
centrifuge), air-dried, redissolved in acetonitrile (30% v/v) in water,
and lyophilized overnight. Lyophilized peptides were redissolved in
distilled water prior to use. Peptide concentration was determined
using the Bio-Rad DC assay, as recommended by the manufacturer.
The isotype of LDS47 was determined to be IgG1, using a commercially available agglutination assay.
When
hybridoma supernatants of LDS47 were tested by ELISA, it was found that
the mAb exhibited a potent reactivity toward reduced, denatured EtxB,
but no reactivity toward EtxB pentamers bound to G (Fig. 1a). For comparative purposes (and as a
control for the assay), we also analyzed the reactivity of another mAb,
118-8, that had previously been found to react with EtxB pentamers, and
to show a weak reactivity toward EtxB monomers (Sandkvist et
al., 1990). The ELISA analysis of 118-8 hybridoma supernatants
confirmed that the antibody recognized EtxB pentamers bound to
G
, and that it showed weak reactivity toward
reduced/denatured EtxB (Fig. 1b). The data on LDS47
suggested that the mAb either recognizes an epitope in the denatured
B-subunit which becomes buried upon formation of the native B-pentamer,
or that it recognizes an epitope in the B-pentamer that is masked by
the binding of G
.
Figure 1:
LDS47 recognizes denatured EtxB.
Supernatants containing mAb LDS47 (a) or mAb 118-8 (b) were serially diluted and tested by ELISA for their
reactivity toward denatured EtxB () or G
-bound
pentameric EtxB (
) as described under ``Experimental
Procedures.'' LDS47 was diluted 2-fold (from a 1 in 50 dilution in
the first well) and was added to plates containing a 5 µg/ml
concentration of either denatured EtxB or G
-bound
pentameric EtxB. 118-8 was diluted 2-fold (from a 1 in 200 dilution in
the first well) and was added to plates containing 5 µg/ml
denatured EtxB or 1 µg/ml G
-bound pentameric
EtxB.
To distinguish between these two
possibilities, we evaluated the capacity of LDS47 to immunoprecipitate
either monomeric EtxB or EtxB pentamers, in solution, in the absence of
G. Two approaches were tested. Firstly, purified
preparations of EtxB pentamers or heat-denatured EtxB (monomers) were
subjected to immunoprecipitation by LDS47. This revealed that
heat-denatured EtxB (monomers) could be readily immunoprecipitated by
LDS47, whereas native EtxB pentamers could not (Fig. 2).
Secondly, a recombinant strain of E. coli expressing EtxB was
radioactively pulse-labeled to generate an in vivo source of
radiolabeled EtxB monomers and pentamers that could be subjected to
immunoprecipitation by LDS47. In this experiment, E. coli G6
pMMB68 was pulse-labeled with [
S]Met for 15 s,
then chased with L-Met, and samples were taken 10 s and 10 min
after addition of the chase. Periplasmic extracts of each sample were
isolated and analyzed by SDS-PAGE to examine the total protein content
of the periplasm and subjected to immunoprecipitation using LDS47. EtxB
has previously been shown to exhibit an unusual property when subjected
to SDS-PAGE (e.g. Hirst and Holmgren (1987a)). If boiled in
SDS sample buffer containing 2-mercaptoethanol prior to
electrophoresis, the protein migrates as a monomer. However, if left at
room temperature in the same buffer, it remains stable and migrates as
a pentamer. SDS-polyacrylamide gel electrophoretic analysis of the
total protein in the periplasmic extracts derived from the pulse-chase
revealed that after 10 s of chase the predominant proportion of
radiolabeled EtxB subunits still migrated as monomers and had not
assembled into stable pentamers (Fig. 3a, lane
2). In contrast, after 10 min of chase, all of the radiolabeled
EtxB subunits in the periplasmic fraction migrated as SDS-stable
pentamers (Fig. 3a, lane 4). The capacity of
LDS47 to precipitate unassembled B-subunits present at the early time
points compared with its capacity to precipitate the fully assembled
pentamer present after long chase times, was examined. Immune complexes
containing LDS47 and EtxB were precipitated with the aid of Protein
G-agarose and mixed with SDS sample buffer and analyzed directly by
SDS-PAGE and autoradiography. Fig. 3b shows that LDS47
clearly recognizes and precipitates in vivo-generated EtxB
monomers which are present in the 10-s chase sample (lane 1).
