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A serine protease isolated from the bristles of the Amazonic caterpillar, Premolis semirufa, is a potent complement system activator.

Villas Boas IM, Pidde-Queiroz G, Magnoli FC, Gonçalves-de-Andrade RM, van den Berg CW, Tambourgi DV - PLoS ONE (2015)

Bottom Line: The caterpillar of the moth Premolis semirufa, commonly named pararama, is found in the Brazilian Amazon region.The protease, named here as Ps82, reduced the haemolytic activity of the alternative and classical pathways and inhibited the lectin pathway.The use of Phenanthroline, metalloprotease inhibitor, in the reactions did not significantly interfere with the activity of the Ps82, whereas the presence of PMSF, serine protease inhibitor, totally blocked the activity.

View Article: PubMed Central - PubMed

Affiliation: Immunochemistry Laboratory, Butantan Institute, São Paulo, SP, Brazil.

ABSTRACT

Background: The caterpillar of the moth Premolis semirufa, commonly named pararama, is found in the Brazilian Amazon region. Accidental contact with the caterpillar bristles causes an intense itching sensation, followed by symptoms of an acute inflammation, which last for three to seven days after the first incident. After multiple accidents a chronic inflammatory reaction, called "Pararamose", characterized by articular synovial membrane thickening with joint deformities common to chronic synovitis, frequently occurs. Although complement mediated inflammation may aid the host defense, inappropriate or excessive activation of the complement system and generation of anaphylatoxins can lead to inflammatory disorder and pathologies. The aim of the present study was to evaluate, in vitro, whether the Premolis semirufa's bristles extract could interfere with the human complement system.

Results: The bristles extract was able to inhibit the haemolytic activity of the alternative pathway, as well as the activation of the lectin pathway, but had no effect on the classical pathway, and this inhibition seemed to be caused by activation and consumption of complement components. The extract induced the production of significant amounts of all three anaphylatoxins, C3a, C4a and C5a, promoted direct cleavage of C3, C4 and C5 and induced a significant generation of terminal complement complexes in normal human serum. By using molecular exclusion chromatography, a serine protease of 82 kDa, which activates complement, was isolated from P. semirufa bristles extract. The protease, named here as Ps82, reduced the haemolytic activity of the alternative and classical pathways and inhibited the lectin pathway. In addition, Ps82 induced the cleavage of C3, C4 and C5 and the generation of C3a and C4a in normal human serum and it was capable to cleave human purified C5 and generate C5a. The use of Phenanthroline, metalloprotease inhibitor, in the reactions did not significantly interfere with the activity of the Ps82, whereas the presence of PMSF, serine protease inhibitor, totally blocked the activity.

Conclusion: These data show that a serine protease present in the Premolis semirufa's bristles extract has the ability to activate the complement system, which may contribute to the inflammatory process presented in humans after envenomation.

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Related in: MedlinePlus

Proteolytic action of the Premolis semirufa’s bristles extract on purified human C-components C3, C4 and C5.Samples of the bristles extract (2.0 μg for C3 and C4 or 3.0 μg for C5) were incubated, in the absence or presence of 10 mM PMSF or 1,10-Phenanthroline (Phen), with human C3 (3 μg) [A], human C4 (3 μg) [B] and human C5 (3 μg) [C] at 37°C for 1 h. Proteolytic activity was examined on 10% polyacrylamide gel under reducing conditions and stained by silver. In the 1st lanes of gels: electrophoretic separation of purified components incubated with PBS as a positive control; 2nd lanes: incubation of purified components with the extract; 3rd lanes: incubation of the mixture with PMSF; 4th lanes: incubation of the mixture with Phenanthroline and 5th lanes: electrophoretic separation of the extract. α (115 kDa) and β (75 kDa) for C3; α (93 kDa), β (75 kDa) and γ (33 kDa) for C4 and α (115 kDa) and β (75 kDa) for C5.
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pone.0118615.g004: Proteolytic action of the Premolis semirufa’s bristles extract on purified human C-components C3, C4 and C5.Samples of the bristles extract (2.0 μg for C3 and C4 or 3.0 μg for C5) were incubated, in the absence or presence of 10 mM PMSF or 1,10-Phenanthroline (Phen), with human C3 (3 μg) [A], human C4 (3 μg) [B] and human C5 (3 μg) [C] at 37°C for 1 h. Proteolytic activity was examined on 10% polyacrylamide gel under reducing conditions and stained by silver. In the 1st lanes of gels: electrophoretic separation of purified components incubated with PBS as a positive control; 2nd lanes: incubation of purified components with the extract; 3rd lanes: incubation of the mixture with PMSF; 4th lanes: incubation of the mixture with Phenanthroline and 5th lanes: electrophoretic separation of the extract. α (115 kDa) and β (75 kDa) for C3; α (93 kDa), β (75 kDa) and γ (33 kDa) for C4 and α (115 kDa) and β (75 kDa) for C5.

