Isolation and characterization of four type 2 ribosome inactivating pulchellin isoforms from Abrus pulchellus seeds Priscila V. Castilho 1,2 , Leandro S. Goto 2 , Lynne M. Roberts 3 and Ana Paula U. Arau ´ jo 1,2 1 Programa de Po ´ s-graduac¸a˜o em Gene ´ tica e Evoluc¸a˜o, Universidade Federal de Sa˜o Carlos, Brazil 2 Instituto de Fı ´ sica de Sa˜o Carlos, Universidade de Sa˜o Paulo, Sa˜o Carlos, Brazil 3 Department of Biological Sciences, University of Warwick, Coventry, UK Ribosome-inactivating proteins (RIPs; rRNA N-glyco- sidases; EC 3.2.2.22) are found predominantly in plants but they may also occur in fungi and bacteria [1]. Collectively, unless mutated, they are all rRNA- specific N-glycosidases capable of selectively cleaving a glycosidic bond to release an adenine within the uni- versally conserved sarcin ⁄ ricin loop of the large rRNA in 60S ribosomal subunits [2]. This modification pre- vents the binding of elongation factors and thereby irreversibly inhibits protein synthesis in eukaryotic cells. Despite this common activity, RIPs can vary in their physical properties and cellular effects [3]. Currently, RIPs are divided into three groups. Type 1 RIPs comprise a single catalytically active sub- unit of 25 ± 30 kDa, whereas type 3 RIPs consist of an amino-terminal domain, resembling type 1 RIPs, linked to a carboxyl-terminal domain with unknown function [4]. In contrast, type 2 RIPs contain at least one ribosome inactivating A-chain and a correspond- ing number of carbohydrate-binding B-chains, with the latter generally showing a preference for b1-4 linked galactosides [3]. It follows that, although type 1 and type 2 RIPs are active against ribosomes in vitro, only the type 2 proteins are cytotoxic due to the presence of a B-chain that mediates surface binding and entry of holotoxin into susceptible cells. From studies of the biosynthesis of type 2 RIPs in their producing tissues, it is apparent that both polypeptides are made in cor- rect stoichiometry by being derived from a single pre- cursor through the excision of a intervening peptide sequence [5]. The two polypeptides remain covalently joined, however, by a disulfide bridge between cysteine Keywords Abrus pulchellus; characterization; cloning; isoforms; ribosome-inactivating protein Correspondence A. P. U. Arau ´ jo, Grupo de Biofı ´ sica Molecular, IFSC, PO Box 369, Zip 13560-970, Sa˜o Carlos, Brazil Fax: +55 16 33715381 Tel: +55 16 33739834 E-mail: anapaula@ifsc.sc.usp.br (Received 14 November 2007, revised 11 December 2007, accepted 20 December 2007) doi:10.1111/j.1742-4658.2008.06258.x Abrus pulchellus seeds contain at least seven closely related and highly toxic type 2 ribosome-inactivating pulchellins, each consisting of a toxic A-chain linked to a sugar binding B-chain. In the present study, four pulchellin isoforms (termed P I, P II, P III and P IV) were isolated by affinity, ion exchange and chromatofocusing chromatographies, and investigated with respect to toxicity and sugar binding specificity. Half maximal inhibitory concentration and median lethal dose values indicate that P I and P II have similar toxicities and that both are more toxic to cultured HeLa cells and mice than P III and P IV. Interestingly, the secondary structural character- istics and sugar binding properties of the respective pairs of isoforms corre- late well with the two toxicity levels, in that P I ⁄ P II and P III ⁄ P IV form two specific subgroups. From the deduced amino acids sequences of the four isoforms, it is clear that the highest similarity within each subgroup is found to occur within domain 2 of the B-chains, suggesting that the disparity in toxicity levels might be attributed to subtle differences in B-chain-mediated cell surface interactions that precede and determine toxin uptake pathways. Abbreviations GalNAc, N-acetylgalactosamine; IC 50 , half maximal inhibitory concentration; LD 50 , median lethal dose; P I, pulchellin isoform I; P II, pulchellin isoform II; P III, pulchellin isoform III; P IV, pulchellin isoform IV; RIP, ribosome-inactivating protein. 948 FEBS Journal 275 (2008) 948–959 ª 2008 The Authors Journal compilation ª 2008 FEBS residues at the C-terminal of the A-chain and the N-terminal of the B-chain. Overall, relatively few type 2 RIPs are known [6]. Ricin and abrin (from Ricinus communis and Abrus precatorius seeds, respectively) were recognized more than a century ago, with others (e.g. mistletoe lectin I [viscumin] from Viscum album [7], modeccin [8] and volkensin [9] from the roots of Adenia digitata and Adenia volkensii, respectively, and pulchellin from Abrus pulchellus seeds [10,11]) being discovered within the last 30 years. The greatest number of RIPs have been found in the Caryophyllaceae, Sambucaceae, Cucurbitaceae, Euphorbiaceae, Phytolaccaceae and Poaceae [1]. Although many are potentially useful in agriculture and medicine because of their antiviral properties [12] and cell killing characteristics (e.g. in ‘immunotoxins’) [13], the complete distribution map, mode of cell entry ⁄ action and the role(s) of RIPs in nature remain only partly understood. Plants commonly produce several RIP isoforms encoded by multigene families that could possess adap- tations related to their specific role in plant tissues [6]. Therefore, widening our knowledge of the occurrence, structural properties