Interaction of sugar stabilised silver nanoparticles with Momordica charantia seed lectin, a type II ribosome inactivating protein.

Sugar-stabilised nanomaterials have received a lot of attention in cancer therapy in recent years due to their pronounced application as specific targeting agents and maximizing their therapeutic potential while bypassing off-target effects. Lectins, the carbohydrate-binding proteins, are capable of binding to receptors present on the target cell/tissue and interact with transformed glycans better than normal cells. Besides some of the lectins exhibit anticancer activity. Conjugating sugar-stabilised NPs with lectins there for is expected to multiply the potential for the early diagnosis of cancer cells and the specific release of drugs into the tumor site. Because of the prospective applications of lectin-sugar-stabilised nanoparticle conjugates, it is important to understand their molecular interaction and physicochemical properties. Momordica charantia Seed Lectin (MCL) is a type II RIP and has been known as an anti-tumor agent. Investigation of the interaction between sugar-stabilised silver nanoparticles and MCL has been performed by fluorescence spectroscopy to explore the possibility of creating an effective biocompatible drug delivery system against cancer cells. In this regard interaction between lectin and NPs should be well-preserved, while recognizing the specific cell surface sugar. Therefore experiments were carried out in the presence and absence of specific sugar galactose. Protein intrinsic fluorescence emission is quenched at ~ 20% at saturation during the interaction without any significant shift in fluorescence emission maximum. Binding experiments reveal a good affinity. Tetrameric MCL binds to a single nanoparticle. Stern-Volmer analysis of the quenching data suggests that the interaction is via static quenching leading to complex formation. Hemagglutination experiments together with interaction studies in the presence of specific sugar show that the sugar-binding site of the lectin is distinct from the nanoparticle-binding site and cell recognition is very much intact even after binding to AgNPs. Our results propose the possibility of developing MCL-silver nanoparticle conjugate with high stability and multiple properties in the diagnosis and treatment of cancer.

Isomorphic Fluorescent Nucleosides Facilitate Real-Time Monitoring of RNA Depurination by Ribosome Inactivating Proteins.

Ribosome-inactivating proteins, a family of highly cytotoxic proteins, interfere with protein synthesis by depurinating a specific adenosine residue within the conserved α-sarcin/ricin loop of eukaryotic ribosomal RNA. Besides being biological warfare agents, certain RIPs have been promoted as potential therapeutic tools. Monitoring their deglycosylation activity and their inhibition in real time have remained, however, elusive. Herein, we describe the enzymatic preparation and utility of consensus RIP hairpin substrates in which specific G residues, next to the depurination site, are surgically replaced with tz G and th G, fluorescent G analogs. By strategically modifying key positions with responsive fluorescent surrogate nucleotides, RIP-mediated depurination can be monitored in real time by steady-state fluorescence spectroscopy. Subtle differences observed in preferential depurination sites provide insight into the RNA folding as well as RIPs' substrate recognition features.

The root-specific NtR12 promoter-based expression of RIP increased the resistance against bacterial wilt disease in tobacco.

BACKGROUND: Tobacco is an important economic crop, but the quality and yield have been severely impaired by bacterial wilt disease (BWD) caused by Ralstonia solanacearum. METHODS AND RESULTS: Here, we describe a transgenic approach to prevent BWD in tobacco plants. A new root-specific promoter of an NtR12 gene was successfully cloned. The NtR12 promoter drove GUS reporter gene expression to a high level in roots but to less extent in stems, and no significant expression was detected in leaves. The Ribosome-inactivating proteins (RIP) gene from Momordica charantia was also cloned, and its ability to inhibit Ralstonia solanacearum was evaluated using RIP protein produced by the prokaryotic expression system. The RIP gene was constructed downstream of the NtR12 promoter and transformed into the tobacco cultivar "Cuibi No. 1" (CB-1), resulting in many descendants. The resistance against BWD was significantly improved in transgenic tobacco lines expressing NtR12::RIP. CONCLUSION: This study confirms that the RIP gene confers resistance to BWD and the NtR12 as a new promoter for its specific expression in root and stem. Our findings pave a novel avenue for transgenic engineering to prevent the harmful impact of diseases and pests in roots and stems.

Lyophyllin, a Mushroom Protein from the Peptidase M35 Superfamily Is an RNA N-Glycosidase.

