Download Targeting of specific domains of diphtheria toxin by site

Journal of General Microbiology (1992), 138, 2197-2203.
Printed in Great Britain
2197
Targeting of specific domains of diphtheria toxin by site-directed
antibodies
DOROTHEA
SESARDIC,*
VANITAKHANand MICHAEL
J. CORBEL
Division of Bacteriology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms,
Hertfordshire EN6 3QG. UK
(Received 19 March 1992; revised 15 June 1992; accepted 23 June 1992)
~
Antibodies highly selective for two functionally distinct regions of diphtheria toxin (DTx) were prepared using
synthetic peptide conjugates as immunogens. Three peptides were selected for synthesis: sequence DTx141-l5 7 on
fragment A, which contains the putative protein elongation factor (EF-2)ADP-ribosyltransferasesite; DTx224237 on fragment B, selected on the basis of forming a predicted surface loop; and D T x ~ on
~ fragment
~ - ~ B,
~ ~
forming a part of the region containing the putative receptor binding domain. All of the anti-peptide antibodies
recognized the corresponding peptide, and also reacted with the toxin, specifically with the fragment containing the
sequence against which they were raised, confirming the utility of this approach in generating fragment-specific
antibodies. The anti-peptide antibody with the highest binding titre both to the peptide and to the native toxin was
the one prepared against the sequence with the highest surface and loop likelihood indices of the three peptides
selected. The similarity of the reactivity profiles with peptide and native and denatured toxin is consistent with the
prediction that the region selected occurs in a surface loop and that the structure of the peptide is similar to the
conformation of this region ia the native protein. The epitopes for two of the anti-peptideantibodies were mapped.
The results indicated that even though the antisera were raised to peptides containing 14 amino acids (aa) they were
directed predominately against a narrow region within the peptide, consisting of only 5-6 aa residues. The
predicted location of the peptide and their epitopes was confirmed by inspection of the X-ray crystallographic
structure of DTx. Antibodies to peptides were selective for the toxin, one binding to DTx some 5-6O-foldbetter
than to diphtheria toxoid, presumably reflecting variability caused by toxoid preparation at this epitope. None of
the antisera produced protected against DTx challenge in the guinea pig intradermal test in aim. Although the
availability of site-specific antibodies that recognize neutralizing epitopes would be very valuable, antibodies such
as those described here should prove extremely useful in the structure-function analysis of DTx.
Introduction
Diphtheria toxin (DTx) is an extremely potent bacterial
protein toxin with M , 58350, synthesized as a single
polypeptide chain by pathogenic strains of Corynebacterium diphtheriae. Following mild trypsin treatment and
reduction, DTx is cleaved into two functionally different
fragments, A and B. Fragment A is a potent NAD+binding enzyme which ADP-ribosylates elongation
factor 2 (EF-2) (Pappenheimer, 1977; Collier et al.,
1964), thereby inhibiting protein synthesis. Fragment B,
on the other hand, is required for recognition of specific
cell surface receptors (Honjo et al., 1968; Uchida et al.,
* Author for correspondence. Tel. (0707) 54753; fax (0707) 46730.
Abbreuiation : KLH, keyhole limpet haemocyanin.
1977), binding to which results in translocation of
fragment A into the cytosol where it exerts toxicity
(Cieplak et al., 1987; Mekada et al., 1988).
The clinical manifestations of diphtheria are not due
to invasive bacterial infection but to release of this potent
cytotoxin. Thus, successful vaccination against diphtheria requires the acquisition of toxin neutralizing
antibodies. There have been several suggestions as to
how modern methods could be applied to create purer or
more effective vaccines against toxin-producing bacteria, including those based on peptides capable of
eliciting neutralizing antibodies (Audibert et al., 1981,
1982). The main obstacle in the development of such
vaccines is lack of understanding of the immunological
specificity of the areas of the toxin responsible for
eliciting protective antibodies. Antibodies raised against
peptides corresponding to the linear protein sequence
0001-7473 O 1992 SGM
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 10 May 2017 19:45:33
2198
D . Sesardic, V . Khan and M . J . Corbel
Table 1. The structure of the peptides chosen for synthesis and their corresponding regions on
diphtheria toxin
The amino acid sequence of diphtheria toxin was predicted from the cDNA sequence (Greenfield etal., 1983)
and is numbered from the N-terminal glycine which is the first amino acid on fragment A.
