First serine protease inhibitor isolated from Rhinella schneideri poison

Background Toad secretions are a source of molecules with potential biotechnological application on a wide spectrum of diseases. Toads from theRhinella family have two kinds of poisonous glands, namely granular and mucous glands. Rhinella schneideritoads produce granular secretions that comprise a great number of molecules, including serine proteases inhibitors. Serine proteases, such as trypsin, chymotrypsin and elastase, are enzymes that have a serine amino acid into its catalytic site and can be found in a large number of vertebrate species and pathogenic microorganisms. Therefore, the present work aims to purify a serine protease inhibitor from Rhinella schneiderigranular secretions.Findings This study presents the protocol used to purify a serine protease inhibitor from the Rhinella schneideri poison. The granular secretion was submitted to dialysis in order to separate the low molecular weight compounds, which were submitted to a reversed phase-fast protein liquid chromatography fractionation step in a C2C18 column. The major fractions were tested over trypsin, chymotrypsin and elastase through colorimetric assay. The inhibition tests were performed with the enzyme in absence (positive control) and presence of fractions, denatured enzyme (negative control) and the respective chromogenic substrate. Rs20 was the compound with the major inhibitory activity over chymotrypsin, inducing a delay in the formation of the chromogenic enzymatic product. The structure characterization of Rs20 was performed by high resolution electronspray ionization-mass spectrometry (HRESI-MS) and gas chromatography coupled with mass spectrometry (GC-MS). HRESI showed an intense signal suggesting the presence of bufadienolide with less than 10 ppm error. In addition, it was observed a low intense signal at m/z399 that could be lithocholic acid, a biosynthetic precursor of bufadienolide. Finally, GC-MS analysis applying NIST library identification reinforced this hypothesis.Conclusions The current study have isolated and partially characterized the function and structure of the first bufadienolide with inhibitory action over chymotrypsin.

Assessment and partial purification of serine protease inhibitors from Rhipicephalus (Boophilus) annulatus larvae / Avaliação e purificação parcial dos inibidores da serina protease de larvas do Rhipicephalus (Boophilus) annulatus

Ticks are rich sources of serine protease inhibitors, particularly those that prevent blood clotting and inflammatory responses during blood feeding. The tick Rhipicephalus (Boophlus) annulatus is an important ectoparasite of cattle. The aims of this study were to characterize and purify the serine protease inhibitors present in R. (B.) annulatus larval extract. The inhibitors were characterized by means of one and two-dimensional reverse zymography, and purified using affinity chromatography on a trypsin-Sepharose column. The analysis on one and two-dimensional reverse zymography of the larval extract showed trypsin inhibitory activity at between 13 and 40 kDa. Through non-reducing SDS-PAGE and reverse zymography for proteins purified by trypsin-Sepharose affinity chromatography, some protein bands with molecular weights between 13 and 34 kDa were detected. Western blotting showed that five protein bands at 48, 70, 110, 130 and 250 kDa reacted positively with immune serum, whereas there was no positive reaction in the range of 13-40 kDa. Serine protease inhibitors from R. (B.) annulatus have anti-trypsin activity similar to inhibitors belonging to several other hard tick species, thus suggesting that these proteins may be useful as targets in anti-tick vaccines.

Carrapatos são uma rica fonte de inibidores da serina protease, particularmente aqueles que previnem coagulação e respostas inflamatórias durante a alimentação com sangue. O carrapato Rhipicephalus (B.) annulatus é um ectoparasita importante de bovinos. O objetivo deste estudo foi caracterizar e purificar os inibidores da serina protease presentes no extrato de larva do R. (B.) annulatus. Os inibidores foram caracterizados através de zimografia reversa uni e bidimensional e purificados com cromatografia de afinidade em uma coluna de sepharose-tripsina. A análise do extrato de larva pela zimografia reversa uni e bidimensional mostrou atividade inibitória de tripsina entre 13 e 40 kDa. Através de SDS-PAGE e zimografia reversa para proteínas purificadas pela cromatografia por sepharose-tripsina, algumas bandas de proteínas com pesos moleculares entre 13 e 34 kDa foram detectadas. Western blotting mostrou que cinco bandas de proteínas a 48, 70, 110, 130 e 250 kDa reagiram positivamente com o soro imune, enquanto não houve reação positiva nas bandas 13-40 kDa. Inibidores da serina protease do R. (B.) annulatus têm atividade antitripsina semelhante àquelas dos inibidores de outras espécies de carrapatos duros, sugerindo, assim, que essas proteínas podem ser úteis como alvo de vacinas contra carrapatos.

Kazal-type serine proteinase inhibitors in the midgut of Phlebotomus papatasi

Sandflies (Diptera: Psychodidae) are important disease vectors of parasites of the genus Leishmania, as well as bacteria and viruses. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2). Analyses of expression profiles indicated that PpKzl1 and PpKzl2 transcripts are both regulated by blood-feeding in the midgut of P. papatasi and are also expressed in males, larva and pupa. We expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells) that was applied to in vitro studies to assess serine proteinase inhibition. Recombinant PpKzl2 inhibited α-chymotrypsin to 9.4% residual activity and also inhibited α-thrombin and trypsin to 33.5% and 63.9% residual activity, suggesting that native PpKzl2 is an active serine proteinase inhibitor and likely involved in regulating digestive enzymes in the midgut. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sandfly midgut. Thus, characterising serine proteinase inhibitors may provide new targets and strategies to prevent transmission of Leishmania.