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Ribosome-inactivating proteins (RIPs) consist of three varieties. Type 1 RIPs are single-chained and approximately 30-kDa in molecular weight. Type 2 RIPs are double-chained and composed of a type 1 RIP chain and a lectin chain. Type III RIPs, such as maize b-32 barley and JIP60 which are produced as single-domain proenzymes, possess an N-terminal domain corresponding to the A domain of RIPs and fused to a C-terminal domain. In addition to the aforementioned three types of RIPs originating from flowering plants, there are recently discovered proteins and peptides with ribosome-inactivating and protein synthesis inhibitory activities but which are endowed with characteristics such as molecular weights distinctive from those of the regular RIPs. These new/unusual RIPs discussed in the present review encompass metazoan RIPs from Anopheles and Culex mosquitos, antimicrobial peptides derived from RIP of the pokeweed Phytolacca dioica, maize RIP (a type III RIP derived from a precursor form), RIPs from the garden pea and the kelp. In addition, RIPs with a molecular weight smaller than those of regular type 1 RIPs are produced by plants in the Cucurbitaceae family including the bitter gourd, bottle gourd, sponge gourd, ridge gourd, wax gourd, hairy gourd, pumpkin, and Chinese cucumber. A small type II RIP from camphor tree (Cinnamomum camphora) seeds and a snake gourd type II RIP with its catalytic chain cleaved into two have been reported. RIPs produced from mushrooms including the golden needle mushroom, king tuber mushroom, straw mushroom, and puffball mushroom are also discussed in addition to a type II RIP from the mushroom Polyporus umbellatus. Bacterial (Spiroplasma) RIPs associated with the fruitfly, Shiga toxin, and Streptomyces coelicolor RIP are also dealt with. The aforementioned proteins display a diversity of molecular weights, amino acid sequences, and mechanisms of action. Some of them are endowed with exploitable antipathogenic activities.

Antimicrobial peptides, also called Host Defence Peptides (HDPs), are effectors of innate immune response found in all living organisms. In a previous report, we have identified by chemical fragmentation, and characterized the first cryptic antimicrobial peptide in PD-L4, a type 1 ribosome inactivating protein (RIP) from leaves of Phytolacca dioica L. We applied a recently developed bioinformatic approach to a further member of the differently expressed pool of type 1 RIPs from P. dioica (PD-L1/2), and identified two novel putative cryptic HDPs in its N-terminal domain. These two peptides, here named IKY31 and IKY23, exhibit antibacterial activities against planktonic bacterial cells and, interestingly, significant anti-biofilm properties against two Gram-negative strains. Here, we describe that PD-L1/2 derived peptides are able to induce a strong dose-dependent reduction in biofilm biomass, affect biofilm thickness and, in the case of IKY31, interfere with cell-to-cell adhesion, likely by affecting biofilm structural components. In addition to these findings, we found that both PD-L1/2 derived peptides are able to assume stable helical conformations in the presence of membrane mimicking agents (SDS and TFE) and intriguingly beta structures when incubated with extracellular bacterial wall components (LPS and alginate). Overall, the data collected in this work provide further evidence of the importance of cryptic peptides derived from type 1 RIPs in host/pathogen interactions, especially under pathophysiological conditions induced by biofilm forming bacteria. This suggests a new possible role of RIPs as precursors of antimicrobial and anti-biofilm agents, likely released upon defensive proteolytic processes, which may be involved in plant homeostasis.

