Inhibition of Escherichia coli heat-labile enterotoxin B subunit pentamer (EtxB5) assembly in vitro using monoclonal antibodies.
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 (EtxB(5)) 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 (EtxB(5)) from newly synthesized B-subunit monomers (EtxB(1)). We previously described and characterized a mAb (LDS47) that was shown to be highly specific for an N-terminal decapeptide region of EtxB(1) (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 EtxB(5) from EtxB(1). 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.
Chung
Wen Yuan
WY
Department of Infection, Immunity and Inflammation, University of Leicester, Leicester LE1 9HN, United Kingdom.
Carter
Rachel
R
Hardy
Tara
T
Sack
Markus
M
Hirst
Timothy R
TR
James
Roger F L
RF
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001427:Bacterial Toxins / Q000737:chemistry; D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D004768:Enterotoxins / Q000737:chemistry / Q000502:physiology*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000502:physiology*; D006825:Hybridomas / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry
D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C027538:heat-labile enterotoxin, E coli