Angiotensin-degrading serine peptidase: A new chymotrypsin-like activity in the venom of Bothrops jararaca partially blocked by the commercial antivenom

Abstract

Snakebite envenomation is considered a highly relevant public health hazard in South America, having an impact in terms of mortality and morbidity. In Brazil, Bothrops (sensu latu) poisoning is responsible for 90% of the snakebites and in patients treated at the Vital Brazil Hospital (Butantan Institute) this index reaches 97.5%. The objective of the present study was to analyze more specifically the ability of the antibothropic antivenom, produced by the Butantan Institute, São Paulo, Brazil, to neutralize metallo-and serine peptidases, known as the major toxins present in Bothrops jararaca venom. A set of Fret peptides (Free Ressonance Energy Transfer) was studied using the BjV (B. jararaca venom) and site-directed inhibitors PMSF, EDTA and 1,10-phenanthroline. Two substrates were reached to be used as specific tools for studies with metallo peptidases, Abz-FASSAQ-EDDnp, and the serine peptidases, Abz-RPPGFSPFRQ-EDDnp. In disagreement with the literature, the use of both substrates and the antibothropic serum showed a weak neutralization of the serine peptidases present in this venom and a strong neutralization of the metallo peptidases. In order to investigate possible mechanisms of action that have not yet been described for the serine peptidases from the BjV, the present study shows for the first time a new tyrosine-specific chymotrypsin-like and angiotensin-degrading serine peptidase activity, that was partially blocked by the antibothropic serum. In conclusion, the antivenom presented a good neutralization of metallo peptidases but not of serine peptidases, indicating that further studies about serine peptidases immunogenicity are necessary to improve the antibothropic serum.

Edema and Nociception Induced by Philodryas patagoniensis Venom in Mice: A Pharmacological Evaluation with Implications for the Accident Treatment

“…Effect of EDTA on PpV-Induced Edema and Nociception. The effectiveness of EDTA treatment as a metalloprotease inhibitor was evaluated using a free resonance energy transfer peptide Abz-RPPGFSPFRQ-EDDnp as previously described in Kuniyoshi et al (2012). Briefly, PpV activity assay was conducted in 7.4 pH phosphate-buffered saline (final volume 100 ml) containing 50 mM phosphate and 20 mM NaCl, using Corning 96-well plates (Oakville, Canada) and a peptide substrate in a final concentration of 5 mM.…”

Eficácia do soro antibotrópico produzido no Instituto Butantan: obtenção, caracterização e neutralização de serinopeptidases de interesse do veneno <i>Bothrops jararaca</i>.

“…The BAV was not able to block the STT hydrolysis, and since the PMSF, which is a serine proteases inhibitor, fully blocked the STT hydrolysis by the venom, once more we assume that the BAV is unable to block at least one serine peptidase from this venom. An identical phenomenon, that is, the failure of the commercial antivenom to block serine peptidases, was observed when we studied the hydrolysis of dynorphin A, angiotensin I and FRET substrates selective for the metallopeptidases and serine peptidases from BjV (Kuniyoshi et al, 2012).…”

“…It is important to note that a family of peptides with ACE inhibitory activity, the BPPs (Bradykinin-Potentiating Peptides), is present in the venom of B. jararaca . Also, while bradykinin is totally resistant and angiotensin-I is degrade by BjV (Kuniyoshi et al, 2012), it contains a serine peptidase able to release bradykinin from the high molecular weight kininogen (KN-BJ) . Together, the data presented here comes to contribute to the better understand the mechanisms of hypotension observed in cases of human envenomation by B. jararaca.…”

“…In contrast, little is known about the activity of these enzymes on biological active peptides that can also be important in envenomation. Notwithstanding, some studies using synthetic substrates, such as chromogenic and fluorescent peptides, can be found in the literature (Faiz et al, 1996;Kuniyoshi et al, 2012;Porto et al, 2007). Although less explored in the literature, we can hypothesized that endogenous peptides can be hydrolyzed by the venom.…”

“…hidrólise da cadeia B da insulina pelo VBj já foi reportada em diversos trabalhos (MANDELBAUM; CARRILLO;, e apesar deste fenômeno não poder ser correlacionado com o envenenamento, este substrato foi uma ótima ferramenta de estudos uma vez a sua hidrólise foi inibida Como os resultados de soroneutralização mostrados acima indicaram uma falha na neutralização de algumas serinopeptidases de VBj atuando sobre estes substratos, acreditamos que este problema possa estar relacionado à dor local persistente descrita por alguns pacientes, até mesmo depois de vários dias após o acidente e o tratamento(CARDOSO et al, 1993). Portanto, as causas desta falha foram mais bem estudadas e os resultados serão demonstrados a seguir.4.2.1 Obtenção e caracterização das serinopeptidases não neutralizadas pelo SABUm dos principais objetivos do presente projeto é estudar as causas da baixa neutralização das serinopeptidases presentes o VBj pelo soro antibotrópico do Instituto Butantan, como pode ser observado nos resultados descritos acima e também no trabalho publicado pelo nosso grupo(KUNIYOSHI et al, 2012). Assim, decidimos pela purificação de serinopeptidases não bloqueadas pelo SAB para servirem como antígenos e obtenção de antissoros experimentais.…”