Characterization of epitopes in native and unfolded cobrotoxin: evidence of an immunodominant C-terminal region related to the production of precipitating and non-precipitating antibodies against cobrotoxin.
Rabbits hyperimmunized with cobrotoxin from Taiwan cobra venom produced non-precipitating as well as precipitating antibodies. Both antibody preparations exhibited higher affinity for native cobrotoxin than for reduced and S-carboxymethylated (RCM) cobrotoxin. This indicated that the epitope structures in cobrotoxin are mostly conformation-dependent. In order to identify the conformational epitopes, native cobrotoxin was hydrolyzed with acid protease A, and 12 peptides were obtained on HPLC. Three peptide fragments, AP-10, AP-11, and AP-12, showed pronounced antigenicities toward precipitating as well as non-precipitating antibodies. AP-10, AP-11, and AP-12 contained a common segment in the C-terminal region of cobrotoxin, residues 43 to 62, with intact disulfide linkages. Complete removal of the C-terminal antibodies from antisera and precipitating antibodies on a C-terminal segment-Sepharose affinity column resulted in the loss of their precipitability with cobrotoxin, whilst restoration of precipitability was observed on the addition of the C-terminal antibodies to the C-terminal antibody-depleted antisera and precipitating antibodies. Studies on the antigenic structures of RCM-cobrotoxin revealed that RCM-cobrotoxin contains an immunodominant epitope at positions 22-38. The N-terminal and C-terminal regions of RCM-cobrotoxin encompass other epitopes which exhibit low reactivities toward anti-RCM-cobrotoxin antibodies. However, no precipitated antigen-antibody complexes were observed with the mixture of anti-RCM-cobrotoxin antibodies and RCM-cobrotoxin. These results suggest that the inherently different immunogenicities with different segments might affect the precipitabilities of the resulting antibodies, and that the notable immunogenecity of the C-terminal region is related to the production of precipitating and non-precipitating antibodies against cobrotoxin.
Chang
L S
LS
Department of Biochemistry, Kaohsiung Medical College, Taiwan.
Lin
J
J
Kuo
K W
KW
Lin
S R
SR
Chang
C C
CC
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000936:Antigen-Antibody Complex; D003039:Cobra Neurotoxin Proteins / Q000737:chemistry / Q000276:immunology*; D017815:Elapidae; D018604:Epitope Mapping; D000939:Epitopes / Q000032:analysis* / Q000276:immunology; D016056:Immunodominant Epitopes / Q000276:immunology*; D008969:Molecular Sequence Data; D017510:Protein Folding; D017434:Protein Structure, Tertiary
D000906:Antibodies; D000936:Antigen-Antibody Complex; D003039:Cobra Neurotoxin Proteins; D000939:Epitopes; D016056:Immunodominant Epitopes