Transformation of a non-enzymatic toxin into a toxoid by genetic engineering.
Curaremimetic toxins are typical non-enzymatic toxins that bind to their target [the nicotinic acetylcholine receptor (AChR)] through multiple residues. Nevertheless, we show that the concomitant substitutions of only three of the ten functionally important residues of such a toxin sufficed to cause an affinity decrease of the toxin for AChR that is higher than four orders of magnitude. Despite these triple mutations, the overall conformation of the mutated protein remains similar to that of a related recombinant toxin, as judged from both circular dichroism analysis and investigation of antigenicity, using monoclonal and polyclonal antibodies. Furthermore, we show that the detoxified toxin is capable of eliciting antibodies that neutralize the binding of a wild-type toxin to AChR. Therefore, transformation of a non-enzymatic toxin into a toxoid can be achieved, like in the case of enzymatic toxins, by introducing a small number of mutations at positions identified to be critical for expression of toxicity.
Fromen-Romano
C
C
Département d'Ingénierie et d'Etudes des Protéines, DSV, CEA Saclay, Gif-sur-Yvette, France.
Maillère
B
B
Drevet
P
P
Lajeunesse
E
E
Ducancel
F
F
Boulain
J C
JC
Ménez
A
A
D000818:Animals; D000915:Antibody Affinity; D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers / Q000737:chemistry; D017815:Elapidae; D004797:Enzyme-Linked Immunosorbent Assay; D004870:Erabutoxins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D007106:Immune Sera / Q000276:immunology / Q000378:metabolism; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009994:Osmolar Concentration; D011485:Protein Binding; D011817:Rabbits; D011978:Receptors, Nicotinic / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D014121:Toxoids / Q000737:chemistry* / Q000235:genetics / Q000276:immunology
D017931:DNA Primers; D007106:Immune Sera; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins; D014121:Toxoids; C038908:erabutoxin A; D004870:Erabutoxins