Peptide
name
DTA
DTB 1
DTB2
Structure
Sequence on DTx
Ala-Glu-Gly-Ser-Ser-Ser-Val-Glu-Tyr-Ile-Asn-Asn-Trp-Glu-Gln-Ala-Lys DTx 141-157
Gly-Pro-Ile-Lys-Asn-Lys-Met-Ser-Glu-Ser-Pro-Asn-Lys-Thr
DTx 224-237
DTx 513-526
Gly-Tyr-Gln-Lys-Thr-Val-Asp-His-Thr-Lys-Val-Asn-Ser-Lys
have very high and predefined specificity, and thus
provide invaluable tools with which to study the
antigenic structure of the protein.
We now report the preparation of site-specific
antibodies to the two functionally different fragments of
DTx, using synthetic peptide conjugates as immunogens.
The peptides were chosen from the primary amino acid
sequence of DTx previously deduced by Greenfield et al.
(1983).
Methods
Reagents and general procedures. All peptide synthesis reagents and
N-a-9-fluorenylmethoxycarbonyl-amino
acids were from Novabiochem UK and were of the highest purity available. Pin Technology
solid-phase peptide synthesis blocks were from Cambridge Research
Biochemicals. HPLC cartridges packed with Spherisorb S50 DS2 were
from Chrompack. SDS-PAGE reagents were from National Diagnostics. Nitrocellulose membrane was from Amersham. Immunochemical
reagents and reagents for tissue culture were from Sigma except for
Auro Dye Forte protein stain which was from Cambio. The reagents
necessary for the preparation of maleimide-activated keyhole limpet
haemocyanin (KLH) and bovine serum albumin (BSA) for peptide
conjugation were from Pierce, and Freund’s complete and incomplete
adjuvants were from Difco. For solid phase immunoassay, 96-well
polystyrene immuno-Nunc plates (Life Technologies) were used and all
plastic-ware for tissue culture was from Falcon. All other reagents were
of the highest grade available. Purified diphtheria toxin J G 1039/10
(5 mg protein ml-l, 1560 Lf ml-I) and J G 1002/10(8 mg ml-l, 2560 Lf
ml-l) were provided by Wellcome Biotechnology Research Laboratories, Beckenham, Kent and freeze-dried preparation 79/1 (1000 Lf
per ampoule) was from the RIVM, The Netherlands.
Choice of peptides, synthesis and coupling to carrier proteins. Peptide
selection was based on computer prediction of likelihood of surface
location and hydrophilicity, using Genetics Computer Group (GCG)
multiple sequence software package (Devereux et al., 1984) and loop
formation (Edwards et al., 1991). Some consideration was also given to
possible involvement of certain regions in toxin function (Tweten et al.,
1985; Rolf et al., 1990). The sequences selected are shown in Table 1.
Peptide DTA was custom synthesized by Cambridge Research
Biochemicals and the anti-peptide antibody prepared essentially as
previously described (Perera & Corbel, 1990). Peptides DTBl and
DTB2 were synthesized using a NovaSyn K R polyamide support and
N-a-9-fluorenylmethoxycarbonyl
amino acids on an LKB Biolynx 4170
peptide synthesizer. Amino acids were coupled as pre-formed
symmetrical anhydrides or as pentafluorophenyl esters. For the
purpose of conjugation to carrier proteins, cysteine was added to the N-
terminus. Cleavage and deprotection was carried out in 13 Mtrifluoroacetic acid in the presence of phenol, 1,Zethanedithiol and
thioanisole as cation scavengers. The purity of the peptides was
confirmed by reversed phase HPLC using a Spherisorb S5 ODs2
column (100 mm x 3 mm) monitored at 210 nm. The peptides had the
expected composition on amino acid analysis, determined as the ophthaldehyde-p-mercaptoethanol derivatives (Jones et al., 1981) and
relative molecular masses as determined by fast atom bombardment
mass spectrometry (M H+ : m / z DTBl calculated = 1529-79 and
m/z
DTB2
calculated = 1707-94
and
found = 1529.70;
found = 1706.90).
m - Maleimidobenzoyl- N- hydroxysuccinamide - ester- derivatized
KLH or BSA was added at 2mg per 1.3 pmol cysteinyl peptide,
determined by the free thiol content of the preparation using Ellman’s
reagent (Ellman, 1959). On average, 60-70% of the peptide was
conjugated. Null conjugates were also prepared, where cysteine was
substituted for the peptide. All conjugates were purified by gel filtration
on a Sephadex (G-50) column and stored in phosphate buffered saline
(PBS : 1a5 mM-potassium phosphate, 8-1 mM-sodium phosphate,
2.7 mM-potassium chloride, 137 mM-sodium chloride, pH 7.5). Protein
concentration was determined by the method of Bradford (1976), using
crystalline BSA, fraction V, as standard.
