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Purification and partial characterization of a ribosome-inactivating protein from the latex of Euphorbia trigona Miller with cytotoxic activity toward human cancer cell lines Judith Villanueva a, Luis Manuel Quirós b, Sonia Castañón a,∗
Q1
a b
Neiker-Tecnalia, Biotechnology Department, Arkaute Agrifood Campus, PO Box 46, 01080 Vitoria-Gasteiz, Álava, Spain University Institute of Oncology of Asturias and Department of Functional Biology, University of Oviedo, Julian Claveria s/n, Oviedo 33006, Spain
a r t i c l e
i n f o
Article history: Received 2 January 2015 Revised 10 March 2015 Accepted 6 April 2015 Available online xxx Keywords: Lectin Antitumoral Cytotoxic Hemagglutination Ribosome-inactivating proteins (RIPs)
a b s t r a c t Background: The objective of this study is to investigate the cytotoxic activity of three isolectins purified from the latex of Euphorbia trigona Miller. Hypothesis: Among lectins are the ribosome-inactivating proteins (RIPs), which are potent inhibitors of protein synthesis in cells and in cell-free systems. Results: Three isolectins, ETR1, ETR2 and ETR3, were purified by anion exchange chromatography. Both ETR1 and ETR3 yielded a single band on SDS–PAGE under reducing conditions, corresponding to a molecular weight of 32 g mol−1 , while ETR2 yielded two bands corresponding to 31 and 33 g mol−1 . When non-reducing conditions were used molecular weight decreased, indicating the presence of intrachain disulfide bonds. Sizeexclusion chromatography revealed proteins of apparent molecular weight of 59–63 g mol−1 , suggesting a dimeric nature, with subunits not being held together by disulfide linkage. ETR1, ETR2 and ETR3 hemagglutinated human, sheep and rat erythrocytes and this hemagglutination was specifically inhibited by galactose and its derivatives. The lectins studied were thermostable up to 60 °C and their observed activity was maintained across pH range 5–12. These lectins, from the latex of Euphorbia trigona, are potent inhibitors of eukaryotic protein synthesis in a cell-free system. Flow cytometry analysis revealed the antiproliferative activity of them toward A549, HeLa, H116, HL-60 cell lines. Conclusion: Euphorbia trigona isolectins are RIPs with cytotoxic activity toward human cancer cell lines. © 2015 Published by Elsevier GmbH.
1
Introduction
2
Lectins are a group of proteins (or glycoproteins) of non-immune origin that bind specifically and reversibly to carbohydrates, resulting in cell agglutination and precipitation (Goldstein et al. 1980). Due to this capacity, lectins are involved in diverse cellular mechanisms (Sharon and Lis 1993). Among the various types of plant lectins are some which pertain to the RIPs. These are toxic N-glycosidases that remove a highly conserved adenine from the 28S ribosomal RNA (rRNA). This depurination inactivates the ribosome and arrests protein synthesis. Type II RIP family is widely represented in plants of different taxonomic
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Abbreviations: RIP(s), ribosome inactivating protein(s); HU, hemagglutination unit; ECA, Euphorbia calcina agglutinin; EDA, Euphorbia dalberi agglutinin; EspA, Euphorbia sp. agglutinin; EML, Euphorbia millii lectin; IC50, concentration at which 50% inhibition is achieved; GalNAc, N-acetyl-d-galactosamine; GlcNAc, N-acetyl-d-glucosamine; PS, phosphatidilserine. ∗ Corresponding author. Tel.: +34 945 121313; fax: +34 945 281422. E-mail address: [email protected] (S. Castañón).
origins such as ricin, viscumin, abrin, volkesin, etc. (for review see Stirpe and Batelli 2006; Van Damme et al. 1998a, 1998b). These proteins consist of two chains, the B chain being responsible for binding to carbohydrates and the A chain, which is an RNA N-glycosidase, for irreversibly inactivating eukaryotic ribosomes. They are particularly interesting because of their cytotoxic activity, determined by the ability of the B chain to bind to galactosyl terminated glycoproteins on the cell surface. This binding permits and facilitates the entry of A chains into cells, where they then exert their enzymatic activity on ribosomes thereby inhibiting protein synthesis. There has, as a result, been considerable interest in type II RIPs due to their potential role in the development of therapeutic agents, preferentially binding to cancer cell membranes or their receptors, causing cytotoxicity, apoptosis and inhibition of tumor growth. Euphorbia trigona Miller is a succulent plant from Africa used as an ornamental and is known as the African milk tree because of its high latex content. Latex is a colloidal suspension which contains an extraordinary array of secondary metabolites and proteins (Agrawal and Konno 2009). Many studies suggest that the latex of the members of Euphorbiaceous is a rich source of lectins, for example
http://dx.doi.org/10.1016/j.phymed.2015.04.006 0944-7113/© 2015 Published by Elsevier GmbH.
Please cite this article as: J. Villanueva et al., Purification and partial characterization of a ribosome-inactivating protein from the latex of Euphorbia trigona Miller with cytotoxic activity towards human cancer cell lines, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2015.04.006
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E. marginata (Stirpe et al. 1993), E. neriifolia (Seshagirirao and Prasad 1995), Synadenium carinatum (Souza et al. 2005). In this work, we report the purification and characterization of three new galactoside-derivatives binding lectins from the latex of Euphorbia trigona. Their RNA N-glicosidase activity was also tested and found to inhibit the proliferation of several human cancer cell lines.
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Materials and methods
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Chemicals and reagents
53
HiTrapۛ desalting, Mono Qۛ 5/50 GL and Superoseۛ 12 10/300 GL chromatographic columns were purchased from GE Healthcare. Carbohydrates for hemagglutination inhibition studies (d-glucose, d-fructose, d-galactose, lactose, N-acetyl-d-galactosamine, N-acetyld-glucosamine, xylose, d-raffinose, melibiose, l-arabinose, d-mannose and d-fucose) and size-exclusion standards were obtained from Sigma–Aldrich. Bovine serum albumin, molecular weight standards for SDS–PAGE and Bradford reactive were procured from Bio-Rad. The rabbit reticulocyte lysate system used for protein synthesis inhibition was purchased from Promega and [3 H]-leucine (specific activity 165.2 Ci mmol−1 ) from PerkinElmer. All other chemicals were obtained from commercial sources and were of analytical grade.
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Biological samples
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Plants of Euphorbia trigona Miller were grown in the greenhouse of Neiker-Tecnalia, Alava, Spain. Venous human blood was obtained from the Basque Biobank for Research, Sondika, Spain. Sheep blood was kindly provided by the Department of Animal Production, Neiker-Tecnalia and rat blood by the Department of Functional Biology of the Faculty of Medicine, Oviedo University, Spain. The cytotoxic effect of the purified lectins was studied in different cell lines: HeLa (cervix adenocarcinoma), A549 (lung carcinoma), H116 (colorectal carcinoma), HL-60 (promyelocytic leukemia), HT-29 (colon adenocarcinoma) and NIH-3T3 (mouse fibroblasts), all obtained from American Type Culture Collection
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Purification of Euphorbia trigona lectin
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56 57 58 59 60 61 62 63 64 65
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Latex was collected by repeated incisions in the stems of the plants with sharp blades and diluted in 10 vol. of PBS, pH 7.4. The subsequent operations were performed at 4 °C. The diluted latex was stirred for 3 h. Afterwards, the sample was centrifuged at 70,000 × g for 1 h and the supernatant was dialyzed overnight against the same buffer. The total soluble protein was fractionated by ammonium sulfate precipitation (40–90% saturation). After desalting, the sample was applied to a Mono Q HR column, previously equilibrated with 25 mM Tris–HCl, pH 8.5. The column was washed until the A280 of the effluent was negligible and the lectins were eluted in a stepwise gradient using 98, 123 and 250 mM NaCl. The protein content of the crude extract and the fractionated protein samples was estimated by Bradford assay using BSA as a standard.
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Hemagglutination assays
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Hemagglutination assays were performed using human (AB0 groups), sheep and rat blood in a U-bottomed 96-well microtiter plate. Fifty-microliter aliquots of 2-fold serially diluted lectins were mixed with an equal volume of 3% (v/v) suspension of erythrocytes in saline solution. After incubation at 37 °C for 1 h, the results were determined visually. Hemagglutination units (HU) were expressed as
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the reciprocal of the highest dilution of the lectin showing detectable hemagglutination.
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Effect of temperature and pH on lectin activity
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The influence of temperature and pH were assessed by studying their effect on lectin activity. The protein samples were incubated for 1 h prior to assay at a temperature ranging from 25 to 90 °C. The pH sensitivity of the lectins was established by incubating the samples for 1 h in different buffers: Sodium acetate (pH 3–5), Tris–HCl (pH 7– 8.5), sodium carbonate (pH 10) and KCl/NaOH (pH 12), followed by the determination of their hemagglutinating activity.