Ribosome-inactivating proteins (RIPs) hydrolyze the N-glycosidic bond and depurinate a specific adenine residue (A-4324 in rat 28S ribosomal RNA, rRNA) in the conserved α-sarcin/ricin loop (α-SRL) of rRNA. In this study, we have purified and characterized lyophyllin, an unconventional RIP from Lyophyllum shimeji, an edible mushroom. The protein resembles peptidase M35 domain of peptidyl-Lys metalloendopeptidases. Nevertheless, protein either from the mushroom or in recombinant form possessed N-glycosidase and protein synthesis inhibitory activities. A homology model of lyophyllin was constructed. It was found that the zinc binding pocket of this protein resembles the catalytic cleft of a classical RIP, with key amino acids that interact with the adenine substrate in the appropriate positions. Mutational studies showed that E122 may play a role in stabilizing the positively charged oxocarbenium ion and H121 for protonating N-3 of adenine. The tyrosine residues Y137 and Y104 may be used for stacking the target adenine ring. This work first shows a protein in the peptidase M35 superfamily based on conserved domain search possessing N-glycosidase activity.

New ribosome-inactivating proteins and other proteins with protein synthesis-inhibiting activities.

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.

Intracellular Transport of Ribosome-Inactivating Proteins Depends on Annexin 13.

In the present study, we assessed the role of annexin 13 membrane-binding protein (ANXA13) in the intracellular transport of vesicles containing type II ribosome-inactivating proteins (RIP-IIs). A modified human intestinal epithelial cell line HT29 was used, in which the expression of ANXA13 was significantly reduced. The cytotoxic effect of ricin and viscumin was evaluated by modification of 28S ribosome RNA. The observed differences in the activity of toxins on the parental and modified HT29 lines indicate that ANXA13 plays a different role in the intracellular transport of vesicles containing the RIP-IIs.

Generality of toxins in defensive symbiosis: Ribosome-inactivating proteins and defense against parasitic wasps in Drosophila.

While it has become increasingly clear that multicellular organisms often harbor microbial symbionts that protect their hosts against natural enemies, the mechanistic underpinnings underlying most defensive symbioses are largely unknown. Spiroplasma bacteria are widespread associates of terrestrial arthropods, and include strains that protect diverse Drosophila flies against parasitic wasps and nematodes. Recent work implicated a ribosome-inactivating protein (RIP) encoded by Spiroplasma, and related to Shiga-like toxins in enterohemorrhagic Escherichia coli, in defense against a virulent parasitic nematode in the woodland fly, Drosophila neotestacea. Here we test the generality of RIP-mediated protection by examining whether Spiroplasma RIPs also play a role in wasp protection, in D. melanogaster and D. neotestacea. We find strong evidence for a major role of RIPs, with ribosomal RNA (rRNA) from the larval endoparasitic wasps, Leptopilina heterotoma and Leptopilina boulardi, exhibiting the hallmarks of RIP activity. In Spiroplasma-containing hosts, parasitic wasp ribosomes show abundant site-specific depurination in the α-sarcin/ricin loop of the 28S rRNA, with depurination occurring soon after wasp eggs hatch inside fly larvae. Interestingly, we found that the pupal ectoparasitic wasp, Pachycrepoideus vindemmiae, escapes protection by Spiroplasma, and its ribosomes do not show high levels of depurination. We also show that fly ribosomes show little evidence of targeting by RIPs. Finally, we find that the genome of D. neotestacea's defensive Spiroplasma encodes a diverse repertoire of RIP genes, which are differ in abundance. This work suggests that specificity of defensive symbionts against different natural enemies may be driven by the evolution of toxin repertoires, and that toxin diversity may play a role in shaping host-symbiont-enemy interactions.

Functional analysis of RIP toxins from the Drosophila endosymbiont Spiroplasma poulsonii.

BACKGROUND: Insects frequently live in close relationship with symbiotic bacteria that carry out beneficial functions for their host, like protection against parasites and viruses. However, in some cases, the mutualistic nature of such associations is put into question because of detrimental phenotypes caused by the symbiont. One example is the association between the vertically transmitted facultative endosymbiont Spiroplasma poulsonii and its natural host Drosophila melanogaster. Whereas S. poulsonii protects its host against parasitoid wasps and nematodes by the action of toxins from the family of Ribosome Inactivating Proteins (RIPs), the presence of S. poulsonii has been reported to reduce host's life span and to kill male embryos by a toxin called Spaid. In this work, we investigate the harmful effects of Spiroplasma RIPs on Drosophila in the absence of parasite infection. RESULTS: We show that only two Spiroplasma RIPs (SpRIP1 and SpRIP2) among the five RIP genes encoded in the S. poulsonii genome are significantly expressed during the whole Drosophila life cycle. Heterologous expression of SpRIP1 and 2 in uninfected flies confirms their toxicity, as indicated by a reduction of Drosophila lifespan and hemocyte number. We also show that RIPs can cause the death of some embryos, including females. CONCLUSION: Our results indicate that RIPs released by S. poulsonii contribute to the reduction of host lifespan and embryo mortality. This suggests that SpRIPs may impact the insect-symbiont homeostasis beyond their protective function against parasites.