+
Immunization. Female New Zealand White rabbits (3 kg body
weight, from Froxfield Farms Ltd, Petersfield, Hampshire, UK) were
immunized with 10C160 pg peptide conjugate in PBS mixed 1 : 1 with
Freund’s complete adjuvant, in a total volume of 0.5 ml, by
subcutaneous and intradermal injections. One booster injection was
administered 14 d later using 50 pg peptide in incomplete adjuvant.
Rabbits were killed by cardiac puncture on day 28. Control (preimmune) blood samples were collected from a marginal ear vein of the
same rabbits before immunization started. Sera were prepared from
clotted blood and stored at -30 “C until required.
Purijkation of antibodies. Antisera were purified by affinity
chromatography as previously described by Edwards et al. (1989).
Immunological analyses. ELISA microtitre plates were coated with
100 p1 peptide (2 pg ml-l), peptide conjugate (1 pg ml-l), purified
diphtheria toxin or diphtheria toxoid (5pg ml-l), in coating buffer
(0.05 M-carbonate buffer, pH 9.6). Washing steps were performed in
PBS/Tween buffer (PBS containing 0.05 % Tween 20), and blocking
steps in 1% (w/v) skimmed milk powder (Marvel) in PBS. The
detecting antibody was anti-rabbit IgG conjugated to horseradish
acid
peroxidase with 2,2’-azino-bis-3-ethylbenzthiazoline-6-sulphonic
and hydrogen peroxide as co-substrates. A405was determined using an
Anthos plate reader, model 2001 (Anthos Labtec Instruments, Austria)
with ARCOM 2.4 software running on an Olivetti 286 PC. The binding
titre was defined as the concentration of antibody that gave halfmaximal binding to an antigen.
Western blotting was performed essentially as described by Towbin
et al. (1979) where, on average, 0.5-2.5 pg purified reduced diphtheria
toxin was applied to 10% (w/v) SDS-PAGE gels and run in a mini-
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 10 May 2017 19:45:33
Anti-diphtheria toxin site-directed antibodies
1
1
2
2
1
2
1
2 199
2
DTx
B
A
Aurodye
KLH-DTA
KLH-DTB 1
DTxd
Fig. 1. Western blotting of diphtheria toxin fragments with anti-peptide and anti-toxoid antisera. Purified diphtheria toxin, 2-5 pg (1)
or 0.5 pg (2) per lane, was subjected to 10% (w/v) SDS-PAGE and immunoblotted with rabbit antisera to peptide DTA (KLH-DTA),
peptide DTBl (KLH-DTB1) or to the whole diphtheria toxoid (DTxd). One nitrocellulose membrane was stained with Auro dye
following electrophoretic transfer. A and B indicate the positions of the respective DTx fragments at M , 20000 and 38000 and DTx
indicates the same for intact toxin at M , 58000.
Table 2 . Binding titre of anti-peptide and anti-diphtheria
toxoid antibodies in ELISA
The serum titres shown are the dilutions, starting with the neat
serum, that gave half-maximal binding. In the case of purified
anti-peptide antibody the titre shown is that dilution that gave
half-maximal binding, starting from a purified antibody
preparation of 100 pg specific IgG ml-l.
Binding titre
Anti-peptide antisera
Antigens
DTA
DTB2
DTBl
Homologous peptide*
KLH-peptide*
DTx native
DTx
urea
1 :32
1 :2200
1:800
1:1600
1 : 1000
1 :10000
>1
1:30
1 :6500
1 :40000
1 :3000
1~5600
+
Purified Ab
DTB 1
1 :2500
1 :10000
1 :2500
Epitope mapping. The General Net Epitope Scanning strategy
(Cambridge Research Biochemicals Pin Technology) was used to
synthesize simultaneously 172 peptides, each of 8 aa residues, with an
overlap of 6, spanning the entire length of DTx fragment B. The
peptides were synthesized on the tips of the derivatized polyethylene
pins in the configuration of 96-well microtitre plates (Geysen et al.,
1987), as detailed in the Epitope Scanning Kit manual provided by the
manufacturers. The N-termini of the peptides thus synthesized were
acetylated, prior to deprotection with trifluoroacetic acid/anisole/ethanedithiol. Finally, the pins were neutralized by sonication in 0.1%
hydrochloric acid in methanol and stored under silica gel as a desiccant.
