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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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Construction of the polypeptide sequence of Eutirucallin.A. Cluster assembled from of peptides obtained by ms/ms. B. The order of peptides in the cluster was established by alignment with ricin. The letters “single letter code” correspond to the amino acids residues. (*) Represent identical residues; (:) conserved residues; and (.) semi-conserved replacements. The arrow (↓) corresponds to the initial residues of the A- (N-glycosidase) and B- (lectin domain) chains, respectively. The signal peptide is indicated in the sequence initial segment. The residues (cys-cys) correspond to the intramolecular and intermolecular disulfide bindings, indicated by the full and dashed lines, respectively.
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pone-0088422-g005: Construction of the polypeptide sequence of Eutirucallin.A. Cluster assembled from of peptides obtained by ms/ms. B. The order of peptides in the cluster was established by alignment with ricin. The letters “single letter code” correspond to the amino acids residues. (*) Represent identical residues; (:) conserved residues; and (.) semi-conserved replacements. The arrow (↓) corresponds to the initial residues of the A- (N-glycosidase) and B- (lectin domain) chains, respectively. The signal peptide is indicated in the sequence initial segment. The residues (cys-cys) correspond to the intramolecular and intermolecular disulfide bindings, indicated by the full and dashed lines, respectively.

Mentions: The 32 kDa band corresponding to the Eutirucallin purified by lactose affinity (Fig. 2C) was submitted to tryptic digestion and mass spectrometry in NanoUPLC-ESI Q-Tof, to establish the protein's identity. Nine tryptic peptides separated by the NanoUPLC were fragmented with manual selection and the sequences were generated by ms/ms (Table 4). The peptide sequence was analyzed in comparison with the NCBI databank. All peptides showed variable identity with type 2 ribosome-inactivating protein (RIPs) of different species (Table 4). The alignment developed with peptides individually evaluated showed variable identity from 30.8 to 69.7%, and similarity from 69.2 to 100% (data not shown) with Ricin (type 2 RIP of Ricinus communis). The peptide sequence was clustered based on the position of each peptide in the alignment with the Ricin (Fig. 5A). Seven of the nine peptides of the cluster showed 83.1% of coverage with the Ricin sequence, all of which in the protein's carboxy-terminal region (Fig. 5B). The partial sequence of the Eutirucallin was compared to the lectin domain regions (B-chain) of eight type 2 RIPs (Ricin, Abrin, Viscumin, Ebulin, Pulchellin, Cinnamomin, Volkensin and type 2 RIP of Iris hollandica) (Fig. 6) and presented identities of 41.5–52% and similarities of 67.4–83.1%. The higher similarity (83.1%) occurred with Ricin and Abrin a (Table 5).


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)

Construction of the polypeptide sequence of Eutirucallin.A. Cluster assembled from of peptides obtained by ms/ms. B. The order of peptides in the cluster was established by alignment with ricin. The letters “single letter code” correspond to the amino acids residues. (*) Represent identical residues; (:) conserved residues; and (.) semi-conserved replacements. The arrow (↓) corresponds to the initial residues of the A- (N-glycosidase) and B- (lectin domain) chains, respectively. The signal peptide is indicated in the sequence initial segment. The residues (cys-cys) correspond to the intramolecular and intermolecular disulfide bindings, indicated by the full and dashed lines, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0088422-g005: Construction of the polypeptide sequence of Eutirucallin.A. Cluster assembled from of peptides obtained by ms/ms. B. The order of peptides in the cluster was established by alignment with ricin. The letters “single letter code” correspond to the amino acids residues. (*) Represent identical residues; (:) conserved residues; and (.) semi-conserved replacements. The arrow (↓) corresponds to the initial residues of the A- (N-glycosidase) and B- (lectin domain) chains, respectively. The signal peptide is indicated in the sequence initial segment. The residues (cys-cys) correspond to the intramolecular and intermolecular disulfide bindings, indicated by the full and dashed lines, respectively.
Mentions: The 32 kDa band corresponding to the Eutirucallin purified by lactose affinity (Fig. 2C) was submitted to tryptic digestion and mass spectrometry in NanoUPLC-ESI Q-Tof, to establish the protein's identity. Nine tryptic peptides separated by the NanoUPLC were fragmented with manual selection and the sequences were generated by ms/ms (Table 4). The peptide sequence was analyzed in comparison with the NCBI databank. All peptides showed variable identity with type 2 ribosome-inactivating protein (RIPs) of different species (Table 4). The alignment developed with peptides individually evaluated showed variable identity from 30.8 to 69.7%, and similarity from 69.2 to 100% (data not shown) with Ricin (type 2 RIP of Ricinus communis). The peptide sequence was clustered based on the position of each peptide in the alignment with the Ricin (Fig. 5A). Seven of the nine peptides of the cluster showed 83.1% of coverage with the Ricin sequence, all of which in the protein's carboxy-terminal region (Fig. 5B). The partial sequence of the Eutirucallin was compared to the lectin domain regions (B-chain) of eight type 2 RIPs (Ricin, Abrin, Viscumin, Ebulin, Pulchellin, Cinnamomin, Volkensin and type 2 RIP of Iris hollandica) (Fig. 6) and presented identities of 41.5–52% and similarities of 67.4–83.1%. The higher similarity (83.1%) occurred with Ricin and Abrin a (Table 5).

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