In-depth characterization of trypsin-like serine peptidases in the midgut of the sugar fed Culex quinquefasciatus

Background: culex quinquefasciatus is a hematophagous insect from the culicidae family that feeds on the blood of humans,dogs,birds and livestock. this species transmits a wide variety of pathogens between humans and animals. the midgut environment is the first location of pathogen-vector interactions for blood-feeding mosquitoes and the expression of specific peptidases in the early stages of feeding could influence the outcome of the infection. trypsin-like serine peptidases belong to a multi-gene family that can be expressed in different isoforms under distinct physiological conditions. however,the confident assignment of the trypsin genes that are expressed under each condition is still a challenge due to the large number of trypsin-coding genes in the culicidae family and most likely because they are low abundance proteins. methods: we used zymography for the biochemical characterization of the peptidase profile of the midgut from c. quinquefasciatus females fed on sugar. protein samples were also submitted to sds-page followed by liquid chromatography-tandem mass spectrometry (lc-ms/ms) analysis for peptidase identification. the peptidases sequences were analyzed with bioinformatics tools to assess their distinct features. results: zymography revealed that trypsin-like serine peptidases were responsible for the proteolytic activity in the midgut of females fed on sugar diet. after denaturation in sds-page,eight trypsin-like serine peptidases were identified by lc-ms/ms. these peptidases have structural features typical of invertebrate digestive trypsin peptidases but exhibited singularities at the protein sequence level such as: the presence of different amino acids at the autocatalytic motif and substrate binding regions as well as different number of disulfide bounds. data mining revealed a group of trypsin-like serine peptidases that are specific to c. quinquefasciatus when compared to the culicids genomes sequenced so far. conclusion: we demonstrated that proteomics approaches combined with bioinformatics tools and zymographic analysis can lead to the functional annotation of trypsin-like serine peptidases coding genes and aid in the understanding of the complexity of peptidase expression in mosquitoes. © 2015 borges-veloso et al.