Biochemical and functional properties of a thrombin-like enzyme isolated from Bothrops pauloensis snake venom

Costa, Fabíola Medeiros da; Rodrigues, Renata Santos; Izidoro, Luíz Fernando Moreira; Menaldo, Danilo L.; Hamaguchi, Amélia; Homsi-Brandeburgo, Maria Inês; Fuly, André Lopes; Soares, Sandro G.; Selistre-de-Araújo, Heloísa Sobreiro; Barraviera, Benedito; Soares, Andreimar Martins; Rodrigues, Veridiana M.

doi:10.1016/j.toxicon.2009.05.040

Cited by 47 publications

(31 citation statements)

References 34 publications

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“…SVSPs are usually glycoproteins presenting variable contents of N-and O-linked glycosylations in non-homologous positions, which can shift the molecular mass of these enzymes in up to 40 kDa (Serrano and Maroun 2005). SVSPs are known to present carbohydrate motif composed by fucose, hexose, sialic acid, Nace ty lg lu cosamine (Glc NAc), m ann ose, and Nacetylneuraminic acid (NauAc) (Pirkle 1998;Koh et al 2001;Wang et al 2001;Bortoleto et al 2002;Oyama and Takahashi 2003;Costa et al 2009;Lin et al 2011). On the other hand, N-glycosylations carried out by yeast are generally polysaccharides composed of 100 or more mannose residues.…”

Section: Discussionmentioning

confidence: 99%

Expression of a new serine protease from Crotalus durissus collilineatus venom in Pichia pastoris and functional comparison with the native enzyme

Boldrini-França

Rodrigues

Santos-Silva

et al. 2015

Appl Microbiol Biotechnol

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Snake venom serine proteases (SVSPs) act primarily on plasma proteins related to blood clotting and are considered promising for the treatment of several hemostatic disorders. We report the heterologous expression of a serine protease from Crotalus durissus collilineatus, named collinein-1, in Pichia pastoris, as well as the enzymatic comparative characterization of the toxin in native and recombinant forms. The complementary DNA (cDNA) encoding collinein-1 was amplified from cDNA library of C. d. collilineatus venom gland and cloned into the pPICZαA vector. The recombinant plasmid was used to transform cells of KM71H P. pastoris. Heterologous expression was induced by methanol and yielded 56 mg of recombinant collinein-1 (rCollinein-1) per liter of culture. The native collinein-1 was purified from C. d. collilineatus venom, and its identity was confirmed by amino acid sequencing. The native and recombinant enzymes showed similar effects upon bovine fibrinogen by releasing preferentially fibrinopeptide A. Although both enzymes have induced plasma coagulation, native Colinein-1 has shown higher coagulant activity. The serine proteases were able to hydrolyze the chromogenic substrates S-2222, S-2238, and S2302. Both enzymes showed high stability on different pH and temperature, and their esterase activities were inhibited in the presence of Zn2+ and Cu2+. The serine proteases showed similar k cat/K m values in enzyme kinetics assays, suggesting no significant differences in efficiency of these proteins to hydrolyze the substrate. These results demonstrated that rCollinein-1 was expressed with functional integrity on the evaluated parameters. The success in producing a functionally active recombinant SVSP may generate perspectives to their future therapeutic applications.

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Section: Discussionmentioning

confidence: 99%

Expression of a new serine protease from Crotalus durissus collilineatus venom in Pichia pastoris and functional comparison with the native enzyme

Boldrini-França

Rodrigues

Santos-Silva

et al. 2015

Appl Microbiol Biotechnol

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“…SVTLE is comprised of various enzymes, including proteinase, phospholipases, ATPase, aspartic and cystine proteinases, metalloproteinases and serine proteinases, as well as a number of unclassified enzymes [17,18]. These enzymes transform fibrin into fiber protein and hydrolysate proteins [19]. Indeed, the data from our study suggest that a mixture of SVTLE and TE buffer (groups 5 and 6) had a significantly higher dissolution rate of calcium oxalate stones compared to TE buffer or EDTA alone (groups 1-4).…”

