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

Tehmina Amin, A. Larkins, R. F L James, T. R. Hirst*

*Corresponding author for this work

Research output: Contribution to journalArticle

8 Citations (Scopus)

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; t( 1/2 ) = 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.

Original languageEnglish
Pages (from-to)20143-20150
Number of pages8
JournalThe Journal of Biological Chemistry
Volume270
Issue number34
DOIs
Publication statusPublished - 1995

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Enterotoxins
Escherichia coli
Hot Temperature
Monoclonal Antibodies
Periplasmic Proteins
Macromolecular Substances
Precipitates
Periplasm
Peptides
Cholera Toxin
Fingers
Epitopes
Proteins
X-Rays
Structural analysis
X rays

ASJC Scopus subject areas

  • Biochemistry

Cite this

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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; t( 1/2 ) = 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.",
author = "Tehmina Amin and A. Larkins and James, {R. F L} and Hirst, {T. R.}",
year = "1995",
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journal = "The Journal of Biological Chemistry",
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T1 - 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

AU - Amin, Tehmina

AU - Larkins, A.

AU - James, R. F L

AU - Hirst, T. R.

PY - 1995

Y1 - 1995

N2 - 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; t( 1/2 ) = 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.

AB - 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; t( 1/2 ) = 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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U2 - 10.1074/jbc.270.34.20143

DO - 10.1074/jbc.270.34.20143

M3 - Article

VL - 270

SP - 20143

EP - 20150

JO - The Journal of Biological Chemistry

JF - The Journal of Biological Chemistry

SN - 0021-9258

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