©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Generation of a Monoclonal Antibody That Recognizes the Amino-terminal Decapeptide of the B-subunit of Escherichia coli Heat-labile Enterotoxin
A NEW PROBE FOR STUDYING TOXIN ASSEMBLY INTERMEDIATES (*)

(Received for publication, March 27, 1995)

Tehmina Amin (1) Audrey Larkins (2) Roger F. L. James (2) Timothy R. Hirst (1)(§)

From the  (1)Research School of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ, United Kingdom and the (2)Department of Surgery, University of Leicester, Leicester, LE2 7LX, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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) (^1)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 beta 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 alpha-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(r) = 28,000) which has ADP-ribosyltransferase activity and five identical B-subunits (M(r) = 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(5) 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.


EXPERIMENTAL PROCEDURES

Materials

All reagents were purchased from Sigma unless otherwise stated. Monoclonal antibody 118-8, which recognizes both pentameric EtxB and oxidized EtxB monomers (Sandkvist et al., 1990), was provided by Dr. H. Person (University of Umea, Sweden).

Hybridoma Production

A recombinant preparation of EtxB, derived from the human enterotoxinogenic strain H74-114 (Leong et al., 1985), was purified as described by Amin and Hirst(1994) and used as the immunogen. 50 µg of EtxB was heated at 95 °C for 5 min in a buffer containing 0.5 M Tris-HCl, pH 6.8, 1% (w/v) sodium dodecyl sulfate (SDS), and 30 mM dithiothreitol and then subjected to SDS-polyacrylamide (14% w/v) gel electrophoresis. The gel was then soaked in blotting buffer (48 mM Tris-HCl, pH 9.2, 39 mM glycine, 1.3 mM SDS, and 20% (v/v) methanol), and the monomeric B-subunit was transferred onto a nitrocellulose membrane (Schleicher and Schuell) using a Bio-Rad Trans-Blot apparatus at 2 mA/cm^2 for 10 min. The nitrocellulose-bound B-subunit was visualized by temporary staining with Ponceau S in 5% acetic acid, washed, excised, and sonicated in phosphate-buffered saline (PBS, 150 mM NaCl, 10 mM sodium phosphate, pH 7.2) using an Ultrasonics sonicator, model W375, at 50% duty cycle for 20 30 s bursts.

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(2) (= 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.

Preparation of Ascites

20 male MF1 BALB/c F1 hybrid mice were injected with 0.5 ml of 2,6,10,14-tetramethylpentadecane (Pristane). One week later, each mouse was injected intraperitoneally with 3 10^6 hybridoma cells, and the ascitic fluid was collected approximately 2 weeks later.

Screening of Hybridoma Supernatants

Two ELISA assays were employed to discriminate between antibodies that recognized denatured EtxB monomers and assembled EtxB pentamers.

G-ELISA

This was performed essentially as described previously by Amin and Hirst(1994). Each hybridoma supernatant was added to the wells of a microtiter plate (Immulon 1, Dynatech) that had been coated with G-ganglioside and 0.2 µg/ml EtxB pentamers in PBS. Antibody binding to EtxB was detected using a goat anti-mouse-horseradish peroxidase conjugate (Jackson Immunoresearch Laboratories).

Non-G-ELISA

Since denatured EtxB subunits do not bind to the receptor G, denatured EtxB monomers were coated directly onto the plastic surface of the microtiter plate. Denatured EtxB monomers were prepared by boiling 5 µg/ml purified EtxB in PBS, 30 mM dithiothreitol for 5 min. Microtiter plates were coated with this preparation for 1 h at 37 °C, and the plate was washed with PBS. Nonspecific binding sites were blocked with 1% (w/v) bovine serum albumin (BSA), and the remainder of the assay was carried out in a manner identical with the G-ELISA. Pentameric EtxB at 5 µg/ml in PBS was also coated directly onto microtiter plates in the absence of G.

ELISAs were also performed using purified cholera toxin B-subunit from List Biologicals (Campbell, CA).