and biological functions of RIPs will contribute to an understanding of their role(s) in vivo. Abrus pulchellus tenuiflorus (Leguminosae-Papi- lionoideae) seeds contain a highly toxic type 2 RIP named pulchellin. It exhibits specificity for galactose and galactose-containing structures, can agglutinate human and rabbit erythrocytes, and kills mice and the microcrustacean Artemia salina at very low concentra- tions [10]. Similar to the RIP in A. precatorius seeds [14], this toxic activity is presented by a mixture of clo- sely related isoforms. In the present study, four pulchel- lin isoforms were isolated, and their amino acids sequences deduced by cDNA cloning and verified by MS. Half maximal inhibitory concentration (IC 50 ) and median lethal dose (LD 50 ) values from HeLa cells and mice divided them into two subgroups: the more toxic forms (P I and P II) and the less toxic forms (P III and P IV). In similar pairwise combinations, their interac- tion with specific sugars was also shown to differ. From a comparison of deduced amino acid sequences within each subgroup, it is striking that the members of each show closest identity in domain 2 of the B-chain. The potential implications of this are discussed. Results Nomenclature of the toxic pulchellin lectins The abbreviation P is followed by the Roman numer- als I, II, III and IV and refers to each pulchellin isoform (P I, P II, P II and P IV). The A-chain of P II was formerly cloned and named recombinant pulchel- lin A-chain [15]. The heterologous expression and refolding of a recombinant pulchellin binding chain was previously reported [16], although this recombi- nant pulchellin binding chain does not correspond to any of the four B-chains presented here. Purification of four pulchellin isoforms from A. pulchellus seeds Using a combination of affinity, ion exchange and chromatofocusing chromatography, four pulchellin isoforms were isolated from A. pulchellus seeds. The protein eluting from an affinity column with lactose suggested protein homogeneity. However, isoeletric focusing revealed multiple bands (data not shown), indicating the presence of related isoforms in the affin- ity-purified preparation. The very distinct differences in isoelectric points suggested that the ion exchange chromatography could be used for the separation of the various isoforms. Using an anion exchanger, four peaks were resolved (Fig. 1A) and proteins were iso- lated. Denaturating gels revealed pulchellins of approx- imately 62 kDa, which, upon reduction, showed a pattern of two bands of approximately 28 and 34 kDa, related to A and B-chains respectively (Fig. 1B). The slight differences in the migration pattern of the A-chains is possibly attributable to glycosylation differences. Lanes 5 and 9 (Fig. 1B) relate to the peak indicated by an asterisk (Fig. 1A) and showed hetero- geneity, which was further confirmed in LC-MS ⁄ MS assays. Although samples from the asterisked peak in Fig. 1A displayed hemagglutination and toxicity toward mice (data not shown), additional efforts to cleanly isolate the isoform were not successful and further characterization was abandoned. A chromato- focusing step was included to separate the P III and P IV isoforms from the eluate P III ⁄ P IV (Fig. 1C). Isoelectric focusing gave pI of 5.8, 5.7, 5.5 and 5.2 for the four isoforms respectively. Secondary structure of the pulchellin isoforms and melting temperature CD-spectral analyses were performed as described in the Experimental procedures. As can be seen from Fig. 2, the far-UV CD spectra of the pulchellin iso- forms suggest only subtle differences in the content of secondary structure, which was confirmed by the spec- tral deconvolution using cdpro software. Thermal sta- bility was also monitored by CD, following changes in each spectrum with increasing temperature. The P. V. Castilho et al. Characterization of four pulchellin isoforms FEBS Journal 275 (2008) 948–959 ª 2008 The Authors Journal compilation ª 2008 FEBS 949 predicted content of secondary structure and melting temperatures found for the four isoforms are given in Table 1. Sequence comparison of pulchellins from A. pulchellus seeds Using RT-PCR and a primer set, full length cDNA clones were prepared and sequenced with primer walk- ers as detailed in the Experimental procedures. Accord- ing to the extent of similarity, seven different sequences were found amongst all analyzed clones. Nanoelectro- spray LC-MS ⁄ MS was also carried out on the individ- ual proteins to demonstrate correspondence between each cloned sequence and a native pulchellin isolated from mature seeds. To generate samples for mass anal- ysis, intact proteins were isolated by chromatographic 0 1020304050 0 50 100 150 200 250 300 * P I Elution volume (mL) Absorbance in 280 nm (a.u.) 0 20 40 60 80 100 % of buffer B 66 45 30 20 kDa B-chain A-chain 0 5 10 15 20 25 0 20 40 60 80 100 0 20 40 60 80 100 P IV P III % of buffer B Absorbance at 280 nm (a.u.) Elution volume (mL) C B A Fig. 1. Mono Q elution profile of pulchellin isoforms. (A) The four isoforms were eluted with a linear gradient of 0–20% 1 M NaCl in 20 m M Tris–HCl, pH 8, for 45 min (dashed line) at a flow rate of 1 mLÆmin )1 . The peaks referring to each isoform are indicated by arrows. The asterisk indicates the peak containing a mixture of other isoforms. (B) Gel visualization