Mentions: Fig. 4 shows that the extract induced cleavage of the alpha chains of all three evaluated components as indicated by slight reductions (8–9 kDa) in their Mrs, most potently in C3, where nearly all alpha chain was reduced in Mr, while the alpha chains of C4 and C5 seemed to be more resistant (Fig. 4, 2nd lanes). The serine protease inhibitor PMSF inhibited the cleavage of the alpha chains of both C3 and C4 (Fig. 4A and B, 3rd lanes), while the cleavage of the C5-alpha chain seemed to be enhanced. Surprisingly, 1,10-phenanthroline increased the cleavage of the C3 alpha and beta chains caused by the bristles extract (Fig. 4A), but had no effect on the cleavage of C4 and C5 (Fig. 4B and C, 4th lanes). These results suggest a complex role for metalloproteases and serine proteases in the activity of the bristles extract. Of note, the serine protease inhibitor PMSF could not be used in the whole serum assays (Figs. 1–3) as it would also inhibit the serine proteases of the complement system and, thus, would have prevented any complement activation.


A serine protease isolated from the bristles of the Amazonic caterpillar, Premolis semirufa, is a potent complement system activator.

Villas Boas IM, Pidde-Queiroz G, Magnoli FC, Gonçalves-de-Andrade RM, van den Berg CW, Tambourgi DV - PLoS ONE (2015)

Proteolytic action of the Premolis semirufa’s bristles extract on purified human C-components C3, C4 and C5.Samples of the bristles extract (2.0 μg for C3 and C4 or 3.0 μg for C5) were incubated, in the absence or presence of 10 mM PMSF or 1,10-Phenanthroline (Phen), with human C3 (3 μg) [A], human C4 (3 μg) [B] and human C5 (3 μg) [C] at 37°C for 1 h. Proteolytic activity was examined on 10% polyacrylamide gel under reducing conditions and stained by silver. In the 1st lanes of gels: electrophoretic separation of purified components incubated with PBS as a positive control; 2nd lanes: incubation of purified components with the extract; 3rd lanes: incubation of the mixture with PMSF; 4th lanes: incubation of the mixture with Phenanthroline and 5th lanes: electrophoretic separation of the extract. α (115 kDa) and β (75 kDa) for C3; α (93 kDa), β (75 kDa) and γ (33 kDa) for C4 and α (115 kDa) and β (75 kDa) for C5.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4356561&req=5

