Amino acid substitutions in alphaA and alphaC of Cyt2Aa2 alter hemolytic activity and mosquito-larvicidal specificity

Boonhiang Promdonkoy; Amporn Rungrod; Patcharee Promdonkoy; Wanwarang Pathaichindachote; Chartchai Krittanai; Sakol Panyim

doi:10.1016/j.jbiotec.2007.10.007

Amino acid substitutions in alphaA and alphaC of Cyt2Aa2 alter hemolytic activity and mosquito-larvicidal specificity

J Biotechnol. 2008 Feb 1;133(3):287-93. doi: 10.1016/j.jbiotec.2007.10.007. Epub 2007 Oct 30.

Authors

Boonhiang Promdonkoy¹, Amporn Rungrod, Patcharee Promdonkoy, Wanwarang Pathaichindachote, Chartchai Krittanai, Sakol Panyim

Affiliation

¹ National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Phahonyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand. [email protected]

PMID: 18054404
DOI: 10.1016/j.jbiotec.2007.10.007

Abstract

Cyt2Aa2 produced by Bacillus thuringiensis subsp. darmstadiensis exhibits in vitro cytolytic activity against broad range of cells but shows specific in vivo toxicity against larvae of Dipteran insects. To investigate the role of amino acids in alphaA and alphaC of this toxin, 3 single-point mutants (A61C, S108C and V109A) were generated. All 3 mutant proteins were highly produced as inclusion bodies that could be solubilized and activated by proteinase K similar to that of the wild type. Hemolytic activity of A61C and S108C mutants was significantly reduced whereas the V109A mutant showed comparable hemolytic activity to the wild type. Interestingly, the A61C mutant exhibited high larvicidal activity to both Aedes aegypti and Culex quinquefasciatus. S108C and V109A mutants showed low activity against C. quinquefasciatus but relatively high toxicity to A. aegypti. These results demonstrated for the first time that amino acids in alphaA and alphaC are involved in the selectivity of the Cyt toxin to the targeted organism.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence
Amino Acid Substitution*
Animals
Bacillus thuringiensis / chemistry*
Bacillus thuringiensis / drug effects
Bacillus thuringiensis Toxins
Bacterial Proteins / chemistry
Bacterial Proteins / metabolism*
Bacterial Toxins / chemistry
Bacterial Toxins / metabolism*
Bacterial Toxins / pharmacology
Cell Membrane / drug effects
Cell Membrane / metabolism
Culicidae / drug effects
Culicidae / physiology*
Endotoxins / chemistry
Endotoxins / metabolism*
Erythrocytes / drug effects
Hemolysin Proteins / chemistry
Hemolysin Proteins / metabolism*
Hemolysis / drug effects
Hemolysis / physiology*
Larva / drug effects
Molecular Sequence Data
Mosquito Control*
Mutagenesis, Site-Directed
Mutant Proteins / chemistry
Mutant Proteins / metabolism
Protein Structure, Quaternary
Protein Structure, Secondary
Protein Subunits / chemistry
Protein Subunits / metabolism*
Sheep
Solubility / drug effects
Species Specificity

Substances

Bacillus thuringiensis Toxins
Bacterial Proteins
Bacterial Toxins
Endotoxins
Hemolysin Proteins
Mutant Proteins
Protein Subunits
insecticidal crystal protein, Bacillus Thuringiensis