of proteins eluted. Lane 1, molecular weight markers. Numbers on the left indicate the M r values of the standards in thousands. Lanes 2–9, 5 lg of peak P I, P II, P III ⁄ P IV and a mixture of other isoforms (*), respec- tively, in the presence (lanes 2–5) or absence (lanes 6–9) of 2-mercaptoethanol. (C) Elution profile of P III and P IV Chromato- focusing chromatography of the P III ⁄ P IV peak previously iso- lated from the Mono Q column. Samples were dialyzed against 10 m M sodium phosphate buffer, pH 7.0. The column was simi- larly equilibrated and P III and P IV were separated by a linear gradient (dashed line) of 10 m M sodium phosphate buffer, pH 5.8 from 0–100% for 20 min, holding for 5 min in 100% buffer B. Flow rate = 1 mLÆmin )1 . The peaks relating to each isoform are indicated by arrows. 200 210 220 230 240 250 –4 –3 –2 –1 0 1 2 3 L·Mol –1 ·cm –1 ) Wavelen g th (nm) Fig. 2. Circular dichroism spectra of P I, P II, P III and P IV. CD spectra of P I (solid), P II (dash), P III (dot) and P IV (dash dot) were measured in the far-UV range (195–250 nm) in 1 mm path length quartz cuvettes and recorded as an average of 16 scans. CD spec- tra were measured in protein solution of 0.125 mgÆmL )1 (Tris 20 m M,pH8,10mM NaCl added). Table 1. Secondary structure content (expressed as %) and melt- ing temperatures found for P I, P II, P III and P IV. Secondary struc- ture values were obtained by the spectral deconvolution using CDPRO software. For all deconvolutions, rmsd values were less than 1. The melting temperatures were calculated based on CD thermal scans (at 232 nm) of the proteins. Secondary structure content (%) P I P II P III P IV Helix 13 12 10 16 b sheet 32 32 30 31 Turn 22 23 24 20 Unordered 33 33 36 33 Melting temperatures (°C) 65.1 63.9 61.7 60.9 Characterization of four pulchellin isoforms P. V. Castilho et al. 950 FEBS Journal 275 (2008) 948–959 ª 2008 The Authors Journal compilation ª 2008 FEBS methods previously described and subjected to tryptic digestion. The acquired masses were then compared with those deduced from peptide sequences encoded by the seven cDNA clones. The deduced precursor pro- teins (prepropulchellins) from the cDNA clones, which were found to correspond with the isolated mature P I, P II, P III and P IV isoforms, are shown in Fig. 3. Stretches of sequence that matched the calculated masses obtained by LC-MS ⁄ MS are underlined. For example, our results showed that the first ten residues of the mature proteins (EDPIKFTTEG) were the same for P I, P III and P IV, but P II differs in that it con- tains an additional arginine as the third residue and has glutamine instead of lysine as the sixth residue: ED- RPIEFTTE. LC-MS ⁄ MS analysis of a tryptic digest of mature P II revealed a peptide mass compatible with a Fig. 3. Deduced amino acid sequences from the cDNA clones of P I, P II, P III and P IV aligned to abrin-a (pdb 1ABR), ricin (pdb 2AAI) and mistletoe lectin I (pdb 1CE7). The peptides selected by the PROTEINLYNX 2.0 software are underlined. As a databank, the program used the seven amino acids sequences deduced from seven pulchellin cDNA clones that contained the immature precursors. The signal peptides were predicted based on the program SIGNAL P. The amino acids numbers were based on the mature proteins. Conserved amino acids are highlighted in gray, conserved residues only amongst pulchellin isoforms are shown in bold. Residues involved in the active site cleft, pre- dicted by homology to abrin and ricin, are indicated by an asterisk. Glycosylation sites have a black background and residues forming the two carbohydrate-binding sites, first (Æ) and second (:), predicted by homology to mistletoe, abrin and ricin [17], are boxed. Ten cysteines that form one interchain and four intrachain disulfide bonds are marked by (^). Dashes denote gaps introduced to obtain maximal homology. P. V. Castilho et al. Characterization of four pulchellin isoforms FEBS Journal 275 (2008) 948–959 ª 2008 The Authors Journal compilation ª 2008 FEBS 951 fragment containing these changes (underlined at the N-terminus of P II). Overall, the four isoforms precursors have 562 (P I), 563 (P II) and 561 (P III and P IV) aminoacyl resi- dues. The protein outside this family to which they showed the highest amino acid identity was abrin, at approximately 94%. Besides abrin, the pulchellin iso- forms were also compared with ricin and mistletoe lec- tin I (Fig. 3), with which they showed approximately 47% amino acid identity to both sequences. The respective A-chains contain 251 (P II) and 250 (P I, P III and P IV) amino acids. P I and P IV A-chains have two N-glycosylation sites, whereas P II and P III only one. In the four pulchellin A-chains, the residues involved in the active site cleft are the same as in abrin and ricin A-chains. This suggests that the catalytic reaction is exactly the same. The sugar binding pulchellin B-chains are 264 (P I and P II) or 263 (P III and P IV) amino acids in length and contain two N-glycosylation sites. Soler et al. [17] defined two homologous carbohydrate binding sites that were shared in mistletoe lectin I, ricin-d and abrin-a B-chains. Based on these previously published observa- tions, we predict residues comprising the two sugar binding pockets in the pulchellins (Fig. 3). In order to compare the similarity of the A- and