Inhibition ELISA for Epitope Mapping

Microtiter plates were coated with a 5 µg/ml concentration of a denatured, reduced preparation of EtxB, and the wells were blocked with BSA, as described above. Synthetic peptides (at various concentrations) were diluted in buffer containing 0.1% (w/v) BSA, 0.05% (v/v) Tween 20, PBS and mixed with an equal volume of a 1/5000 dilution of purified LDS47 (in the same buffer). The mixture was added to wells and analyzed in quadruplicate. A series of control wells, in which LDS47 was added without a synthetic peptide, provided a measure of 100% mAb binding in this assay.

Antibody Isotyping and Purification

Antibody isotyping was determined using an agglutination assay (Serotec Ltd., Oxford, UK) using the conditions recommended by the manufacturer.

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.

Immunoprecipitation

Immunoprecipitation of EtxB

0.05 ml of a purified preparation of EtxB (20 µg/ml) in 10 mM Tris-HCl, pH 7.6, was either kept at room temperature or heat-denatured by boiling for 5 min and then added to 0.35 ml of 1% (v/v) Triton X-100, 0.005% (w/v) SDS, 5 mM magnesium acetate, 60 mM ammonium chloride, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride, 10 mM Tris-HCl, pH 7.6 (solution 1). 0.5 ml of mAb LDS47 supernatant (diluted 2.5-fold in solution 1) was added to each sample. After a 30-min incubation on ice, 100 µl of a 10% (v/v) slurry of Protein A-Sepharose in 10 mM Tris-HCl, pH 7.6, was added, and incubation continued for an additional 30 min. The Protein A beads were centrifuged at 13,000 rpm at 4 °C for 3 min in a Sigma 2K10 centrifuge, and the supernatant was discarded. The pellet was then washed sequentially with the following solutions: 1 ml of ice-cold 0.15 M NaCl, 0.5% (v/v) Triton X-100, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM Tris-HCl, pH 7.6 (solution 2), 1 ml of 0.5 M NaCl, 0.5% (v/v) Triton X-100, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM Tris-HCl, pH 7.6 (solution 3), and 1 mM phenylmethylsulfonyl fluoride, 10 mM Tris-HCl, pH 7.6 (solution 4). 50 µl of SDS-PAGE sample buffer was added to each pellet and then heated to 95 °C for 5 min prior to their application to a polyacrylamide gel. Gels were stained using the Bio-Rad silver stain kit as recommended by the manufacturer.

Immunoprecipitation of Radiolabeled EtxB

50-µl samples of periplasmic extracts from radiolabeled cells were mixed with antibodies LDS47 (5 µl of purified IgG), 118-8 (10 µl of supernatant), or distilled water (no antibody control) and made up to a total of 0.85 ml with solution 1 (above) for 30-40 min on ice. Insoluble periplasmic material was removed by centrifugation at 13,000 rpm for 3 min at 4 °C in a Sigma 2K10 centrifuge. 100 µl of a 10% (v/v) Protein G-agarose slurry was added to the resulting supernatant, and the mixture was incubated and washed with 1 ml of ice-cold solutions 2, 3, and 4. Immune complexes were dissociated from the Protein G-agarose by addition of 50 µl of 1 SDS-PAGE sample buffer for approximately 10 min at room temperature, and the supernatant was analyzed by SDS-PAGE.

SDS-Polyacrylamide Gel Electrophoresis and Western Blotting

SDS-polyacrylamide gel electrophoresis was performed using 14% T and 3.3% C on a Bio-Rad Protean II system, according to the conditions recommended by the manufacturer. Low molecular mass range markers supplied by Bio-Rad included rabbit muscle phosphorylase b (97.4 kDa), BSA (66.2 kDa), hen egg white ovalbumin (45 kDa), bovine carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), and hen egg white lysozyme (14.4 kDa) and were loaded onto each gel. Gels containing radiolabeled, immunoprecipitated EtxB samples were soaked in Amplify (Amersham) for 30 min prior to drying. The dried gels were exposed to Hyperfilm MP (Amersham) for 4 to 5 days.

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.

