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Eutirucallin, a RIP-2 type lectin from the latex of Euphorbia tirucalli L. presents proinflammatory properties.

Santana SS, Gennari-Cardoso ML, Carvalho FC, Roque-Barreira MC, Santiago Ada S, Alvim FC, Pirovani CP - PLoS ONE (2014)

Bottom Line: The analysis of the trypsin-digested Eutirucallin by ms/ms in ESI-Q-TOFF resulted in nine peptides similar to type 2 ribosome-inactivating protein (type-2 RIP).It's partial sequence showed a similarity of 67.4 - 83.1% for the lectin domain of type-2 RIP [Ricin and Abrin (83.1%), Viscumin, Ebulin, Pulchellin, Cinnamomin, Volkensin and type-2 RIP Iris hollandica].Our data suggest that Eutirucallin is a new member of type 2 ribosome-inactivating protein and presents biotechnological potential.

View Article: PubMed Central - PubMed

Affiliation: Universidade Estadual de Santa Cruz, Centro de Biotecnologia e Genética, Ilhéus, Bahia, Brasil.

ABSTRACT
Lectins are carbohydrate-binding proteins that recognize and modulate physiological activities and have been used as a toll for detection and identification of biomolecules, and therapy of diseases. In this study we have isolated a lectin present in the latex of Euphorbia tirucalli, and named it Eutirucallin. The latex protein extract was subjected to ion exchange chromatography and showed two peaks with haemagglutinating activity. Polypeptides of 32 kDa protein extract strongly interacted with immobilized galactose (α-lactose > D-N-acetylgalactosamine). The Eutirucallin was obtained with a yield of 5.6% using the α-lactose column. The lectin domain has 32 kDa subunits and at least two of which are joined by disulfide bridges. The agglutinating capacity for human erythrocytes A(+), B(+) and O(+) is inhibited by D-galactose. The haemagglutinating activity of Eutirucallin was independent of Ca(2+) and maintained until the temperature of 55°C. Eutirucallin presented biological activities such as neutrophils recruitment and cytokine prodution by macrophages. The analysis of the trypsin-digested Eutirucallin by ms/ms in ESI-Q-TOFF resulted in nine peptides similar to type 2 ribosome-inactivating protein (type-2 RIP). It's partial sequence showed a similarity of 67.4 - 83.1% for the lectin domain of type-2 RIP [Ricin and Abrin (83.1%), Viscumin, Ebulin, Pulchellin, Cinnamomin, Volkensin and type-2 RIP Iris hollandica]. Our data suggest that Eutirucallin is a new member of type 2 ribosome-inactivating protein and presents biotechnological potential.

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Influence of the heat treatment and EDTA on the haemagglutinating activity of the Eutirucallin.A. Eutirucallin (1.8 µg) was incubated for 20 min at 25, 35, 45, 55, 65, 75, 85 and 95°C. After 60 min of sedimentation at room temperature the haemagglutinating activity was evaluated. B. The Eutirucallin (1.8 µg) was incubated with EDTA in the concentrations of 10, 50 and 100 mM, and kept for 18 h at 4°C. Following to that the haemagglutinating activity was evaluated. Human erythrocytes A+ at 2% were used in both assays. The positive haemagglutination was indicated by the formation of a uniform coating of red blood cells in the surface of the U plates, and its absence was determined by the sedimentation of the cells in the bottom of the plates.
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pone-0088422-g003: Influence of the heat treatment and EDTA on the haemagglutinating activity of the Eutirucallin.A. Eutirucallin (1.8 µg) was incubated for 20 min at 25, 35, 45, 55, 65, 75, 85 and 95°C. After 60 min of sedimentation at room temperature the haemagglutinating activity was evaluated. B. The Eutirucallin (1.8 µg) was incubated with EDTA in the concentrations of 10, 50 and 100 mM, and kept for 18 h at 4°C. Following to that the haemagglutinating activity was evaluated. Human erythrocytes A+ at 2% were used in both assays. The positive haemagglutination was indicated by the formation of a uniform coating of red blood cells in the surface of the U plates, and its absence was determined by the sedimentation of the cells in the bottom of the plates.

Mentions: To assess the thermal stability of the Eutirucallin, protein aliquots were submitted to different temperatures before the haemagglutination assay (Fig. 3A). The Eutirucallin tolerated temperature below 55°C, preserving the haemagglutinating activity. The full haemagglutinating activity inhibition was observed at 65°C (Fig. 3A).