92
90
93 94 95 96 97 98
Sugar inhibition of hemagglutination
99
Carbohydrate-binding specificity of the lectins was assessed by the ability of each of them to inhibit hemagglutination. Serial 2-fold dilutions of sugars were prepared in saline solution. An equal volume of each lectin containing 4 HU was mixed in each well. The mixtures were incubated for 30 min at room temperature and, afterwards, the hemagglutination assay was performed. Inhibitory activity was determined as the lowest concentration of the carbohydrate required for the complete inhibition of the hemagglutination.
100
Electrophoretic analysis and molecular weight determination
108
SDS–PAGE was carried out using a 10–15% (w/v) gradient slab gel in the presence or absence of 2-mercaptoethanol. Native-PAGE using a discontinuous buffer system was carried out on a 7.5% (w/v) gel at pH 8.8. The gels were stained with Coomassie Brilliant Blue. Size-exclusion chromatography was used to determine the molecular weight of the native proteins. It was calibrated with protein standards in the range from 12.4 to 200 g mol−1 and equilibrated and eluted with 50 mM Tris–HCl containing 100 mM KCl and 200 mM lactose, pH 7.5. The flow rate was adjusted to 0.4 ml min−1 . Detection was achieved made by UV detector at 280 nm.
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Amino acid sequencing
119
Purified proteins were analyzed and sequenced by mass spectrometry at the Proteomics Platform Service of the CIC-Biogune. Peptides with reliable scores were compared with public databases using the BLAST algorithm.
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Protein synthesis inhibition
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The effects of lectins on cell-free protein synthesis were determined with a rabbit reticulocyte lysate, following the manufacturer’s instructions. Biological activity was determined by each lectin’s ability to inhibit incorporation of [3 H]-leucine into proteins. Lectins were analyzed in the range of 0–5 nM. Samples (5 μl) were added to a protein synthesis reaction mixture along with 17.5 μl of rabbit reticulocyte lysate, 0.25 μl of a mixture of all amino acids (minus leucine) at 1 mM, 0.5 μl of RNas in ribonuclease inhibitor and 0.5 μl of luciferase control RNA at 1 μg μl−1 which had previously been denatured for 3 min at 65 °C. The reaction mixture was incubated at 30 °C for 90 min and placed on ice. After this, 5 μl of the reaction mixture was mixed with 95 μl of NaOH 1 M and H2 O2 2% and incubated for 10 min at 37 °C at the end of which the resultant protein was precipitated using 900 μl ice-cold TCA 25%/casamino acids 2% for 30 min on ice. Afterwards, 250 μl of this solution were collected by vacuum filtering in a glass fiber filter, washed with acetone and transferred to vials with scintillation fluid (PerkinElmer). Radioactivity was determined in a Wallac 1409 liquid-scintillation spectrometer. This assay was repeated at least three times.
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Please cite this article as: J. Villanueva et al., Purification and partial characterization of a ribosome-inactivating protein from the latex of Euphorbia trigona Miller with cytotoxic activity towards human cancer cell lines, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2015.04.006
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Cytotoxic activity
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Inhibitory potential of the isolated lectins was tested against six cell lines: HeLa, A549, H116, HL-60, HT-29 (tumoral cell lines) and NIH-3T3 (non-tumoral cell line). These cell lines were maintained in DMEM (Dulbecco modified Eagle’s medium) supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 1 mM sodium piruvate and 1 mM amino acids at 37 °C in a humidified atmosphere of 5% CO2 . In these conditions, 20.000 cells were seeded into a 6-well culture plate and, the following day, the cells were co-incubated with increasing amounts of ETR1, ETR2 and ETR3 lectins for 24 h under the same culture conditions. As negative control we used 25 mM Tris–HCl, pH 8.5. Following these cells were harvested by trypsinization and then resuspended in Annexin V staining solution (containing Annexin V fluorescein and propidium iodide) and incubated for 10 min at room temperature. The samples were analyzed with a Cytomics FC500 (Beckman Coulter).
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Results and discussion Purification of Euphorbia trigona lectin Three lectins were purified by anion-exchange chromatography on a Mono Q column using a stepwise gradient for elution. Lectins are usually purified by affinity chromatography but, in this case, this technique was not resolutive, so we used this strategy. Prior to purification, crude protein extract of the latex from E. trigona was subjected to an ammonium sulfate fractionation to obtain the clarified protein extract. After dialyzing, the fractions with maximum lectin activity (40–90% ammonium sulfate) were employed in the anionexchange chromatography. The absence of lectin activity in the flowthrough indicated that complete adsorption had occurred. Initially a linear gradient was used to elute the bound proteins but this was not sufficiently resolutive as it resulted in overlapping peaks. Thus, in order to improve the separation of the bound lectins, a stepwise gradient was used for the elution. The NaCl concentrations used for this stepwise gradient were selected from the linear gradient elution previously performed and resulted in three absorbance peaks at 98, 123 and 250 mM NaCl (Fig. 1). All the peak fractions tested were able to hemagglutinate human erythrocytes, indicating that lectins were present. The first peak was named ETR1, the second ETR2 and the third ETR3. Those fractions containing pure protein in high concentrations were pooled and concentrated for further studies. In this 0,2
100 A595 H.U. NaCl
0,16
80
0,12
60
0,08
40
0,04
Activity (H.U.) -2 [NaCl] (10 M)
147
A595
146
20
0 1
28
40
54
64
80
96
0 110
Fraction Fig. 1. Anion exchange chromatography of a dialyzed protein fraction from the latex of E. trigona after ammonium sulfate precipitation (40–90%) on a Mono Q. Hemagglutinating Units, H.U. Experimental details are outlined in the text.
3
manner, 25% of the activity was recovered. Similarly, it was determined that 15.3% of the soluble protein content of the latex after ammonium sulfate precipitation corresponded to lectins. This percentage is double that of the lectin obtained from the latex of E. marginata (Stirpe et al. 1993) and similar to the percentage from the latex of E. Neriifolia (Seshagirirao and Prasad 1995).
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Characterization
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ETR1 gave a single band in SDS–PAGE under reducing conditions, having a molecular weight of 32 g mol−1 , ETR2 showed two bands of 33 and 31 g mol−1 and ETR3 a single band of 32 g mol−1 (Fig. 2A). However, when non-reducing conditions were used, the result was different: ETR1 gave a single band of 29 g mol−1 , ETR2 gave two bands of 31 and 27 g mol−1 , and ETR3 also gave two, of 29 and 31 g mol−1 along with a minor peak of approximately 27 g mol−1 (Fig. 2A). The lower molecular weight of unreduced samples in comparison with reduced ones suggests the presence of intrachain disulfide bonds, as have been described previously in other lectins as ECA (Euphorbia calcina agglutinin) and EDA (Euphorbia dalberi agglutinin) or Euticurallin (Nsimba-Lubaki et al. 1986; Silva Santana et al. 2014). These molecular sizes are also consistent with the results obtained for other lectins of the Euphorbiaceae family, which are composed by two or more subunits of 28–32 g mol−1 (Nsimba-Lubaki et al. 1986; Silva Santana et al. 2014, Seshagirirao and Prasad 1995; Souza et al. 2005). Most plant lectins are composed of several subunits. For example, Ricin, a type II RIP lectin, is a dimeric protein (Mw 63 g mol−1 ) formed by a 32 g mol−1 chain and another of 34 g mol−1 (Van Damme et al. 1998b). The molecular weight of native ETR1, ETR2 and ETR3 was determined by size-exclusion chromatography. In all cases, a single peak was obtained showing molecular weights of 59 g mol−1 , 66 g mol−1 and 63 g mol−1 , respectively. These results clearly suggest that lectins of the latex from E. trigona are dimers which are not held together by disulfide linkages as happens with ECA, EDA and EspA (Euphorbia sp. agglutinin) (Nsimba-Lubaki et al. 1986). However, there are several lectins from the latex of other Euphobiaceae, for instance, Euticurallin, a type II RIP lectin from the latex of Euphorbia tirucalli L. with at least two 32 g mol−1 subunits joined by disulfide bridges (Silva Santana et al. 2014). The analysis of the isolated proteins by native-PAGE at pH 8.8 demonstrated the purity of the major proteins originally present in the latex (Fig. 2B). Each protein moved to a different position in the gel, indicating the presence of different isoforms with different pIs. The existence of lectin isoforms may be due to variation in oligosaccharide chains (Hayes and Goldstein 1974) or may stem from changes in the amino acid sequence, resulting in different charged proteins (Van Damme et al. 1992). Most RIPs isolated from the same plant are isomers with similar primary protein structure. Purified lectins from E. trigona showed strong hemagglutination of erythrocytes from different species (human, sheep and rat). The isoforms varied in their hemagglutination potential, expressed in terms of HU. ETR1, ETR2 and ETR3 each had strong hemagglutination ability against all human blood groups (AB0), showing no specificity for one in particular as happens in other previously described latex lectins of other Euphorbiaceae (Lynn and Clevette-Radford 1986; Souza et al. 2005). 0.78 μg ml−1 of any of the ETR lectins being necessary to hemagglutinate them. With respect to erythrocytes of other species: ETR1 required 12.5 μg ml−1 to agglutinate sheep erythrocytes and 3.12 μg ml−1 to hemagglutinate rat blood, ETR2 needed 1.56 and 6.25 μg ml−1 to hemagglutinate sheep and rat erythrocytes and ETR3 1.56 and 3.12 μg ml−1 , respectively. These variations in hemagglutination potential of ETR isoforms suggest that they might also differ in their sugar specificities and cell recognition. To ascertain the stability of ETR isoforms under different physicochemical conditions, their biological properties in terms of lectin
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Fig. 2. Gel electrophoresis of the proteins purified from E. trigona. (A) Purified proteins analyzed by SDS–PAGE under both reducing and non-reducing conditions. Lanes 1, 2, 3 show ETR1, ETR2, ETR3, respectively, under non-reducing conditions. Lanes 4, 5, 6 show the same proteins under reducing conditions. M: molecular weight marker. (B) Purified proteins analyzed by native PAGE. Lanes 1, 2, 3 show ETR1, ETR2, ETR3, respectively. FS: dialyzed protein fraction after ammonium sulfate precipitation.
