Biochemical studies of serine proteases inhibitors purified from Triatoma infestans eggs
deMarco, R.1, Campos, I.T.N.1 and Tanaka, A.S1
1Departamento de Bioquímica, UNIFESP – EPM, São Paulo. E-mail: demarco36@grad.unifesp.br
In the last decade, a new serine protease inhibitor family called pacifastins has been described in arthropods. Eight members of this family were purified from locusts and share a conserved cysteine pattern (Cys-Xaa9−12-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa2−3-Gly-Xaa3−6-Cys-Thr-Xaa3-Cys) with nine inhibitory domains of the light chain of the crayfish protease inhibitor, pacifastin (PLDs; pacifastin light chain domains). At RNAm level, several pacifastin-related precursors have been identified, encoding additional PLD-related peptides in different arthropods species. In the present study for the first time, we identified and purified two PDLs in blood-sucking bug, Triatoma infestans. The T. infestans PDLs purification was done using a T. infestans eggs crude extract and affinity chromatography on chymotrypsin-Sepharose column. The eluted chymotrypsin inhibitory activity was further purified by reverse-phase chromatography on C18 column which separated two peaks containing inhibitory activities toward human neutrophil elastase. The peaks were named TiEi-1 and TiEi-2 (Triatoma infestans Elastase Inhibitor). The TiEi-1 and TiEi-2 molecular masses determined by MALDI-TOF mass spectrometry were 4257 and 4024 Da, respectively. Both purified TiEi-1 and TiEi-2 presented dissociation constants (Kis) in the pM and nM range for elastase and chymotrypsin, respectively, but they did not inhibit subtilisin A. TiEi-1 and TiEi-2 were submitted to N-terminal amino acid sequencing and presented the same amino acid sequence. Based on this amino acid sequence degenerated oligonucleotides were synthesized and used in a PCR with fat body cDNA preparation. The amplified DNA fragment was sequenced and analyzed in a database search using BLAST. The DNA fragment encodes a polypeptide chain containing two predicted pacifastin-type domains. Sequence analyses of these inhibitors are described in this work. Supported by: FAPESP and CNPq.
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