Engineering of spectator glycocalyx structures to evaluate molecular interactions at crowded cellular boundaries.

In the mucosal epithelium, the cellular glycocalyx can project tens to hundreds of nanometers into the extracellular space, erecting a physical barrier that provides protective functions, mediates the exchange of nutrients and regulates cellular interactions. Little is understood about how the physical properties of the mucosal glycocalyx influence molecular recognition at the cellular boundary. Here, we report the synthesis of PEG-based glycopolymers with tunable glycan composition, which approximate the extended architecture of mucin glycoproteins, and tether them to the plasma membranes of red blood cells (RBC) to construct an artificial mucin brush-like glycocalyx. We evaluated the association of two lectins, ConA and SNA, with their endogenous glycan ligands on the surface of the remodelled cells. The extended glycocalyx provided protection against agglutination of RBCs by both lectins; however, the rate and magnitude of ConA binding were attenuated to a greater degree in the presence of the glycopolymer spectators compared to those measured for SNA. The different sensitivity of ConA and SNA to glycocalyx crowding likely arises from the distinct presentation of their mannoside and sialoside receptors, respectively, within the native RBC glycocalyx.

A ribosome-inactivating protein in a Drosophila defensive symbiont.

Vertically transmitted symbionts that protect their hosts against parasites and pathogens are well known from insects, yet the underlying mechanisms of symbiont-mediated defense are largely unclear. A striking example of an ecologically important defensive symbiosis involves the woodland fly Drosophila neotestacea, which is protected by the bacterial endosymbiont Spiroplasma when parasitized by the nematode Howardula aoronymphium. The benefit of this defense strategy has led to the rapid spread of Spiroplasma throughout the range of D. neotestacea, although the molecular basis for this protection has been unresolved. Here, we show that Spiroplasma encodes a ribosome-inactivating protein (RIP) related to Shiga-like toxins from enterohemorrhagic Escherichia coli and that Howardula ribosomal RNA (rRNA) is depurinated during Spiroplasma-mediated protection of D. neotestacea. First, we show that recombinant Spiroplasma RIP catalyzes depurination of 28S rRNAs in a cell-free assay, as well as Howardula rRNA in vitro at the canonical RIP target site within the α-sarcin/ricin loop (SRL) of 28S rRNA. We then show that Howardula parasites in Spiroplasma-infected flies show a strong signal of rRNA depurination consistent with RIP-dependent modification and large decreases in the proportion of 28S rRNA intact at the α-sarcin/ricin loop. Notably, host 28S rRNA is largely unaffected, suggesting targeted specificity. Collectively, our study identifies a novel RIP in an insect defensive symbiont and suggests an underlying RIP-dependent mechanism in Spiroplasma-mediated defense.

Biological and antipathogenic activities of ribosome-inactivating proteins from Phytolacca dioica L.

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.

Binding of a type 1 RIP and of its chimeric variant to phospholipid bilayers: evidence for a link between cytotoxicity and protein/membrane interactions.

Ribosome-inactivating proteins (RIPs) are enzymes, almost all identified in plants, able to kill cells by depurination of rRNAs. Recently, in order to improve resistance to proteolysis of a type 1 RIP (PD-L4), we produced a recombinant chimera combining it with a wheat protease inhibitor (WSCI). Resulting chimeric construct, named PD-L4UWSCI, in addition to present the functions of the two domains, shows also an enhanced cytotoxic action on murine cancer cells when compared to PD-L4. Since different ways of interaction of proteins with membranes imply different resulting effects on cells, in this study we investigate conformational stability of PD-L4 and PD-L4UWSCI and their interaction with membrane models (liposomes). Circular dichroism analysis and differential scanning calorimetry measurements indicate that PD-L4 and PD-L4UWSCI present high and similar conformational stability, whereas analysis of their binding to liposomes, obtained by isothermal titration calorimetry and differential scanning calorimetry, clearly indicate that chimera is able to interact with biomembranes more effectively. Overall, our data point out that WSCI domain, probably because of its flexibility in solution, enhances the chimeric protein interaction with membrane lipid surfaces without however destabilizing the overall protein structure. Analysis of interactions between RIPs or RIP based conjugates and lipid surfaces could provide novel insights in the search of more effective selective membrane therapeutics.