A
100
80 HU (%)
80 HU (%)
B
100
60 40
ETR 1 ETR 2
20 25
35
45
40
ETR 1 ETR 2
20
ETR 3
0
60
ETR 3
0 55
65
75
85
4
6
Tª (ºC)
8
10
12
pH
Fig. 3. Influence of physicochemical parameters. (A) Effect of temperature and (B) pH on the hemagglutinating activity of E. trigona lectin isoforms. Percentage activity was calculated considering maximum measured activity to be 100%. Hemagglutination units, H.U.
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activity were analyzed at a range of temperatures between 25 and 90 °C and at pHs ranging from 5 to 12, for 1 h. All three lectins isoforms from E. trigona were heat stable and retained complete biological activity up to 60 °C. Above that temperature, lectin activity was gradually lost and almost totally inactivated at 90 °C (Fig. 3A). These results are comparable to those obtained for other lectins (Mishra et al. 2004; Zhang et al. 2010). In addition, lectin activity was unchanged in the pH range 5–12 (Fig. 3B), showing ETRs to be more resistant to pH than other latex lectins of Euphorbiaceae which rapidly lost their activity at pH values above 10 (Nsimba-Lubaki et al. 1986). The hemagglutination activity of all three purified lectins was inhibited by d-galactose and N-acetyl-d-galactosamine (GalNAc), as happens with other lectins derived from Euphorbiaceae (Silva Santana et al. 2014, Stirpe et al. 1993) and with most B chains of the type II RIPs, such as ricin and viscumin (Van Damme et al. 1998a, 1998b). The activity was inhibited to a lesser degree by disaccharides and trisaccharides like lactose, melibiose and raffinose, all of which are saccharides with a terminal non-reducing galactose (Table 1). The hemagglutination produced by ETR1 was inhibited by 0.73 mM galactose or GalNAc. That produced by ETR2 was inhibited by 1.46 mM galactose or 2.92 mMGalNAc and that by ETR3 by 1.46 mM for each sugar. This small difference in the concentration necessary to inhibit hemagglutination could be due to the presence of slight differences in cell recognition. Besides the aforementioned carbohydrates, we also observed inhibition of the hemagglutination when we incubated the lectins with fucose. There was not, however, any inhibitory effect using sugars such as glucose, fructose or N-acetyl-d-glucosamine
Table 1 Inhibition by sugar of hemagglutinating activity of E. trigona lectins. Carbohydrate
M.I.C.a (mM) ETRI 1
Glucose Fructose Galactose Lactose N-Acetylgalactosamine N-Acetylglucosamine Xylose Raffinose Melibiose Arabinose Mannose Fucose Heparin Chondroitin sulfate A Chondroitin sulfate B Chondroitin sulfate C
b
N.I. N.I. 073 219 073 N.I. N.I. 583 583 N.I. N.I. 875 N.I. N.I. N.I. N.I.
ETRI 2
ETRI 3
N.I. N.I. 146 583 292 N.I. N.I. 2333 1167 N.I. N.I. 4666 N.I. N.I. N.I. N.I.
N.I. N.I. 146 583 146 N.I. N.I. 2333 1167 N.I. N.I. 2333 N.I. N.I. N.I. N.I.
a M.I.C.: minimum concentration required for 50% inhibition of hemagglutination. b N.I.: no inhibition.
(GlcNAc). This suggests that the axial C4 hydroxyl position is a critical binding region, as is the case with EML (Euphorbia milii lectin) (Irazoqui et al. 2005). Contrary to what occurs with the latex lectins from other Euphorbiaceous, d-fucose (6-deoxy-d-galactose) showed
Please cite this article as: J. Villanueva et al., Purification and partial characterization of a ribosome-inactivating protein from the latex of Euphorbia trigona Miller with cytotoxic activity towards human cancer cell lines, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2015.04.006
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Table 2 Results of the sequence analysis of peptides of ETR1, ETR2 and ETR3 obtained by mass spectrometry. Number represents different peptides appearing in each protein. Peptides with the same number are homologous. Conserved amino acid residues are underlined and amino acid residues of the active site are detailed in bold. Number
Peptide ETR1 (29 kDa)
1
VLVLTCGLGPSSQR
2 3 4
ALLVYPFTGNPNQK QQWYFYPDGTLGVPK VPLVYDETGGGPTLR
Number
Peptide ETR2 (31 kDa)
2 5
LALVYPFTGNPNQK LVMDVAQSKPSLK
6
DPVLTCGLGALSQR
Number
Peptide ETR2 (27 kDa)
6
DPVLTCGLGALSQR
Number
Peptides ETR3 (29 kDa)
1
VVLTCGLGPSSQR
2 3 5
LALVYPFTGNPNQK QQWYFYPDGTLGVPK VGEDVAQSDLALK
Identity
Protein
Species
Access number
RIP Type II RIP precursor Ricin-agglutinin Type II RIP Protein with lectin domain Ricin precursor
Panax ginseng Sambucus nigra Ricinus communis Momordica charantia Oryza sativa Ricinus communis
ABG76785 AAC49754 EEF30192 BAH05018 ABA97653 8287993 RCOM_2159910
Identity
Protein
Species
Access number
9/14 11/12 9/12 8/14 7/14
Ricin-agglutinin Precursor lectina Agglutinin I RIP Lectin
Ricinus communis Viscum album Ricinus communis Panax ginseng Medicago truncatula
EEF30192 AY377892 AAB22584 ABG76785 ABN08398
Identity
Protein
Species
Access number
8/14 7/14
RIP Lectin
Panax ginseng Medicago truncatula
ABG76785 ABN08398
Identity
Protein
Species
Access number
RIP Type II RIP precursor Ricin-agglutinin Type II RIP Agglutinin I
Panax ginseng Sambucus nigra Ricinus communis Momordica charantia Ricinus communis
ABG76785 AAC49754 EEF30192 BAH05018 AAB22584
8/14 8/14 9/14 8/16 8/15 7/15
7/14 7/14 9/14 8/16 9/12
Fig. 4. Effects of the lectins studied on protein synthesis. (A) Protein synthesis inhibition on cell-free system produced by purified lectin isoforms ETR1, ETR2, ETR3. (B) Comparison of the inhibition rate of E. trigona lectins with other cytotoxic type II RIP proteins from the literature.
278 279 280 281 282 283 284 285 286 287 288 289 290 291 292
less inhibition of hemagglutination than galactose, indicating the importance of the C6 hydroxyl for binding, especially for ETR2 and ETR3, which seems to have no influence in other Euphorbiaceous lectins (Nsimba-Lubaki et al. 1986). ETR lectins have 2- to 5-fold greater affinity for lactose than for raffinose or melibiose, which suggests they may be more specific to the β linkage than to the α (Nsimba-Lubaki et al. 1986; Seshagirirao and Prasad 1995). No inhibitory effect was observed using other sugars such as xylose, arabinose or mannose. Amino acid sequencing We obtained partial sequences of different proteins after the mass spectrometry analysis of ETR1, ETR2 and ETR3. Six of these peptides showed 50–75% identity with various plant lectins. All the sequenced peptides could be aligned to the B chain of type II RIPs (Table 2). Ye et al. (2006) highlighted that the B chains of type II RIPs show highly
conserved sequences and, therefore, in their 3D structure. However, their sugar binding specificity varies. The sequences obtained from the three purified proteins exhibited a high degree of similarity (indicated by the same number in Table 2), which supports the idea that they are isoforms. According to Wacker et al. (2005), among the most conserved domains in the B chain, we found the amino acids that comprise the two active sites, which are, firstly, D22, Q35, S37, N46, and Q47, and secondly, D234, I246, Y248, N255, and Q256. A detailed comparison between the carbohydrate-binding site of ricin, viscumin and volkensin and the sequences obtained here indicate that the peptide identified as number 5 in Table 2 contained amino acid D234 which is part of the carbohydrate-binding site. Likewise, peptide 2 in Table 2 contains the amino acids Y248, N255 and Q256 from the same binding site (Chambery et al. 2004; Wacker et al. 2005). Thus, it is suggested that the purified proteins of the latex from E. trigona are lectins belonging to the type II RIP family.
