Proteomic Analysis of Glutathione Transferases from Lucilia Cuprina

The glutathione transferases are a family of multifunctional enzymes involved in detoxification of xenobiotic and endogenous electrophilic compounds. Interest in insect GSTs has primarily focused on their role in insecticide resistance. The sheep blowfly, Lucilia cuprina is a major economic problem for the sheep meat and wool industries in Australasia and hence this thesis has attempted the study of the Lucilia cuprina GST family, using proteomics, with a view to eventually determining their role in insecticide resistance. Combinations of different affinity matrices (glutathione-Sepharose matrix (GSH) followed by dinitrophenyl-glutathione-Sepharose matrix (DNP-GSH)) and two-dimensional electrophoresis has successfully isolated members from major four insect GST classes: Sigma, Delta, Epsilon and Omega. Drosophila melanogaster has been used as a model insect throughout as a basis for comparison. To characterise Lucilia GSTs, the whole metazoan fragmentation database was used for sequence alignment with Lucilia peptides. This approach is broad and speculative but predicts a possible classification of the GSTs based on % similarity and % identity. This method of characterisation yielded match scores that provided a basis for classification, which must at present be regarded as tentative and in need of confirmation. In D. melanogaster and L. cuprina, GSH affinity-purified extracts showed the presence of only Sigma and Delta GSTs. In D. melanogaster, the DNP-GSH affinity-purified GSTs showed mostly the presence of Epsilon and Omega GSTs whereas in L. cuprina no Omega GSTs were detected. In both species, the migration pattern of Delta GST on 2D PAGE gel indicated possible post-translational modification. The results from analysis of LC-MS/MS data by the software PEAKS suggested deamidation at asparagine and glutamine residues in a limited number of the matched peptides of Delta GST. GST activity was present in all developmental stages of L. cuprina. The number of isoenzymes and their extent of expression vary as the insect develops. Delta GSTs were present in all developmental stages. The Sigma GST started expressing from the larval stage and was abundantly present in adult stage. The DNP-GSH affinity matrix purified GSTs which have been tentatively classified as Mu-like GSTs were present in egg, larvae and pupae but totally absent in adult stage. The GST families were characterised by proteomics in the main body sections of L. cuprina. Higher GST activity towards 1-chloro-2, 4-dinitrobenzene (CDNB) was found in the thorax (65.2 %) followed by the abdomen (19.6%) and the head (15.2%). The cytosolic GSTs of a resistant strain (PY81) of L. cuprina had significantly higher (2.26- and 2.6- fold) activity than the susceptible strains (NSW and CSIRO) towards CDNB and 2, 3-dichloro, 4-nitrobenzene (DCNB) respectively. The proteomic analysis of DNP-GSH purified extract from susceptible and resistant strains showed quantitatively higher expression of GSTs on 2D PAGE gel of the PY81 strain. The in vitro interaction of purified GSTs and model insecticides studied by high performance liquid chromatography revealed that Delta and DNP-GSH affinity-purified GSTs catalyse the conjugation of the insecticides to reduced glutathione but Sigma GST had almost no activity.