title: 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.
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-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.
authors.affiliation: Research School of Biosciences, University of Kent, Canterbury, United Kingdom.
authors.forename: T
authors.initials: T
authors.lastname: Amin
authors.forename: A
authors.initials: A
authors.lastname: Larkins
authors.forename: R F
authors.initials: RF
authors.lastname: James
authors.forename: T R
authors.initials: TR
authors.lastname: Hirst
mesh_terms: 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
chemical_list: 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