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IN-DEPTH CHARACTERIZATION OF TRYPSIN-LIKE SERINE PEPTIDASES IN THE MIDGUT OF THE SUGAR FED CULEX QUINQUEFASCIATUS
Author
Affilliation
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa em Leishmaniose. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil.
Universidade Federal do Rio de Janeiro. Instituto de Química. Laboratório de Química de Proteínas. Unidade de Proteômica. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa em Leishmaniose. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil / Universidade Federal de São João del Rei. Faculdade de Medicina. Departamento de Medicina. São João del Rey, MG, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa em Leishmaniose. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil.
Universidade Federal do Rio de Janeiro. Instituto de Química. Laboratório de Química de Proteínas. Unidade de Proteômica. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa em Leishmaniose. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ, Brasil / Universidade Federal de São João del Rei. Faculdade de Medicina. Departamento de Medicina. São João del Rey, MG, Brasil.
Abstract
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.
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