Solution Structure of an Active Mutant of Maize Ribosome-Inactivating Protein (MOD) and Its Interaction with the Ribosomal Stalk Protein P2

Ribosome-inactivating proteins (RIPs) are N-glycosidases that depurinate a specific adenine residue in the conserved sarcin/ricin loop of ribosomal RNA. This modification renders the ribosome unable to bind the elongation factors, thereby inhibiting the protein synthesis. Maize RIP, a type III RIP, is unique compared to the other type I and type II RIPs because it is synthesized as a precursor with a 25-residue internal inactivation region, which is removed in order to activate the protein. In this study, we describe the first solution structure of this type of RIP, a  28-kDa active mutant of maize RIP (MOD). The overall protein structure of MOD is comparable to those of the other type I RIPs and the A-chain of type II RIPs but shows significant differences in specific regions, including (1) shorter β6 and αB segments, probably for accommodating easier substrate binding, and (2) an α-helix instead of an antiparallel β-sheet in the C-terminal domain, which has been reported to be involved in binding ribosomal protein P2 in some RIPs. Furthermore, NMR chemical shift perturbation experiments revealed that the P2 binding site on MOD is located at the N-terminal domain near the internal inactivation region. This relocation of the P2 binding site can be rationalized by concerted changes in the electrostatic surface potential and 3D structures on the MOD protein and provides vital clues about the underlying molecular mechanism of this unique type of RIP.     

Pokeweed antiviral protein regulates the stability of its own mRNA by a mechanism that requires depurination but can be separated from depurination of the alpha-sarcin/ricin loop of rRNA

Pokeweed antiviral protein (PAP), a single chain ribosome-inactivating protein (RIP) isolated from pokeweed plants (Phytolacca americana), removes specific adenine and guanine residues from the highly conserved, alpha-sarcin/ricin loop in the large rRNA, resulting in inhibition of protein synthesis. We recently demonstrated that PAP could also inhibit translation of mRNAs and viral RNAs that are capped by binding to the cap structure and depurinating the RNAs downstream of the cap. Cell growth is inhibited when PAP cDNA is expressed in the yeast Saccharomyces cerevisiae under the control of the galactose-inducible GAL1 promoter. Here, we show that overexpression of wild type PAP in yeast leads to a decrease in PAP mRNA abundance. The decrease in mRNA levels is not observed with an active site mutant, indicating that it is due to the N-glycosidase activity of the protein. PAP expression had no effect on steady state levels of mRNA from four different endogenous yeast genes examined, indicating specificity. We demonstrate that PAP can depurinate the rRNA in trans in a translation-independent manner. When rRNA is depurinated and translation is inhibited, the steady state levels of PAP mRNA increase dramatically relative to the U3 snoRNA. Using a PAP variant which depurinates rRNA, inhibits translation but does not destabilize its mRNA, we demonstrate that PAP mRNA is destabilized after its levels are up-regulated by a mechanism that occurs independently of rRNA depurination and translation. We quantify the extent of rRNA depurination in vivo using a novel primer extension assay and show that the temporal pattern of rRNA depurination is similar to the pattern of PAP mRNA destabilization, suggesting that they may occur by a common mechanism. These results provide the first in vivo evidence that a single chain RIP targets not only the large rRNA but also its own mRNA. These findings have implications for understanding the biological function of RIPs.     

Characterization of pokeweed antiviral protein binding to mRNA cap analogs: competition with nucleotides and enhancement by translation initiation factor iso4G