Ribosome-inactivating proteins are plant cytotoxic enzymes, also present in fungi, algae and bacteria, mainly known for their ability to inhibit protein synthesis. We previously purified and structurally characterized three type 1 RIPs (PD-S1-3) from Phytolacca dioica seeds and four type 1 RIPs (PD-L1-4) from adult plant leaves. Two additional RIPs, named dioicin 1 and dioicin 2, were isolated from leaves of young plants and developing leaves of adult plants. The evidence that P. dioica synthesizes and accumulates these RIPs isoforms suggests that these proteins have been conserved during evolution. Though several aspects of P. dioica type 1 RIP characterization have been studied, some important questions remain to be answered especially with respect to Phytolaccaceae RIP evolution. One of the major problems encountered in approaching RIPs phylogeny concerns the availability of their sequences. In this study, we report the characterization of biological and structural properties of dioicin 1, including the determination of its primary structure by using a combined approach based on Edman degradation, de novo sequencing by ESI-Q-TOF-MS/MS and peptide mapping by MALDI-TOF MS. Knowledge of dioicin 1 primary structure provide us a mean to deepen Phytolaccaceae's RIPs phylogeny. We speculate that both dioicins 1 and 2 share common ancestors with PAP-II and PAP icos-II and that dioicin 1 is not closely related to other members of this clade, thus shedding lights on evolutionary relationships among type 1 RIPs from Phytolaccaceae. Ribosome-inactivating proteins are plant cytotoxic enzymes, also present in fungi, algae and bacteria, mainly known for their ability to inhibit protein synthesis. We previously purified and structurally characterized three type 1 RIPs (PD-S1-3) from Phytolacca dioica seeds and four type 1 RIPs (PD-L1-4) from adult plant leaves. Two additional RIPs, named dioicin 1 and dioicin 2, were isolated from leaves of young plants and developing leaves of adult plants. The evidence that P. dioica synthesizes and accumulates these RIPs isoforms suggests that these proteins have been conserved during evolution. Though several aspects of P. dioica type I RIP characterization have been studied, some important questions remain to be answered especially with respect to Phytolaccaceae RIP evolution. One of the major problems encountered in approaching RIPs phylogeny concerns the availability of their sequences.In this study, we report the characterization of biological and structural properties of dioicin 1, including the determination of its primary structure by using a combined approach based on Edman degradation, de novo sequencing by ESI-Q-TOF-MS/MS and peptide mapping by MALDI-TOF MS. Knowledge of dioicin 1 primary structure provide us a mean to deepen Phytolaccaceae's RIPs phylogeny. We speculate that both dioicins 1 and 2 share common ancestors with PAP-II and PAP icos-II and that dioicin 1 is not closely related to other members of this clade, thus shedding lights on evolutionary relationships among type 1 RIPs from Phytolaccaceae. (C) 2015 Elsevier Inc. All rights reserved.

Aims: Ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RubisCO) catalyses a key reaction by which inorganic carbon is assimilated into organic carbon found in the biosphere. The present study was aimed to isolate this enzyme from leaves of Phytolacca dioica L. Study Design: In this work, first crude extracts from leaves at different stages of development were assayed to isolate this enzyme, then young leaves of P. dioica were used considering high value of specific activity. Methodology: Classical methods for protein isolation have been used to characterise RubisCO from P. dioica leaves. Results: RubisCO was isolated from young leaves by gel-filtration. The pure RubisCO showed two predominant bands (56- and 15-kDa) by SDS-PAGE. N-terminal sequences data on large (56 kDa) and Small (15 kDa) subunits obtained by automatic Edman degradation show a high percentage of identity with large and small subunit of other RubisCo enzymes. Moreover, the N- Original Research Article

Background The species from the genus Phytolacca constitute one of the best sources of ribosome-inactivating proteins (RIPs) that have been used both in the therapy against virus and tumors and in the construction of transgenic plants resistant to virus, bacteria, fungi and insects. Here we investigate new activities of three representative RIPs from Phytolacca dioica (dioicin 2, PD-S2 and PD-L4). Results The three RIPs displayed, in addition to already reported activities, rRNA N-glycosylase activities against plant, bacterial and fungal ribosomes. Additionally dioicin 2 and PD-L4 displayed endonuclease activity on a supercoiled plasmid DNA, and dioicin 2 and PD-S2 arrested the growth of the fungus Penicillium digitatum. Furthermore, dioicin 2 induced caspase activation and apoptosis in cell cultures. Conclusions The different activities of the RIPs from Phytolacca dioica may explain the antipathogenic properties attributed to these RIPs in plants and their antiviral and antitumoral effects. In spite of the similarity in their rRNA N-glycosylase and DNA polynucleotide:adenosine glycosylase activities, they differed in their activities against viral RNA, plasmid DNA, fungi and animal cultured cells. This suggests that the presence of isoforms might optimize the response of the plant against several types of pathogens. General significance RIPs from Phytolacca can induce plant resistance or tumor cell death not only by means of ribosome inactivation but also by the activities found in this report. Furthermore, the induction of cell death by different mechanisms turns these RIPs into more useful tools for cancer treatment rendering the selection of RIP-resistant mutants impossible.

