title: Serine carboxypeptidases of Triatoma brasiliensis (Hemiptera, Reduviidae): Sequence characterization, expression pattern and activity localization.
abstract: Using specific oligonucleotides, 5'- and 3'-RACE and sequencing, two cDNAs encoding serine carboxypeptidases (tbscp-1 and tbscp-2) from the midgut of the blood sucking heteropteran Triatoma brasiliensis were identified. Both cDNAs with an open reading frame of 1389bp, encode serine carboxypeptidase precursors of 463 amino acid residues, which possess a signal peptide cleavage site after Ala19. Analysis of tbscp-1 and tbscp-2 genomic DNA showed an absence of introns in both sequences and the presence of a further intron-free SCP encoding gene (tbscp-2b). By reverse transcription polymerase chain reaction (RT-PCR), tbscp-1 and tbscp-2 transcript abundance was found similarly in fifth instar nymphs at different days after feeding (daf), high in the posterior midgut (small intestine), lower in the anterior midgut (stomach) and fat body and almost undetectable in the salivary glands. In the anterior, middle and posterior regions of the small intestine at 5daf the transcript abundance of both genes was almost identical. Also in adult female and male insects at 5daf both genes showed the strongest signal in the posterior midgut. Molecular modeling suggested that TBSCP-1 has carboxypeptidase D activity; activities against Hippuryl-Phenylalanine and Hippuryl-Arginine were also located at the posterior midgut, both were induced after blood feeding. Treatment of the posterior midgut extracts with the serine protease inhibitor PMSF strongly reduced carboxypeptidase activity. These findings suggest that triatomines might use serine carboxypeptidases, which are usually found in lysosomes, as digestive enzymes in the posterior midgut lumen, from which TBSCP-1 and TBSCP-2 are possible candidates to fulfill this function.
authors.affiliation: Laboratório de Biologia de Tripanosomatídeos, Instituto Oswaldo Cruz, FIOCRUZ/RJ, Av Brasil 4365, CEP 21045-900 Rio de Janeiro, RJ, Brazil; Laboratório de Bioquímica e Fisiologia de Insetos, Instituto Oswaldo Cruz, FIOCRUZ/RJ, Av Brasil 4365, CEP 21045-900 Rio de Janeiro, RJ, Brazil. Electronic address: peter.waniek@cityweb.de.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Waniek
authors.affiliation: Laboratório de Biologia de Tripanosomatídeos, Instituto Oswaldo Cruz, FIOCRUZ/RJ, Av Brasil 4365, CEP 21045-900 Rio de Janeiro, RJ, Brazil.
authors.forename: Catarina A C
authors.initials: CA
authors.lastname: Araújo
authors.affiliation: Laboratório de Bioquímica e Fisiologia de Insetos, Instituto Oswaldo Cruz, FIOCRUZ/RJ, Av Brasil 4365, CEP 21045-900 Rio de Janeiro, RJ, Brazil.
authors.forename: Marisa M
authors.initials: MM
authors.lastname: Momoli
authors.affiliation: Laboratório de Bioquímica e Fisiologia de Insetos, Instituto Oswaldo Cruz, FIOCRUZ/RJ, Av Brasil 4365, CEP 21045-900 Rio de Janeiro, RJ, Brazil.
authors.forename: Patricia
authors.initials: P
authors.lastname: Azambuja
authors.affiliation: Laboratório de Biologia de Tripanosomatídeos, Instituto Oswaldo Cruz, FIOCRUZ/RJ, Av Brasil 4365, CEP 21045-900 Rio de Janeiro, RJ, Brazil.
authors.forename: Ana M
authors.initials: AM
authors.lastname: Jansen
authors.affiliation: Laboratório de Bioquímica e Fisiologia de Insetos, Instituto Oswaldo Cruz, FIOCRUZ/RJ, Av Brasil 4365, CEP 21045-900 Rio de Janeiro, RJ, Brazil.
authors.forename: Fernando A
authors.initials: FA
authors.lastname: Genta
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002268:Carboxypeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D043402:Cathepsin A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D010802:Phylogeny; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D014018:Tissue Distribution; D014224:Triatoma / Q000235:genetics* / Q000378:metabolism
chemical_list: D019476:Insect Proteins; D002268:Carboxypeptidases; D043402:Cathepsin A; C022633:serine carboxypeptidase
keywords: Digestive enzymes; Hematophagy; RT-PCR; Serine carboxypeptidase; Triatoma brasiliensis