Please cite this article as: J. Villanueva et al., Purification and partial characterization of a ribosome-inactivating protein from the latex of Euphorbia trigona Miller with cytotoxic activity towards human cancer cell lines, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2015.04.006
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HeLa A549 H116 HL-60 HT-29 NIH-3T3
IC50 a (nM) ETRI 1
ETRI 2
ETRI 3
11 32 40 75 N.I.b 875
12 32 18 44 N.I. 480
10 18 21 44 N.I. 480
a
IC50 : Minimum concentration of lectin required for causing 50% of cell death. b N.I.: IC50 is not reached at the tested protein concentration.
310
Inhibition of protein synthesis in cell-free system
311
Some galactose-binding lectins, which are type II RIPs, have been reported to inhibit protein synthesis in cell-free systems (Barbieri et al. 1979). All three lectins purified here significantly inhibited protein synthesis in a cell-free system (Fig. 4A). ETR2 was the most active isoform: this lectin showed an IC50 value (concentration at which 50% inhibition is achieved) at 7.5 nM. ETR3 inhibited protein synthesis in a more moderate way: 50% inhibition required 12 nM and ETR1 was the least active isoform with its IC50 value was reached at 16 nM. According to the literature, very toxic lectins such as ricin, abrin and volkensin have an IC50 ranging from 40 to 85 nM (Stirpe and Barbieri 1986). These concentrations are higher than found in the
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current study so it places ETR1, ETR2 and ETR3 among the most toxic type II RIPs (Fig. 4B). In contrast, there are some lectins from Euphorbiaceae which do not inhibit protein synthesis, for example, E. heterophylla seed lectin (Nsimba-Lubaki et al. 1983) and E. neriifolia latex lectin (Seshagirirao and Prasad 1995). There are lectins from other Euphorbiaceous species in the literature but biological assays of specific inhibition of ribosomal activity are required to conduct a thorough analysis and compare them. These results show that lectins from the latex of E. trigona possess similar chemical composition in several aspects to that obtained from other Euphorbia species (stability under different physico-chemical conditions, the sugar specificity, lack of specificity for blood groups) with the exceptions mentioned earlier. However, ETR lectins are different from those due to the presence of isolectins in the latex, the amino acid composition and the ability to inhibit protein synthesis in rabbit reticulocytes.
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Cytotoxicity
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Some plant lectins are highly cytotoxic and are able to induce apoptosis (Kim et al. 1993). We evaluated the cytotoxic effect of the three purified proteins on different cell lines: HeLa, A549, H116, HL60, HT-29 (tumoral cell lines) and NIH-3T3 (non-tumoral cell line) (Table 3). Cell viability was determined by flow-cytometry. A dosedependent decrease in cell viability was observed with increasing concentration of each sample. Using ETR1 as an example, Fig. 5 demonstrates that after Annexin V-FITC and propidium iodide (PI)
Fig. 5. Flow cytometry analysis of HL-60 cells after treatment with ETR1 for 24 h, followed by staining with Annexin V-FITC.
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staining, the HL-60 cells in the lower left quadrant of the profile shifted toward the lower right quadrant as ETR1 increased. This shift is indicative of phosphatidilserine (PS) externalization, suggesting that HL-60 cells were undergoing the early stage of apoptosis. Cells located at the upper right quadrant also increased with increasing ETR1, indicating that some HL-60 cells had entered the late stage of apoptosis. Authors, using the same technique to evaluate antiproliferative effects, obtained similar results with lectins at μM concentrations (Chan and Ng 2013). But some authors determine cytotoxicity in terms of inhibition of protein synthesis in whole cells (Stirpe and Barbieri 1986). In this way, the IC50 value for ricin was established at 1 pM, for abrin and at 3.4 pM and for viscumin at 8 pM when tested against HeLa cells. The difference between the order of magnitude of these IC50 values is evident. This difference could be attributed to the fact that inhibition of protein synthesis is an event which occurs prior to loss of cell viability (Stirpe et al. 2007) and therefore, the minimal concentration necessary to inhibit protein synthesis, as considered in other studies, is far less than that required to inhibit cell viability, the focus of this study. All the isoforms purified here were inactive against the HT-29 cell line at the maximum concentration studied (100 nM). These results concur with the idea of a particular lectin being able to show more toxicity with respect to certain cells than to others (Wang et al. 2000). The reason for these differences is not known exactly, but is thought to be related to the binding of the proteins and their entry into the cells (Stirpe and Batelli 2006). Malignant cells have carbohydrate alterations, and it is at this point that lectin-mediated cytotoxicity might play a role in antitumor defense (Gorelik et al. 2001). It has been speculated that the selective cytotoxicity of lectins against tumoral cells is determined by their differing affinity for specific sugars, the recognition and binding of the B chain to sugars on the cell surface which is essential for the entry of the cytotoxic A chain (Ye et al. 2006). It is of note that 875 nM of ETR1, ETR2 or ETR3 were necessary to achieve the IC50 value in non-tumoral cells (NIH-3T3), almost two orders of magnitude higher than with HeLa cells (10–12 nM). This accords with the work of Lin et al. (1970), who found that certain tumoral cells were more sensitive to type II RIPs than normal cells.
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Conclusions
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In this study, three lectins (isoforms) were purified from the latex of Euphorbia trigona by anion exchange chromatography. These lectins bind specifically to galactose-derivative sugars and inhibit protein synthesis in a cell-free system which strongly suggests that the purified lectins belong to the type II RIP family and situates ETR1, ETR2 and ETR3 among the most toxic. These lectins also show remarkable cytotoxic activity against certain tumoral cell lines.
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Conflict of interest
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We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
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Acknowledgments
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This work was financially supported by the Department of Environment, Land Planning, Agriculture and Fisheries of the Basque Government. We are grateful to Dr. Marcos Garcia Ocaña for sharing his expertise with human cell culture and his skilled technical assistance. The University Institute of Oncology of Asturias is supported by Obra Social Cajastur, Asturias, Spain.
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Cancer Metastasis Rev. 20, 245– 277. Hayes, E., Goldstein, I.J., 1974. An α -d-galactosyl-binding lectin from Bandeirae simplicifolia seeds: isolation by affinity chromatography and characterization. J. Biol. Chem 249, 1904–1914. Irazoqui, F.J., Vozari-Hampe, M.M., Lardone, R.D., Villarreal, M.A., Sendra, V.G., Montich, G.G., Trindade, V.M., Clausen, H., Nores, G.A., 2005. Fine carbohydrate recognition of Euphorbia milii lectin. Biochem. Biophys. Res. Commun. 333, 14–21. Kim, M., Rao, M.V., Tweardy, D.J., Prakash, M., Galili, U., Gorelik, E., 1993. Lectin-induced apoptosis of tumour cells. Glycobiology 3 (5), 447–453. Lin, J.Y., Tserng, K.Y., Chen, C.C., Lin, L.T., Tung, T.C., 1970. Abrin and ricin: new antitumour substances. Nature 227, 292–293. Lynn, K.R., Clevette-Radford, N.A., 1986. Lectins from lattices of Euphorbia and Elaeophorbia species. Phytochemistry 25 (7), 1553–1557. Mishra, V., Sharma, R.S., Yadav, S., Babu, C.R., Singh, T.P., 2004. Purification and characterization of four isoforms of Himalayan mistletoe ribosome-inactivating protein from Viscum album having unique sugar affinity. Arch. Biochem. Biophys 423, 288– 301. Nsimba-Lubaki, M., Peumans, W.J., Carlier, A.R., 1983. Isolation and partial characterization of a lectin from Euphorbia heterophylla seeds. Biochem. J. 215, 141–145. Nsimba-Lubaki, M., Allen, A.K., Peumans, W.J., 1986. Isolation and partial characterization of latex lectins from three species of the genus Euphorbia (Euphorbiaceae). Physiol. Plant. 