The structure of the highly glycosylated type 1 ribosome inactivating protein PD-L1 was determined by X-ray crystallography. This protein belongs to a group of four PD-Ls (PD-L1-4) expressed in Phytolacca dioica leaves. Of these, PD-L1 and PD-L2 are endowed with the ability to cleave double strand DNA, a property which is not shared by the other two components of the family. Single crystals of native PD-L1, the most glycosylated, were obtained using seeding techniques and phase determination was achieved using molecular replacement. To investigate the role of glycosylation in the different functionality of these proteins, we performed DNA cleavage assays on the E. coli plasmid pBR322. These experiments revealed that DNA cleaving ability does not depend on the level of glycosylation of PD-L1, since there is no difference in the activities displayed by native PD-L1 and a recombinant non-glycosylated form. Besides, confirming that DNA cleavage by PD-L1 cannot be attributed to contaminations, these data unambiguously show that functional changes between PD-L1 and PD-L4 are solely to be attributed to their sequence differences. On the basis of the comparison of PD-L1 and PD-L4 crystal structures, we propose possible structural determinants responsible for their different functional behavior. © 2009 Wiley Periodicals, Inc.

The amino acid sequence and glycan structure of PD-L1, PD-L2 and PD-L3, type 1 ribosome-inactivating proteins isolated from Phytolacca dioica L. leaves, were determined using a combined approach based on peptide mapping, Edman degradation and ESI-Q-TOF MS in precursor ion discovery mode. The comparative analysis of the 261 amino acid residue sequences showed that PD-L1 and PD-L2 have identical primary structure, as it is the case of PD-L3 and PD-L4. Furthermore, the primary structure of PD-Ls 1-2 and PD-Ls 3-4 have 81.6% identity (85.1% similarity). The ESI-Q-TOF MS analysis confirmed that PD-Ls 1-3 were glycosylated at different sites. In particular, PD-L1 contained three glycidic chains with the well known paucidomannosidic structure (Man)3 (GlcNAc)2 (Fuc)1 (Xyl)1 linked to Asn10, Asn43 and Asn255. PD-L2 was glycosylated at Asn10 and Asn43, and PD-L3 was glycosylated only at Asn10. PD-L4 was confirmed to be not glycosylated. Despite an overall high structural similarity, the comparative modeling of PD-L1, PD-L2, PD-L3 and PD-L4 has shown potential influences of the glycidic chains on their adenine polynucleotide glycosylase activity on different substrates. © 2008 Elsevier Masson SAS. All rights reserved.

The ribosome inactivating protein PD-L4 from Phytolacca dioica is a N-β-glycosidase, probably involved in plant defence. The crystal structures of wild type PD-L4 and of the S211A PD-L4 mutant with significantly decreased catalytic activity were determined at atomic resolution. To determine the structural determinants for the reduced activity of S211A PD-L4, both forms have also been co-crystallized with adenine, the major product of PD-L4 catalytic reaction. In the structure of the S211A mutant, the cavity formed by the lack of the Ser hydroxyl group is filled by a water molecule; the insertion of this non-isosteric group leads to small albeit concerted changes in the tightly packed active site of the enzyme. These changes have been correlated to the different activity of the mutant enzyme. This work highlights the importance of atomic resolution studies for the deep understanding of enzymatic properties. Proteins 2008. © 2007 Wiley-Liss, Inc.

We investigated the antimicrobial activity of PD-L4, a type 1 RIP from Phytolacca dioica. We found that this protein is active on different bacterial strains both in a native and denatured/alkylated form and that this biological activity is related to a cryptic peptide, named PDL440-65, identified by chemical fragmentation. This peptide showed the same antimicrobial activity of full-length protein and possessed, similarly to several antimicrobial peptides, an immunomodulatory effect on human cells. It assumes an alpha-helical conformation when interact with mimic membrane agents as TFE and likely bacterial membranes are a target of this peptide. To date PDL440-65 is the first antimicrobial peptide identified in a type 1 RIP. We investigated the antimicrobial activity of PD-L4, a type 1 RIP from Phytolacca dioica. We found that this protein is active on different bacterial strains both in a native and denatured/alkylated form and that this biological activity is related to a cryptic peptide, named PDL4(40-65), identified by chemical fragmentation. This peptide showed the same antimicrobial activity of full-length protein and possessed, similarly to several antimicrobial peptides, an immunomodulatory effect on human cells. It assumes an alpha-helical conformation when interact with mimic membrane agents as TFE and likely bacterial membranes are a target of this peptide. To date PDL4(40-65) is the first antimicrobial peptide identified in a type 1 RIP. (C) 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Ribosome-inactivating proteins isolated fromPhytolacca dioicaL. leaves are rRNA-N-glycosidases, as well as adenine polynucleotide glycosylases. Here we report that some of them cleave supercoiled pBR322 dsDNA, generating relaxed and linear molecules. PD-L1, the glycosylated major form isolated from the winter leaves of adultP.dioicaplants, produces both free 3′-OH and 5′-P termini randomly distributed along the DNA molecule, as suggested by labelling experiments with [α-32P]dCTP and [γ-32P]dATP. Moreover, when the reaction is carried out under low-salt conditions, cleavage is observed mainly at a specific site, located downstream of the ampicillin resistance gene (close to position 3200), ending with the deletion of a fragment of approximately 70 nucleotides. This cleavage pattern is similar to that obtained under the same conditions with mung bean nuclease, a single-strand endonuclease. Furthermore, pBR322 DNA treated with PD-L1 shows reduced transforming activity withE.coliHB101 competent cells in comparison to untreated control plasmid DNA.

