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.
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-1; t1/2 = 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.
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Research School of Biosciences, University of Kent, Canterbury, United Kingdom.
Larkins
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James
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Hirst
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D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D003429:Cross Reactions; D004768:Enterotoxins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D008958:Models, Molecular; D015335:Molecular Probes; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D004768:Enterotoxins; D000939:Epitopes; D029968:Escherichia coli Proteins; D015335:Molecular Probes; D010446:Peptide Fragments; C027538:heat-labile enterotoxin, E coli