However, the mAb failed to precipitate the small amount of B-pentamer
present in that fraction (compare Fig. 3b, lane
1, with Fig. 3a, lane 2), suggesting that
LDS47 has no specificity for the native EtxB pentamer. This was further
substantiated by the inability of LDS47 to precipitate any EtxB from
the 10-min chase sample (Fig. 3b, lane 4), in
which only fully assembled EtxB pentamers are present. For comparison,
mAb 118-8 precipitated both unassembled and pentameric EtxB (Fig. 3b, lanes 2 and 5). We conclude
that LDS47 recognizes an epitope in EtxB that becomes inaccessible when
the B-subunit assembles into a native pentameric complex.
Figure 2: LDS47 precipitates denatured EtxB but not native EtxB pentamers. Heat-denatured EtxB and pentameric EtxB were subjected to immunoprecipitation with LDS47. Immune complexes were dissociated from the Protein A-Sepharose by boiling in SDS-containing sample buffer and then analyzed by SDS-PAGE. Lane 1, immunoprecipitate of heat-denatured EtxB; lane 2, immunoprecipitate of pentameric EtxB. The migration positions of the EtxB subunit and the heavy (H) and light (L) chains of IgG are indicated. The position of the molecular weight markers are indicated on the right-hand side of the figure.
Figure 3: LDS47 recognizes and precipitates in vivo-derived EtxB monomers. a, periplasmic proteins from E. coli G6 pMMB68 were extracted from radiolabeled cells, sampled 10 s and 10 min after addition of the chase, and analyzed by SDS-PAGE. The samples were either boiled or kept at room temperature prior to electrophoresis. Lane 1, 10-s sample (boiled); lane 2, 10-s sample (unheated); lane 3, 10-min sample (boiled); and lane 4, 10-min sample (unheated). The migration positions of the EtxB pentamer (EtxB5) and EtxB monomer (EtxB1) are indicated. b, periplasmic extracts from cells chased for 10 s (lanes 1-3) or 10 min (lanes 4-6) were subjected to immunoprecipitation using the following mAbs: lanes 1 and 4, LDS47; lanes 2 and 5, 118-8; and lanes 3 and 6, no antibody (control). Immunoprecipitated proteins were released from the immune complexes by adding SDS-containing sample buffer at room temperature and then analyzed by SDS-PAGE and autoradiography.
Figure 4:
LDS47 recognizes the amino-terminal
decapeptide of EtxB. Overlapping synthetic peptides corresponding to
the entire EtxB polypeptide were analyzed in a competitive ELISA for
their capacity to inhibit LDS47 binding to unfolded EtxB as described
under ``Experimental Procedures.'' a, peptides used
were: A, residues 1-20; B, residues
11-30; C, residues 21-40; D, residues
31-50; E, residues 41-60; F, residues
51-70; G, residues 61-80; H, residues
71-90; I, residues 81-100; and J,
residues 91-103. The addition of no peptide or denatured EtxB (1
µM; EtxB1) served as negative and positive
controls, respectively. b, peptides of varying length from the
amino-terminal 20 amino acids of EtxB were tested by competitive ELISA
for their effect on LDS47 binding to unfolded EtxB. The level of LDS47
binding to EtxB1 in the absence of any added peptide (no peptide
control) was interpolated from the A to be 100%
binding. The percent binding (n = 4, ±S.D. from
the mean) of LDS47 in the presence of different peptides (at 20
µM) is shown. The minimal inhibitory peptide,
corresponding to the amino-terminal 10 residues of EtxB, is indicated
in bold.
To investigate whether LDS47 recognized an epitope located within the amino-terminal 20 amino acids of EtxB, a series of shorter peptides were synthesized corresponding to 6-, 8-, 9-, 10-, 12-, and 15-mers from the amino terminus. Competitive ELISAs using peptide concentrations of 20 and 4 µM demonstrated that the minimum peptide sequence, capable of completely inhibiting LDS47 binding, comprised the first 10 amino acids of EtxB (Fig. 4b). Only approximately 50% inhibition of mAb binding was caused by the amino-terminal 9-amino-acid peptide, suggesting that the 10th residue (Ser) is either a component of the epitope or contributes to stabilizing the epitope. An analysis of additional peptides lacking one or more amino-terminal residues demonstrated that the amino-terminal Ala residue was necessary for inhibition of LDS47 binding (Fig. 4b). We conclude that the epitope recognized by LDS47 is located within the amino-terminal decapeptide of EtxB.