The ribosome inactivating protein BE27 displays several biological activities in vitro that could result in a broad action against several types of pathogens. Beetin 27 (BE27), a ribosome-inactivating protein (RIP) from sugar beet (Beta vulgaris L.) leaves, is an antiviral protein induced by virus and signaling compounds such as hydrogen peroxide and salicylic acid. Its role as a defense protein has been attributed to its RNA polynucleotide:adenosine glycosidase activity. Here we tested other putative activities of BE27 that could have a defensive role against pathogens finding that BE27 displays rRNA N-glycosidase activity against yeast and Agrobacterium tumefaciens ribosomes, DNA polynucleotide:adenosine glycosidase activity against herring sperm DNA, and magnesium-dependent endonuclease activity against the supercoiled plasmid PUC19 (nicking activity). The nicking activity could be a consequence of an unusual conformation of the BE27 active site, similar to that of PD-L1, a RIP from Phytolacca dioica L. leaves. Additionally, BE27 possesses superoxide dismutase activity, thus being able to produce the signal compound hydrogen peroxide. BE27 is also toxic to COLO 320 cells, inducing apoptosis in these cells by either activating the caspase pathways and/or inhibiting protein synthesis. The combined effect of these biological activities could result in a broad action against several types of pathogens such as virus, bacteria, fungi or insects.

Phytolacca dioica L. leaves produce at least two type-I ribosome-inactivating proteins. Each polypeptide chain is subjected to different post-translational modifications giving rise to PD-L1 and PD-L2, and PD-L3 and PD-L4, each polypeptide pair having the same primary structure. With the aim of exploiting the cytotoxic properties of these proteins as potential biological phytodrugs, a gene encoding PD-L4 was designed based on criteria expected to maximize the translation efficiency in tomato. The gene was constructed from 18 oligonucleotides and preliminarily expressed in Escherichia coli, using the T7 promoter system. The protein produced was insoluble and accumulated in inclusion bodies to about 300 mg/l of culture. Ribosome-inactivating activity was generated by controlled oxidation of the reduced and denatured protein. The recombinant protein was indistinguishable from natural PD-L4 as isolated from leaves of Phytolacca dioica, in both catalytic activity and primary structure.

A peroxidase (PD-cP; 0.47 mg/100 g leaves) was purified from autumn leaves of Phytolacca dioica L. and characterized. PD-cP was obtained by acid precipitation followed by gel-filtration and cation exchange chromatography. Amino acid composition and N-terminal sequence of PD-cP up to residue 15 were similar to that of Spinacia oleracea (N-terminal pairwise comparison showing four amino acid differences). PD-cP showed a molecular mass of approx. 36 kDa by SDS-PAGE, pH and temperature optima at 3.0 and 50.0°C, respectively and seasonal variation. The Michaelis-Menten constant (K(M)) for H(2)O(2) was 5.27 mM, and the velocity maximum (V(max)) 1.31 nmol min(-1), while the enzyme turnover was 0.148 s(-1). Finally, the presence of Ca(2+) and Mg(2+) enhanced the PD-cP activity, with Mg(2+) 1.4-fold more effective than Ca(2+)

Evolution of Plant Ribosome-Inactivating Proteins.- RNA N-Glycosidase Activity of Ribosome-Inactivating Proteins.- Enzymatic Activities of Ribosome-Inactivating Proteins.- Type I Ribosome-Inactivating Proteins from Saponaria officinalis.- Type 1 Ribosome-Inactivating Proteins from the Ombu Tree (Phytolacca dioica L.).- Sambucus Ribosome-Inactivating Proteins and Lectins.- Ribosome-Inactivating Proteins from Abrus pulchellus.- Ribosome-Inactivating Proteins in Cereals.- Ribosome Inactivating Proteins and Apoptosis.- The Synthesis of Ricinus communis Lectins.- How Ricin Reaches its Target in the Cytosol of Mammalian Cells.- Ribosome-Inactivating Protein-Containing Conjugates for Therapeutic Use.