Immunoassay of the peptides on pins was performed by the
modification of the ELISA procedure described above. Non-specific
binding sites were saturated by overnight incubation of pins with 2%
(w/v) BSA in PBS. Following use, the pins were regenerated by
sonication as detailed in the Epitope Scanning Kit manual.
ND
* Results shown are for the orthologous peptide sequence only. None
of the antisera reacted with any of the heterologous peptide sequences.
ND, not determined.
Protean I1 (Bio Rad) electrophoresis cell. The co-substrates for Western
blotting were 4-chloro-a-naphthol and hydrogen peroxide.
In vivo toxin neutralization assay. Female Dunkin Hartley guinea pigs
(450-500 g) from Harlan Olac (Bicester, Oxon, UK) were shaved and
injected intradermally (0.2 ml) with mixtures containing diphtheria
toxin, test antibody or both. The animals were observed for 48 h. The
protective effect of a test antiserum was assessed by its ability to
neutralize a dermonecrotic dose of DTx at the site of injection (B. P.
Vol I1 Appendix XIV B2).
Results
Antisera from rabbits immunized with the three different peptides conjugated to KLH all reacted with the
respective immunogen with high titre, as well as to the
corresponding peptide in ELISA (Table 2). The binding
titre of anti-peptide DTBl to the orthologous peptide
conjugate was 4-18 fold higher than that of the other two
preparations, Following affinity purification, the binding titre of the anti-peptide antibodies for the orthologous immunogen was 1000-fold greater than for the
carrier protein. Although there were large differences in
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 10 May 2017 19:45:33
2200
D . Sesardic, V . Khan and M . J . Corbel
0.8
0.6
<
wl
0.4
0.2
I
I
I
I
I
I
0
30
60
90
Peptide
120
150
30
60
L
180
1.5
1.2
0.9
<
YI
0.6
0.3
90
Peptide
120
150
180
Fig. 2. Reactivity of antisera to peptide DTBl (a) and DTB2 (b) to multiple peptides encompassing the whole of DTx fragment B. A
modified ELISA procedure was used to screen the reactivity of two of the anti-peptide antisera, a-DTB1 (a) and a-DTB2 (b), against 172
overlapping octapeptides, spanning the whole of DTx fragment B. Peptides were synthesized using Pin Technology (Cambridge
Research Biochemicals) as described in Methods.
the binding titre of the antibodies to the peptides alone,
this could reflect differences in the ability of the peptides
to bind to the microtitre plate.
Of the three anti-peptide antibodies prepared, two also
reacted strongly with native DTx, in ELISA. The antipeptide DTBl antibody had the highest titre of binding
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 10 May 2017 19:45:33
Anti-diphtheria toxin site-directed antibodies
1.5
1.2
0.9
8
pl
0.6
0.3
**
*t
9
t
0.8
(b1
0.6
<
0.4
0.2
a
y* y
**
$I
*
*9
&
>
c a
>
id c
D
*
**
**
$
**
**
*
2
Fig. 3. Mapping of epitope for anti-DTB1 (a) and anti-DTB2 (b)
antisera. Only the regions of maximum response from Fig. 2a and 2b
are shown for reactivity of anti-DTB1 (a) and anti-DTB2 (b) antisera
against peptides of DTx fragment B. One letter symbols represent
amino acids contained in the peptide to which the antiserum was
originally produced and * denotes other amino acids in the sequence.
to peptide, conjugate and to the toxin. None of the antipeptide antibodies showed a reduction in binding titre
following denaturation of the DTx with 8M-urea.
Indeed, the antiserum to peptide DTB2 bound to DTx
only after such denaturation of the antigen (Table 2). The
220 1
antiserum to DTA bound much better to DTx, both
native and denatured, than did the anti-DTB2
antiserum.