Radiolabeling of in Vivo Synthesized EtxB

E. coli G6 pMMB68 (EtxB, Ap^R; Sandkvist et al.(1987)) was cultured in a modified M9 minimal medium at 37 °C on a shaking incubator (Infors, Switzerland), as reported previously (Sandkvist et al., 1990). When the cultures reached an A of 0.35, expression of EtxB was induced by the addition of 0.5 mM isopropyl-1-thio-beta-D-galactopyranoside. After 30 min of induction, the cells were radiolabeled with 3700 kBq/ml (100 µCi/ml) [S]methionine (specific activity > 37 TBq/mmol; ICN Flow) for 15 s, followed by addition of a chase of 1 mM nonradioactive L-methionine. Samples (1 ml) were removed at different time intervals, ranging from 10 s to 30 min, into ice-cold glass vials. The cells were pelleted at 13,000 rpm at 4 °C for 3 min in a Sigma 2K10 centrifuge, washed in 1 volume of ice-cold PBS, and repelleted as before. Washed cells were resuspended in 0.2 ml of ice-cold 0.1 M phosphate buffer, pH 7.6, containing 0.3 M sucrose. Lysozyme and EDTA were added to final concentrations of 20 µg/ml and 5 mM, respectively, and the mixture was incubated on ice for 15 min, with occasional mixing. Cells were pelleted, as before, and the supernatant (containing periplasmic proteins) was removed and placed on ice for a maximum of 45 min prior to immunoprecipitation. A portion of each periplasmic sample was also mixed with SDS-containing sample buffer for analysis by SDS-PAGE.

Peptide Synthesis

Peptides were synthesized using Fmoc chemistry on a Shimadzu PSSM-8 (Fields and Noble, 1990). Resins used were TGA Tentagel with carboxyl-terminal amino acids attached. Fmoc amino acids (from Novabiochem) were activated using O-benzotriazol-1-yl-N,N,N`,N`-tetramethyluronium hexafluorophosphate and N-hy-droxybenzotriazolebulletH(2)O. Fmoc groups were removed during synthesis using piperidine. Four cleavage mixtures were used for different classes of peptides. N1 (general) consisted of 94% (v/v) trifluoroacetic acid, 5% (v/v) anisole, and 1% (v/v) ethanedithiol. N2 (for Trp-containing peptides) was 94% (v/v) trifluoroacetic acid, 3% (v/v) anisole, and 5 mg of 2-methylindole. N3 (for Arg-containing peptides) was 82% (v/v) trifluoroacetic acid, 5% (v/v) H(2)O, 5% (v/v) thioanisole, 3% (v/v) ethanedithiol, 2% (v/v) ethylmethylsulfide, and 3% (w/v) phenol. N4 (for peptides containing both Trp and Arg) was N3 with 5 mg of 2-methylindole added.

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.


RESULTS

Identification of a mAb with a Specificity for Denatured (Monomeric) EtxB

Prior to the identification of LDS47, 44 hybridoma fusion experiments on lymphocytes derived from BALB/c mice repeatedly immunized with either native EtxB pentamers or denatured EtxB monomers were performed. However, none of the hybridomas obtained from these experiments produced antibodies that selectively recognized denatured EtxB monomers. In an attempt to overcome these difficulties, mice were immunized with a reduced, denatured preparation of EtxB that had been electroblotted onto nitrocellulose. This resulted in the identification of one hybridoma that produced antibodies recognizing denatured EtxB monomers (that had been applied directly to microtiter wells) but not EtxB pentamers (that had been captured in wells coated with G). The hybridoma was cloned by limiting dilution and designated mAb LDS47.

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 (bullet) 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.



Mapping of the Epitope Recognized by LDS47

Since LDS47 recognizes heat-denatured EtxB, this suggested that it binds to an epitope comprising a linear portion of the EtxB polypeptide. To identify the epitope, an inhibition ELISA was established in which the binding of LDS47 to heat-denatured, reduced EtxB (coated onto a microtiter plate) was tested in the presence of overlapping synthetic peptides from the complete EtxB sequence. Initially, nine 20-mer peptides and one 13-mer peptide (each overlapping by 10 residues) were prepared and tested (Fig. 4a). The results shown in Fig. 4a are a representative set of data from 3 independent ELISA experiments, in which the synthetic peptides were added at a concentration of 20 µM. Peptide A, corresponding to the extreme amino-terminal 20 amino acids of EtxB, caused a significant (75%) reduction in the binding of LDS47 (Fig. 4a). mAb binding was also reduced, albeit to a lesser extent, in the presence of peptide G, corresponding to amino acid residues 71-90. The possibility that peptide G was not a part of the epitope recognized by LDS47 was suggested by the observations that peptide G failed to inhibit LDS47 binding when tested at a concentration of 10 µM, and that a heterologous peptide, prepared using the same N3 cleavage mixture used in preparing peptide G, also reduced the binding of LDS47 (data not shown). In contrast, peptide A at 4 µM inhibited mAb binding by at least 35%.


