HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 281, Issue 51, 39465-39470, December 22, 2006

This Article Full Text Full Text (PDF) All Versions of this Article: 281/51/39465    most recent M606038200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Chung, W. Y. Articles by James, R. F. L. Search for Related Content PubMed PubMed Citation Articles by Chung, W. Y. Articles by James, R. F. L. Social Bookmarking                      What's this?

Inhibition of Escherichia coli Heat-labile Enterotoxin B Subunit Pentamer (EtxB₅) Assembly in Vitro Using Monoclonal Antibodies^*

Wen Yuan Chung, Rachel Carter, Tara Hardy, Markus Sack^¶, Timothy R. Hirst^||, and Roger F. L. James¹

From the Department of Infection, Immunity and Inflammation, University of Leicester, Leicester LE1 9HN, United Kingdom, The Cancer Research UK Clinical Centre, Southampton, S016 6YD, United Kingdom, ^¶Die Leitseite der Rheinisch-Westfälischen Hochschule, 52056 Aachen, Germany, and the ^||Departments of Pathology and Microbiology, University of Bristol, BS8 1TD, United Kingdom

Heat-labile enterotoxin (Etx) produced by certain strains of Escherichia coli is a major virulence factor related to cholera toxin. Both are hexameric proteins comprising one A-subunit and five B-subunits. The pentameric B-subunit of E. coli has a high affinity for G_M1-ganglioside receptors on gut epithelial cells and is directly responsible for toxin entry. The pentameric B-subunit (EtxB₅) is an exceptionally stable protein, being able to maintain its quaternary structure over a wide pH range (2.0- 11.0). However, little is known about the formation of the pentameric structure (EtxB₅) from newly synthesized B-subunit monomers (EtxB₁). We previously described and characterized a mAb (LDS47) that was shown to be highly specific for an N-terminal decapeptide region of EtxB₁ (Amin, T., Larkins, A., James, R. F. L., and Hirst, T. R. (1995) J. Biol. Chem. 270, 20143-20150). Here we also describe a mAb (LDS16) with exquisite specificity for pentameric EtxB. In this study, we have used these two mAbs, in combination, to probe the in vitro assembly of EtxB₅ from EtxB₁. EtxB pentamers disassemble in highly acidic conditions, giving rise to monomeric B-subunits that can reassemble if placed in buffers of neutral pH. Using this in vitro assembly model, it was found that at a molar ratio of 1:1; LDS47:EtxB, 50% of reassembly was inhibited, and that this inhibition increased to 90% at a ratio of 2:1. These results infer that the N-terminal decapeptide region (APQSITELCS) defined by the LDS47 antibody is crucial for competent pentameric B-subunit assembly and stabilization.

Received for publication, June 23, 2006 , and in revised form, September 7, 2006.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 44-116-223-1406; Fax: 44-116-252-5030; E-mail: rj1{at}leicester.ac.uk.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?