Abstract
Snake venom serine proteases (SVSPs) represent an essential group of enzymatic toxins involved in several pathophysiological effects on blood homeostasis. Some findings suggest the involvement of this class of enzymatic toxins in inflammation. In this paper, we purified and isolated a new gyroxin isoform from the Crotalus durissus terrificus (Cdt) venom, designated as Cdtsp 2, which showed significant proinflammatory effects in a murine model. In addition, we performed several studies to elucidate the main pathway underlying the edematogenic effect induced by Cdtsp 2. Enzymatic assays and structural analysis (primary structure analysis and three-dimensional modeling) were closely performed with pharmacological assays. The determination of edematogenic activity was performed using Cdtsp 2 isolated from snake venom, and was applied to mice treated with protein kinase C (PKC) inhibitor, phospholipase C (PLC) inhibitor, dexamethasone (Dexa), antagonists for protease-activated receptors (PARs), or saline (negative control). Additionally, we measured the levels of cyclooxygenase 2 (COX-2), malondialdehyde (MDA), and prostaglandin E2 (PGE2). Cdtsp 2 is characterized by an approximate molecular mass of 27 kDa, an isoelectric point (pI) of 4.5, and significant fibrinolytic activity, as well as the ability to hydrolyze Nα-benzoyl-l-arginine 4-nitroanilide (BAPNA). Its primary and three-dimensional structures revealed Cdtsp 2 as a typical snake venom serine protease that induces significant edema via the metabolism of arachidonic acid (AA), involving PARs, PKC, PLC, and COX-2 receptors, as well as inducing a significant increase in MDA levels. Our results showed that Cdtsp 2 is a serine protease with significant enzymatic activity, and it may be involved in the degradation of PAR1 and PAR2, which activate PLC and PKC to mobilize AA, while increasing oxidative stress. In this article, we provide a new perspective for the role of SVSPs beyond their effects on blood homeostasis.
Highlights
Some studies showed that the venom of Crotalus durissus ssp., including Crotalus durissus terrificus (Cdt), contains two serine-protease fractions, in addition to gyroxin
Crotalus durissus collilineatus venom revealed the presence of two serine-protease fractions that were able to induce mild inflammation, and the results suggested that their activity did not involve the presence of gyroxin
The chromatographic run was monitored at an absorbance of 280 nm (A280) and each fraction collected were assayed for secretory phospholipase A2 using chromogenic substrate 4-nitro-3-benzoic acid (NOB) monitored at 425 nm (A425)
Summary
Some studies showed that the venom of Crotalus durissus ssp., including Crotalus durissus terrificus (Cdt), contains two serine-protease fractions, in addition to gyroxin. Serine proteases isolated from Crotalus durissus cumanensis venom showed the presence of two serine-protease fractions through a combination of molecular exclusion on a Sephacryl S-200 column and reverse-phase HPLC on a semi-preparative C8 column [3] In both cases, this new fraction was found to be structurally different from gyroxin purified from the venom of Cdt. In both cases, this new fraction was found to be structurally different from gyroxin purified from the venom of Cdt Both fractions were eluted together with the crotoxin. These two enzymes are single-chain proteins of similar molecular masses that induce the coagulation of plasma In terms of their biochemical characteristics, these enzymes are similar to other snake venom thrombin-like serine proteases and fibrinogenases, with characteristics unlike gyroxin. Crotoxin, in addition to being the major fraction of Crotalus durissus ssp., revealed the presence of crotoxin B (CB), and contained a crotapotin fraction with essential serine proteases
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