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.



Cross-reactivity of LDS47

LDS47 was also tested for its reactivity toward CtxB using an ELISA in which denatured CtxB was applied directly to the plastic surface of a microtiter plate or in which CtxB pentamers were coated onto G. This showed that LDS47 recognized denatured CtxB but not G-bound CtxB pentamers. However, when the titer of the LDS47 supernatant toward 5 µg/ml denatured CtxB or 5 µg/ml denatured EtxB was determined by ELISA, it was found that there was an 80-fold difference in mAb reactivity toward CtxB versus EtxB, with titers of 1:125 and 1:10,000, respectively. This suggests that while LDS47 recognizes an epitope that is present in CtxB as well as EtxB, one or more of the amino acid differences in the amino-terminal decapeptides of CtxB and EtxB influences the efficiency of mAb binding.

The Epitope Recognized by LDS47 Becomes Accessible When the B-Pentamer Binds to Plastic or Nitrocellulose Surfaces

When a purified preparation of EtxB pentamers was bound directly to the plastic surface of an ELISA plate, LDS47 showed limited recognition of the antigen (Fig. 7a). This contrasted with the earlier finding that LDS47 lacks an ability to recognize the native B-pentamer in solution (Fig. 3) or B-pentamer bound to G-coated ELISA plates (Fig. 1). In addition, LDS47 was found to recognize both EtxB monomers and pentamers in a Western blot (Fig. 7b). This suggests that a conformational change occurs upon binding of EtxB pentamers to plastic or nitrocellulose surfaces that exposes the epitope recognized by LDS47. Similar conformational changes resulting in exposure of epitopes have been noted for other antigens (Friguet et al., 1984; Geysen, 1985).


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.''



Use of LDS47 to Monitor the Turnover of Assembling EtxB Subunits in Vivo

A pulse-chase experiment, similar to that described in Fig. 3, was performed with samples taken from 10 s to 30 min after addition of the chase. The resulting periplasms were subjected to immunoprecipitation with LDS47 and 118-8, and the immune complexes were incubated with SDS-containing sample buffer (at room temperature) prior to analysis by SDS-PAGE and autoradiography (Fig. 8a). The radiolabeled EtxB subunits immunoprecipitated by LDS47 migrated as monomers, whereas those precipitated by 118-8 comprised both SDS-stable EtxB pentamers and a small proportion of monomers. A densitometric analysis of the distribution of EtxB monomers (precipitated by LDS47) and EtxB pentamers (precipitated by 118-8) revealed that the turnover of monomers occurred concurrently with the appearance of B pentamers, with rates that approximated to a first order process, with k = 0.317 ± 0.170 min (Fig. 8b). This represents a t of approximately 2.2 min for the rate of turnover and assembly of newly synthesized EtxB subunits in the periplasm of E. coli, a finding consistent with previous estimates of the rate of toxin assembly in vivo (Hardy et al., 1988).


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 (bullet; 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.


DISCUSSION

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)^2 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. (^2)

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 beta2 subunit of E. coli tryptophan synthase. When hybridomas raised against the beta2 subunit were screened in ELISAs in which the native beta2 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 alpha-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 alpha-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.


FOOTNOTES

*
This work was supported by Grants 032215/Z/90 and 037867/Z/93 from The Wellcome Trust and a grant from The Royal Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Research School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, United Kingdom. Tel.: 441-227-764-000; Fax: 441-227-763-912.

(^1)
The abbreviations used are: mAb, monoclonal antibody; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; Fmoc, N-(9-fluorenyl)methoxycarbonyl.

(^2)
T. Amin, R. F. L. James and T. R. Hirst, unpublished observations.


ACKNOWLEDGEMENTS

We thank the Biomedical Services Department, University of Leicester, for assistance, Dr. R. Hlodan and J. Hardy for help in the preparation of synthetic peptides, Dr. L. Ruddock for assistance with the Raster model of EtxB, and S. Ruston and C. Cheesman for technical support. We also thank Drs. T. O. Nashar and L. W. Ruddock for critically reading the manuscript.


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