Immunoblotting of purified, reduced diphtheria toxin
with the anti-peptide antibodies showed that the antisera
were highly selective for the corresponding toxin
fragment. In contrast, a rabbit antiserum to DTxd
reacted with both fragments (Fig. 1.). Antiserum to
peptide DTB2 also blotted only to DTx fragment B, but
at a lower dilution than for antiserum to peptide DTBl
(data not shown).
The antiserum to peptide DTBl (and to peptide DTA)
bound better to DTx than to DTxd in ELISA, using three
different preparations of DTx and six DTxds. Whereas
there was no significant difference between the binding
of a polyclonal DTxd antiserum to DTx or to any of the
DTxd preparations, the antiserum to peptide DTB 1
bound significantly (p < 0.001) better to DTx (average of
60-fold)than to the DTxd preparations (data not shown).
The results of epitope mapping of antisera to peptides
DTBl and DTB2 on to the DTx fragment B sequence
indicated that each of the anti-peptide antisera is highly
specific to a cluster of 4-5 different peptides (Fig. 2a, b),
from the total of 172 analysed.
The antiserum to synthetic peptide DTBl bound to 8
peptides, with maximum binding to two with the
sequences MSESPNKT and ESPNKTVS (Fig. 3a),
from which it can be deduced that the epitope for this
antiserum is predominantly in the C-terminal part of the
peptide, sequence SESPNK. Similarly, the antiserum to
peptide DTB2 bound only to 4 peptides, with maximum
binding to one containing the sequence KTVDHTKV
(Fig. 3 b). It is therefore deduced that the epitope for this
antiserum is localized to the sequence KTVDH, which
occurs towards the middle of the peptide.
The affinity-purified antibodies were tested for their
ability to protect against DTx challenge in the guinea pig
intradermal test in vivo. Rabbit polyclonal antiserum to
DTxd was used as a positive control. None of the antipeptide antibodies was protective when used singly or in
combination (data not shown).
Discussion
Site-directed antibodies with high reactivity and specificity against the two functionally distinct fragments of
DTx (A and B) have been prepared. These antibodies
reacted specifically with the fragment containing the
sequence against which they were raised, confirming the
utility of the approach in generating fragment-specific
antibodies.
Often the most suitable region against which to direct
an anti-peptide antibody is an amino acid sequence
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 10 May 2017 19:45:33
2202
D . Sesardic, V . Khan and M . J . Corbel
predicted to occur in a loop region (i.e. a linear sequence
of residues), with a high hydrophilicity index, i.e. most
likely to occur at the surface of the protein. Such regions
are often very antigenic, and an antibody against such a
region should recognize both native and denatured
protein, as well as the immunizing peptide. A suitable
region on DTx fragment B was identified on this basis,
peptide DTB 1, and the anti-peptide antibody produced
did indeed have the highest binding titre to the native
toxin, amongst the anti-peptide antibodies obtained.
This is consistent with the fact that DTBl has the highest
surface and loop likelihood indices of the three peptides.
The ability of this antibody to bind to the toxin as well as
to the immunizing peptide, and the similarity between
the reactivity profiles of the antiserum with native and
denatured toxin is consistent with the prediction that the
region selected occurs in a surface loop region and that
the structure of the peptide is similar to the conformation
of this region in the native protein, i.e. a linear epitope.
This prediction was supported by epitope mapping using
overlapping peptides to fragment B. Anti-peptide DTB 1
antibody reacted only with 6 C-terminal aa of the 14-mer
immunizing peptide (cys-gpiknmSESPNKt). This 6 aa
sequence has the highest probability of forming a loop.
As the peptide was coupled to carrier protein via its Nterminal cys, these C-terminal residues, with a high
probability of forming a surface loop, would be most
accessible for antibody generation, and would be highly
accessible for antibody-binding in both the native and
denatured toxin. The location of the predicted epitope
for anti-DTB1 antibody, to occur in a surface loop region
of DTx, was recently confirmed by X-ray crystallography
(Choe et al., 1992).
