Generation of a Monocloal-Antibody that Recognizes the Amino-Terminal Decapeptide of the B-Subunit of Rscheichia-Coli Heat-Labile Enterotoxin - a New Probe for Studying Toxn Assemy Intermediates

Amin, Tehmina and Larkins, Audrey and James, Roger F. L. and Hirst, Timothy R. (1995) Generation of a Monocloal-Antibody that Recognizes the Amino-Terminal Decapeptide of the B-Subunit of Rscheichia-Coli Heat-Labile Enterotoxin - a New Probe for Studying Toxn Assemy Intermediates. Journal of Biological Chemistry, 270 (34). pp. 20143-20150. ISSN 0021-9258. (The full text of this publication is not available from this repository)

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Official URL
http://dx.doi.org/10.1074/jbc.270.34.20143

Abstract

Cholera toxin and the related Escherichia coil heat-labile enterotoxin are hexameric proteins comprising one B-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. coil 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 off. coil 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. Mel. 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.

Item Type: Article
Subjects: Q Science > QP Physiology (Living systems) > QP517 Biochemistry
Divisions: Faculties > Science Technology and Medical Studies > School of Biosciences
Depositing User: P. Ogbuji
Date Deposited: 09 Jun 2009 08:38
Last Modified: 23 May 2014 13:45
Resource URI: http://kar.kent.ac.uk/id/eprint/19667 (The current URI for this page, for reference purposes)
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