B-chains of the four isoforms, a pairwise alignment was performed and the values of identity expressed in Fig. 3. (Continued). Characterization of four pulchellin isoforms P. V. Castilho et al. 952 FEBS Journal 275 (2008) 948–959 ª 2008 The Authors Journal compilation ª 2008 FEBS percentage are shown in Table 2. The B-chain domains were predicted by comparison with ricin domains (pdb 2aai). Domains are defined by a repeating pattern of disulfide-bonded loops in each half of the polypeptide, analogous to those described for the ricin B-chain, which suggested that this lectin arose as a product of gene duplication [18]. Domain 1 comprises residues 251–387 (P I, P III and P IV) or 252––388 (P II), and domain 2 comprises 388–514 (P I), 389–515 (P II) or 388–513 (P III and P IV). In vivo toxicity The addition of pulchellin isoforms to cultures of HeLa cells resulted in high inhibition of protein syn- thesis (Fig. 4). The IC 50 values showed that P I and P II have similar toxicity [21.7 ngÆmL )1 (0.375 nm) and 22.7 ngÆmL )1 (0.391 nm), respectively] and are approxi- mately five-fold more toxic then the others [101.9 ngÆmL )1 (1.76 nm) for P III and 98.4 ngÆmL )1 (1.7 nm) for P IV]. LD 50 experiments also showed vari- ability in the toxicity to mice, with the most potent toxin being P II (15 lgÆkg )1 ), followed by P I (25 lgÆkg )1 ), P IV (60 lgÆkg )1 ) and P III (70 lgÆkg )1 ). These results indicate that the pulchellin isoforms are highly toxic, but not as much as mistletoe lectin (LD 50 5–10 lgÆkg )1 ) [19], ricin (IC 50 0.001 nm and LD 50 2.6 lgÆkg )1 ) [20] and abrin (IC 50 0.0037 nm and LD 50 0.56 lgÆkg )1 ) [21]. Agglutination and carbohydrate-binding of the B-chains It has been observed on several occasions that different type 2 RIPs from a single plant differ from each other with respect to their agglutination activity and ⁄ or spec- ificity [22,23]. To check whether this also holds true for pulchellin isoforms, the agglutination properties and carbohydrate-binding affinity were studied in some detail. The pulchellin isoforms were examined for their hemagglutination potential using blood of three spe- cies: human (types A + ,B + and O + ), rabbit and horse where they showed blood group specificity and distinct hemagglutination activity. P I and P II promoted hem- agglutination of human erythrocytes at 22.5 and 27.5 ngÆmL )1 , respectively. Although P I showed only activity towards human erythrocytes, P II was able to agglutinate rabbit (27.5 ngÆmL )1 ) and horse (41.7 ngÆmL )1 ) erythrocytes. P III and P IV aggluti- nated only rabbit blood (18.5 ngÆmL )1 and 12.3 ngÆmL )1 , respectively). To determine their carbohydrate binding specificity, a series of hemagglutination inhibition assays were carried out using 14 sugars of three classes. Whereas agglutination was inhibited by galactose and its deriva- tives [such as N-acetylgalactosamine (GalNAc), methyl-a-d-galactopyranoside], it was evident that, at doses up to 100 mm, glucose, mannose, a-methylman- noside, fucose, maltose, xylose and saccharose did not inhibit agglutination (Table 3). All four pulchellins were shown to interact with ga- lactosides, although the minimum sugar concentration that promoted inhibition of hemagglutination varied. The failure to bind glucose, mannose, a-methylmanno- side, fucose, maltose, xylose and saccharose shows that an axial hydroxyl group at C4 is not only an impor- tant binding group for the lectin, but also that a reversed configuration at this position might prevent sugar recognition. P I and P II were able to inhibit hemagglutination in the presence of GalNAc whereas Table 2. Identity of pulchellin isoforms in a pairwise alignments. Values are expressed as a percentage (%). The B-chain domains were defined by comparison with ricin B-chain (PDB: 2aai). Domain 1 comprises residues 251–387 (P I, P III and P IV) or 252–388 (P II), and domain 2 comprises residues 388–514 (P I), 389–515 (P II) or 388–513 (P III and P IV). A-chain B-chain Domain 1 Domain 2 PI· P II 77.6 79.4 100 P III · P IV 79.2 83.8 99.2 PI· P III 79.2 77.2 78.7 PI· P IV 100 93.4 79.5 PII· P III 98.4 89 78.7 PII· P IV 77.6 72.8 79.5 5 15 25 35 45 55 65 75 85 95 0.1 1 10 100 1000 [toxin] (ng·mL –1 ) Protein synthesis (% of control) Fig. 4. Inhibition of protein synthesis in HeLa cells. Each isoform was diluted serially in DMEM ⁄ fetal bovine serum and added to HeLa cells at the concentrations shown. The incorporation of [ 35 S]methionine into new cellular proteins was subsequently deter- mined as described in the Experimental procedures. Each value is the mean for triplicate samples. h,PI; , P II; s, P III; d, P IV. P. V. Castilho et al. Characterization of four pulchellin isoforms FEBS Journal 275 (2008) 948–959 ª 2008 The Authors Journal compilation ª 2008 FEBS 953 P III and P IV did not. The hemagglutination inhibi- tion caused by methyl- a -d-galactopyranoside suggests that the -OH on C2, C3 and C4, which have the same configuration as those in galactose and lactose, are responsible for the strong interaction with the iso- forms. Interestingly, P II was the only isoform with affinity for rhamnose. As a result, P II lacked the galactose and ⁄ or N-acetyl galactosamine specificity that is a characteristic feature of the archetypal type 2 RIP (with few exceptions). The most striking difference in sugar binding prefer- ence