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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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Related in: MedlinePlus

Cleavage of purified human component C3.[A] Samples of purified human C3 (3 µg) were incubated with increasing concentrations of C-SVMP (0.01–0.32 µg) or PBS for 30 min at 37°C. Cleavage was visualized by SDS-PAGE under reducing conditions followed by silver staining. [B] C3 is formed by two protein chains, β and α (1,641 amino-acid residues). The arrow indicates the site of cleavage for C-SVMP and the amino acids determined by Edman degradation are underlined. Anaphylatoxin Domain (ANA).
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pntd-0002519-g003: Cleavage of purified human component C3.[A] Samples of purified human C3 (3 µg) were incubated with increasing concentrations of C-SVMP (0.01–0.32 µg) or PBS for 30 min at 37°C. Cleavage was visualized by SDS-PAGE under reducing conditions followed by silver staining. [B] C3 is formed by two protein chains, β and α (1,641 amino-acid residues). The arrow indicates the site of cleavage for C-SVMP and the amino acids determined by Edman degradation are underlined. Anaphylatoxin Domain (ANA).

Mentions: The action of purified C-SVMP on C3 was assessed by SDS-PAGE. C-SVMP dose-dependently hydrolyzed the alpha chain of purified human C3, resulting in a slightly lower Mr, while the beta chain was not affected (Fig. 3A). Analysis by Edman degradation of the cleaved C3 alpha chain revealed the N-terminal amino acid sequence Ser-Asn-Leu-Asp-Glu, suggesting that C-SVMP cleaves the C3 alpha chain at the same site as C3 convertases after Complement activation, thus generating C3b and C3a (Fig. 3B).


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)

Cleavage of purified human component C3.[A] Samples of purified human C3 (3 µg) were incubated with increasing concentrations of C-SVMP (0.01–0.32 µg) or PBS for 30 min at 37°C. Cleavage was visualized by SDS-PAGE under reducing conditions followed by silver staining. [B] C3 is formed by two protein chains, β and α (1,641 amino-acid residues). The arrow indicates the site of cleavage for C-SVMP and the amino acids determined by Edman degradation are underlined. Anaphylatoxin Domain (ANA).
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0002519-g003: Cleavage of purified human component C3.[A] Samples of purified human C3 (3 µg) were incubated with increasing concentrations of C-SVMP (0.01–0.32 µg) or PBS for 30 min at 37°C. Cleavage was visualized by SDS-PAGE under reducing conditions followed by silver staining. [B] C3 is formed by two protein chains, β and α (1,641 amino-acid residues). The arrow indicates the site of cleavage for C-SVMP and the amino acids determined by Edman degradation are underlined. Anaphylatoxin Domain (ANA).
Mentions: The action of purified C-SVMP on C3 was assessed by SDS-PAGE. C-SVMP dose-dependently hydrolyzed the alpha chain of purified human C3, resulting in a slightly lower Mr, while the beta chain was not affected (Fig. 3A). Analysis by Edman degradation of the cleaved C3 alpha chain revealed the N-terminal amino acid sequence Ser-Asn-Leu-Asp-Glu, suggesting that C-SVMP cleaves the C3 alpha chain at the same site as C3 convertases after Complement activation, thus generating C3b and C3a (Fig. 3B).

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