67, 193–198. Silva Santana, S., Gennari-Cardoso, M.L., Carvalho, F.C., Roque-Barreira, M.C., da Silva Santiago, A., Cerqueira Alvim, F., Priminho Pirovani, C., 2014. Eutirucallin, a RIP-2 type lectin from the latex of Euphorbia tirucalli L. Presents proinflammatory properties. PLOS ONE 9 (2), 1–12. Sharon, N., Lis, H., 1993. Carbohydrates in cell recognition. Sci. Am. 268, 82–89. Seshagirirao, K., Prasad, M.N., 1995. Purification and partial characterization of a lectin from Euphorbia neriifolia latex. Biochem. Mol. Biol. Int. 35, 1199–1204. Souza, M.A., Amâncio-Pereira, F., Barros Cardoso, C.R, Gomes da Silva, A., Gomes Silva, E., Resende Andrade, L., Oliveira Pena, J.D., Lanza, H., Afonso-Cardoso, S.R., 2005. Isolation and partial characterization of a d-galactose-binding lectin from the latex of Synadenium carinatum. Braz. Arch. Biol. Technol. 48, 705–716. Stirpe, F., Barbieri, L., 1986. Ribosome-inactivating proteins up to date. FEBS Lett. 195, 1–8. Stirpe, F., Battelli, M.G., 2006. Ribosome-inactivating proteins: progress and problems. Cell. Mol. Life Sci. 63, 1850–1866. Stirpe, F., Bolognesi, A., Bortolotti, M., Farini, V., Lubelli, P., Pelosi, E., Polito, L., Dozza, B., Strocchi, P., Chambery, A., Parente, A., Barbieri, L., 2007. Characterization of highly toxic type 2 ribosome-inactivating proteins from Adenia lanceolata and Adenia stenodactyla (Passifloraceae). Toxicon 50, 94–105. Stirpe, F., Licastro, F., Morini, M.C., Parente, A., Savino, G., Abbondanza, S., Bolognesi, A., Falasca, A.I., Rossi, C.A., 1993. Purification and partial characterization of mitogenic lectin from the latex of Euphorbia marginata. Biochem. Biophys. Acta 1158, 33–39. Van Damme, J.E., Smeets, K., Torrekens, S., Van Leuven, F., Goldstein, I.J., Peumans, W.J., 1992. The closely related homomeric and heterodimeric mannose-binding lectins from garlic are encoded by one-domain and two-domain lectin genes, respectively. Eur. J. Biochem. 206 (2), 413–420. Van Damme, E.J.M., Peumans, W.J., Barre, A., Rougé, P., 1999a. Plant lectins: a composite of several distinct families of structurally and evolutionary related proteins with diverse biological roles. Crit. Rev. Plant Sci. 17, 575–692. Van Damme, E.J.M., Peumans, W.J., Pusztai, A., Bardocz, S., 1999b. Handbook of Plant Lectins: Properties and Biomedical Applications. John Wiley and Sons, Chichester, England. Wacker, R., Stoeva, S., Betzel, C., Voelter, W., 2005. Complete structure determination of N-acetylgalactosamine-binding mistletoe lectin-3 from Viscum album L. album. J. Peptide Sci. 11, 289–302. Wang, H., Ng, T.B., Ooi, V.E.C., Liu, W.K., 2000. Effects of lectins with different carbohydrate-binding specificities on hepatoma, choriocarcinoma, melanoma and osteosarcoma cell lines. Int. J. Biochem. Cell Biol. 32, 365–372. Ye, W., Nanga, R.P., Kang, C.B., Song, J.H., Song, S.K., Yoon, H.S., 2006. Molecular characterization of the recombinant A-chain of a type II ribosome-inactivating protein (RIP) from Viscum album coloratum and structural basis on its ribosomeinactivating activity and the sugar-binding properties of the B-chain. J. Biochem. Mol. Biol. 39 (5), 560–570. Zhang, G., Sun, J., Wang, H., Ng, T.B., 2010. First isolation and characterization of a novel lectin with potent antitumor activity from Russula mushroom. Phytomedicine 17 (10), 775–781.
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Contents lists available at ScienceDirect
Phytomedicine journal homepage: www.elsevier.com/locate/phymed
Purification and partial characterization of a ribosome-inactivating protein from the latex of Euphorbia trigona Miller with cytotoxic activity toward human cancer cell lines Judith Villanueva a, Luis Manuel Quirós b, Sonia Castañón a,∗
Q1
a b
Neiker-Tecnalia, Biotechnology Department, Arkaute Agrifood Campus, PO Box 46, 01080 Vitoria-Gasteiz, Álava, Spain University Institute of Oncology of Asturias and Department of Functional Biology, University of Oviedo, Julian Claveria s/n, Oviedo 33006, Spain
a r t i c l e
i n f o
Article history: Received 2 January 2015 Revised 10 March 2015 Accepted 6 April 2015 Available online xxx Keywords: Lectin Antitumoral Cytotoxic Hemagglutination Ribosome-inactivating proteins (RIPs)
a b s t r a c t Background: The objective of this study is to investigate the cytotoxic activity of three isolectins purified from the latex of Euphorbia trigona Miller. Hypothesis: Among lectins are the ribosome-inactivating proteins (RIPs), which are potent inhibitors of protein synthesis in cells and in cell-free systems. Results: Three isolectins, ETR1, ETR2 and ETR3, were purified by anion exchange chromatography. Both ETR1 and ETR3 yielded a single band on SDS–PAGE under reducing conditions, corresponding to a molecular weight of 32 g mol−1 , while ETR2 yielded two bands corresponding to 31 and 33 g mol−1 . When non-reducing conditions were used molecular weight decreased, indicating the presence of intrachain disulfide bonds. Sizeexclusion chromatography revealed proteins of apparent molecular weight of 59–63 g mol−1 , suggesting a dimeric nature, with subunits not being held together by disulfide linkage. ETR1, ETR2 and ETR3 hemagglutinated human, sheep and rat erythrocytes and this hemagglutination was specifically inhibited by galactose and its derivatives. The lectins studied were thermostable up to 60 °C and their observed activity was maintained across pH range 5–12. These lectins, from the latex of Euphorbia trigona, are potent inhibitors of eukaryotic protein synthesis in a cell-free system. Flow cytometry analysis revealed the antiproliferative activity of them toward A549, HeLa, H116, HL-60 cell lines. Conclusion: Euphorbia trigona isolectins are RIPs with cytotoxic activity toward human cancer cell lines. © 2015 Published by Elsevier GmbH.
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Introduction
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Lectins are a group of proteins (or glycoproteins) of non-immune origin that bind specifically and reversibly to carbohydrates, resulting in cell agglutination and precipitation (Goldstein et al. 1980). Due to this capacity, lectins are involved in diverse cellular mechanisms (Sharon and Lis 1993). Among the various types of plant lectins are some which pertain to the RIPs. These are toxic N-glycosidases that remove a highly conserved adenine from the 28S ribosomal RNA (rRNA). This depurination inactivates the ribosome and arrests protein synthesis. Type II RIP family is widely represented in plants of different taxonomic
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Abbreviations: RIP(s), ribosome inactivating protein(s); HU, hemagglutination unit; ECA, Euphorbia calcina agglutinin; EDA, Euphorbia dalberi agglutinin; EspA, Euphorbia sp. agglutinin; EML, Euphorbia millii lectin; IC50, concentration at which 50% inhibition is achieved; GalNAc, N-acetyl-d-galactosamine; GlcNAc, N-acetyl-d-glucosamine; PS, phosphatidilserine. ∗ Corresponding author. Tel.: +34 945 121313; fax: +34 945 281422. E-mail address: [email protected] (S. Castañón).
origins such as ricin, viscumin, abrin, volkesin, etc. (for review see Stirpe and Batelli 2006; Van Damme et al. 1998a, 1998b). These proteins consist of two chains, the B chain being responsible for binding to carbohydrates and the A chain, which is an RNA N-glycosidase, for irreversibly inactivating eukaryotic ribosomes. They are particularly interesting because of their cytotoxic activity, determined by the ability of the B chain to bind to galactosyl terminated glycoproteins on the cell surface. This binding permits and facilitates the entry of A chains into cells, where they then exert their enzymatic activity on ribosomes thereby inhibiting protein synthesis. There has, as a result, been considerable interest in type II RIPs due to their potential role in the development of therapeutic agents, preferentially binding to cancer cell membranes or their receptors, causing cytotoxicity, apoptosis and inhibition of tumor growth. Euphorbia trigona Miller is a succulent plant from Africa used as an ornamental and is known as the African milk tree because of its high latex content. Latex is a colloidal suspension which contains an extraordinary array of secondary metabolites and proteins (Agrawal and Konno 2009). Many studies suggest that the latex of the members of Euphorbiaceous is a rich source of lectins, for example
http://dx.doi.org/10.1016/j.phymed.2015.04.006 0944-7113/© 2015 Published by Elsevier GmbH.
Please cite this article as: J. Villanueva et al., Purification and partial characterization of a ribosome-inactivating protein from the latex of Euphorbia trigona Miller with cytotoxic activity towards human cancer cell lines, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2015.04.006
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E. marginata (Stirpe et al. 1993), E. neriifolia (Seshagirirao and Prasad 1995), Synadenium carinatum (Souza et al. 2005). In this work, we report the purification and characterization of three new galactoside-derivatives binding lectins from the latex of Euphorbia trigona. Their RNA N-glicosidase activity was also tested and found to inhibit the proliferation of several human cancer cell lines.
