Kazal-type inhibitors in the stomach of Panstrongylus megistus (Triatominae, Reduviidae)

Triatomines inhibit the clotting of ingested blood in the stomach (anterior midgut). After verifying this phenomenon in Panstrongylus megistus using coagulation assays, a full-length cDNA encoding a Kazal-like inhibitor was amplified by PCR. The open reading frame encodes a putative precursor protein of 412 amino acid residues, which was named PmStKaz and contains seven Kazal-like domains forming four Kazal-type inhibitors. A single domain inhibitor and three double-domain inhibitors possess sequence identities of up to 91% to the respective domains of Kazal-type inhibitors from other triatomines. The gene is expressed in the stomach (anterior midgut) but not in the small intestine (posterior midgut), salivary glands or haemocytes. After hydrophobic interaction chromatography of the stomach contents, four fractions (numbers 1–4) inhibited the activity of trypsin, fraction 2 that of subtilisin A, fractions 1, 3 and 4 that of plasmin, and fractions 3 and 4 that of thrombin. After ion exchange chromatography, MALDI-TOF-MS analysis of the intact proteins in fractions 3 and 4 showed diverse masses correlating to PmStKaz IV–V and PmStKaz II–III, respectively. Both proteins seem to be present in several isoforms with variant amino- and carboxy-terminal ends. In reverse zymography of the proteins of the stomach contents after separation by isoelectric focusing and non-reducing SDS-PAGE, much higher concentrations of isoforms of PmStKaz II–III and IV–V were evident than of PmStKaz I and VI–VII.