N-glycosylation is one of the major naturally occurring covalent co-translational modifications of proteins in plants, being involved in proteins structure, folding, stability and biological activity. In the present work the influence of carbohydrate moieties on the structure-function relationships of type 1 ribosome-inactivating proteins (RIPs) was investigated. To this aim, PD-Ls, RIPs isolated from Phytolacca dioica L. leaves, differing for their glycosylation degree, were used as an experimental system. In particular, comparative structural and biological analyses were performed using native and unglycosylated recombinant PD-L1, the most glycosylated P. dioica RIP isoform. The glycans influence on protein synthesis inhibition and adenine polynucleotide glycosidase activity was investigated. The interaction with adenine, the product of the de-adenylation reaction, was also investigated for native and recombinant PD-L1 by fluorescence spectroscopy. Furthermore, the crystal structure of PD-L1 in complex with adenine was determined. Our data confirm that the absence of glycan moieties did not affect the biological activity in terms of protein synthesis inhibition. However, the removal of carbohydrate chains significantly increased the deadenylation capability, likely as a consequence of the increased accessibility of substrates to the active site pocket. Furthermore, preliminary data on cellular uptake showed that all PD-L isoforms were internalized and, for the first time, that the vesicular distribution within cells could be influenced by the protein glycosylation degree. © 2010 The Royal Society of Chemistry.

The toxicity of plant proteins, later identified as ribosome-inactivating proteins (RIPs), was described more than a century ago and their enzymatic activity was established more than 30 years ago. However, their physiological role and related biological activities are still uncertain. Therefore, despite the body of literature, research on RIPs is ongoing. This review deals with new RIPs being purified, sequenced, characterized, and cloned, and an increasing number of 3D-structures that are determined at high resolution. This is the case of the five type 1 RIPs (PD-S1-3, PD-L1/2, PD-L3/4, dioicin 1, and dioicin 2) from seeds and leaves of the ombú tree (Phytolacca dioica L.), native of the grassy pampas of Argentina. The data collected so far will contribute to our understanding of important issues of RIP research: (1) identifying structural determinants responsible for new enzymatic activities such as the DNA cleaving activity; (2) glycosylation and its influence on the catalytic and biological activities; (3) cellular localization of endogenous RIPs and their physiological role(s). © Springer-Verlag Berlin Heidelberg 2010.

Despite the widespread use of Phytolacca dioica L. in folklore medicine of South Africa, there is dearth of information on its safety/toxicity. The aim of this study was to evaluate the toxicological effect of aqueous leaf and berry extracts of the plant at different dosages for 14 days on the liver and kidney function indices in male Wistar rats. Phytochemical screening indicated that the extracts are rich in phytonutrients including alkaloid, tannin, saponins, phenolics, lectins and flavonoids; while triterpenoids and phlobatanins were absent. The extracts significantly reduced the body and absolute organ weights of the animals at all the dosages investigated. Whereas, significant increase was observed in the serum levels of alkaline phosphatase (ALP), gamma glutamyl transferase (GGT), alanine transaminase (ALT), aspartate transaminase (AST), bilirubin, calcium, creatinine, urea and uric acid; the serum concentration of total protein, albumin and globulin were reduced in the serum following treatment with the extracts. Generally, the effect of the herb appeared to be dosage dependent. This investigation clearly showed that P. dioica can impair normal hepatic and renal functions. This is an indication that the extracts may not be completely safe in male rats when continuously administered for 14 days.