was observed with GalNAc (Table 3). We there- fore performed cytotoxicity assays in which the various pulchellins were pre-incubated or not with free GalNAc to determine whether this sugar can prevent surface binding of toxin in a manner that might indi- cate a possible basis for the distinctive subgroup potencies (Fig. 4). For P I and P II, we observed improved levels of cellular protein synthesis as the concentration of pre-mixed GalNAc was increased (Fig. 5). The reason for the different plateaus seen with P I (where the rescue of protein synthesis reaches a maximum of approximately 50%) and P II (where res- cue of protein synthesis reaches a maximum of approx- imately 85%) is not known, but the variation may reflect suboptimal binding of GalNAc in one or both binding pockets of P I. However, in striking contrast with the rescue of protein synthesis observed for P I and P II, protection against P III and P IV was mar- ginal, even when toxin was pre-treated with 100 mm GalNAc. Taken together with the inability of P III and P IV to inhibit hemagglutination in vitro in the presence of this sugar, these data suggest that the bind- ing and uptake of these two isoforms does not require receptors containing GalNAc. The pulchellin isoforms P I and P II are clearly different since, in the presence of this sugar, both hemagglutination and cytotoxicity are inhibited. Discussion The present study reports the isolation and initial char- acterization of four pulchellin type 2 RIPs and their encoding cDNA sequences. Seven cDNAs were com- pletely sequenced and four were correlated with the iso- forms isolated from mature seeds. Since the pulchellin isoforms contain both A-chain and B-chain sequences connected in sequence (Fig. 3), they are clearly made as precursors. This is compatible with other type 2 RIPs. The precursors contain a very similar 34 residue N-terminal pre-sequence, and a short intervening linker peptide joining the A- and B-chains, that must be removed during protein maturation upon their biosyn- thesis. The pre-sequence resembles a true endoplasmic signal peptide to direct the proteins into the secretory pathway. The additional N-terminal sequence may function in a manner akin to the N-terminal propeptide found in preproricin [24]. It is most likely cleaved after an Asn residue once the protein is deposited in vacu- oles. The intervening linker peptides are also extremely similar and, by analogy to that of preproricin, may well contain a vacuolar targeting signal [25]. Alignment of the immature polypeptide sequences (Fig. 3) shows that some residues are conserved only amongst the pulchellin isoforms (Fig. 3). Although Table 3. Carbohydrate-binding specifity of P I, P II, P III and P IV. In the first well, 100 lL of each sugar at 100 m M was placed and 50 lL was taken and serially two-fold diluted in wells containing 50 lL of NaCl ⁄ P i . Then, 50 lL of each isoform solution (112 lgÆmL )1 ) was added to the wells. Following incubation, 50 lL of a 1% erythrocyte solution was added. Numbers indicate the minimal concentration that inhibits agglutination. Sugar Minimum concentration for inhibition (m M) PI PII PIII PIV Lactose 0.78 1.56 12.5 12.5 N-acetyl- D-galactosamine 25 25 – – Galactose 6.25 1.56 100 25 Raffinose 25 3.12 – 50 Methyl-a- D-galactopyranoside 1.56 3.12 100 25 L-Rhamnose – 12.5 – – Melibiose – – 100 50 0 10 20 30 40 50 60 70 80 90 110 [N -acetyl B- D-galactosamine] (mM) Protein synthesis (% of control) 100 Fig. 5. Competition of pulchellin entry by N-acetyl-D-galactosamine. A single dose of toxin (200 ngÆmL -1 P I and P II, or 800 ngÆmL )1 P III and P IV), previously shown capable of inhibiting 90% protein synthesis within 4 h, was used in all preincubations. Each toxin was mixed with increasing concentrations of GalNAc in DMEM ⁄ FCS for 30 min. at 37 °C. The mixtures were added to cells for 4 h and remaining protein synthesis determined as detailed in the Experimental procedures. Each value is the mean for tripli- cate samples. h,PI; , P II; s, P III; d, P IV. Characterization of four pulchellin isoforms P. V. Castilho et al. 954 FEBS Journal 275 (2008) 948–959 ª 2008 The Authors Journal compilation ª 2008 FEBS three isoforms (P I, P II and P IV) have the nine con- served cysteines in the B-chains, P III has only eight of these and lacks Cys506, indicating that it must lack one of the usual four intra-chain disulfide bridges. The primary sequences of the catalytic A-chains were found to be only slightly different (Fig. 3) but not in the pairwise manner indicated from cytotoxities (Fig. 4). Indeed, virtually all of the changes within the pulchellin A-chains revealed pairwise identity of P II ⁄ P III and P I ⁄ P IV (Table 2). However, these differ- ences lie outside the residues that are known, from other ribosome inactivating proteins, to determine the major folds and the catalytic site (Asn71, Tyr73, Tyr112, Arg123, Gln159, Glu163, Arg166, Glu194, Asn195, Trp197, P I numbering) [26]. Indeed, these residues are retained in positions corresponding exactly to those in the A-chains of ricin and abrin [26]. Over- all, it is therefore unlikely that the A-chains differ sig- nificantly in catalytic activity. The toxicity values found for the pulchellin isolectins divided them into two subgroups, the more toxic forms (P I and P II) and the less toxic forms (P III