Serine carboxypeptidases of Triatoma brasiliensis (Hemiptera, Reduviidae): Sequence characterization, expression pattern and activity localization Peter J. Waniek a,b,⇑ , Catarina A.C. Araújo a , Marisa M. Momoli b , Patricia Azambuja b , Ana M. Jansen a , Fernando A. Genta b a Laboratório de Biologia de Tripanosomatídeos, Instituto Oswaldo Cruz, FIOCRUZ/RJ, Av Brasil 4365, CEP 21045-900 Rio de Janeiro, RJ, Brazil b Laboratório de Bioquímica e Fisiologia de Insetos, Instituto Oswaldo Cruz, FIOCRUZ/RJ, Av Brasil 4365, CEP 21045-900 Rio de Janeiro, RJ, Brazil article info Article history: Received 28 September 2013 Received in revised form 6 February 2014 Accepted 7 February 2014 Available online 15 February 2014 Keywords: Triatoma brasiliensis Digestive enzymes Serine carboxypeptidase RT-PCR Hematophagy abstract Using specific oligonucleotides, 5 0 - and 3 0 -RACE and sequencing, two cDNAs encoding serine carboxypep- tidases (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 1389 bp, encode serine carboxypeptidase precur- sors 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 pos- terior regions of the small intestine at 5 daf the transcript abundance of both genes was almost identical. Also in adult female and male insects at 5 daf 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 ser- ine carboxypeptidases, which are usually found in lysosomes, as digestive enzymes in the posterior mid- gut lumen, from which TBSCP-1 and TBSCP-2 are possible candidates to fulfill this function. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction Triatoma brasiliensis is an epidemiological important triatomine species, because in the north-eastern, semi-arid region of Brazil this insect is mainly responsible for the transmission of Trypano- soma cruzi, the causative agent of Chagas disease (Dias et al., 2002). T. brasiliensis occupies sylvatic, domestic and peridomestic habitats and is therefore more difficult to control than strictly domestic insects such as Triatoma infestans in Brazil (Diotaiuti et al., 2000; Soares et al., 2000). Therefore knowledge about triato- mine digestive physiology might be important for an efficient in- sect control and new approaches of disease management in the future. Insects which live on plant diets rich in trypsin inhibitors have developed two different mechanisms of efficient protein digestion. While Lepidoptera express a high number of different serine prote- ase isoforms with different active site structures to avoid inhibi- tion, Hemiptera developed a digestive system based on cysteine proteases, with maximum activity in acid conditions (Terra et al., 1996; Lopez-Ordoñez et al., 2001; Foissac et al., 2002). Since Red- uviidae seem to originate from those plant sucking hemipterans, which during evolution have lost their ability to synthesize tryp- sin-like proteases in the midgut, triatomine digestion is also based on acid cathepsin-like proteases (Garcia and Garcia, 1977; Schofield 1996). However, in other organs, for example salivary glands, tri- atomines synthesize alkaline serine proteases (Amino et al., 2001; Meiser et al., 2010). http://dx.doi.org/10.1016/j.jinsphys.2014.02.003 0022-1910/Ó 2014 Elsevier Ltd. All rights reserved. ⇑ Corresponding author at: Laboratório de Bioquímica e Fisiologia de Insetos, Instituto Oswaldo Cruz, FIOCRUZ/RJ, Av Brasil 4365, CEP 21045-900 Rio de Janeiro, RJ, Brazil. Tel.: +55 21 2598 4324; fax: +55 21 2560 6572. E-mail address: peter.waniek@cityweb.de (P.J. Waniek). Journal of Insect Physiology 63 (2014) 9–20 Contents lists available at ScienceDirect Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys