The full-length cDNA of anticoagulant protein infestin revealed a novel releasable Kazal domain, a neutrophil elastase inhibitor lacking anticoagulant activity.

Infestins are Kazal-type serine proteinase inhibitors found in the midgut of the Chagas' disease vector, Triatoma infestans. In previous studies, we characterized two double-headed infestins with potent anticoagulant activity; infestin 1-2, which inhibits thrombin and infestin 3-4, a factor XIIa inhibitor. In the present work, we have cloned the full-length cDNA of infestins' precursor. The translated cDNA predicted a polypeptide containing a signal peptide and seven Kazal-type domains, four domains from infestin 1-2 and infestin 3-4, and three new domains. Northern blot analysis confirmed that infestins are synthesized in a single transcript (approximately 1,800 bp) in the insect midgut, but not in salivary glands. Based on the cDNA sequence, the three new Kazal domains were named infestin 1R, 2R and 3R. Infestin 2R-3R has 77% amino acid sequence identity to infestin 1-2 and the same basic amino acid residue at P1 position in the inhibitory reactive site suggesting that these two proteins have a similar inhibitory specificity. In contrast, infestin 1R has two different characteristics when compared to the other infestins: i) a hydrophobic amino acid residue at P1 position in the inhibitory reactive site and ii) a prediction to be processed as a single Kazal domain. These two characteristics were experimentally demonstrated by the purification of native infestin 1R from T. infestans midgut. Native infestin 1R was shown to be processed as a single Kazal domain by mass spectrometry and it was able to inhibit neutrophil elastase, subtilisin A and chymotrypsin. To further characterize infestin 1R inhibitory activity, it was expressed as a recombinant protein in bacteria. Recombinant infestin 1R inhibited neutrophil elastase with the same K(i) of the native inhibitor. Moreover, it inhibited subtilisin A, chymotrypsin and proteinase K but did inhibit neither thrombin nor coagulation assays. In conclusion, unlike the other described infestins, infestin 1R did not present anticoagulant activity and is processed as a single Kazal domain with inhibitory specificity towards proteases that hydrolyze peptide bonds after hydrophobic amino acid residues.