and P IV). It was suggested that the presence ⁄ absence of a carbohydrate chain close to the RNA-binding sites could influence the different toxicities found for mistle- toe lectins I and III [27]. P I and P IV A-chain have two N-glycosylation sites, whereas P II and P III have only one. In this sense, and in contrast to mistletoe lectins, no correspondence between glycosylated ⁄ nonglycosylated A-chain and their biological activities could be found. The amino acids residues most likely involved in the two B-chain sugar binding sites also vary, although only very slightly (Fig. 3). Although the first putative sugar binding site is the same in P II and P III, it dif- fers by a single residue (Trp instead of Tyr) in both P I and P IV. This may be analogous to ricin B-chain in which Trp and Tyr side chains have been reported to provide a flat binding surface for galactose, although they do not make more specific interactions with the sugar [28]. If similar to the present study, then this sub- tle difference between sugar binding site 1 in P II and P III may have no functional consequence in relation to carbohydrate binding. From the putative C-terminal sugar binding site (identical in P I and P II but differ- ing by a single residue (Trp instead of Tyr) in both P III and P IV), the same logic may also apply. In the present study, the absence of any marked difference between the actual sugar binding residues suggests that the simplest explanation for the different haemaggluti- nation and cytotoxicities between the two subgroups (Table 3, Figs 4 and 5), is that flanking residues may be critical in preventing a P III ⁄ P IV interaction with GalNAc. This hypothesis was also raised for the mistletoe lectin I [29]. The pairwise alignment of the isoforms reveals that, although the highest primary sequence similarity of each subgroup is found in the C-terminal half of the B-chains (domain 2; Table 2), and that there is only a single conserved aromatic sub- stitution in the residues that make up the putative sugar binding pockets, there is some interesting varia- tion in the flanking regions around the second sugar binding pocket that could influence the P III ⁄ PIV specificity and binding properties. Of particular interest is the substitution G488R presented by P III ⁄ P IV. In summary, our data describe a preliminary charac- terization of a family of pulchellins and reveal a num- ber of clear differences in B-chain behaviour. We speculate that variations within domain 2 (C-terminal half) of these lectins may be relevant for the different patterns of cell surface binding that are likely to influ- ence receptor clustering, entry of these toxins into cells and ultimately their toxicities. Further studies aim to investigate the proposed structure–function relation- ships experimentally. Experimental procedures Abrus pulchellus seeds were obtained from a plant culti- vated in the garden of our laboratory, in Sa ˜ o Carlos-SP, Brazil. Escherichia coli DH5-a (Promega, Madison, WI, USA) was used for plasmid amplification. Oligonucleotide synthesis was produced by IDT, Inc. (Coralville, IA, USA). Restriction endonucleases and DNA ladders were obtained from Promega. Immobilized d-galactose was purchased from Pierce (Rockford, IL, USA). Mono Q 5 ⁄ 50 and Mono P5⁄ 50 were purchased from GE Healthcare (GE Health- care, Little Chalfont, UK). Sugars were purchased from Sigma (St Louis, MO, USA). All other chemicals used were of analytical grade. Isolation of pulchellin isoforms Dehulled seeds of A. pulchellus were ground in a mixer and the fine flour obtained was suspended (1 : 10, w ⁄ v) in the extraction buffer (20 mm Tris–HCl, pH 8, containing 150 mm NaCl) for 3 h at 4 °C and centrifuged at 12 000 g for 20 min at 4 °C. The supernatant was loaded onto an immobilized d-galactose column, previously equilibrated with the same buffer. The unbound material was eluted from the column with the extraction buffer, whereas the adsorbed proteins were obtained in a single peak after elu- tion with a solution containing 150 mm NaCl and 100 mm lactose. The fractions containing the pulchellin were dia- lyzed against 5% acetic acid in order to remove the lactose. To isolate the isoforms, the samples were dialyzed against 20 mm Tris–HCl, pH 8, containing 10 mm NaCl (buffer A) P. V. Castilho et al. Characterization of four pulchellin isoforms FEBS Journal 275 (2008) 948–959 ª 2008 The Authors Journal compilation ª 2008 FEBS 955 and 1 mL of sample containing approximately 1 mg was loaded onto a Mono Q 5 ⁄ 50 column previously equili- brated with the same buffer. Elution was performed with a linear gradient of buffer B (20 mm Tris–HCl, pH 8, con- taining 1 m NaCl) from 0% to 20% for 40 min followed by 20–100% in 5 min. The corresponding peaks of P I and P I) were collected, dialyzed and freezed. The peaks related to the P III and P IV were dialyzed against 10 mm sodium phosphate, pH 7 (buffer A from Mono P) and submitted to a second chromatographic step in a Mono P 5 ⁄ 50 chro- matofocusing column previously equilibrated with the same buffer. One milliliter samples containing P III and P IV (approximately 0.5 mg) were isolated by an elution gradient of 0–100% of buffer B (10 mm sodium phosphate, pH 5.7) for 20 min, holding for 5 min in 100% buffer B. The corre- sponding peaks of P III and P IV were collected, dialyzed