Effectively enhancing cytotoxic and apoptotic effects of alpha-momorcharin by integrating a heparin-binding peptide.

Alpha-momorcharin (α-MMC), a type I ribosome-inactivating protein, has attracted a great deal of attention because of its antitumor activity. However, the cytotoxicity of α-MMC is limited due to insufficient cellular internalization in cancer cells. To enhance the cytotoxicity of α-MMC, a heparin-binding domain derived from heparin-binding epidermal growth factor (named heparin-binding peptide [HBP]) was used as a cell-penetrating peptide and fused to the C-terminus of α-MMC. This novel α-MMC-HBP fusion protein was expressed and purified with a Ni2+ -resin. The N-glycosidase activity and DNase activity assay indicated that the introduction of HBP did not interfere with the intrinsic bioactivities of α-MMC. HBP was able to efficiently carry α-MMC into the tested cancer cells and significantly enhance the cytotoxic effects of α-MMC in a dose-dependent manner. This enhanced cytotoxic ability occurred due to the higher level of cell apoptosis induced by α-MMC-HBP, which was demonstrated in western blot analysis in which α-MMC-HBP triggered caspase 8, caspase 9, casapase 3, and PARP more intensely than α-MMC alone. α-MMC-HBP led to an upregulation of cleaved PARP and an increase in the Bax/Bcl-2 ratio. Our study provided a new practical way to greatly improve the antitumor activity of α-MMC, which could significantly expand the pharmaceutical applications of α-MMC.

A comparison between constitutive and inducible transgenic expression of the PhRIP I gene for broad-spectrum resistance against phytopathogens in potato.

OBJECTIVES: To engineer broad spectrum resistance in potato using different expression strategies. RESULTS: The previously identified Ribosome-Inactivating Protein from Phytolacca heterotepala was expressed in potato under a constitutive or a wound-inducible promoter. Leaves and tubers of the plants constitutively expressing the transgene were resistant to Botrytis cinerea and Rhizoctonia solani, respectively. The wound-inducible promoter was useful in driving the expression upon wounding and fungal damage, and conferred resistance to B. cinerea. The observed differences between the expression strategies are discussed considering the benefits and features offered by the two systems. CONCLUSIONS: Evidence is provided of the possible impact of promoter sequences to engineer BSR in plants, highlighting that the selection of a suitable expression strategy has to balance specific needs and target species.

Special Issue: Ribosome-Inactivating Proteins-Commemorative Issue in Honor of Professor Fiorenzo Stirpe.

The family of ribosome-inactivating proteins (RIPs) groups all enzymes (EC.3.2.2.22) with a so-called RIP domain which comprises N-glycosidase activity and enables these proteins to catalytically inactivate ribosomes.[...].

Lectin inhibits antigen-antibody reaction in a glycoform-specific manner: Application for detecting α2,6sialylated-carcinoembryonic antigen.

Carcinoembryonic antigen (CEA) is a glycoprotein marker, which is widely used for diagnosing various cancers, especially colon adenocarcinoma. In addition, CEA mediates homotypic adhesion of colon adenocarcinoma cells, which appears to favor hematogenous metastasis. CEA carries α2,6sialyl residues on its N-glycans whereas a normal counterpart, normal fecal antigen-2, does α2,3sialyl residues, suggesting that cancer-specific  α2,6sialylation on CEA may play a role for cell invasion and metastasis. A simple and rapid estimation of α2,6sialyled CEA in detergent extracts from formalin-fixed colon adenocarcinoma by "lectin inhibition" is reported. In the lectin inhibition method, Sambucus sieboldiana Agglutinin (SSA) lectin, an α2,6sialic acid binder, was used as a glycoform-specific inhibitor for antigen-antibody reaction in ELISA. Detergent extracts from colon adenocarcinoma showed a fair amount of ELISA signal in the absence of SSA whereas the signal was markedly reduced (45≈74%) in the presence of SSA, suggesting that the extracts contains α2,6sialyled CEA. The presence of α2,6sialyled CEA in the extracts was confirmed by lectin microarray, in which SSA, Sambucus nigra agglutinin, and Trichosanthes japonica agglutinin I lectins were used as α2,6sialyl binders. Thus lectin inhibition is a simple and rapid method for detecting α2,6sialyled CEA even in crude detergent extracts from formalin-fixed adenocarcinoma tissue.

Responsive Fluorescent PNA Analogue as a Tool for Detecting G-quadruplex Motifs of Oncogenes and Activity of Toxic Ribosome-Inactivating Proteins.

