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P-I snake venom metalloproteinase is able to activate the complement system by direct cleavage of central components of the cascade.

Pidde-Queiroz G, Magnoli FC, Portaro FC, Serrano SM, Lopes AS, Paes Leme AF, van den Berg CW, Tambourgi DV - PLoS Negl Trop Dis (2013)

Bottom Line: The aim of the present study was to isolate and characterize the C-activating protease from Bothrops pirajai venom.The mass spectrometric identification of this protein, named here as C-SVMP, revealed peptides that matched sequences from the P-I class of SVMPs.C-SVMP activated the alternative, classical and lectin C-pathways by cleaving the α-chain of C3, C4 and C5, thereby generating anaphylatoxins C3a, C4a and C5a.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Imunoquímica, Instituto Butantan, São Paulo, Brazil.

ABSTRACT

Background: Snake Venom Metalloproteinases (SVMPs) are amongst the key enzymes that contribute to the high toxicity of snake venom. We have recently shown that snake venoms from the Bothrops genus activate the Complement system (C) by promoting direct cleavage of C-components and generating anaphylatoxins, thereby contributing to the pathology and spread of the venom. The aim of the present study was to isolate and characterize the C-activating protease from Bothrops pirajai venom.

Results: Using two gel-filtration chromatography steps, a metalloproteinase of 23 kDa that activates Complement was isolated from Bothrops pirajai venom. The mass spectrometric identification of this protein, named here as C-SVMP, revealed peptides that matched sequences from the P-I class of SVMPs. C-SVMP activated the alternative, classical and lectin C-pathways by cleaving the α-chain of C3, C4 and C5, thereby generating anaphylatoxins C3a, C4a and C5a. In vivo, C-SVMP induced consumption of murine complement components, most likely by activation of the pathways and/or by direct cleavage of C3, leading to a reduction of serum lytic activity.

Conclusion: We show here that a P-I metalloproteinase from Bothrops pirajai snake venom activated the Complement system by direct cleavage of the central C-components, i.e., C3, C4 and C5, thereby generating biologically active fragments, such as anaphylatoxins, and by cleaving the C1-Inhibitor, which may affect Complement activation control. These results suggest that direct complement activation by SVMPs may play a role in the progression of symptoms that follow envenomation.

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Proteolytic activity of C-SVMP on C1-Inh and analysis of its regulatory activity.[A] Human purified C1-Inh samples (3 µg) were incubated with purified C-SVMP (0.5 µg) or Bothrops venom (1 µg) in the presence or absence of 1,10 phenanthroline (Phe - 15 mM). [B] Alternatively, samples of purified human C1-Inh (1 µg) were incubated with increasing concentrations of C-SVMP (0.5 µg, 1 µg and 2 µg). The cleavage was visualized by SDS-PAGE (10%) under reducing conditions followed by silver staining. [C] C1-Inh activity was determined using the MicroVue ELISA kit (***p<0.001). Data are representative of two separate experiments.
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pntd-0002519-g006: Proteolytic activity of C-SVMP on C1-Inh and analysis of its regulatory activity.[A] Human purified C1-Inh samples (3 µg) were incubated with purified C-SVMP (0.5 µg) or Bothrops venom (1 µg) in the presence or absence of 1,10 phenanthroline (Phe - 15 mM). [B] Alternatively, samples of purified human C1-Inh (1 µg) were incubated with increasing concentrations of C-SVMP (0.5 µg, 1 µg and 2 µg). The cleavage was visualized by SDS-PAGE (10%) under reducing conditions followed by silver staining. [C] C1-Inh activity was determined using the MicroVue ELISA kit (***p<0.001). Data are representative of two separate experiments.

Mentions: The cleavage of C1-Inh was completely inhibited by 1,10-phenanthroline after treatment with either C-SVMP or crude venom (Fig. 6A), suggesting that C-SVMP may be solely responsible for cleavage of C1-Inh. Moreover, C-SVMP also dose dependently cleaved C1-Inh, generating a fragment of ∼83 kDa, which was positively associated with the dose dependent reduction in the level of the functionally active C1-Inhibitor protein (Fig. 6B,C).