Pokeweed antiviral protein (PAP) is a type I ribosomal inactivating protein (RIP). PAP binds to and depurinates the sarcin/ricin loop (SRL) of ribosomal RNA resulting in the cessation of protein synthesis. PAP has also been shown to bind to mRNA cap analogs and depurinate mRNA downstream of the cap structure. The biological role of cap binding and its possible role in PAP activity are not known. Here we show the first direct quantitative evidence for PAP binding to the cap analog m(7)GTP. We report a binding affinity of 43.3+/-0.1 nM at 25 degrees C as determined by fluorescence quenching experiments. This is similar to the values reported for wheat cap-binding proteins eIFiso4E and eIFiso4F. van't Hoff analysis of m(7)GTP-PAP equilibrium reveals a binding reaction that is enthalpy driven and entropy favored with TDeltaS degrees contributing 15% to the overall value of DeltaG degrees . This is in contrast to the wheat cap-binding proteins which are enthalpically driven in the DeltaG degrees for binding. Competition experiments indicate that ATP and GTP compete for the cap-binding site on PAP with slightly different affinities. Fluorescence studies of PAP-eIFiso4G binding reveal a protein-protein interaction with a K(d) of 108.4+/-0.3 nM. eIFiso4G was shown to enhance the interaction of PAP with m(7)GTP cap analog by 2.4-fold. These results suggest the involvement of PAP-translation initiation factor complexes in RNA selection and depurination.     

Correlation between the activities of five ribosome-inactivating proteins in depurination of tobacco ribosomes and inhibition of tobacco mosaic virus infection

The rRNA depurination activities of five ribosome-inactivating proteins (RIPs) were compared in vitro using yeast and tobacco leaf ribosomes as substrates. All of the RIPs (pokeweed antiviral protein (PAP), dianthin 32, tritin, barley RIP and ricin A-chain) were active on yeast ribosomes. PAP and dianthin 32 were highly active and ricin A-chain weakly active on tobacco ribosomes, whereas tritin and barley RIP were inactive. PAP and dianthin 32 were highly effective in inhibiting the formation of local lesions caused by tobacco mosaic virus (TMV) on tobacco leaves, whereas tritin, barley RIP and ricin A-chain were ineffective. The apparent anomaly between the in vitro rRNA depurination activity, but lack of antiviral activity of ricin A-chain was further investigated by assaying for rRNA depurination in situ following the topical application of the RIP to leaves. No activity was detected, a finding consistent with the apparent lack of antiviral activity of this RIP. Thus, it is concluded that there is a positive correlation between RIP-catalysed depurination of tobacco ribosomes and antiviral activity which gives strong support to the hypothesis that the antiviral activity of RIPs works through ribosome inactivation.     

The C-terminus of pokeweed antiviral protein has distinct roles in transport to the cytosol, ribosome depurination and cytotoxicity

Pokeweed antiviral protein (PAP) produced by pokeweed plants is a single-chain (type I) ribosome-inactivating protein (RIP) that depurinates ribosomes at the alpha-sarcin/ricin loop of the large rRNA, resulting in inhibition of translation. Unlike the type II RIPs, which have an active and a binding moiety, PAP has only the active moiety. The mechanism by which toxins without a binding moiety gain access to cytosolic ribosomes is not known. We set up yeast as a simple and genetically tractable system to investigate how PAP accesses ribosomes and showed that the mature form of PAP is targeted to the cytosol from the endomembrane system in yeast. In the present study, we performed a systematic deletion analysis to identify the signal required for transport of PAP to the cytosol. We demonstrate here that processing of the C-terminal extension and sequences at the C-terminus of the mature protein are critical for its accumulation in the cytosol. Using a series of PAP mutants, we identified the C-terminal signal and demonstrated that it is distinct from the sequences required for ribosome depurination and cytotoxicity. The C-terminal motif showed sequence similarity to type II RIPs that retrotranslocate from the endoplasmic reticulum to the cytosol. These results demonstrate that a conserved sequence at the C-terminus of a type I RIP mediates its transport to the cytosol and suggest that type I and II RIPs may use a common signal to enter the cytosol.     

