Monoclonal antibody 11E10, which neutralizes shiga toxin type 2 (Stx2), recognizes three regions on the Stx2 A subunit, blocks the enzymatic action of the toxin in vitro, and alters the overall cellular distribution of the toxin.
Monoclonal antibody (MAb) 11E10 recognizes the Shiga toxin type 2 (Stx2) A(1) subunit. The binding of 11E10 to Stx2 neutralizes both the cytotoxic and lethal activities of Stx2, but the MAb does not bind to or neutralize Stx1 despite the 61% identity and 75% similarity in the amino acids of the A(1) fragments. In this study, we sought to identify the segment or segments on Stx2 that constitute the 11E10 epitope and to determine how recognition of that region by 11E10 leads to inactivation of the toxin. Toward those objectives, we generated a set of chimeric Stx1/Stx2 molecules and then evaluated the capacity of 11E10 to recognize those hybrid toxins by Western blot analyses and to neutralize them in Vero cell cytotoxicity assays. We also compared the amino acid sequences and crystal structures of Stx1 and Stx2 for stretches of dissimilarity that might predict a binding epitope on Stx2 for 11E10. Through these assessments, we concluded that the 11E10 epitope is comprised of three noncontiguous regions surrounding the Stx2 active site. To determine how 11E10 neutralizes Stx2, we examined the capacity of 11E10/Stx2 complexes to target ribosomes. We found that the binding of 11E10 to Stx2 prevented the toxin from inhibiting protein synthesis in an in vitro assay but also altered the overall cellular distribution of Stx2 in Vero cells. We propose that the binding of MAb 11E10 to Stx2 neutralizes the effects of the toxin by preventing the toxin from reaching and/or inactivating the ribosomes.
Smith
Michael J
MJ
Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799, USA.
Melton-Celsa
Angela R
AR
Sinclair
James F
JF
Carvalho
Humberto M
HM
Robinson
Cory M
CM
O'Brien
Alison D
AD
D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002522:Chlorocebus aethiops; D018604:Epitope Mapping; D008297:Male; D008958:Models, Molecular; D009500:Neutralization Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D022641:Shiga Toxin 2 / Q000037:antagonists & inhibitors* / Q000276:immunology* / Q000378:metabolism; D014709:Vero Cells
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D022641:Shiga Toxin 2