The IC (50% inhibitory
concentration) of the amino-terminal decapeptide, required for
inhibition of LDS47 binding, was calculated to be 0.28 µM (Fig. 5). For comparison, a peptide corresponding to the
amino-terminal 8 amino acids of EtxB exhibited no inhibitory effect,
when tested at concentrations ranging from 0.01 to 30 µM (Fig. 5). Fig. 6illustrates the location of the
amino-terminal decapeptide within the three-dimensional structure of
EtxB.
Figure 5:
Amino-terminal decapeptide inhibition of
LDS47 binding to EtxB. The effect on LDS47 binding to denatured EtxB
was determined by competitive ELISA in the presence of the inhibitory
amino-terminal decapeptide, APQSITELCS, and a noninhibitory peptide
corresponding to the amino-terminal octapeptide, APQSITEL. The peptides
were serially diluted 3-fold, mixed with LDS47, and applied to
EtxB1-coated ELISA plates. The mean A (n = 2, ±S.D.) is plotted against peptide
concentration.
Figure 6: Location of the amino-terminal decapeptide in the crystal structure of EtxB. A Raster model of the porcine EtxB pentamer is shown with two neighboring B-subunits in mid- and dark gray, and the remaining three subunits in light gray. The amino-terminal decapeptide of the dark gray subunit is shown in black. Plots were generated using Quanta on Silicon Graphics.
Figure 7:
Binding of pentameric EtxB to plastic or
nitrocellulose causes the epitope recognized by LDS47 to become
accessible. a, microtiter plates were coated with a 5
µg/ml concentration of either denatured EtxB (EtxB1) or
pentameric EtxB (EtxB5 - GM1) or coated with G before addition of pentameric EtxB (EtxB5 + GM1).
The binding of LDS47 (1:50 dilution of supernatant) to these proteins
was tested by ELISA. The mean A
(n = 2, ±S.D.) is given for each protein. b,
Western blotting of LDS47 to EtxB1 (lane 1) and EtxB5 (lane 2). The blot was probed with LDS47 supernatant at a
1:100 dilution, as described under ``Experimental
Procedures.''
Figure 8:
Kinetics of EtxB turnover and assembly in vivo. a, LDS47 (lanes 1-8) or 118-8 (lanes 8-16) were used to immunoprecipitate EtxB from
periplasmic fractions from a culture of E. coli G6 pMMB68 that
had been radiolabeled for 15 s and then chased for the following
lengths of time: lanes 1 and 9, 0.16 min; lanes 2 and 10, 0.5 min; lanes 3 and 11, 1 min; lanes 4 and 12, 2 min; lanes 5 and 13, 5 min; lanes 6 and 14, 10 min; lanes
7 and 15, 20 min; and lanes 8 and 16,
30 min. Immunoprecipitated proteins were released from the immune
complexes and analyzed by SDS-PAGE and autoradiography as described in
the legend to Fig. 3. The migration positions of unassembled
EtxB monomers (EtxB1) and assembled EtxB pentamers (EtxB5) are indicated. b, the amount of unassembled
EtxB (; in lanes 1-7) and assembled EtxB pentamers
(
; in lanes 9-15) were quantified by densitometric
scanning of the autoradiogram and analyzed using Bioimage image
analysis software. The amounts of labeled B-subunits are given in
arbitrary integration units and are plotted against the time at which
the samples were removed during the pulse-chase (with zero time
representing the initiation of the chase). The lines of best fit are to
a first order process.
The data in Fig. 8a (lanes 1-8) also show that no additional proteins (other than unassembled EtxB subunits), were precipitated by LDS47. This finding lends credence to the view that the periplasm may not possess a folding factor or chaperone that stably interacts with the toxin subunits during their folding and assembly into a macromolecular complex.
