Effect of site directed mutagenesis on the activity of recombinant trimucrotoxin, a neurotoxic phospholipase from Trimeresurus mucrosquamatus venom.
Trimucrotoxin, the basic phospholipase A2 from Trimeresurus mucrosquamatus venom, is neurotoxic and myotoxic, and structurally similar to crotoxin B subunit. To investigate the amino acid residues responsible for its neurotoxicity, we have mutated its interface-recognition residues including a conserved Asn6 in all the Crotalinae neurotoxic phospholipases. The wild-type and the mutants were expressed in E. coli as fusion-proteins and activated in vitro by factor Xa cleavage after folding. The completion of folding and activation were checked with electrospray ionization mass spectrometry and circular dichroism measurement. Enzymatic activities and neurotoxicities toward the chick tissue of four trimucrotoxin mutants (N6A, N6E, N6R and 6E7T8L) were compared with those of the wild type which was as active as that was isolated from the venom. Mutants N6A and N6E retained more than half of the original enzymatic activity but their neurotoxicities reduced to 33% and 10% that of the wild type, respectively. Mutants N6R and 6E7T8L retained 20-25% of the enzyme activity toward the anionic micellar substrate but were inactive toward the zwitterionic micellar substrate, and their neurotoxicities were less than 3% of that of the wild type. These results demonstrate the importance of residues 6-8 in trimucrotoxin for its neuronal specificity and the specificity toward potential substrates.
Tsai
I H
IH
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
Wang
Y M
YM
D000595:Amino Acid Sequence; D000818:Animals; D003439:Crotoxin / Q000235:genetics / Q000302:isolation & purification / Q000633:toxicity*; D004926:Escherichia coli; D054501:Group II Phospholipases A2; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009498:Neurotoxins / Q000235:genetics / Q000302:isolation & purification / Q000633:toxicity*; D010741:Phospholipases A / Q000235:genetics / Q000302:isolation & purification / Q000633:toxicity*; D054467:Phospholipases A2; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000633:toxicity; D017386:Sequence Homology, Amino Acid; D017841:Trimeresurus
D009498:Neurotoxins; D011994:Recombinant Proteins; D003439:Crotoxin; D010741:Phospholipases A; D054501:Group II Phospholipases A2; D054467:Phospholipases A2; C096805:trimucrotoxin