pone.0118615.g004: Proteolytic action of the Premolis semirufa’s bristles extract on purified human C-components C3, C4 and C5.Samples of the bristles extract (2.0 μg for C3 and C4 or 3.0 μg for C5) were incubated, in the absence or presence of 10 mM PMSF or 1,10-Phenanthroline (Phen), with human C3 (3 μg) [A], human C4 (3 μg) [B] and human C5 (3 μg) [C] at 37°C for 1 h. Proteolytic activity was examined on 10% polyacrylamide gel under reducing conditions and stained by silver. In the 1st lanes of gels: electrophoretic separation of purified components incubated with PBS as a positive control; 2nd lanes: incubation of purified components with the extract; 3rd lanes: incubation of the mixture with PMSF; 4th lanes: incubation of the mixture with Phenanthroline and 5th lanes: electrophoretic separation of the extract. α (115 kDa) and β (75 kDa) for C3; α (93 kDa), β (75 kDa) and γ (33 kDa) for C4 and α (115 kDa) and β (75 kDa) for C5.
Mentions: Fig. 4 shows that the extract induced cleavage of the alpha chains of all three evaluated components as indicated by slight reductions (8–9 kDa) in their Mrs, most potently in C3, where nearly all alpha chain was reduced in Mr, while the alpha chains of C4 and C5 seemed to be more resistant (Fig. 4, 2nd lanes). The serine protease inhibitor PMSF inhibited the cleavage of the alpha chains of both C3 and C4 (Fig. 4A and B, 3rd lanes), while the cleavage of the C5-alpha chain seemed to be enhanced. Surprisingly, 1,10-phenanthroline increased the cleavage of the C3 alpha and beta chains caused by the bristles extract (Fig. 4A), but had no effect on the cleavage of C4 and C5 (Fig. 4B and C, 4th lanes). These results suggest a complex role for metalloproteases and serine proteases in the activity of the bristles extract. Of note, the serine protease inhibitor PMSF could not be used in the whole serum assays (Figs. 1–3) as it would also inhibit the serine proteases of the complement system and, thus, would have prevented any complement activation.

Bottom Line: The caterpillar of the moth Premolis semirufa, commonly named pararama, is found in the Brazilian Amazon region.The protease, named here as Ps82, reduced the haemolytic activity of the alternative and classical pathways and inhibited the lectin pathway.The use of Phenanthroline, metalloprotease inhibitor, in the reactions did not significantly interfere with the activity of the Ps82, whereas the presence of PMSF, serine protease inhibitor, totally blocked the activity.

View Article: PubMed Central - PubMed

Affiliation: Immunochemistry Laboratory, Butantan Institute, São Paulo, SP, Brazil.

ABSTRACT

Background: The caterpillar of the moth Premolis semirufa, commonly named pararama, is found in the Brazilian Amazon region. Accidental contact with the caterpillar bristles causes an intense itching sensation, followed by symptoms of an acute inflammation, which last for three to seven days after the first incident. After multiple accidents a chronic inflammatory reaction, called "Pararamose", characterized by articular synovial membrane thickening with joint deformities common to chronic synovitis, frequently occurs. Although complement mediated inflammation may aid the host defense, inappropriate or excessive activation of the complement system and generation of anaphylatoxins can lead to inflammatory disorder and pathologies. The aim of the present study was to evaluate, in vitro, whether the Premolis semirufa's bristles extract could interfere with the human complement system.

Results: The bristles extract was able to inhibit the haemolytic activity of the alternative pathway, as well as the activation of the lectin pathway, but had no effect on the classical pathway, and this inhibition seemed to be caused by activation and consumption of complement components. The extract induced the production of significant amounts of all three anaphylatoxins, C3a, C4a and C5a, promoted direct cleavage of C3, C4 and C5 and induced a significant generation of terminal complement complexes in normal human serum. By using molecular exclusion chromatography, a serine protease of 82 kDa, which activates complement, was isolated from P. semirufa bristles extract. The protease, named here as Ps82, reduced the haemolytic activity of the alternative and classical pathways and inhibited the lectin pathway. In addition, Ps82 induced the cleavage of C3, C4 and C5 and the generation of C3a and C4a in normal human serum and it was capable to cleave human purified C5 and generate C5a. The use of Phenanthroline, metalloprotease inhibitor, in the reactions did not significantly interfere with the activity of the Ps82, whereas the presence of PMSF, serine protease inhibitor, totally blocked the activity.

Conclusion: These data show that a serine protease present in the Premolis semirufa's bristles extract has the ability to activate the complement system, which may contribute to the inflammatory process presented in humans after envenomation.

Show MeSH
Related in: MedlinePlus