In this paper we describe the successful generation and
characterization of a monoclonal antibody that recognizes the monomeric
B-subunit of E. coli heat-labile enterotoxin but which fails
to precipitate the native B-subunit pentamer. Evidence is presented,
and is discussed further below, indicating that the mAb interacts with
the amino-terminal decapeptide of EtxB. The rationale in seeking to
obtain such a monoclonal antibody was to provide an investigative tool
for monitoring the intermediates of enterotoxin biogenesis. The
previous lack of suitable reagents for studying toxin biogenesis was
exacerbated by the finding that polyclonal antitoxin antisera
recognizes preferentially the pentameric B-subunit (Palva et
al., 1981) and that the available mAbs generated by
this and other laboratories recognized B-pentamers, although some also
recognized the denatured subunit as well (Remmers et al.,
1982; Robb et al., 1982; Holmes and Twiddy, 1983; Lindholm et al., 1983; Belisle et al., 1984a, 1984b;
Svennerholm et al., 1986; Finkelstein et al., 1987;
Kazemi and Finkelstein, 1990). In this study, a large number of
hybridomas had to be screened before one was identified that exhibited
the desired specificity. This may reflect the fact that antibodies to
EtxB appear to be primarily directed against conformational
determinants, a finding that may stem from the potent immunogenicity of
the assembled B-pentamer compared with that of the denatured B-subunit.
Only after EtxB had been denatured and electroblotted onto
nitrocellulose (for use as the immunogen) were we successful in
generating a mAb against the denatured B-subunit. The binding of EtxB
to nitrocellulose most likely served to adjuvant the immune response,
since the level of serum antibodies against denatured EtxB was higher
than in mice immunized with denatured EtxB alone. (
)
The
screening procedure used in the identification of mAb LDS47 relied on
an ELISA technique in which hybridoma supernatants were tested for
their differential reactivity toward denatured EtxB (bound directly to
the plastic) and EtxB pentamers (captured on plastic that had been
coated with G). This established that LDS47 bound to
denatured EtxB but not to G
-bound EtxB pentamers. The
subsequent finding that the mAb recognizes B-pentamers coated directly
onto plastic (albeit less efficiently) as well as EtxB pentamers
electroblotted onto nitrocellulose raised the prospect that the epitope
recognized by LDS47 was part of the receptor binding site and was
masked by the binding of EtxB to G
. Qu and Finkelstein
(1993) have reported mAbs against CtxB which exhibit similar
characteristics and have concluded that since G
binding
prevented the reactivity of mAbs toward CtxB pentamers, the epitope
must be in the G
binding site. However, based upon the
observation that LDS47 is unable to precipitate native EtxB pentamers
in solution (in the absence of G
), an alternative
explanation is suggested. It is proposed that the process of binding
pentamer directly to ELISA plates or to nitrocellulose causes a
conformational change in the protein leading to exposure of the epitope
only normally accessible in the monomeric subunit. Similar conclusions
have also been drawn from studies on the
2 subunit of E. coli tryptophan synthase. When hybridomas raised against the
2
subunit were screened in ELISAs in which the native
2 subunit had
been applied to the plastic surface, mAbs were identified that
preferentially recognized the denatured protein (Friguet et
al., 1984).
In order to seek an explanation for the apparent
discrepancy between the failure of LDS47 to precipitate native EtxB
pentamers compared with newly synthesized EtxB monomers, we undertook a
series of epitope mapping experiments to investigate the identity of
the epitope recognized by LDS47. Since LDS47 binds to reduced,
denatured EtxB we postulated that the epitope probably comprised a
linear stretch of amino acids. The inhibitory effect of overlapping
synthetic EtxB peptides on LDS47 binding to denatured EtxB revealed
that a peptide corresponding to the amino-terminal 10 amino acids of
EtxB was sufficient to inhibit EtxB-LDS47 recognition. Peptides
corresponding to residues 2-10 or 1-8 of EtxB exhibited no
inhibitory effect, while a peptide corresponding to residues 1-9
caused partial inhibition. The size of the epitope required to inhibit
LDS47 binding was somewhat larger than might have been expected, since
linear epitopes typically consist of 4-8 residues (Geysen, 1985).
This suggests that the epitope may possess a degree of secondary
structure, such as the formation of a partial or complete -helical
segment within the 10-amino-acid sequence of the epitope. Similar
phenomena of apparent discontinuous epitopes in linear sequences have
been reported for other peptide/antibody interactions (Appel et
al., 1990). The finding that the amino-terminal 20-mer peptide was
less efficient at inhibiting mAb binding to EtxB than the
amino-terminal decapeptide could be due to peptide folding partially
masking the epitope.