Binding interactions between the active center cleft of recombinant pokeweed antiviral protein and the alpha-sarcin/ricin stem loop of ribosomal RNA

Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein that catalytically cleaves a specific adenine base from the highly conserved alpha-sarcin/ricin loop of the large ribosomal RNA, thereby inhibiting protein synthesis at the elongation step. Recently, we discovered that alanine substitutions of the active center cleft residues significantly impair the depurinating and ribosome inhibitory activity of PAP. Here we employed site-directed mutagenesis combined with standard filter binding assays, equilibrium binding assays with Scatchard analyses, and surface plasmon resonance technology to elucidate the putative role of the PAP active center cleft in the binding of PAP to the alpha-sarcin/ricin stem loop of rRNA. Our findings presented herein provide experimental evidence that besides the catalytic site, the active center cleft also participates in the binding of PAP to the target tetraloop structure of rRNA. These results extend our recent modeling studies, which predicted that the residues of the active center cleft could, via electrostatic interactions, contribute to both the correct orientation and stable binding of the substrate RNA molecules in PAP active site pocket. The insights gained from this study also explain why and how the conserved charged and polar side chains located at the active center cleft of PAP and certain catalytic site residues, that do not directly participate in the catalytic deadenylation of ribosomal RNA, play a critical role in the catalytic removal of the adenine base from target rRNA substrates by affecting the binding interactions between PAP and rRNA.     

The 1.4 anstroms structure of dianthin 30 indicates a role of surface potential at the active site of type 1 ribosome inactivating proteins

Ribosome inactivating proteins (RIPs) are plant proteins with enzymatic activity identified as rRNA N-glycosidase (EC 3.2.2.22), which cleaves the N-glycosidic bond of a specific adenine on the ricin/sarcin region of rRNA, thus causing inhibition of protein synthesis. They also depurinate extensively DNA and other polynucleotides. The three-dimensional structure of dianthin 30, a type 1 (single-chain) RIP of Dianthus caryophyllus (leaves), is now described at 1.4 angstroms, a resolution never achieved before for any RIP. The fold typical of RIPs is conserved, despite some differences in the loop regions. The general structure comparison by superimposed alpha-carbon (249 atoms) and the sequence alignment by structure for dianthin 30 and saporin-S6 give a root mean square deviation of 0.625 angstroms. Despite the differences reported for the biological activities of the two RIPs, their structures fit quite well and both show a protein segment containing strands beta7, beta8, and beta9 shorter than other RIPs. However, the surface electrostatic potential in the active site region neatly distinguishes dianthin 30 from saporin-S6. The possible relationship between the charge distribution and the behavior of the proteins toward different substrates is discussed.     

Pokeweed antiviral protein cleaves double-stranded supercoiled DNA using the same active site required to depurinate rRNA

Ribosome-inactivating proteins (RIPs) are N-glycosylases that remove a specific adenine from the sarcin/ricin loop of the large rRNA in a manner analogous to N-glycosylases that are involved in DNA repair. Some RIPs have been reported to remove adenines from single-stranded DNA and cleave double-stranded supercoiled DNA. The molecular basis for the activity of RIPs on double-stranded DNA is not known. Pokeweed antiviral protein (PAP), a single-chain RIP from Phytolacca americana, cleaves supercoiled DNA into relaxed and linear forms. Double-stranded DNA treated with PAP contains apurinic/apyrimidinic (AP) sites due to the removal of adenine. Using an active-site mutant of PAP (PAPx) which does not depurinate rRNA, we present evidence that double-stranded DNA treated with PAPx does not contain AP sites and is not cleaved. These results demonstrate for the first time that PAP cleaves supercoiled double-stranded DNA using the same active site that is required for depurination of rRNA.     