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Materials and methods
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Chemicals and reagents
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HiTrapۛ desalting, Mono Qۛ 5/50 GL and Superoseۛ 12 10/300 GL chromatographic columns were purchased from GE Healthcare. Carbohydrates for hemagglutination inhibition studies (d-glucose, d-fructose, d-galactose, lactose, N-acetyl-d-galactosamine, N-acetyld-glucosamine, xylose, d-raffinose, melibiose, l-arabinose, d-mannose and d-fucose) and size-exclusion standards were obtained from Sigma–Aldrich. Bovine serum albumin, molecular weight standards for SDS–PAGE and Bradford reactive were procured from Bio-Rad. The rabbit reticulocyte lysate system used for protein synthesis inhibition was purchased from Promega and [3 H]-leucine (specific activity 165.2 Ci mmol−1 ) from PerkinElmer. All other chemicals were obtained from commercial sources and were of analytical grade.
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Biological samples
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Plants of Euphorbia trigona Miller were grown in the greenhouse of Neiker-Tecnalia, Alava, Spain. Venous human blood was obtained from the Basque Biobank for Research, Sondika, Spain. Sheep blood was kindly provided by the Department of Animal Production, Neiker-Tecnalia and rat blood by the Department of Functional Biology of the Faculty of Medicine, Oviedo University, Spain. The cytotoxic effect of the purified lectins was studied in different cell lines: HeLa (cervix adenocarcinoma), A549 (lung carcinoma), H116 (colorectal carcinoma), HL-60 (promyelocytic leukemia), HT-29 (colon adenocarcinoma) and NIH-3T3 (mouse fibroblasts), all obtained from American Type Culture Collection
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Purification of Euphorbia trigona lectin
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Latex was collected by repeated incisions in the stems of the plants with sharp blades and diluted in 10 vol. of PBS, pH 7.4. The subsequent operations were performed at 4 °C. The diluted latex was stirred for 3 h. Afterwards, the sample was centrifuged at 70,000 × g for 1 h and the supernatant was dialyzed overnight against the same buffer. The total soluble protein was fractionated by ammonium sulfate precipitation (40–90% saturation). After desalting, the sample was applied to a Mono Q HR column, previously equilibrated with 25 mM Tris–HCl, pH 8.5. The column was washed until the A280 of the effluent was negligible and the lectins were eluted in a stepwise gradient using 98, 123 and 250 mM NaCl. The protein content of the crude extract and the fractionated protein samples was estimated by Bradford assay using BSA as a standard.
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Hemagglutination assays
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Hemagglutination assays were performed using human (AB0 groups), sheep and rat blood in a U-bottomed 96-well microtiter plate. Fifty-microliter aliquots of 2-fold serially diluted lectins were mixed with an equal volume of 3% (v/v) suspension of erythrocytes in saline solution. After incubation at 37 °C for 1 h, the results were determined visually. Hemagglutination units (HU) were expressed as
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the reciprocal of the highest dilution of the lectin showing detectable hemagglutination.
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Effect of temperature and pH on lectin activity
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The influence of temperature and pH were assessed by studying their effect on lectin activity. The protein samples were incubated for 1 h prior to assay at a temperature ranging from 25 to 90 °C. The pH sensitivity of the lectins was established by incubating the samples for 1 h in different buffers: Sodium acetate (pH 3–5), Tris–HCl (pH 7– 8.5), sodium carbonate (pH 10) and KCl/NaOH (pH 12), followed by the determination of their hemagglutinating activity.
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Sugar inhibition of hemagglutination
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Carbohydrate-binding specificity of the lectins was assessed by the ability of each of them to inhibit hemagglutination. Serial 2-fold dilutions of sugars were prepared in saline solution. An equal volume of each lectin containing 4 HU was mixed in each well. The mixtures were incubated for 30 min at room temperature and, afterwards, the hemagglutination assay was performed. Inhibitory activity was determined as the lowest concentration of the carbohydrate required for the complete inhibition of the hemagglutination.
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Electrophoretic analysis and molecular weight determination
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SDS–PAGE was carried out using a 10–15% (w/v) gradient slab gel in the presence or absence of 2-mercaptoethanol. Native-PAGE using a discontinuous buffer system was carried out on a 7.5% (w/v) gel at pH 8.8. The gels were stained with Coomassie Brilliant Blue. Size-exclusion chromatography was used to determine the molecular weight of the native proteins. It was calibrated with protein standards in the range from 12.4 to 200 g mol−1 and equilibrated and eluted with 50 mM Tris–HCl containing 100 mM KCl and 200 mM lactose, pH 7.5. The flow rate was adjusted to 0.4 ml min−1 . Detection was achieved made by UV detector at 280 nm.
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Amino acid sequencing
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Purified proteins were analyzed and sequenced by mass spectrometry at the Proteomics Platform Service of the CIC-Biogune. Peptides with reliable scores were compared with public databases using the BLAST algorithm.
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Protein synthesis inhibition
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The effects of lectins on cell-free protein synthesis were determined with a rabbit reticulocyte lysate, following the manufacturer’s instructions. Biological activity was determined by each lectin’s ability to inhibit incorporation of [3 H]-leucine into proteins. Lectins were analyzed in the range of 0–5 nM. Samples (5 μl) were added to a protein synthesis reaction mixture along with 17.5 μl of rabbit reticulocyte lysate, 0.25 μl of a mixture of all amino acids (minus leucine) at 1 mM, 0.5 μl of RNas in ribonuclease inhibitor and 0.5 μl of luciferase control RNA at 1 μg μl−1 which had previously been denatured for 3 min at 65 °C. The reaction mixture was incubated at 30 °C for 90 min and placed on ice. After this, 5 μl of the reaction mixture was mixed with 95 μl of NaOH 1 M and H2 O2 2% and incubated for 10 min at 37 °C at the end of which the resultant protein was precipitated using 900 μl ice-cold TCA 25%/casamino acids 2% for 30 min on ice. Afterwards, 250 μl of this solution were collected by vacuum filtering in a glass fiber filter, washed with acetone and transferred to vials with scintillation fluid (PerkinElmer). Radioactivity was determined in a Wallac 1409 liquid-scintillation spectrometer. This assay was repeated at least three times.
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Please cite this article as: J. Villanueva et al., Purification and partial characterization of a ribosome-inactivating protein from the latex of Euphorbia trigona Miller with cytotoxic activity towards human cancer cell lines, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2015.04.006
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Cytotoxic activity
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Inhibitory potential of the isolated lectins was tested against six cell lines: HeLa, A549, H116, HL-60, HT-29 (tumoral cell lines) and NIH-3T3 (non-tumoral cell line). These cell lines were maintained in DMEM (Dulbecco modified Eagle’s medium) supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 1 mM sodium piruvate and 1 mM amino acids at 37 °C in a humidified atmosphere of 5% CO2 . In these conditions, 20.000 cells were seeded into a 6-well culture plate and, the following day, the cells were co-incubated with increasing amounts of ETR1, ETR2 and ETR3 lectins for 24 h under the same culture conditions. As negative control we used 25 mM Tris–HCl, pH 8.5. Following these cells were harvested by trypsinization and then resuspended in Annexin V staining solution (containing Annexin V fluorescein and propidium iodide) and incubated for 10 min at room temperature. The samples were analyzed with a Cytomics FC500 (Beckman Coulter).
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Results and discussion Purification of Euphorbia trigona lectin Three lectins were purified by anion-exchange chromatography on a Mono Q column using a stepwise gradient for elution. Lectins are usually purified by affinity chromatography but, in this case, this technique was not resolutive, so we used this strategy. Prior to purification, crude protein extract of the latex from E. trigona was subjected to an ammonium sulfate fractionation to obtain the clarified protein extract. After dialyzing, the fractions with maximum lectin activity (40–90% ammonium sulfate) were employed in the anionexchange chromatography. The absence of lectin activity in the flowthrough indicated that complete adsorption had occurred. Initially a linear gradient was used to elute the bound proteins but this was not sufficiently resolutive as it resulted in overlapping peaks. Thus, in order to improve the separation of the bound lectins, a stepwise gradient was used for the elution. The NaCl concentrations used for this stepwise gradient were selected from the linear gradient elution previously performed and resulted in three absorbance peaks at 98, 123 and 250 mM NaCl (Fig. 1). All the peak fractions tested were able to hemagglutinate human erythrocytes, indicating that lectins were present. The first peak was named ETR1, the second ETR2 and the third ETR3. Those fractions containing pure protein in high concentrations were pooled and concentrated for further studies. In this 0,2
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Activity (H.U.) -2 [NaCl] (10 M)
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Fraction Fig. 1. Anion exchange chromatography of a dialyzed protein fraction from the latex of E. trigona after ammonium sulfate precipitation (40–90%) on a Mono Q. Hemagglutinating Units, H.U. Experimental details are outlined in the text.
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manner, 25% of the activity was recovered. Similarly, it was determined that 15.3% of the soluble protein content of the latex after ammonium sulfate precipitation corresponded to lectins. This percentage is double that of the lectin obtained from the latex of E. marginata (Stirpe et al. 1993) and similar to the percentage from the latex of E. Neriifolia (Seshagirirao and Prasad 1995).