The crystallographic structure of Etx (Sixma et al., 1993; Fig. 6) revealed that the amino-terminal
decapeptide appears to resemble a curved finger that clasps the
adjacent subunit in the B-pentamer. Consequently, this might be
expected to be fully exposed in the B-monomer or heat-denatured
subunit, but to become partially solvent-inaccessible in the assembled
B-pentamer. It is also noteworthy that this segment of EtxB does not
form part of the G binding site (Sixma et al.,
1993; Merritt et al., 1994), so binding to G
would be unlikely to mask the epitope. Although this region is
involved in subunit-subunit interaction, it does not form part of the
major subunit interfaces. It is thus easy to see how the B-pentamer
might maintain its oligomeric structure when bound to plastic or
nitrocellulose, but that such interactions might weaken the clasp of
the amino-terminal decapeptide to reveal the epitope for recognition by
LDS47. The crystal structure also revealed that the first 10 amino
acids of EtxB form a secondary structural element comprised of a short
-helix from residues 5-10 (Sixma et al., 1993).
This finding is consistent with the proposal that the epitope
recognized by LDS47 needs to adopt a degree of secondary structure.
The extensive packing of the B-subunits against one another in the assembled crystal structure was evaluated by Sixma et al. (1993) by comparing the difference in the accessible surface of monomer and dimer subunits. This revealed that residues 1, 2, 3, and 8 show a significant difference in solvent exposure upon association with an adjacent subunit. Other residues within the amino-terminal decapeptide, namely residues 6, 7, and 10, are equally exposed in both the monomer and dimer. Thus, we would speculate that one or more of the residues that interacts with the neighboring subunit is likely to interact with the antibody combining site of LDS47.
A comparison of the amino acid sequences of EtxB and CtxB reveals a number of substitutions in the first 10 residues of the polypeptide; Ala/Thr at residue 1, Ser/Asn at residue 4, Glu/Asp at residue 7, and Ser/Ala at residue 10, respectively. Since LDS47 recognizes EtxB more efficiently than CtxB, one or more of these residues are likely to be involved in interacting with the antibody combining site.
The availability of a mAb, such as LDS47, whose specificity has been defined, represents a valuable investigative tool. In recent years, protein folding and assembly in living cells has been shown to be facilitated by molecular chaperones and catalysts of protein folding (Gething and Sambrook, 1992). Chaperones, with broad specificity capable of assisting the folding of a range of different proteins, have been identified in various cellular compartments, such as the cytoplasm, cytosol, endoplasmic reticulum, and in mitochondria and chloroplasts (Gething and Sambrook, 1992). However, the periplasm of Gram-negative bacteria, which represents a compartment where newly exported proteins fold and assemble, has, as yet, not been shown to contain a general chaperone, although it possesses catalysts of protein disulfide bond formation, peptidyl prolyl cis-trans isomerization, and specialized chaperones involved in the assembly of macromolecular structures, such as pili (Lui and Walsh, 1990; Bardwell et al., 1991; Hayano et al., 1991; Hultgren et al., 1991; Yu et al., 1992; Missiakis et al., 1993, 1994). We have previously speculated that enterotoxin assembly could require a chaperone to facilitate either subunit folding or assembly, particularly since the sites of subunit-subunit interaction are predominantly hydrophobic. Hofstra and Witholt(1985) found that a polyclonal antitoxin antiserum caused the co-precipitation of newly synthesized toxin subunits and an 8-kDa polypeptide (of unknown identity). This suggested that the 8-kDa protein may interact with unassembled toxin subunits, prior to formation of assembled holotoxin. By using LDS47, we have been able to re-evaluate whether newly synthesized, unassembled EtxB monomers in the periplasm of E. coli are associated with another protein, possibly a molecular chaperone. Similar strategies have recently been employed in studying the T-cell receptor complex in eukaryotic cells where monoclonal antibodies against the individual subunits of the receptor were used to show that before they assembled they associate with an endoplasmic reticulum resident protein (IP90) (Hochstenbach et al., 1992; David et al., 1993). However, when LDS47 was used to precipitate unassembled EtxB monomers from the periplasm of E. coli, no other protein(s) were apparently co-precipitated, suggesting that, at least under conditions of this analysis, unassembled EtxB subunits are not associated with a folding factor or chaperone. However, since such associations may be weak or may rely on association with proteins located in the cytoplasmic membrane, it will be necessary to develop ways to stabilize any putative chaperone interactions before it will be possible to unequivocally establish whether the periplasm possesses a generalized chaperone, able to facilitate enterotoxin biogenesis.
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