The expression of type 1 ribosome-inactivating proteins (RIPs) in Phytolacca dioica L. leaves was investigated. Fully expanded leaves of young P. dioica plants (up to 3 years old) expressed two novel RIPs, dioicin 1 and dioicin 2. The former was also found in developing leaves from adult P. dioica within about two and a half weeks after leaf development, and the latter continuously synthesized, with no seasonal or ontogenetic constraint. Fully expanded leaves from adult P. dioica expressed four RIPs (PD-Ls1-4) exhibiting seasonal variation. RIPs were localized in the extracellular space, in the vacuole and in the Golgi apparatus of mesophyll cells. Dioicin 1 and dioicin 2 showed rRNA N-β-glycosidase activity and displayed the following properties, respectively: (1) Mr values of 30,047.00 and 29,910.00, (2) pIs of 8.74 and 9.37, and (3) IC50 values of 19.74 (0.658 nM) and 6.85 ng/mL (0.229 nM). Furthermore, they showed adenine polynucleotide glycosylase activity and nicked pBR322 dsDNA. The amino acid sequence of dioicin 2 had 266 amino acid residues, and the highest percentage identity (81.6%) and similarity (84.6%) with PAP-II from Phytolacca americana, while its identity with other RIPs from Phytolaccaceae was around 40%. © 2008 Springer-Verlag.

PD-L4, a type 1 ribosome inactivating protein from Phytolacca dioica leaves, has been successfully crystallized using vapour diffusion methods and PEG 4000 as a precipitant agent. In addition, crystals of a PD-L4 mutant, which has been recently observed to have a lower polynucleotide-adenosine glycosidase activity on DNA, rRNA and poly (A) substrates, have been obtained. To gather information on PD-L4 reaction mechanism both forms have been co-crystallized with adenine, the major product of their catalytic reaction. Diffraction patterns extend to atomic resolution and crystals belong to the orthorhombic P212121 space group, with one molecule in the asymmetric unit. Structure determination has been achieved using molecular replacement; preliminary electron density maps have clearly given evidence of adenine binding. © 2007 Bentham Science Publishers Ltd.

PD-L1 is a highly glycosylated type 1 ribosome inactivating protein, from Phytolacca dioica leaves, with the peculiarity to act also as a DNase. PD-L1 has been successfully crystallized using vapour diffusion and seeding techniques. Crystals belong to the monoclinic C2 space group, with unit cell dimensions a=161.01, b=34.73, c=120.63 Å, β=127.99°. Two molecules are present in the asymmetric unit. Phase determination has been achieved using molecular replacement. © 2007 Bentham Science Publishers Ltd.

Leaves from Phytolacca heterotepala H. Walter (Mexican pokeweed) contain at least 10 type 1 RIP isoforms, named heterotepalins. Their Mr values are included in the range 28,000-36,000, as shown by SDS-PAGE performed under reduced conditions and the pI values in the pH range 8.50-9.50. Some heterotepalins are glycosylated. ESI-QTOF mass spectrometry provides the accurate Mr of heterotepalin 4 (29,326.00) and heterotepalin 5b (30,477.00), two isoforms purified to homogeneity by conventional chromatographic techniques. The N-terminal sequences up to residue 35, show that heterotepalins exhibit an high percentage identity with other type 1 RIPs isolated from Phytolaccaceae. Some heterotepalins cross-react with antisera raised against RIPs isolated from Phytolacca dioica leaves. The complete amino acid sequence of heterotepalin 4 matches that of Phytolacca heterotepala anti-viral protein PAP (RIP1), deduced from the cDNA sequence of PhRIP1 gene (AC: AY327475), with one exception concerning residue 245 which, in the native protein, is Ile instead of Met. This substitution, found by mass spectrometry mapping, has been directly confirmed by Edman degradation sequencing of the C-terminal tryptic peptide 242-262. The results show the high potential of mass spectrometry and Edman degradation to verify and to uncover possible amino acid substitutions between native proteins and their cDNA deduced sequences. © 2006 Elsevier Ltd. All rights reserved.