P-I snake venom metalloproteinase is able to activate the complement system by direct cleavage of central components of the cascade.

Pidde-Queiroz G, Magnoli FC, Portaro FC, Serrano SM, Lopes AS, Paes Leme AF, van den Berg CW, Tambourgi DV - PLoS Negl Trop Dis (2013)

Proteolytic activity of C-SVMP on C1-Inh and analysis of its regulatory activity.[A] Human purified C1-Inh samples (3 µg) were incubated with purified C-SVMP (0.5 µg) or Bothrops venom (1 µg) in the presence or absence of 1,10 phenanthroline (Phe - 15 mM). [B] Alternatively, samples of purified human C1-Inh (1 µg) were incubated with increasing concentrations of C-SVMP (0.5 µg, 1 µg and 2 µg). The cleavage was visualized by SDS-PAGE (10%) under reducing conditions followed by silver staining. [C] C1-Inh activity was determined using the MicroVue ELISA kit (***p<0.001). Data are representative of two separate experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814341&req=5

pntd-0002519-g006: Proteolytic activity of C-SVMP on C1-Inh and analysis of its regulatory activity.[A] Human purified C1-Inh samples (3 µg) were incubated with purified C-SVMP (0.5 µg) or Bothrops venom (1 µg) in the presence or absence of 1,10 phenanthroline (Phe - 15 mM). [B] Alternatively, samples of purified human C1-Inh (1 µg) were incubated with increasing concentrations of C-SVMP (0.5 µg, 1 µg and 2 µg). The cleavage was visualized by SDS-PAGE (10%) under reducing conditions followed by silver staining. [C] C1-Inh activity was determined using the MicroVue ELISA kit (***p<0.001). Data are representative of two separate experiments.
Mentions: The cleavage of C1-Inh was completely inhibited by 1,10-phenanthroline after treatment with either C-SVMP or crude venom (Fig. 6A), suggesting that C-SVMP may be solely responsible for cleavage of C1-Inh. Moreover, C-SVMP also dose dependently cleaved C1-Inh, generating a fragment of ∼83 kDa, which was positively associated with the dose dependent reduction in the level of the functionally active C1-Inhibitor protein (Fig. 6B,C).

Bottom Line: The aim of the present study was to isolate and characterize the C-activating protease from Bothrops pirajai venom.The mass spectrometric identification of this protein, named here as C-SVMP, revealed peptides that matched sequences from the P-I class of SVMPs.C-SVMP activated the alternative, classical and lectin C-pathways by cleaving the α-chain of C3, C4 and C5, thereby generating anaphylatoxins C3a, C4a and C5a.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Imunoquímica, Instituto Butantan, São Paulo, Brazil.

ABSTRACT

Background: Snake Venom Metalloproteinases (SVMPs) are amongst the key enzymes that contribute to the high toxicity of snake venom. We have recently shown that snake venoms from the Bothrops genus activate the Complement system (C) by promoting direct cleavage of C-components and generating anaphylatoxins, thereby contributing to the pathology and spread of the venom. The aim of the present study was to isolate and characterize the C-activating protease from Bothrops pirajai venom.

Results: Using two gel-filtration chromatography steps, a metalloproteinase of 23 kDa that activates Complement was isolated from Bothrops pirajai venom. The mass spectrometric identification of this protein, named here as C-SVMP, revealed peptides that matched sequences from the P-I class of SVMPs. C-SVMP activated the alternative, classical and lectin C-pathways by cleaving the α-chain of C3, C4 and C5, thereby generating anaphylatoxins C3a, C4a and C5a. In vivo, C-SVMP induced consumption of murine complement components, most likely by activation of the pathways and/or by direct cleavage of C3, leading to a reduction of serum lytic activity.

Conclusion: We show here that a P-I metalloproteinase from Bothrops pirajai snake venom activated the Complement system by direct cleavage of the central C-components, i.e., C3, C4 and C5, thereby generating biologically active fragments, such as anaphylatoxins, and by cleaving the C1-Inhibitor, which may affect Complement activation control. These results suggest that direct complement activation by SVMPs may play a role in the progression of symptoms that follow envenomation.

Show MeSH
Related in: MedlinePlus