title: In-depth characterization of trypsin-like serine peptidases in the midgut of the sugar fed Culex quinquefasciatus.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ, Brazil. abv@ioc.fiocruz.br.
authors.forename: André
authors.initials: A
authors.lastname: Borges-Veloso
authors.affiliation: Laboratorio de Pesquisa em Leishmaniose, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365, Manguinhos, Pav. Leônidas Deane, Sala 509, CEP: 21040-360, Rio de Janeiro, RJ, Brazil. leovahia@ioc.fiocruz.br.
authors.forename: Leonardo
authors.initials: L
authors.lastname: Saboia-Vahia
authors.affiliation: Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ, Brazil. geovane.dl@gmail.com.
authors.forename: Geovane
authors.initials: G
authors.lastname: Dias-Lopes
authors.affiliation: Unidade de Proteômica, Laboratório de Química de Proteínas, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. gilberto@iq.ufrj.br.
authors.forename: Gilberto B
authors.initials: GB
authors.lastname: Domont
authors.affiliation: Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ, Brazil. cbritto@ioc.fiocruz.br.
authors.forename: Constança
authors.initials: C
authors.lastname: Britto
authors.affiliation: Laboratorio de Pesquisa em Leishmaniose, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365, Manguinhos, Pav. Leônidas Deane, Sala 509, CEP: 21040-360, Rio de Janeiro, RJ, Brazil. pcuervo@fiocruz.br.
authors.forename: Patricia
authors.initials: P
authors.lastname: Cuervo
authors.affiliation: Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ, Brazil. jbj@ioc.fiocruz.br.|Departamento de Medicina, Faculdade de Medicina, Universidade Federal de São João del Rei, São João del Rei, MG, Brasil. jbj@ioc.fiocruz.br.
authors.forename: Jose B
authors.initials: JB
authors.lastname: De Jesus
mesh_terms: D000595:Amino Acid Sequence; D000821:Animal Feed; D000818:Animals; D003465:Culex / Q000201:enzymology*; D005260:Female; D041981:Gastrointestinal Tract / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D013395:Sucrose / Q000378:metabolism*
chemical_list: D019476:Insect Proteins; D013395:Sucrose; D012697:Serine Endopeptidases