Biological characterization of compounds from Rhinella schneideri poison that act on the complement system.
BACKGROUND
The skin secretions of toads of the family Bufonidae contain biogenic amines, alkaloids, steroids (bufotoxins), bufodienolides (bufogenin), peptides and proteins. The poison of Rhinella schneideri, formerly classified as Bufo paracnemis, presents components that act on different biological systems, including the complement system. The aim of this study was to isolate and examine the activity of Rhinella schneideri poison (RsP) components on the complement system.


METHODS
The components active on the complement system were purified in three chromatographic steps, using a combination of cation-exchange, anion-exchange and gel filtration chromatography. The resulting fractions were analyzed by SDS-PAGE and screened for their activity in the hemolytic assay of the classical/lectin complement pathways. Fractions active on the complement system were also assessed for their ability to generate C3 fragments evaluated by two dimensional immunoelectrophoresis assay, C3a and C5a by neutrophil chemotaxis assay and SC5b-9 complex by ELISA assay.


RESULTS
The fractionation protocol was able to isolate the component S5 from the RsP, as demonstrated by SDS-PAGE and the RP-FPLC profile. S5 is a protein of about 6000 Da, while S2 presents components of higher molecular mass (40,000 to 50,000 Da). Fractions S2 and S5 attenuated the hemolytic activity of the classical/lectin pathways after preincubation with normal human serum. Both components stimulated complement-dependent neutrophil chemotaxis and the production of C3 fragments, as shown by two-dimensional immunoelectrophoresis. S2 showed a higher capacity to generate the SC5b - 9 complex than the other fractions. This action was observed after the exposure of normal human serum to the fractions.


CONCLUSIONS
This is the first study to examine the activity of RsP components on the complement system. Fractions S2 and S5 reduced the complement hemolytic activity, stimulated complement-dependent neutrophil chemotaxis and stimulated the production of C3 fragments, indicating that they were able to activate the complement cascade. Furthermore, fraction S2 was also able to generate the SC5b-9 complex. These components may be useful tools for studying dysfunction of the complement cascade.
Department of Physics and Chemistry, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo (USP), Avenida do Café, s/n, Ribeirão Preto, 14.040-903 SP Brazil.
Fernando A P
FA
Anjolette
Department of Physics and Chemistry, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo (USP), Avenida do Café, s/n, Ribeirão Preto, 14.040-903 SP Brazil.
Flávia P
FP
Leite
Department of Physics and Chemistry, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo (USP), Avenida do Café, s/n, Ribeirão Preto, 14.040-903 SP Brazil.
Karla C F
KC
Bordon
Department of Physics and Chemistry, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo (USP), Avenida do Café, s/n, Ribeirão Preto, 14.040-903 SP Brazil.
Ana Elisa C S
AE
Azzolini
Department of Clinical Analyses, Toxicology and Food Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo (USP), Avenida do Café, s/n, Ribeirão Preto, 14.040-903 SP Brazil.
Juliana C
JC
Pereira
Department of Clinical Analyses, Toxicology and Food Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo (USP), Avenida do Café, s/n, Ribeirão Preto, 14.040-903 SP Brazil.
Luciana S
LS
Pereira-Crott
Department of Physics and Chemistry, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo (USP), Avenida do Café, s/n, Ribeirão Preto, 14.040-903 SP Brazil.
Eliane C
EC
Arantes
Complement system; Hemolytic activity; Neutrophil chemotaxis; Rhinella schneideri; Terminal complement complex (SC5b-9); Toad poison