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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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Carbohydrate specific identification and purification of Eutirucallin.A. Binding assay protein-carbohydrate with agarose beads adsorbed with different carbohydrates. “Beads” impregnated with different carbohydrates were incubated with the protein extract, later eluted from the resin after addition of 2-mercaptoethanol (2ME+) followed by boiling; supernatants were evaluate4d by SDS-PAGE. Lactose, mannose, N-acetyl-D-galactosamine (GalNAc) and N-acetylglucosamine (GlcNAc) correspond to the binding proteins for the corresponding sugars. B. Lectin purification by affinity chromatography. Approximately 2.41 mg of total protein was applied in 2 mL of α-D-Lactose column previously balanced with PBS, and eluted with galactose (0.4 M). The fractions collected during the purification were spectrophotometrically monitored (A280nm). C. Evaluation of proteins of the protein extract (PE) and Eutirucallin in bland and highly denaturating conditions. 2ME+ and 2ME- correspond, respectively, to the presence and absence of 2-mercaptoethanol. Mr, corresponds to the molecular pattern, whose numbers on the left indicate the mass values.
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pone-0088422-g002: Carbohydrate specific identification and purification of Eutirucallin.A. Binding assay protein-carbohydrate with agarose beads adsorbed with different carbohydrates. “Beads” impregnated with different carbohydrates were incubated with the protein extract, later eluted from the resin after addition of 2-mercaptoethanol (2ME+) followed by boiling; supernatants were evaluate4d by SDS-PAGE. Lactose, mannose, N-acetyl-D-galactosamine (GalNAc) and N-acetylglucosamine (GlcNAc) correspond to the binding proteins for the corresponding sugars. B. Lectin purification by affinity chromatography. Approximately 2.41 mg of total protein was applied in 2 mL of α-D-Lactose column previously balanced with PBS, and eluted with galactose (0.4 M). The fractions collected during the purification were spectrophotometrically monitored (A280nm). C. Evaluation of proteins of the protein extract (PE) and Eutirucallin in bland and highly denaturating conditions. 2ME+ and 2ME- correspond, respectively, to the presence and absence of 2-mercaptoethanol. Mr, corresponds to the molecular pattern, whose numbers on the left indicate the mass values.

Mentions: To confirm the presence of lectin in the E. tirucalli latex we performed a capture assay with different agarose-immobilized sugars (Fig. 2A). The analysis of the binding fraction with SDS-PAGE revealed a polypeptide of approximately 32 kDa able to interact with all carbohydrates used with variable affinity (Fig. 2A). Band intensity was: α-lactose > N-acetyl-D-galactosamine > N-acetyl-D-glucosamine > D-mannose. The galactosidase showed increased protein retention capacity while the others showed a discrete interaction according to the intensity of the bands in SDS-PAGE.


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)

Carbohydrate specific identification and purification of Eutirucallin.A. Binding assay protein-carbohydrate with agarose beads adsorbed with different carbohydrates. “Beads” impregnated with different carbohydrates were incubated with the protein extract, later eluted from the resin after addition of 2-mercaptoethanol (2ME+) followed by boiling; supernatants were evaluate4d by SDS-PAGE. Lactose, mannose, N-acetyl-D-galactosamine (GalNAc) and N-acetylglucosamine (GlcNAc) correspond to the binding proteins for the corresponding sugars. B. Lectin purification by affinity chromatography. Approximately 2.41 mg of total protein was applied in 2 mL of α-D-Lactose column previously balanced with PBS, and eluted with galactose (0.4 M). The fractions collected during the purification were spectrophotometrically monitored (A280nm). C. Evaluation of proteins of the protein extract (PE) and Eutirucallin in bland and highly denaturating conditions. 2ME+ and 2ME- correspond, respectively, to the presence and absence of 2-mercaptoethanol. Mr, corresponds to the molecular pattern, whose numbers on the left indicate the mass values.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0088422-g002: Carbohydrate specific identification and purification of Eutirucallin.A. Binding assay protein-carbohydrate with agarose beads adsorbed with different carbohydrates. “Beads” impregnated with different carbohydrates were incubated with the protein extract, later eluted from the resin after addition of 2-mercaptoethanol (2ME+) followed by boiling; supernatants were evaluate4d by SDS-PAGE. Lactose, mannose, N-acetyl-D-galactosamine (GalNAc) and N-acetylglucosamine (GlcNAc) correspond to the binding proteins for the corresponding sugars. B. Lectin purification by affinity chromatography. Approximately 2.41 mg of total protein was applied in 2 mL of α-D-Lactose column previously balanced with PBS, and eluted with galactose (0.4 M). The fractions collected during the purification were spectrophotometrically monitored (A280nm). C. Evaluation of proteins of the protein extract (PE) and Eutirucallin in bland and highly denaturating conditions. 2ME+ and 2ME- correspond, respectively, to the presence and absence of 2-mercaptoethanol. Mr, corresponds to the molecular pattern, whose numbers on the left indicate the mass values.
Mentions: To confirm the presence of lectin in the E. tirucalli latex we performed a capture assay with different agarose-immobilized sugars (Fig. 2A). The analysis of the binding fraction with SDS-PAGE revealed a polypeptide of approximately 32 kDa able to interact with all carbohydrates used with variable affinity (Fig. 2A). Band intensity was: α-lactose > N-acetyl-D-galactosamine > N-acetyl-D-glucosamine > D-mannose. The galactosidase showed increased protein retention capacity while the others showed a discrete interaction according to the intensity of the bands in SDS-PAGE.

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