Active center cleft residues of pokeweed antiviral protein mediate its high-affinity binding to the ribosomal protein L3

Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein (RIP) which catalytically cleaves a specific adenine base from the highly conserved alpha-sarcin/ricin loop (SRL) of the large ribosomal RNA and thereby inhibits the protein synthesis. The ribosomal protein L3, a highly conserved protein located at the peptidyltransferase center of the ribosomes, is involved in binding of PAP to ribosomes and subsequent depurination of the SRL. We have recently discovered that recombinant PAP mutants with alanine substitution of the active center cleft residues (69)NN(70) (FLP-4) and (90)FND(92) (FLP-7) that are not directly involved in the catalytic depurination at the active site exhibit >150-fold reduced ribosome inhibitory activity [(2000) J. Biol. Chem. 275, 3382--3390]. We hypothesized that the partially exposed half of the active site cleft could be the potential docking site for the L3 molecule. Our modeling studies presented herein indicated that PAP residues 90--96, 69--70, and 118--120 potentially interact with L3. Therefore, mutations of these residues were predicted to result in destabilization of interactions with rRNA and lead to a lower binding affinity with L3. In the present structure-function relationship study, coimmunoprecipitation assays with an in vitro synthesized yeast ribosomal protein L3 suggested that these mutant PAP proteins poorly interact with L3. The binding affinities of the mutant PAP proteins for ribosomes and recombinant L3 protein were calculated from rate constants and analysis of binding using surface plasmon resonance biosensor technology. Here, we show that, compared to wild-type PAP, FLP-4/(69)AA(70) and FLP-7/(90)AAA(92) exhibit significantly impaired affinity for ribosomes and L3 protein, which may account for their inability to efficiently inactivate ribosomes. By comparison, recombinant PAP mutants with alanine substitutions of residues (28)KD(29) and (111)SR(112) that are distant from the active center cleft showed normal binding affinity to ribosomes and L3 protein. The single amino acid mutants of PAP with alanine substitution of the active center cleft residues N69 (FLP-20), F90 (FLP-21), N91 (FLP-22), or D92 (FLP-23) also showed reduced ribosome binding as well as reduced L3 binding, further confirming the importance of the active center cleft for the PAP--ribosome and PAP--L3 interactions. The experimental findings presented in this report provide unprecedented evidence that the active center cleft of PAP is important for its in vitro binding to ribosomes via the L3 protein.     

Small-molecule inhibitor leads of ribosome-inactivating proteins developed using the doorstop approach

Ribosome-inactivating proteins (RIPs) are toxic because they bind to 28S rRNA and depurinate a specific adenine residue from the α-sarcin/ricin loop (SRL), thereby inhibiting protein synthesis. Shiga-like toxins (Stx1 and Stx2), produced by Escherichia coli, are RIPs that cause outbreaks of foodborne diseases with significant morbidity and mortality. Ricin, produced by the castor bean plant, is another RIP lethal to mammals. Currently, no US Food and Drug Administration-approved vaccines nor therapeutics exist to protect against ricin, Shiga-like toxins, or other RIPs. Development of effective small-molecule RIP inhibitors as therapeutics is challenging because strong electrostatic interactions at the RIP•SRL interface make drug-like molecules ineffective in competing with the rRNA for binding to RIPs. Herein, we report small molecules that show up to 20% cell protection against ricin or Stx2 at a drug concentration of 300 nM. These molecules were discovered using the doorstop approach, a new approach to protein•polynucleotide inhibitors that identifies small molecules as doorstops to prevent an active-site residue of an RIP (e.g., Tyr80 of ricin or Tyr77 of Stx2) from adopting an active conformation thereby blocking the function of the protein rather than contenders in the competition for binding to the RIP. This work offers promising leads for developing RIP therapeutics. The results suggest that the doorstop approach might also be applicable in the development of other protein•polynucleotide inhibitors as antiviral agents such as inhibitors of the Z-DNA binding proteins in poxviruses. This work also calls for careful chemical and biological characterization of drug leads obtained from chemical screens to avoid the identification of irrelevant chemical structures and to avoid the interference caused by direct interactions between the chemicals being screened and the luciferase reporter used in screening assays.     

Cloning of the mistletoe lectin gene and characterization of the recombinant A-chain.