Fluorescent oligomers that are resistant to enzymatic degradation and report their binding to target oligonucleotides (ONs) by changes in fluorescence properties are highly useful in developing nucleic-acid-based diagnostic tools and therapeutic strategies. Here, we describe the synthesis and photophysical characterization of fluorescent peptide nucleic acid (PNA) building blocks made of microenvironment-sensitive 5-(benzofuran-2-yl)- and 5-(benzothiophen-2-yl)-uracil cores. The emissive monomers, when incorporated into PNA oligomers and hybridized to complementary ONs, are minimally perturbing and are highly sensitive to their neighboring base environment. In particular, benzothiophene-modified PNA reports the hybridization process with significant enhancement in fluorescence intensity, even when placed in the vicinity of guanine residues, which often quench fluorescence. This feature was used in the turn-on detection of G-quadruplex-forming promoter DNA sequences of human proto-oncogenes (c-myc and c-kit). Furthermore, the ability of benzothiophene-modified PNA oligomer to report the presence of an abasic site in RNA enabled us to develop a simple fluorescence hybridization assay to detect and estimate the depurination activity of ribosome-inactivating protein toxins. Our results demonstrate that this approach with responsive PNA probes will provide new opportunities to develop robust tools to study nucleic acids.

Structures and Ribosomal Interaction of Ribosome-Inactivating Proteins.

Ribosome-inactivating proteins (RIPs) including ricin, Shiga toxin, and trichosanthin, are RNA N-glycosidases that depurinate a specific adenine residue (A-4324 in rat 28S ribosomal RNA, rRNA) in the conserved α-sarcin/ricin loop (α-SRL) of rRNA. RIPs are grouped into three types according to the number of subunits and the organization of the precursor sequences. RIPs are two-domain proteins, with the active site located in the cleft between the N- and C-terminal domains. It has been found that the basic surface residues of the RIPs promote rapid and specific targeting to the ribosome and a number of RIPs have been shown to interact with the C-terminal regions of the P proteins of the ribosome. At present, the structural basis for the interaction of trichosanthin and ricin-A chain toward P2 peptide is known. This review surveys the structural features of the representative RIPs and discusses how they approach and interact with the ribosome.

Plants Producing Ribosome-Inactivating Proteins in Traditional Medicine.

Ribosome-inactivating proteins (RIPs) are enzymes that deadenylate nucleic acids and are broadly distributed in the plant kingdom. Many plants that contain RIPs are listed in the pharmacopoeias of folk medicine all over the world, mostly because of their toxicity. This review analyses the position occupied in traditional medicine by plants from which RIPs have been isolated. The overview starts from the antique age of the Mediterranean area with ancient Egypt, followed by the Greek and Roman classic period. Then, the ancient oriental civilizations of China and India are evaluated. More recently, Unani medicine and European folk medicine are examined. Finally, the African and American folk medicines are taken into consideration. In conclusion, a list of RIP-expressing plants, which have been used in folk medicine, is provided with the geographical distribution and the prescriptions that are recommended by traditional healers. Some final considerations are provided on the present utilization of such herbal treatments, both in developing and developed countries, often in the absence of scientific validation. The most promising prospect for the medicinal use of RIP-expressing plants is the conjugation of purified RIPs to antibodies that recognise tumour antigens for cancer therapy.

Ribosome Inactivating Proteins from Rosaceae.

Ribosome-inactivating proteins (RIPs) are widespread among higher plants of different taxonomic orders. In this study, we report on the RIP sequences found in the genome/transcriptome of several important Rosaceae species, including many economically important edible fruits such as apple, pear, peach, apricot, and strawberry. All RIP domains from Rosaceae share high sequence similarity with conserved residues in the catalytic site and the carbohydrate binding sites. The genomes of Malus domestica and Pyrus communis contain both type 1 and type 2 RIP sequences, whereas for Prunus mume, Prunus persica, Pyrus bretschneideri, and Pyrus communis a complex set of type 1 RIP sequences was retrieved. Heterologous expression and purification of the type 1 as well as the type 2 RIP from apple allowed to characterize the biological activity of the proteins. Both RIPs from Malus domestica can inhibit protein synthesis. Furthermore, molecular modelling suggests that RIPs from Rosaceae possess three-dimensional structures that are highly similar to the model proteins and can bind to RIP substrates. Screening of the recombinant type 2 RIP from apple on a glycan array revealed that this type 2 RIP interacts with terminal sialic acid residues. Our data suggest that the RIPs from Rosaceae are biologically active proteins.