and freezed. In the two last chromatographic steps (ion exchange and chromatofocusing), the flow rate was main- tained at 1 mLÆmin )1 , the protein level was monitored at 280 nm and the pressure was maintained under 5.5 MPa. SDS ⁄ PAGE was used to monitor the isolation as well as the estimation of the apparent molecular weights and struc- tural properties of the pulchellin isoforms. Isoeletric focusing Isoelectric focusing of the proteins was carried out on Phast System (Pharmacia, Uppsala, Sweden). Samples reconstitu- ted in MilliQ water were applied to Phast Gel IEF, pH 3–9, and run according to the standard program. Gels were stained with Comassie brilliant blue. The range of pI values of each protein was estimated by using standard markers. CD measurements CD experiments were performed on Jasco J-715 Spectro- polarimeter (Jasco Inc., Tokyo, Japan) equipped with a thermoelectrically controlled cell holder. CD spectra of the four isolated isoforms were measured in the far-UV range (195–250 nm) in 1 mm path length quartz cuvettes, recorded as the average of 16 scans. CD spectra were measured in 0.125 mgÆmL )1 of protein solution (20 mm Tris, pH 8, 10 mm NaCl added). Analyses of the protein CD spectra for determining the secondary structure fractions were performed using cdpro software, comprising the three programs: selcon3, cdsstr and contin [30]. CD thermal scans were used to examine the melting tem- perature of the proteins. Spectra were measured at 5 °C intervals in the temperature range 20–100 °C with an aver- age time of 3 s, an equilibration time of 3 min, and a band width of 1 nm. The CD signal at 232 nm was recorded as a function of temperature, h 232 (T). The wavelength 232 nm was chosen because of the maximal difference between the denatured and the native protein spectra observed at this wavelength. cDNA cloning and amino acid sequence dedution of the isoforms from A. pulchellus Total RNA was isolated from immature A. pulchellus seeds previously frozen in liquid nitrogen, using the RNAeasy Plant Mini Kit (Qiagen, Hilden, Germany). Total RNA was quantified at 260 nm (Hitachi U-2000 spectrophotometer; Hitachi, Vienna, Austria). RT-PCR (Super Script Choice System for DNA Synthesis, Gibco BRL., Paisley, UK) was performed in two steps. In the first step, for cDNA single strand synthesis, 600 ng of RNA, 0.5 lg of oligo(dT) primer and 10 mm of dNTPs were incubated for 5 min at 65 °C. Subsequently, 4 lL of the first strand buffer 5 X and 2 lL of dithiothreitol (0.1 m) was added and the reaction was incubated for 2 min at 42 °C. Finally 1 lL of Superscript II was added and the reaction was incubated for an additional 1 h at 65 °C. After the cDNA synthesis, the reaction was precipitated with ethanol [31]. In the second RT-PCR step, in order to isolate and amplify the cDNAs of the pulchellin isoforms, the whole amount of the cDNA obtained in the reaction described above was used. Several primer designs, based on the N-terminal amino acid sequence of the iso- forms and on the DNA sequence of pulchellin A- [15] and B- [16] chains, were tested. These included: pair 1: primer sense PulcA (5¢-GTC CAG TTT CAA ATG GAC AAA AC-3¢) and primer anti-sense Oligo (dT)12–18 (Invitrogen, Carlsbad, CA, USA) and pair 2: primer sense Nterm (5¢-ATG GAC AAA ACT TTG AAR CTA CTG ATT TTA TG-3¢) and anti-sense Cterm (5¢-TTA AAA CAA AGT AAG CCA TAT TTG RTT NGG YTT-3¢). The reaction mixtures [75 mm of MgSO 4 , 100 pmol of each primer, 10 mm of dNTPs (Promega), 5 lL of buffer HiFi 10 X (Invitrogen), 2 U of Taq Platinum (Invitrogen), and MilliQ water to a final volume of 50 lL] were submitted to PCR. The conditions were initial denaturation of 2 min at 94 °C followed by 40 cycles of denaturation (94 °C for 30 s), annealing (50 °C for 30 s) and extension (68 °C for 2 min) and a final extension of 68 °C for 7 min. The amplified products were resolved in agarose gels and the DNA was eluted using Perfectprep Gel Cleanup kit (Eppendorf, Westbury, NY, USA). For the ligation mix- ture, 25 ng of each amplified product were ligated to 50 ng of TOPO-TA (pCR 2.1) (Invitrogen) following the manu- facturer’s protocol. E. coli DH5- a cells were transformed [31], plasmids were isolated and the positive clones were screened by EcoRI digestion. DNA sequencing Plasmids were sequenced [32] using an ABI Prism 377 auto- mated DNA sequencer (Perkin Elmer, Waltham, MA, USA) following the manufacturer’s protocol. The primers used for sequencing each whole cDNA sequence were M13 Forward and Reverse (Invitrogen), and six primer walkers: Asense (5¢-CTA GGG TTA CAG GCC TTG AC-3¢), Bsense ⁄ Xho Characterization of four pulchellin isoforms P. V. Castilho et al. 956 FEBS Journal 275 (2008) 948–959 ª 2008 The Authors Journal compilation ª 2008 FEBS (5¢- CCG CTC GAG TTA AAA CAA ATG AAG-3¢), pul- cintFW1 (5¢-CCT GTG CTT CGA GAT CCA AC-3¢), pulcintFW2 (5¢-GCA TCT ACC TAC CTT TTC AC-3¢), pulcintRW1 (5¢-CAC CCA TCG TTG GCT AGC CC-3¢) and pulcintRW2 (5¢-GTA AAG TGC CCA TTG CTG CTC-3¢). Each isoform whole sequence was submitted to a BLAST script databank search [33], which returned the highest sequence identity to preproabrin. The predicted protein sequence was aligned using clustal w software (http://www.ebi.ac.uk/clustalw/) in the default set up to prep- roabrin, proricin and mistletoe lectin I for identity analysis. The nucleotide