Use of monoclonal antibodies as probes for the structure and biological activity of botulinum neurotoxin.
Experiments were done to help clarify the structure-function relationships that govern the interaction between botulinum neurotoxin and the cholinergic neuromuscular junction. Work was done with type E toxin in three different states: 1) unactivated (post-translational product before proteolytic processing), 2) activated (proteolytically modified product) and 3) denatured. Four different monoclonal antibodies were studied (E3, E14, E17 and E32), three of which were capable of diminishing the potency of the toxin. All four antibodies had approximately equivalent affinity for the unactivated and the activated forms of the toxin. Monoclonals E17 and E32 had little ability to interact with denatured toxin, suggesting they recognized conformational epitopes; monoclonals E3 and E14 retained partial ability to bind to denatured toxin, suggesting they recognized both conformational and linear determinants. When phrenic nerve-hemidiaphragm preparations were exposed to toxin under conditions that allowed binding but retarded internalization, the toxin remained accessible to antibodies. However, when tissues were stimulated in an effort to promote endocytosis, the toxin disappeared from accessibility to antibodies. The data indicate that various antigenic domains remain exposed after binding and suggest that certain parts of the toxin molecule undergo little or no conformational change during binding. The data further indicate that the molecular domains recognized by E14, E17 and E32 are internalized simultaneously.
Department of Medicine, Jefferson Medical College, Philadelphia, Pennsylvania.
L L
LL
Simpson
Y
Y
Kamata
S
S
Kozaki
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001905:Botulinum Toxins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D066298:In Vitro Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D009469:Neuromuscular Junction / Q000187:drug effects / Q000378:metabolism; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D001905:Botulinum Toxins