While the anti-peptide DTB2 antibody bound well to
the immunizing peptide, this antibody reacted only
weakly with the toxin, and then only when it was
denatured with 8 M-Urea, suggesting that the epitope for
this antibody is not readily accessible in the toxin,
possibly because the immunizing peptide adopts a
conformation different from that of the corresponding
region of the protein. Some evidence that this might be so
was obtained by epitope mapping. Although it is
possible, and perhaps even likely, that a mixture of
antibodies is present in the polyclonal anti-peptide
antiserum, most appear to be directed against a narrow
region within the peptide, the 5 aa situated centrally in
the 14-mer (cys-gpyqKTVDHtkvnsk). However, it is the
C-terminal 6 aa residues that have a high surface
prediction. Hence, the epitope that is most antigenic in
the peptide is not the most accessible in the toxin. This is
consistent with the X-ray crystallographic structure of
DTx, in which the predicted epitope for anti-DTB2
forms part of a P-sheet adjacent to a short surface
loop/turn region (Choe et al., 1992).
The anti-peptide DTA antibody bound well to the
toxin, both native and denatured. Interestingly, the Nterminal 6 aa of DTA have a high probability of forming
part of a surface loop sequence. Whilst the epitope for
this antibody has not been mapped, the ability of the
antibody to bind to both native and denatured toxin
suggests that the dominant immunogenic epitope on the
peptide is accessible and contiguous within the toxin.
Inspection of the X-ray crystallographic structure of DTx
(Choe et al., 1992) reveals that, in fact, peptide DTA
includes two loop regions, one at each end of the
sequence.
Binding of the anti-peptide antibodies to DTx was
significantly greater, 60-fold in the case of anti-DTB 1,
than to various preparations of DTxd, presumably
reflecting variable modification of the epitopes during
toxoid preparation. It is of interest that an anti-toxoid
antiserum did not recognize any of the synthetic
peptides. Such toxin-selective antibodies could be of
considerable value in monitoring the loss of specific
surface epitopes during toxoiding.
One of the primary objectives of raising site-specific
antibodies against toxins is to obtain reagents that affect
toxin function, particularly by neutralization. However,
none of the anti-peptide antibodies reported here
protected against DTx challenge, in an in vivo intradermal challenge test in guinea pigs. This indicates that
these regions (more specifically their epitopes) do not
represent protective epitopes, although it is still possible
that they form part of such an epitope, which is nonlinear. Clearly, studies such as these will help to delineate
the structure-function relationships of DTx, particularly
when allied with recent information on the X-ray
crystallographic structure of the toxin.
The uniqueness of the deduced epitopes for the antipeptide DTBl and DTB2 antisera was determined by
searching the PIR protein database (release 28.0, March
1991) using the GCG sequence analysis (Devereux et al.,
1984) for matching sequences. No sequence identical
with the anti-DB1 epitope was found, whereas two
sequences, in a hypothetical protein of yeast, identical
with the anti-DTB2 epitope were found amongst a total
of 28,232 sequences analysed. If a single mismatch was
allowed in the matching algorithm, then 20 sequences
were identified for the anti-DTBl epitope and more than
200 for the anti-DTB2 epitope. This demonstrates the
unique specificity of the antiserum to peptide DTBl for
DTx fragment B.
We thank Mr P. A. Knight and Dr A. Whitaker of Wellcome
Research Laboratories for supplying the diphtheria toxin used in this
work. We are grateful to Dr P. H. Corran (Division of Chemistry,
NIBSC) and Dr R. Wait (Division of Pathology, Centre for Applied
Microbiology and Research, Porton) for performance of HPLC, amino
acid analyses and mass spectrometric analyses on the peptides,
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 10 May 2017 19:45:33
Anti-diphtheria toxin site-directed antibodies
respectively. We are also indebted to Dr R. J. Edwards of the
Department of Clinical Pharmacology, RPMS, London for his help
with predictions for the presence of loop regions within the diphtheria
toxin.
References
AUDIBERT,
F., JOLIVET,
M., CHEDID,L., ALOUF,J. E., BOQUET,P.,
RIVAILLE,
P. & SIFFERT,
0. (1981). Active antitoxic immunization by
a diphtheria toxin synthetic oligopeptide. Nature, London 289,
593-594.
AUDIBERT,
F., JOLIVET,
M., CHEDID,L., ARNON,R. & SELA,M. (1982).
Successful immunization with a totally synthetic diphtheria vaccine.
Proceedings of the National Academy of Sciences of the United States of
America 79, 5042-5046.
BRADFORD,
M. M. (1976). A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Analytical Biochemistry 72,
248-254.
CHOE,S., BENNETT,
M. J., FUJII,G., CURMI,P. M. G., KANTARDJIEFF,
K. A., COLLIER,
R. J. & EISENBERG,
D. (1992). The crystal structure
of diphtheria toxin. Nature, London 357, 216-222.