Mistletoe lectin I (MLI) is the major active constituent of mistletoe extracts, which are widely used for adjuvant tumour therapy. The 66-kDa heterodimeric disulphide-linked glycoprotein is classified as type II ribosome-inactivating protein (RIP) due to the rRNA-cleaving enzyme activity of the A-subunit, also referred to as toxic entity. MLI and the close relative ricin both belong to the family of the two-chain plant type II RIP proteins. Isolation of the glycosylated proteins from plant material yield inhomogeneous material probably due to post-translational modifications. The aim of this study was to prepare pure and homogeneous protein as a prerequisite for structural and mechanistic studies in order to gain insight into the mode of action of this cytotoxic plant protein on tumour and immune cells. Of particular interest was to explain whether the differences in toxicity of ML and ricin are the result of variations of their enzymatic activities. By investigating the sequence homologies between the active sites of different RIPs we were able to deduce a set of primers which were suitable for specific amplification of the mistletoe lectin gene. Applying this PCR strategy the full-length 1923 nucleotide DNA sequence coding for the prepro-protein was obtained showing the existence of a single intron-free gene. In order to elucidate the molecular basis for the observed differences in cytotoxicity within the family of RIP the enzymatic A-subunit was expressed in a heterologous system. Expression of the A-chain in E. coli BL21/pT7 resulted in production of insoluble inclusion bodies constituting 20-30% of total protein. Refolding led to a pure and homogeneous protein species with an apparent molecular mass of 27 kDa and a pI value of 6.4. The ribosome-inactivating activity of the unglycosylated recombinant A-chain (IC50 20.5 pM) protein was in the same range as that of the glycosylated plant-derived ML A-chain (IC50 3.7 pM), which was very similar to that of ricin A-chain (IC50 4.9 pM). Thus, the higher cytotoxicity of ricin cannot be accountable for differences in the enzymatic activities of the type II RIP A-chains.     

Evidence for retro-translocation of pokeweed antiviral protein from endoplasmic reticulum into cytosol and separation of its activity on ribosomes from its activity on capped RNA

Pokeweed antiviral protein (PAP) is a single-chain ribosome inactivating protein (RIP) that binds to ribosomes and depurinates the highly conserved alpha-sarcin/ricin loop (SRL) of the large subunit rRNA. Catalytic depurination of a specific adenine has been proposed to result in translation arrest and cytotoxicity. Here, we show that both precursor and mature forms of PAP are localized in the endoplasmic reticulum (ER) in yeast. The mature form is retro-translocated from the ER into the cytosol where it escapes degradation unlike the other substrates of the retro-translocation pathway. A mutation of a highly conserved asparagine residue at position 70 (N70A) delays ribosome depurination and the onset of translation arrest. The ribosomes are eventually depurinated, yet cytotoxicity and loss of viability are markedly absent. Analysis of the variant protein, N70A, does not reveal any decrease in the rate of synthesis, subcellular localization, or the rate of transport into the cytosol. N70A destabilizes its own mRNA, binds to cap, and blocks cap dependent translation, as previously reported for the wild-type PAP. However, it cannot depurinate ribosomes in a translation-independent manner. These results demonstrate that N70 near the active-site pocket is required for depurination of cytosolic ribosomes but not for cap binding or mRNA destabilization, indicating that the activity of PAP on capped RNA can be uncoupled from its activity on rRNA. These findings suggest that the altered active site of PAP might accommodate a narrower range of substrates, thus reducing ribotoxicity while maintaining potential therapeutic benefits.     

Structural analysis of toxic volkensin, a type 2 ribosome inactivating protein from Adenia volkensii Harm (kilyambiti plant): Molecular modeling and surface analysis by computational methods and limited proteolysis

Volkensin, isolated from Adenia volkensii, is one of the most toxic type 2 ribosome-inactivating protein (RIP), exerting its biological function by inhibiting protein synthesis. Despite the high sequence identity with type 2 RIPs, including ricin, volkensin shows interesting peculiar properties.In this work a computational model building of volkensin was performed. The volkensin electrostatic potential charge distribution, the hydrophobic profile and the surface topology analyses were also carried out to aid the understanding of structure–function relationships of this potent toxin. Volkensin surface topology was probed by applying a limited proteolysis approach with the aim to gain insights into volkensin conformational features.     

