0) environment, whereas the posterior part (small intestine) was reddish, indicating an acid milieu (pH ∼5.0). The transition between these midgut regions was abrupt ( Fig. 1). After PCR Galunisertib with degenerate oligonucleotides, 5′- and 3′-RACE and alignment of the nucleotide sequences, two 1112 and 1093 bp cathepsin L-like proteinase encoding
cDNAs (tbcatL-1 and tbcatL-2) were obtained (NCBI accession nos. EU643472 and JN099751). Both sequences contained open reading frames of 990 bp, encoding 330 amino acid residues ( Fig. 2), 61 and 48 bp of putative 5′-non-coding region and 13 and 35 bp of putative 3′-non-coding region between the stop codon (TAA) and the polyadenylation signal (AATAAA), respectively. The predicted TBCATL-1 and TBCATL-2 precursors had a molecular weight of 36.8 and 37.1 kDa, respectively. Both deduced enzyme precursors contained a putative signal peptide cleavage site (pre-region) between positions 16 Nivolumab price and 17 in the amino acid sequence, a pro-region of 97 amino acid residues and a predicted mature protein of 217 amino acid residues, resulting in a theoretical molecular weights of 23.4 and 23.7 kDa, respectively (Fig. 2). The active triad was formed by Cys25, His164 and Asn184 in both mature proteins (Fig. 2). Six cysteine residues forming
three disulfide bridges were located at positions 22, 56, 65, 98, 157 and 206 in the mature enzymes. The two motifs, ERFNIN and GNDF, characteristic for cathepsin L-like cysteine proteinases, were found in the pre-proregion at positions 43–62 and 75–81
of the cathepsin L precursor, respectively. Phenylethanolamine N-methyltransferase The second motif was modified to MNFD in TBCATL-1and KNFD in TBCATL-2, respectively. The structurally important motif GCNGG was located at position 64–68 in both mature proteins, modified to GCEGG within the amino acid sequence of both mature enzymes (Fig. 2). Mature TBCATL-1 had an identity of 90.3% to TBCATL-2. When compared with homologous genes available in the GenBank database (blastx using nr database), TBCATL-1 had between 64.7% and 75.7% identity with precursors of cathepsin L like cysteine proteinases from other insects, 76.0% to CatL of T. infestans and 83.9% to cathepsin L of R. prolixus ( Fig. 2). In the dendrogram of putative mature cathepsin L sequences of different arthropods, both outgroup crustacean cathepsin L amino acid sequences were separated from those of the insects (Fig. 3). All triatomine sequences clustered together in a branch with Aedes aegypti cathepsin L 1 and these four taxa were distinctly separated from all other insect cathepsin L mature amino acid sequences with high bootstrap support. R. prolixus cathepsin L closely grouped with TBCATL-1 and TBCATL-2 with good bootstrap support, whereas the T. infestans cathepsin was more distant to the other three triatomine cathepsin L sequences ( Fig. 3).