Mosquitoes are vectors of several serious diseases such as West Nile fever. Since there are no effective cures for these diseases, it is important to control the population of mosquitoes to reduce the occurrence of these diseases.
One of the methods to control the mosquito population is to use mosquitocidal Cry proteins, which are synthesized by the bacteria Bacillus thuringiensis. Although we know Cry proteins kill larvae of susceptible insects, we are unsure about the mechanism of toxicity of Cry proteins but it is clearly receptor mediated. The purpose of this study was to discover receptor(s) for mosquitocidal Cry proteins in the larvae of mosquito Culex pipiens by using photochemical-cross-linking. Discovering receptors will help us better understand the mechanism of action of Cry toxins, and aid in making more specific and effective mosquitocidal Cry proteins by protein engineering.
We attempted to use a photo-cross-linking approach involving photo-leucine, an unnatural amino acid. This photo-leucine has a diazirine group that can be activated by UV light to generate a highly reactive carbene, which then inserts into nearby molecules forming covalent bonds. Our initial attempt to synthesize photo-leucine failed because of the lack of detail in the methods used to synthesize the photoactivatable leucine. After reviewing the latest methods in the literature for the synthesis of the diazirine group, we came up with a revised strategy and successfully made photo-leucine. We attempted to incorporate photo-leucine into a Cry protein in a Bacillus subtilis strain auxotrophic for leucine. The cells grew in the presence of photo-leucine, but failed to sporulate or make Cry protein even in media containing a mixture of photo-leucine and leucine. As an alternative, we chose to label lysine residues of Cry protein using N-hydroxysuccinimide (NHS)-diazirine.
Cry19Aa was labeled with NHS-diazirine. These NHS-diazirine labeled Cry proteins were then mixed with BBMV (Brush border membrane vesicles) proteins and activated with UV light to induce cross-linking. Cross-linked Cry19Aa proteins were pulled down using anti-Cry19Aa antibody and protein A-containing magnetic beads. Cross-linked bands were excised from the gel and analyzed by mass spectrometry. The mass spectrometry data showed that this cross-linked band contained a sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). SERCA transcription level has previously been reported to be decreased in Cry protein-resistant larvae, providing circumstantial evidence for SERCA being a Cry protein receptor.
We constructed (His)6-tagged Cry19Aa. Bioassays with C. pipiens larvae showed that N-terminally (His)6-tagged Cry19Aa has three-fold lower toxicity compared to the untagged Cry19Aa, while C-terminally (His)6-tagged Cry19Aa retained the same level of toxicity as Cry19Aa; we therefore chose to use C-terminally (His)6-tagged Cry19Aa in pull-down assays. To verify SERCA as a binding protein of Cry19Aa, the pull-down experiment using BBMV with diazirine-modified Cry19Aa was performed. The pulled-down proteins were probed with SERCA antibody using a western blot. Although SERCA was found to be a binding partner, consistent with our earlier mass spectrometry results, firm support for this finding requires further in-depth experimentation includes controls to ascertain specificity of interaction.