Inhibition of Pokeweed Antiviral Protein (PAP) by Turnip Mosaic Virus Genome-linked Protein (VPg)

Pokeweed antiviral protein (PAP) from Phytolacca americana is a ribosome-inactivating protein (RIP) and an RNA N-glycosidase that removes specific purine residues from the sarcin/ricin loop of large rRNA, arresting protein synthesis at the translocation step. PAP is also a cap-binding protein and is a potent antiviral agent against many plant, animal, and human viruses. To elucidate the mechanism of RNA depurination, and to understand how PAP recognizes and targets various RNAs, the interactions between PAP and turnip mosaic virus genome-linked protein (VPg) were investigated. VPg can function as a cap analog in cap-independent translation and potentially target PAP to uncapped IRES-containing RNA. In this work, fluorescence spectroscopy and HPLC techniques were used to quantitatively describe PAP depurination activity and PAP-VPg interactions. PAP binds to VPg with high affinity (29.5 nm); the reaction is enthalpically driven and entropically favored. Further, VPg is a potent inhibitor of PAP depurination of RNA in wheat germ lysate and competes with structured RNA derived from tobacco etch virus for PAP binding. VPg may confer an evolutionary advantage by suppressing one of the plant defense mechanisms and also suggests the possible use of this protein against the cytotoxic activity of ribosome-inactivating proteins.     

Baicalin Inhibits the Lethality of Ricin in Mice by Inducing Protein Oligomerization

Toxic ribosome-inactivating proteins abolish cell viability by inhibiting protein synthesis. Ricin, a member of these lethal proteins, is a potential bioterrorism agent. Despite the grave challenge posed by these toxins to public health, post-exposure treatment for intoxication caused by these agents currently is unavailable. In this study, we report the identification of baicalin extracted from Chinese herbal medicine as a compound capable of inhibiting the activity of ricin. More importantly, post-exposure treatment with baicalin significantly increased the survival of mice poisoned by ricin. We determined the mechanism of action of baicalin by solving the crystal structure of its complex with the A chain of ricin (RTA) at 2.2 Å resolution, which revealed that baicalin interacts with two RTA molecules at a novel binding site by hydrogen bond networks and electrostatic force interactions, suggesting its role as molecular glue of the RTA. Further biochemical and biophysical analyses validated the amino acids directly involved in binding the inhibitor, which is consistent with the hypothesis that baicalin exerts its inhibitory effects by inducing RTA to form oligomers in solution, a mechanism that is distinctly different from previously reported inhibitors. This work offers promising leads for the development of therapeutics against ricin and probably other ribosome-inactivating proteins.     

Anti-HIV and anti-tumor protein MAP30, a 30 kDa single-strand type-I RIP, shares similar secondary structure and beta-sheet topology with the A chain of ricin, a type-II RIP

MAP30 is a 30 kDa single-stranded, type-I ribosome inactivating protein (RIP) possessing anti-tumor and anti-HIV activities. It binds both ribosomal RNA and the HIV-1 long-terminal repeat DNA. To understand the structural basis for MAP30 activities, we undertook the study of MAP30 by solution NMR spectroscopy. We report nearly complete 1H, 13C, and 15N chemical shift assignments of its 263 amino acids. Based upon an analysis of secondary 13C chemical shifts, 3J(HNHA) coupling constants, hydrogen exchange data, and nuclear Overhauser effect patterns, we find that the secondary structure and beta-sheet topology of MAP30 are very similar to those of the ricin A chain, a subunit of the well-known type-II RIP, even though two proteins display distinct activities. We therefore suggest that MAP30 and ricin A chain share a similar three-dimensional fold, and that the reported functional differences between two proteins arise primarily from differences in local three-dimensional structure and other structural properties such as surface electrostatic potentials.     

