Mapping of two "neutralizing" epitopes of a snake curaremimetic toxin by proton nuclear magnetic resonance spectroscopy.
Two monoclonal antibodies, called M alpha 1 and M alpha 2,3, have been previously shown to neutralize the toxic activity of the curaremimetic toxin alpha from Naja nigricollis. In this paper, we report the mapping of the two corresponding epitopes, using affinity chromatography and proton 2D-NMR spectroscopy. The H-D exchange rates of labile amide hydrogens have been measured in toxin alpha bound to each antibody and in toxin alpha alone. Analysis of the exchange data revealed two regions containing amide hydrogens with decreased exchange rates in the bound toxin compared to the free toxin. These two regions correspond to the sites of interaction with M alpha 1 and M alpha 2,3, respectively. They are consistent with prior biochemical mapping studies, and they include several residues that were not previously identified. Thus, the two antigenic sites are found to be centered on two different loops of toxin alpha. Comparison of these antigenic sites with the active site of toxin alpha allows us to delineate the molecular events associated with the two neutralization processes.
Zinn-Justin
S
S
Département d'Ingénierie et d'Etude des Protéines, CE-Saclay, Gif-sur-Yvette, France.
Roumestand
C
C
Drevet
P
P
Ménez
A
A
Toma
F
F
D000577:Amides / Q000737:chemistry; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology; D003039:Cobra Neurotoxin Proteins / Q000737:chemistry / Q000276:immunology*; D000939:Epitopes / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D011522:Protons
D000577:Amides; D000911:Antibodies, Monoclonal; D003039:Cobra Neurotoxin Proteins; D000939:Epitopes; D011522:Protons; C025154:alpha-cobratoxin