Section: Discussionmentioning

confidence: 99%

In vitro Dissolution of Calcium Oxalate Stones with Ethylenediaminetetraacetic Acid and Snake Venom Thrombin-Like Enzyme

Zhou¹,

Zhang²,

Ci³

et al. 2013

Urol Int

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Objective: The aim of this study was to determine the feasibility of using snake venom thrombin-like enzyme (SVTLE) and/or ethylenediaminetetraacetic acid (EDTA) to dissolve calcium oxalate stones in vitro. Methods: Seven calcium oxalate stones were incubated with various chemolytic agents [EDTA, Tris-HCl/EDTA (TE) buffer or SVTLE diluted in TE buffer]. The pH, calcium concentration, stone weight and stone surface integrity were recorded, as well as related pathological changes to bladder mucosae. Results: Compared to all other solutions, those containing SVTLE and buffered EDTA had higher concentrations of mobilized calcium and caused significantly more stone weight loss, stone fragility and gaps in the calcium crystals. Also, there were no adverse pathological effects on rabbit bladder mucosae from any of the solutions. Conclusions: The data indicate that buffered EDTA and SVTLE can be used to dissolve calcium oxalate stones and, at the concentrations used here, do not damage tissue.

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“…These are primarily responsible for well-known biological effects and employed in several diagnostic and therapeutic approaches Costa et al, 2009). The toxicity of the snake venom has been attributed to a class of toxins, the L-amino acid oxidases (svLAAO), although the precise mechanism of action of these enzymes and their role in snake toxicity are not well understood.…”

Section: Discussionmentioning

confidence: 99%

Bothrops pirajai snake venom L-amino acid oxidase: in vitro effects on infection of Toxoplasma gondii in human foreskin fibroblasts

Izidoro¹,

Alves²,

Rodrigues³

et al. 2011

Rev. bras. farmacogn.

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Abstract:The effect of an L-amino acid oxidase isolated from Bothrops pirajai snake venom (BpirLAAO-I) was investigated on infection of Toxoplasma gondii in human foreskin fibroblasts (HFF). The cytotoxic activity of BpirLAAO-I on HFF cells showed a dose-dependent toxicity with median cytotoxic dose (TD50) of 11.8 µg/mL. BpirLAAO-I induced considerable dose-dependent decrease in the T. gondii infection index under two different conditions, treatment of tachyzoites before infection or treatment of HFF cells after infection. A maximal inhibition of infection (56%) was found for treatment before infection, with a median inhibitory dose (ID50) at 1.83 µg/mL and selectivity index (SI) at 6.45. For treatment after infection, it was observed a maximal inhibition of infection at 65%, ID50 of 1.20 µg/mL and SI of 9.83. The treatment before infection was also effective to reduce intracellular parasitism up to 62%, although presenting higher values of ID50 (3.14 µg/mL) and lower values of SI (3.76). However, treatment after infection was not effective, suggesting that the enzyme seems to have no effect on the parasite intracellular replication for this condition. In conclusion, BpirLAAO-I was more effective to inhibit the infection of neighboring cells and consequently parasite dissemination than primary infection and parasite replication. Thus, the effect of BpirLAAO-I described herein could be taken into account for the development of new synthetic anti-parasite therapeutic agents.

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Biochemical and functional properties of a thrombin-like enzyme isolated from Bothrops pauloensis snake venom

Cited by 47 publications

References 34 publications

Expression of a new serine protease from Crotalus durissus collilineatus venom in Pichia pastoris and functional comparison with the native enzyme

Expression of a new serine protease from Crotalus durissus collilineatus venom in Pichia pastoris and functional comparison with the native enzyme

In vitro Dissolution of Calcium Oxalate Stones with Ethylenediaminetetraacetic Acid and Snake Venom Thrombin-Like Enzyme

Bothrops pirajai snake venom L-amino acid oxidase: in vitro effects on infection of Toxoplasma gondii in human foreskin fibroblasts

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