sequences of the four isoforms were deposited in Genbank, with the accession numbers (EU008735, EU008736, EU008737 and EU008738, for P I, P II, P III and P IV respectively). Amino acid sequence analysis Samples of each pulchellin isoform were submitted to SDS ⁄ PAGE and electroblotted on a poly(vinylidene difluo- ride) membrane. Polypeptides were excised from the blots and the N-terminal region was sequenced on an Applied Biosystems model 477A protein sequencer interfaced with an Applied Biosystems model 120A online analyzer (Applied Biosystems, Weiterstadt, Germany). The standard Edman degradation procedure was used [34]. LC-MS ⁄ MS analysis of tryptic peptides Pulchellin isoforms (P I, P II, P III and P IV) (100 lg) were desalted and dried in a SpeedVac SPD12P concentrator (Thermo Savant, Holbrook, NY, USA). The samples were solved in 25 lL of 50% (v ⁄ v) acetonitrile and 50 mm NH 4 HCO 3 ; subsequently 5 lLof45mm dithiothreitol were added to each sample. After incubation for 1 h at 56 °C, 5 lL of 100 mm iodoacetamide were added followed by 2 h of incubation in the dark at room temperature. After five- fold dilution with 100 mm NH 4 HCO 3 , samples were treated with 2 U of trypsin (sequencing grade, modified, Promega) for 24 h at 37 °C and frozen until MS analysis. LC-MS ⁄ MS analyses were performed in a Q-TOF ultima API mass spectrometer (Micromass, Manchester, UK) cou- pled to a capillary liquid chromatography system (CapLC, Waters, Milford, MA, USA). A nanoflow ESI source was used with a lockspray source for lockmass measurement during all chromatographic runs. The digested protein was desalted online using a Waters Opti-Pack C18 trap column. The mixture of trapped peptides was then separated by elu- tion with a water ⁄ acetonitrile ⁄ formic acid gradient through a Nanoease C18 (75 lm inner diameter) capillary column. The column was washed with 90% A solution (0.1% formic acid) and 10% B solution (90% acetonitrile with 0.1% formic acid) for 20 min. Peptides were eluted by a 60 min linear gradient from 10–50% B solution holding for 40 min in 50% B. Data were acquired in a data-dependent mode, and multiplycharged peptide ions (+2 and +3) were automatically mass selected and dissociated in MS ⁄ MS experiments. Typical LC and ESI conditions were: flow of 200 nLÆmin )1 , nanoflow capillary voltage of 3 kV, block temperature of 100 °C and a cone voltage of 100 V. The MS ⁄ MS spectra were processed using proteinlynx 2.0 software (Waters). Search parameters used the fixed cys- teine carbamidomethylation and the variable methionine oxidation as modifications. The PKL file generated was used to perform a database search using the deduced pep- tide sequences provided by the sequences previously cloned. Cytotoxicity assays HeLa cells were maintained in DMEM ⁄ fetal bovine serum (10%). Cells were seeded at 1.5 · 10 4 ⁄ well in a 96-well tis- sue culture plate, allowed to grow overnight and incubated for 4 h with 100 mL DMEM ⁄ fetal bovine serum containing graded concentrations of pulchellin isoforms. Subsequently, cells were washed twice with NaCl ⁄ P i and incubated in NaCl ⁄ P i containing 10 lCiÆmL )1 [ 35 S] methionine for 30 min. Labelled proteins were precipitated with three washes in 5% (w ⁄ v) trichloroacetic acid and the amount of radiolabel incorporated was determined after the addition of 100 mL ⁄ well of scintillation fluid, by scintillation count- ing in a Micro-Beta 1450 Trilux counter (Perkin Elmer, Waltham, MA, USA). For each value, the level of protein synthesis was taken as a percentage of toxin-free control cells, and the mean from four replicate samples was calcu- lated. Where appropriate, toxins were pre-incubated with increasing concentrations of N-acetyl-d-galactosamine. Toxicity to mice The toxic activity of the pulchellin isoforms was determined by simple intraperitoneal injection in female Swiss mice. All animal procedures were performed in accordance with the US National Research Council’s guidelines for care and use of laboratory animals and this work was approved by the Animal Experimentation Ethics Commission of the Federal University of Sa ˜ o Carlos. The protein samples were dilluted in buffer 20 mm Tris–HCl, pH 8, containing 10 mm NaCl. Groups of six animals were used in each different dose and the group received the same proportion of toxin ⁄ body mass. The mice had free access to food and water and tests were carried out over 48 h. The results were evaluated as the median lethal dose. Each assay had an animal control that received only the dilution buffer described above. 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Falini B & Stirpe F (1998) Evaluation of immunotoxins containing single-chain ribosome- inactivating proteins and an ati CD -22 monoclonal antibody (OM- 124 ): in vitro and in vivo studies Br J Haematol 101, 170–188 Hegde R, Maiti TK & Podder SK (1991) Purification and characterisation of three toxins and two agglutinins from Abrus precatorius seed by using lactamyl-Sepharose affinity chromatography Anal . Isolation and characterization of four type 2 ribosome inactivating pulchellin isoforms from Abrus pulchellus seeds Priscila V. Castilho 1 ,2 , Leandro. to any of the four B-chains presented here. Purification of four pulchellin isoforms from A. pulchellus seeds Using a combination of affinity, ion exchange and chromatofocusing