Molecular structure and mechanism of action of the crotoxin inhibitor from Crotalus durissus terrificus serum.
An antivenom protein has been identified in the blood of the snake Crotalus durissus terrificus and proved to act by specifically neutralizing crotoxin, the main lethal component of rattlesnake venoms. The aim of this study was to purify the crotoxin inhibitor from Crotalus serum (CICS), and to analyze its mechanism of action. CICS has been purified from blood serum of the Crotalus snake by gel filtration on Sephadex G-200, ion-exchange chromatography on DEAE-Sephacel, and FPLC gel filtration on a Superose 12 column. It is an oligomeric glycoprotein of 130 kDa, made by the non-covalent association of 23-25-kDa subunits. Two different subunit peptides were identified by SDS/PAGE, however, their N-terminal sequences are identical. They are characterized by the absence of methionine residues and a high content of acidic, hydrophobic and cysteine residues. The neutralizing effect of purified CICS towards the neurotoxic effects of crotoxin has been demonstrated in vivo by lethality assays. CICS binds to the phospholipase subunit CB of crotoxin, but not to the acidic chaperon subunit CA; it efficiently inhibits the phospholipase activity of crotoxin and its isolated CB subunit and evokes the dissociation of the crotoxin complex. The molecular mechanism of the interaction between CICS and crotoxin seems to be very similar to that of crotoxin with its acceptor. It is, therefore, tempting to suggest that CICS acts physiologically as a false crotoxin acceptor that would retain the toxin in the vascular system, thus preventing its action on the neuromuscular system.
Perales
J
J
Departamento de Fisiologia e Farmacodinâmica, Instituto Oswaldo Cruz, Rio de Janeiro, Brasil.
Villela
C
C
Domont
G B
GB
Choumet
V
V
Saliou
B
B
Moussatché
H
H
Bon
C
C
Faure
G
G
D000595:Amino Acid Sequence; D000818:Animals; D003439:Crotoxin / Q000037:antagonists & inhibitors*; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000037:antagonists & inhibitors; D011487:Protein Conformation; D030162:Reptilian Proteins; D017386:Sequence Homology, Amino Acid; D017819:Viperidae / Q000097:blood*
D006023:Glycoproteins; D030162:Reptilian Proteins; C087438:phospholipase A2 inhibitor protein, Crotalus; D003439:Crotoxin; D010741:Phospholipases A