Four type-1 (single-chain) ribosome-inactivating proteins (RIPs), with isoelectric points between 9.5 and 9.7, were isolated from leaves of Phytolacca dioica L. The purification procedure furnished the four proteins with an overall yield of about 16% and separated them from a protein of 29,407 +/- 2 Da, as determined by electrospray mass spectrometry, whose N-terminal amino acid sequence differed from that of pokeweed (Phytolacca americana L.) leaf chitinase (PLC-B) by only one amino acid (R17I). The four RIPs (PD-L1 to PD-L4) inhibited protein synthesis by a rabbit reticulocyte lysate with 50% inhibition at the picomolar level, and produced the beta-fragment, diagnostic of the specific enzymatic action of RIPs, on yeast ribosomes. Comparison of their N-terminal sequences, up to residue 45, showed that PD-L1 is identical to PD-L2 [designated the isoleucine (Ile) form from the N-terminal residue] and PD-L3 is identical to PD-L4 [designated the valine (Val) form from the N-terminal residue] and that there are 35 identical residues between the two forms. Furthermore, the Val form presents the same number of identical residues as PD-S2, an RIP isolated from the seeds of the same plant. With the exception of PD-L4, the purified RIPs gave a positive reaction when stained for sugars on SDS-PAGE gels and, when analyzed by electrospray mass spectrometry, had M(r) values of 32,715 +/- 1 (PD-L1), 31,542 +/- 1 (PD-L2), 30,356 +/- 1 (PD-L3) and 29,185 +/- 1 Da (PD-L4). The 1171 kDa difference in M(r), within the same RIP form, could be due to glycosylation. Like leaf saporins and many other RIPs, the four RIPs released several adenines from poly(A), herring sperm DNA and rRNA 16S + 23S, thus acting as polynucleotide:adenosine glycosidases. This property was less pronounced in PD-L1 and PD-L3 than in PD-L2 and PD-L4, respectively. The proteins PD-L1 and PD-L4 showed 3.7% reactivity with the antiserum anti-dianthin 32 and no reactivity with antisera to PAP-R saporin-S6, momordin 1 and even PD-S2, an RIP isolated from the seeds of the same plant. Protein PD-L4 showed 12.5% cross-reactivity with anti-PD-L1, while the opposite cross-reactivity was 100%.

The primary structure has been determined for PD-S2, a new type 1 ribosome-inactivating protein (RIP), isolated from the seeds of Phytolacca dioica L. PD-S2 has 265 amino-acid residues, and a molecular mass of 29586 Da. The polypeptide chain contains four amino-acid residues more than PAP-S, a type-I RIP isolated from the seeds of the taxonomically related plant Phytolacca americana L. We have compared the amino-acid sequence of PD-S2 with those of two other RIPs with known three-dimensional structure: PAP-S and ricin A-chain (RTA), the active chain of the best known type-2 RIP. This analysis shows an identity of 76% and 33% with PAP-S and RTA respectively, and a similarity of 82% and 54%. Comparison with the PAP sequence, isolated from leaves of P. americana, shows an even higher identity (80%) and similarity (87%). Furthermore, the amino-acid residues reported in other RIPs to be invariant and participate in the definition of the active site (Tyr-76, Tyr-127, Glu-179, Arg-182 and Trp-211; PD-S2 numbering) are all present. Asn-74, Arg-138, Gln-175, and Glu-208 are also conserved, while Asn-209 is substituted by Glu, all residues located in the active-site cleft of RIPs (Tahirov, T.H., Lu, T.-H., Liaw, Y.-C., Chen, J.L. and Lin, J.Y. (1995) Crystal structure of abrin-a at 2.14 A, J. Mol. Biol. 250, 354-367). The polypeptide chain of PD-S2 contains two N-glycosylation sites at Asn-112 and Asn-120, the second of which appears to be linked to sugars. Like PAP-S, PD-S2 does not contain free sulfhydryl groups. The four cysteinyl residues of the two proteins have corresponding sequence positions, most likely with identical S-S pairing.

Three ribosome-inactivating proteins (RIPs) similar to those already known (Stirpe et al. (1992) Bio/Technology 10, 405–412) were purified from the seeds of Phytolacca dioica . These proteins, called Phytolacca dioica RIPs (PD-S1, PD-S2 and PD-S3 RIPs), are glycoproteins, with M r approx. 30 000, inhibit protein synthesis by a rabbit reticulocyte lysate and phenylalanine polymerization by isolated ribosomes, and depurinate rat liver rRNA in an apparently identical manner as the A-chain of ricin and other RIPs (Endo et al. (1987) J. Biol. Chem. 262, 5908–5912). Part of the purified rat liver ribosomes appeared resistant to the action of PD-S RIPs. The most abundant protein, PD-S2 RIP, gave a weak or nil cross-reaction with sera against various other RIPs, including a pokeweed antiviral protein from the roots of Phytolacca americana . PD-S2 RIP was linked to a monoclonal antibody (Ber-H2) against the CD30 human lymphocyte antigen and the resulting immunotoxin was selectively toxic to the CD30+ Hodgkin's lymphoma-derived L540 cell line.