CIEPLAK,
W., GAUDIN,H. M.& EIDELS,L. (1987). Diphtheria toxin
receptor. Identification of specific diphtheria toxin-binding proteins
on the surface of Vero and Bs-C-1 cells. Journal of Biological
Chemistry 262, 13246-13253.
COLLIER,
R. J. & PAPPENHEIMER,
A. M. (1964). Studies on the mode of
action of diphtheria toxin. I1 Effect of toxin on amino acid
incorporation in cell-free systems. Journal of Experimental Medicine
120, 1019-1039.
DEVEREUX,
J., HAEBERLI,
P. & SMITHIES,
0. (1984). A comprehensive
set of sequence analysis programs for the VAX. Nucleic Acids
Research 24, 387-395.
EDWARDS,
R. J., SINGLETON,
A. M., BOOBIS,
A. & DAVIES,
D. S. (1989).
Cross-reaction of antibodies to coupling groups used in the
, production of anti-peptide antibodies. Journal of Immunological
Methods 117, 215-220.
EDWARDS,
R. J . , MURRAY,
B. P. & BOOBIS,A. R. (1991). Anti-peptide
antibodies in studies of cytochrome P450IA. Methods in Enzymology
206, 220-233.
2203
ELLMAN,
G. L. (1959). Tissue sulfhydryl groups. Archives of Biochemistry and Biophysics 82, 70-77.
GEYSEN,
H. M., RODDA,S. J., MASON,T. J., TRIBBICK,
G. &SCHOOFS,
P. G. (1987). Strategies for epitope analysis using peptide synthesis.
Journal of Immunological Methods 102, 259-274.
GREENFIELD,
L., BJORN, M. J., HORN,G., FONG,D., BUCK,G. A.,
COLLIER,
R.J. & KAPLAN,D. A. (1983). Nucleotide sequence of the
structural gene for diphtheria toxin carried by corynebacteriophage
p. Proceedings of the National Academy of Sciences of the United States
of America 80, 6853-6857.
HONJO,T., NISHIZUKA,
Y., HAYAISHI,
0.& KATO,I. (1968). Diphtheria
toxin-dependent adenosine diphosphate ribosylation of aminoacyl
transferase I1 and inhibition of protein synthesis. Journal of
Biological Chemistry 243, 3553-35 55.
JONES,B. N., PAABO,S. & STEIN,S. (1981). Amino acid analysis and
enzymatic sequence determination of peptides by an improved ophthaldialdehyde precolumn labelling procedure. Journal of Liquid
Chromatography 4, 565-586.
MEKADA,E., OKADA,Y. & UCHIDA,T. (1988). Identification of
diphtheria toxin receptor and a nonproteinous diphtheria toxinbinding molecule in Vero cell membrane. Journalof Cell Biology 107,
511-519.
PAPPENHEIMER,
A. M. (1977). Diphtheria toxin. Annual Review in
Biochemistry 46, 69-94.
PERERA,V. Y. & CORBEL,
M. J. (1990). Human antibody response to
fragments A and B of diphtheria toxin and a synthetic peptide of
amino acid residues 141-157 of fragment A. Epidemiology and
Infection 105, 457468.
ROLF,J. M., GAUDIN,H. M. & EIDELS,L. (1990). Localization of the
diphtheria toxin receptor-binding domain to the carboxyl-terminal
M, x6000 region of the toxin. Journal of Biological Chemistry 265,
1331-1 3 31.
TOWBIN,H., STAEHELIN,
T. & GORDON,J. (1979). Electrophoretic
transfer from polyacrylamide gels to nitrocellulose sheets : procedure
and some applications. Proceedings of the National Academy of
Sciences of the United States of America 76, 43504354.
TWETEN,R. K., BARBIERI,
J. T. & COLLIER,R. J. (1985). Effect of
substituting aspartic acid for glutamic acid 148 on ADP-ribosyltransferase activity. Journal of Biological Chemistry 260, 10392-10394.
UCHIDA,T., YAMAIZUMI,
M. & OKADA,Y. (1977). Reassembled HVJ
(Sendai virus) envelopes containing non-toxic mutant proteins of
diphtheria toxin show toxicity to mouse L cell. Nature, London 266,
839-840.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Wed, 10 May 2017 19:45:33