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Characterization
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ETR1 gave a single band in SDS–PAGE under reducing conditions, having a molecular weight of 32 g mol−1 , ETR2 showed two bands of 33 and 31 g mol−1 and ETR3 a single band of 32 g mol−1 (Fig. 2A). However, when non-reducing conditions were used, the result was different: ETR1 gave a single band of 29 g mol−1 , ETR2 gave two bands of 31 and 27 g mol−1 , and ETR3 also gave two, of 29 and 31 g mol−1 along with a minor peak of approximately 27 g mol−1 (Fig. 2A). The lower molecular weight of unreduced samples in comparison with reduced ones suggests the presence of intrachain disulfide bonds, as have been described previously in other lectins as ECA (Euphorbia calcina agglutinin) and EDA (Euphorbia dalberi agglutinin) or Euticurallin (Nsimba-Lubaki et al. 1986; Silva Santana et al. 2014). These molecular sizes are also consistent with the results obtained for other lectins of the Euphorbiaceae family, which are composed by two or more subunits of 28–32 g mol−1 (Nsimba-Lubaki et al. 1986; Silva Santana et al. 2014, Seshagirirao and Prasad 1995; Souza et al. 2005). Most plant lectins are composed of several subunits. For example, Ricin, a type II RIP lectin, is a dimeric protein (Mw 63 g mol−1 ) formed by a 32 g mol−1 chain and another of 34 g mol−1 (Van Damme et al. 1998b). The molecular weight of native ETR1, ETR2 and ETR3 was determined by size-exclusion chromatography. In all cases, a single peak was obtained showing molecular weights of 59 g mol−1 , 66 g mol−1 and 63 g mol−1 , respectively. These results clearly suggest that lectins of the latex from E. trigona are dimers which are not held together by disulfide linkages as happens with ECA, EDA and EspA (Euphorbia sp. agglutinin) (Nsimba-Lubaki et al. 1986). However, there are several lectins from the latex of other Euphobiaceae, for instance, Euticurallin, a type II RIP lectin from the latex of Euphorbia tirucalli L. with at least two 32 g mol−1 subunits joined by disulfide bridges (Silva Santana et al. 2014). The analysis of the isolated proteins by native-PAGE at pH 8.8 demonstrated the purity of the major proteins originally present in the latex (Fig. 2B). Each protein moved to a different position in the gel, indicating the presence of different isoforms with different pIs. The existence of lectin isoforms may be due to variation in oligosaccharide chains (Hayes and Goldstein 1974) or may stem from changes in the amino acid sequence, resulting in different charged proteins (Van Damme et al. 1992). Most RIPs isolated from the same plant are isomers with similar primary protein structure. Purified lectins from E. trigona showed strong hemagglutination of erythrocytes from different species (human, sheep and rat). The isoforms varied in their hemagglutination potential, expressed in terms of HU. ETR1, ETR2 and ETR3 each had strong hemagglutination ability against all human blood groups (AB0), showing no specificity for one in particular as happens in other previously described latex lectins of other Euphorbiaceae (Lynn and Clevette-Radford 1986; Souza et al. 2005). 0.78 μg ml−1 of any of the ETR lectins being necessary to hemagglutinate them. With respect to erythrocytes of other species: ETR1 required 12.5 μg ml−1 to agglutinate sheep erythrocytes and 3.12 μg ml−1 to hemagglutinate rat blood, ETR2 needed 1.56 and 6.25 μg ml−1 to hemagglutinate sheep and rat erythrocytes and ETR3 1.56 and 3.12 μg ml−1 , respectively. These variations in hemagglutination potential of ETR isoforms suggest that they might also differ in their sugar specificities and cell recognition. To ascertain the stability of ETR isoforms under different physicochemical conditions, their biological properties in terms of lectin
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Fig. 2. Gel electrophoresis of the proteins purified from E. trigona. (A) Purified proteins analyzed by SDS–PAGE under both reducing and non-reducing conditions. Lanes 1, 2, 3 show ETR1, ETR2, ETR3, respectively, under non-reducing conditions. Lanes 4, 5, 6 show the same proteins under reducing conditions. M: molecular weight marker. (B) Purified proteins analyzed by native PAGE. Lanes 1, 2, 3 show ETR1, ETR2, ETR3, respectively. FS: dialyzed protein fraction after ammonium sulfate precipitation.
A
100
80 HU (%)
80 HU (%)
B
100
60 40
ETR 1 ETR 2
20 25
35
45
40
ETR 1 ETR 2
20
ETR 3
0
60
ETR 3
0 55
65
75
85
4
6
Tª (ºC)
8
10
12
pH
Fig. 3. Influence of physicochemical parameters. (A) Effect of temperature and (B) pH on the hemagglutinating activity of E. trigona lectin isoforms. Percentage activity was calculated considering maximum measured activity to be 100%. Hemagglutination units, H.U.
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activity were analyzed at a range of temperatures between 25 and 90 °C and at pHs ranging from 5 to 12, for 1 h. All three lectins isoforms from E. trigona were heat stable and retained complete biological activity up to 60 °C. Above that temperature, lectin activity was gradually lost and almost totally inactivated at 90 °C (Fig. 3A). These results are comparable to those obtained for other lectins (Mishra et al. 2004; Zhang et al. 2010). In addition, lectin activity was unchanged in the pH range 5–12 (Fig. 3B), showing ETRs to be more resistant to pH than other latex lectins of Euphorbiaceae which rapidly lost their activity at pH values above 10 (Nsimba-Lubaki et al. 1986). The hemagglutination activity of all three purified lectins was inhibited by d-galactose and N-acetyl-d-galactosamine (GalNAc), as happens with other lectins derived from Euphorbiaceae (Silva Santana et al. 2014, Stirpe et al. 1993) and with most B chains of the type II RIPs, such as ricin and viscumin (Van Damme et al. 1998a, 1998b). The activity was inhibited to a lesser degree by disaccharides and trisaccharides like lactose, melibiose and raffinose, all of which are saccharides with a terminal non-reducing galactose (Table 1). The hemagglutination produced by ETR1 was inhibited by 0.73 mM galactose or GalNAc. That produced by ETR2 was inhibited by 1.46 mM galactose or 2.92 mMGalNAc and that by ETR3 by 1.46 mM for each sugar. This small difference in the concentration necessary to inhibit hemagglutination could be due to the presence of slight differences in cell recognition. Besides the aforementioned carbohydrates, we also observed inhibition of the hemagglutination when we incubated the lectins with fucose. There was not, however, any inhibitory effect using sugars such as glucose, fructose or N-acetyl-d-glucosamine
Table 1 Inhibition by sugar of hemagglutinating activity of E. trigona lectins. Carbohydrate
M.I.C.a (mM) ETRI 1
Glucose Fructose Galactose Lactose N-Acetylgalactosamine N-Acetylglucosamine Xylose Raffinose Melibiose Arabinose Mannose Fucose Heparin Chondroitin sulfate A Chondroitin sulfate B Chondroitin sulfate C
b
N.I. N.I. 073 219 073 N.I. N.I. 583 583 N.I. N.I. 875 N.I. N.I. N.I. N.I.
ETRI 2
ETRI 3
N.I. N.I. 146 583 292 N.I. N.I. 2333 1167 N.I. N.I. 4666 N.I. N.I. N.I. N.I.
N.I. N.I. 146 583 146 N.I. N.I. 2333 1167 N.I. N.I. 2333 N.I. N.I. N.I. N.I.
a M.I.C.: minimum concentration required for 50% inhibition of hemagglutination. b N.I.: no inhibition.
(GlcNAc). This suggests that the axial C4 hydroxyl position is a critical binding region, as is the case with EML (Euphorbia milii lectin) (Irazoqui et al. 2005). Contrary to what occurs with the latex lectins from other Euphorbiaceous, d-fucose (6-deoxy-d-galactose) showed
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Table 2 Results of the sequence analysis of peptides of ETR1, ETR2 and ETR3 obtained by mass spectrometry. Number represents different peptides appearing in each protein. Peptides with the same number are homologous. Conserved amino acid residues are underlined and amino acid residues of the active site are detailed in bold. Number
Peptide ETR1 (29 kDa)
1
VLVLTCGLGPSSQR
2 3 4
ALLVYPFTGNPNQK QQWYFYPDGTLGVPK VPLVYDETGGGPTLR
Number
Peptide ETR2 (31 kDa)
2 5
LALVYPFTGNPNQK LVMDVAQSKPSLK
6
DPVLTCGLGALSQR
Number
Peptide ETR2 (27 kDa)
6
DPVLTCGLGALSQR
Number
Peptides ETR3 (29 kDa)
1
VVLTCGLGPSSQR
2 3 5
LALVYPFTGNPNQK QQWYFYPDGTLGVPK VGEDVAQSDLALK
Identity
Protein
Species
Access number
RIP Type II RIP precursor Ricin-agglutinin Type II RIP Protein with lectin domain Ricin precursor
Panax ginseng Sambucus nigra Ricinus communis Momordica charantia Oryza sativa Ricinus communis
ABG76785 AAC49754 EEF30192 BAH05018 ABA97653 8287993 RCOM_2159910
Identity
Protein
Species
Access number
9/14 11/12 9/12 8/14 7/14
Ricin-agglutinin Precursor lectina Agglutinin I RIP Lectin
Ricinus communis Viscum album Ricinus communis Panax ginseng Medicago truncatula
EEF30192 AY377892 AAB22584 ABG76785 ABN08398
Identity
Protein
Species
Access number
8/14 7/14
RIP Lectin
Panax ginseng Medicago truncatula
ABG76785 ABN08398
Identity
Protein
Species
Access number
RIP Type II RIP precursor Ricin-agglutinin Type II RIP Agglutinin I
Panax ginseng Sambucus nigra Ricinus communis Momordica charantia Ricinus communis
ABG76785 AAC49754 EEF30192 BAH05018 AAB22584
8/14 8/14 9/14 8/16 8/15 7/15
7/14 7/14 9/14 8/16 9/12
Fig. 4. Effects of the lectins studied on protein synthesis. (A) Protein synthesis inhibition on cell-free system produced by purified lectin isoforms ETR1, ETR2, ETR3. (B) Comparison of the inhibition rate of E. trigona lectins with other cytotoxic type II RIP proteins from the literature.