Eutirucallin, a RIP-2 type lectin from the latex of Euphorbia tirucalli L. presents proinflammatory properties.

Santana SS, Gennari-Cardoso ML, Carvalho FC, Roque-Barreira MC, Santiago Ada S, Alvim FC, Pirovani CP - PLoS ONE (2014)

Influence of the heat treatment and EDTA on the haemagglutinating activity of the Eutirucallin.A. Eutirucallin (1.8 µg) was incubated for 20 min at 25, 35, 45, 55, 65, 75, 85 and 95°C. After 60 min of sedimentation at room temperature the haemagglutinating activity was evaluated. B. The Eutirucallin (1.8 µg) was incubated with EDTA in the concentrations of 10, 50 and 100 mM, and kept for 18 h at 4°C. Following to that the haemagglutinating activity was evaluated. Human erythrocytes A+ at 2% were used in both assays. The positive haemagglutination was indicated by the formation of a uniform coating of red blood cells in the surface of the U plates, and its absence was determined by the sedimentation of the cells in the bottom of the plates.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3928152&req=5

pone-0088422-g003: Influence of the heat treatment and EDTA on the haemagglutinating activity of the Eutirucallin.A. Eutirucallin (1.8 µg) was incubated for 20 min at 25, 35, 45, 55, 65, 75, 85 and 95°C. After 60 min of sedimentation at room temperature the haemagglutinating activity was evaluated. B. The Eutirucallin (1.8 µg) was incubated with EDTA in the concentrations of 10, 50 and 100 mM, and kept for 18 h at 4°C. Following to that the haemagglutinating activity was evaluated. Human erythrocytes A+ at 2% were used in both assays. The positive haemagglutination was indicated by the formation of a uniform coating of red blood cells in the surface of the U plates, and its absence was determined by the sedimentation of the cells in the bottom of the plates.
Mentions: To assess the thermal stability of the Eutirucallin, protein aliquots were submitted to different temperatures before the haemagglutination assay (Fig. 3A). The Eutirucallin tolerated temperature below 55°C, preserving the haemagglutinating activity. The full haemagglutinating activity inhibition was observed at 65°C (Fig. 3A).

Bottom Line: The analysis of the trypsin-digested Eutirucallin by ms/ms in ESI-Q-TOFF resulted in nine peptides similar to type 2 ribosome-inactivating protein (type-2 RIP).It's partial sequence showed a similarity of 67.4 - 83.1% for the lectin domain of type-2 RIP [Ricin and Abrin (83.1%), Viscumin, Ebulin, Pulchellin, Cinnamomin, Volkensin and type-2 RIP Iris hollandica].Our data suggest that Eutirucallin is a new member of type 2 ribosome-inactivating protein and presents biotechnological potential.

View Article: PubMed Central - PubMed

Affiliation: Universidade Estadual de Santa Cruz, Centro de Biotecnologia e Genética, Ilhéus, Bahia, Brasil.

ABSTRACT
Lectins are carbohydrate-binding proteins that recognize and modulate physiological activities and have been used as a toll for detection and identification of biomolecules, and therapy of diseases. In this study we have isolated a lectin present in the latex of Euphorbia tirucalli, and named it Eutirucallin. The latex protein extract was subjected to ion exchange chromatography and showed two peaks with haemagglutinating activity. Polypeptides of 32 kDa protein extract strongly interacted with immobilized galactose (α-lactose > D-N-acetylgalactosamine). The Eutirucallin was obtained with a yield of 5.6% using the α-lactose column. The lectin domain has 32 kDa subunits and at least two of which are joined by disulfide bridges. The agglutinating capacity for human erythrocytes A(+), B(+) and O(+) is inhibited by D-galactose. The haemagglutinating activity of Eutirucallin was independent of Ca(2+) and maintained until the temperature of 55°C. Eutirucallin presented biological activities such as neutrophils recruitment and cytokine prodution by macrophages. The analysis of the trypsin-digested Eutirucallin by ms/ms in ESI-Q-TOFF resulted in nine peptides similar to type 2 ribosome-inactivating protein (type-2 RIP). It's partial sequence showed a similarity of 67.4 - 83.1% for the lectin domain of type-2 RIP [Ricin and Abrin (83.1%), Viscumin, Ebulin, Pulchellin, Cinnamomin, Volkensin and type-2 RIP Iris hollandica]. Our data suggest that Eutirucallin is a new member of type 2 ribosome-inactivating protein and presents biotechnological potential.

Show MeSH
Related in: MedlinePlus