The ribosomal stalk is required for ribosome binding,depurination of the rRNA and cytotoxicity of ricin A chain in Saccharomyces cerevisiae

Ribosome inactivating proteins (RIPs) like ricin, pokeweed antiviral protein (PAP) and Shiga‐like toxins 1 and 2 (Stx1 and Stx2) share the same substrate, the α‐sarcin/ricin loop, but differ in their specificities towards prokaryotic and eukaryotic ribosomes. Ricin depurinates the eukaryotic ribosomes more efficiently than the prokaryotic ribosomes, while PAP can depurinate both types of ribosomes. Accumulating evidence suggests that different docking sites on the ribosome might be used by different RIPs, providing a basis for understanding the mechanism underlying their kingdom specificity. Our previous results demonstrated that PAP binds to the ribosomal protein L3 to depurinate the α‐sarcin/ricin loop and binding of PAP to L3 was critical for its cytotoxicity. Here, we used surface plasmon resonance to demonstrate that ricin toxin A chain (RTA) binds to the P1 and P2 proteins of the ribosomal stalk in Saccharomyces cerevisiae. Ribosomes from the P protein mutants were depurinated less than the wild‐type ribosomes when treated with RTA in vitro. Ribosome depurination was reduced when RTA was expressed in the ΔP1 and ΔP2 mutants in vivo and these mutants were more resistant to the cytotoxicity of RTA than the wild‐type cells. We further show that while RTA, Stx1 and Stx2 have similar requirements for ribosome depurination, PAP has different requirements, providing evidence that the interaction of RIPs with different ribosomal proteins is responsible for their ribosome specificity.     

The lower cytotoxicity of cinnamomin (a type II RIP) is due to its B-chain

The cytotoxicity of intact cinnamomin (a type II ribosome-inactivating protein, RIP) and the RNA N-glycosidase activity of cinnamomin A-chain have been studied and compared with those of ricin. Cinnamomin A-chain exhibits a similar RNA N-glycosidase activity in inhibiting in vitro protein synthesis compared with that of ricin, whereas the cytotoxicity to BA/F3beta cells of intact cinnamomin is markedly lower than intact ricin. In order to demonstrate that it is the B-chains of the two RIPs that bear the difference in cytotoxicity, two hybrid RIPs are prepared from the purified A-/B-chains of cinnamomin and ricin by the disulfide exchange reaction. It has been found that hybrid RIP constructed from cinnamomin A-chain and ricin B-chain is more toxic to BA/F3beta cells than the native cinnamomin, and equivalent to the native ricin. However, the cytotoxicity to BA/F3beta cells of the hybrid RIP constructed from the ricin A-chain and cinnamomin B-chain is lower than ricin, equivalent to the native cinnamomin. Furthermore, the bound amounts of two B-chains on the cell surface are determined by the method of direct cellular ELISA and Scatchard analysis of the binding of the two B-chains indicates that cinnamomin and ricin share similar binding sites with different affinity.     

2.8-A crystal structure of a nontoxic type-II ribosome-inactivating protein, ebulin l

Ebulin l is a type-II ribosome-inactivating protein (RIP) isolated from the leaves of Sambucus ebulus L. As with other type-II RIP, ebulin is a disulfide-linked heterodimer composed of a toxic A chain and a galactoside-specific lectin B chain. A normal level of ribosome-inactivating N-glycosidase activity, characteristic of the A chain of type-II RIP, has been demonstrated for ebulin l. However, ebulin is considered a nontoxic type-II RIP due to a reduced cytotoxicity on whole cells and animals as compared with other toxic type-II RIP like ricin. The molecular cloning, amino acid sequence, and the crystal structure of ebulin l are presented and compared with ricin. Ebulin l is shown to bind an A-chain substrate analogue, pteroic acid, in the same manner as ricin. The galactoside-binding ability of ebulin l is demonstrated crystallographically with a complex of the B chain with galactose and with lactose. The negligible cytotoxicity of ebulin l is apparently due to a reduced affinity for galactosides. An altered mode of galactoside binding in the 2gamma subdomain of the lectin B chain primarily causes the reduced affinity.