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less inhibition of hemagglutination than galactose, indicating the importance of the C6 hydroxyl for binding, especially for ETR2 and ETR3, which seems to have no influence in other Euphorbiaceous lectins (Nsimba-Lubaki et al. 1986). ETR lectins have 2- to 5-fold greater affinity for lactose than for raffinose or melibiose, which suggests they may be more specific to the β linkage than to the α (Nsimba-Lubaki et al. 1986; Seshagirirao and Prasad 1995). No inhibitory effect was observed using other sugars such as xylose, arabinose or mannose. Amino acid sequencing We obtained partial sequences of different proteins after the mass spectrometry analysis of ETR1, ETR2 and ETR3. Six of these peptides showed 50–75% identity with various plant lectins. All the sequenced peptides could be aligned to the B chain of type II RIPs (Table 2). Ye et al. (2006) highlighted that the B chains of type II RIPs show highly
conserved sequences and, therefore, in their 3D structure. However, their sugar binding specificity varies. The sequences obtained from the three purified proteins exhibited a high degree of similarity (indicated by the same number in Table 2), which supports the idea that they are isoforms. According to Wacker et al. (2005), among the most conserved domains in the B chain, we found the amino acids that comprise the two active sites, which are, firstly, D22, Q35, S37, N46, and Q47, and secondly, D234, I246, Y248, N255, and Q256. A detailed comparison between the carbohydrate-binding site of ricin, viscumin and volkensin and the sequences obtained here indicate that the peptide identified as number 5 in Table 2 contained amino acid D234 which is part of the carbohydrate-binding site. Likewise, peptide 2 in Table 2 contains the amino acids Y248, N255 and Q256 from the same binding site (Chambery et al. 2004; Wacker et al. 2005). Thus, it is suggested that the purified proteins of the latex from E. trigona are lectins belonging to the type II RIP family.
Please cite this article as: J. Villanueva et al., Purification and partial characterization of a ribosome-inactivating protein from the latex of Euphorbia trigona Miller with cytotoxic activity towards human cancer cell lines, Phytomedicine (2015), http://dx.doi.org/10.1016/j.phymed.2015.04.006
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HeLa A549 H116 HL-60 HT-29 NIH-3T3
IC50 a (nM) ETRI 1
ETRI 2
ETRI 3
11 32 40 75 N.I.b 875
12 32 18 44 N.I. 480
10 18 21 44 N.I. 480
a
IC50 : Minimum concentration of lectin required for causing 50% of cell death. b N.I.: IC50 is not reached at the tested protein concentration.
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Inhibition of protein synthesis in cell-free system
311
Some galactose-binding lectins, which are type II RIPs, have been reported to inhibit protein synthesis in cell-free systems (Barbieri et al. 1979). All three lectins purified here significantly inhibited protein synthesis in a cell-free system (Fig. 4A). ETR2 was the most active isoform: this lectin showed an IC50 value (concentration at which 50% inhibition is achieved) at 7.5 nM. ETR3 inhibited protein synthesis in a more moderate way: 50% inhibition required 12 nM and ETR1 was the least active isoform with its IC50 value was reached at 16 nM. According to the literature, very toxic lectins such as ricin, abrin and volkensin have an IC50 ranging from 40 to 85 nM (Stirpe and Barbieri 1986). These concentrations are higher than found in the
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current study so it places ETR1, ETR2 and ETR3 among the most toxic type II RIPs (Fig. 4B). In contrast, there are some lectins from Euphorbiaceae which do not inhibit protein synthesis, for example, E. heterophylla seed lectin (Nsimba-Lubaki et al. 1983) and E. neriifolia latex lectin (Seshagirirao and Prasad 1995). There are lectins from other Euphorbiaceous species in the literature but biological assays of specific inhibition of ribosomal activity are required to conduct a thorough analysis and compare them. These results show that lectins from the latex of E. trigona possess similar chemical composition in several aspects to that obtained from other Euphorbia species (stability under different physico-chemical conditions, the sugar specificity, lack of specificity for blood groups) with the exceptions mentioned earlier. However, ETR lectins are different from those due to the presence of isolectins in the latex, the amino acid composition and the ability to inhibit protein synthesis in rabbit reticulocytes.
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Cytotoxicity
338
Some plant lectins are highly cytotoxic and are able to induce apoptosis (Kim et al. 1993). We evaluated the cytotoxic effect of the three purified proteins on different cell lines: HeLa, A549, H116, HL60, HT-29 (tumoral cell lines) and NIH-3T3 (non-tumoral cell line) (Table 3). Cell viability was determined by flow-cytometry. A dosedependent decrease in cell viability was observed with increasing concentration of each sample. Using ETR1 as an example, Fig. 5 demonstrates that after Annexin V-FITC and propidium iodide (PI)
Fig. 5. Flow cytometry analysis of HL-60 cells after treatment with ETR1 for 24 h, followed by staining with Annexin V-FITC.
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staining, the HL-60 cells in the lower left quadrant of the profile shifted toward the lower right quadrant as ETR1 increased. This shift is indicative of phosphatidilserine (PS) externalization, suggesting that HL-60 cells were undergoing the early stage of apoptosis. Cells located at the upper right quadrant also increased with increasing ETR1, indicating that some HL-60 cells had entered the late stage of apoptosis. Authors, using the same technique to evaluate antiproliferative effects, obtained similar results with lectins at μM concentrations (Chan and Ng 2013). But some authors determine cytotoxicity in terms of inhibition of protein synthesis in whole cells (Stirpe and Barbieri 1986). In this way, the IC50 value for ricin was established at 1 pM, for abrin and at 3.4 pM and for viscumin at 8 pM when tested against HeLa cells. The difference between the order of magnitude of these IC50 values is evident. This difference could be attributed to the fact that inhibition of protein synthesis is an event which occurs prior to loss of cell viability (Stirpe et al. 2007) and therefore, the minimal concentration necessary to inhibit protein synthesis, as considered in other studies, is far less than that required to inhibit cell viability, the focus of this study. All the isoforms purified here were inactive against the HT-29 cell line at the maximum concentration studied (100 nM). These results concur with the idea of a particular lectin being able to show more toxicity with respect to certain cells than to others (Wang et al. 2000). The reason for these differences is not known exactly, but is thought to be related to the binding of the proteins and their entry into the cells (Stirpe and Batelli 2006). Malignant cells have carbohydrate alterations, and it is at this point that lectin-mediated cytotoxicity might play a role in antitumor defense (Gorelik et al. 2001). It has been speculated that the selective cytotoxicity of lectins against tumoral cells is determined by their differing affinity for specific sugars, the recognition and binding of the B chain to sugars on the cell surface which is essential for the entry of the cytotoxic A chain (Ye et al. 2006). It is of note that 875 nM of ETR1, ETR2 or ETR3 were necessary to achieve the IC50 value in non-tumoral cells (NIH-3T3), almost two orders of magnitude higher than with HeLa cells (10–12 nM). This accords with the work of Lin et al. (1970), who found that certain tumoral cells were more sensitive to type II RIPs than normal cells.
386
Conclusions
387
393
In this study, three lectins (isoforms) were purified from the latex of Euphorbia trigona by anion exchange chromatography. These lectins bind specifically to galactose-derivative sugars and inhibit protein synthesis in a cell-free system which strongly suggests that the purified lectins belong to the type II RIP family and situates ETR1, ETR2 and ETR3 among the most toxic. These lectins also show remarkable cytotoxic activity against certain tumoral cell lines.
394
Conflict of interest
395 397
We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
398
Acknowledgments
399
This work was financially supported by the Department of Environment, Land Planning, Agriculture and Fisheries of the Basque Government. We are grateful to Dr. Marcos Garcia Ocaña for sharing his expertise with human cell culture and his skilled technical assistance. The University Institute of Oncology of Asturias is supported by Obra Social Cajastur, Asturias, Spain.
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