Ligand blot analysis of juvenile hormone esterase binding proteins in Manduca sexta L.

Biotinylated recombinant juvenile hormone esterase (JHE) was used for ligand blotting of proteins from fat body tissue and pericardial athrocytes of Manduca sexta. Proteins were separated by SDS-polyacrylamide gel electrophoresis or by two-dimensional electrophoresis. Eight putative JHE binding proteins were detected in fat body tissue and in pericardial athrocytes of both M. sexta and Heliothis virescens. The predominant bands were 29, 72, 75, 125 and 240kDa, with minor bands at 50, 80 and 205kDa. All putative JHE binding proteins were present from the second through to the fifth instar larvae of M. sexta. On wide-range isoelectric focusing, the 29kDa JHE binding protein separated into three species with isoelectric points of 6.5, 6.6 and 6.8. Biotinylated-JHE did not bind recombinant M. sexta-derived juvenile hormone binding protein. The mutant JHE with mutations K29R and K524R binds weakly to the JHE binding protein P29, relative to binding of wild-type JHE [Shanmugavelu et al., J. Biol. Chem., 275 (2000) 1802-1806]. A similar reduction in binding was not seen for the 29kDa binding protein identified here in pericardial athrocytes by ligand blot. This result is discussed.

Identification of the functional site in the mosquito larvicidal binary toxin of Bacillus sphaericus 1593M by site-directed mutagenesis.

To study the mode of action of the binary toxin (51- and 42-kDa) of Bacillus sphaericus, amino acid residues were substituted at selected sites of the N- and C-terminal regions of both peptides. Bioassay results of the mutant binary toxins tested against mosquito larvae, Culex quinquefasciatus, revealed that most of the substitutions made on both peptides led to either decrease or total loss of the activity. Furthermore, receptor binding studies carried out for some of the mutants of the 42-kDa peptide showed mutations in N- and C-terminal regions of the 42-kDa peptide did not affect the binding of the binary toxin to brush border membrane vesicles of mosquito larvae. One of the mutants having a single amino acid substitution at the C-terminal region ((312)R) of the 42-kDa peptide completely abolished the biological activity, implicating the role of this residue in membrane pore formation. These results indicate the importance of the C-terminal region of the 42-kDa of binary toxin, in general, and particularly the residue (312)R for biological activity against mosquito larvae.

A novel protein that binds juvenile hormone esterase in fat body tissue and pericardial cells of the tobacco hornworm Manduca sexta L.

Juvenile hormone esterase degrades juvenile hormone, which acts in conjunction with ecdysteroids to control gene expression in insects. Circulating juvenile hormone esterase is removed from insect blood by pericardial cells and degraded in lysosomes. In experiments designed to characterize proteins involved in the degradation of juvenile hormone esterase, a pericardial cell cDNA phage display library derived from the tobacco hornworm moth Manduca sexta L. was constructed and screened for proteins that bind juvenile hormone esterase. A 732-base pair cDNA encoding a novel 29-kDa protein (P29) was isolated. Western and Northern analyses indicated that P29 is present in both pericardial cell and fat body tissues and is expressed in each larval instar. In immunoprecipitation experiments, P29 bound injected recombinant juvenile hormone esterase taken up by pericardial cells and native M. sexta juvenile hormone esterase in fat body tissue, where the enzyme is synthesized. Binding assays showed that P29 bound juvenile hormone esterase more strongly than it did a mutant form of the enzyme with mutations that perturb lysosomal targeting. Based on these data, we propose that P29 functions in pericardial cells to facilitate lysosomal degradation of juvenile hormone esterase.

Functional complementation of nontoxic mutant binary toxins of Bacillus sphaericus 1593M generated by site-directed mutagenesis.

Alanine residues were substituted by site-directed mutagenesis at selected sites of the N- and C-terminal regions of the binary toxin (51- and 42-kDa peptides) of B. sphaericus 1593M, and the mutant toxins were cloned and expressed in Escherichia coli. Bioassays with mosquito larvae, using binary toxins derived from individual mutants, showed that the substitution of alanine at some sites in both the 51-kDa and the 42-kDa peptides resulted in a total loss of activity. Surprisingly, after mixing two nontoxic derivatives of the same peptide, i.e., one mutated at the N-terminal end and the other mutated at the C-terminal end of either the 51-kDa or the 42-kDa peptide, the toxicity was restored. This result indicates that the altered binary toxins can functionally complement each other by forming oligomers.

Polymerase chain reaction and non-radioactive gene probe based identification of mosquito larvicidal strains of Bacillus sphaericus and monitoring of B. sphaericus 1593M, released in the environment.

In attempts towards an accurate monitoring in the environment, the status of the deliberate release of Bacillus sphaericus, a powerful mosquito larvicidal agent, as well as to evolve a rapid method of screening for potent isolates of B. sphaericus, we have developed a polymerase chain reaction (PCR) method of analysis. Using specific primers spanning the 5' and 3' ends of the coding sequences of these two mosquito larvicidal genes, a 1.3 and a 1.1 kb product from gene A and a 2.6 kb product from gene B have been amplified by PCR. The primers and the products amplified from them in PCR are highly specific for B. sphaericus. We used digoxigenin based non-radioactive chemiluminescence method for the detection of PCR products and the sensitivity of the method was high enough to detect the presence of 1 to 5 cells of B. sphaericus. A simple and inexpensive sample processing procedure has also been developed for direct PCR amplification of B. sphaericus DNA from field samples collected from areas where it had been applied. Several highly toxic to non-toxic strains of B. sphaericus were screened with these primers by PCR for the presence of either or both of these toxin genes. The results indicate that there is a good correlation between the presence of both genes with higher toxicity of the strains.