Primary amino acid sequence of alpha-trichosanthin and molecular models for abrin A-chain and alpha-trichosanthin

Ricin A-chain, abrin A-chain, and alpha-trichosanthin are members of a larger group of proteins called ribosome-inactivating proteins. These proteins all function to catalytically inactivate eukaryotic 60 S ribosomal subunits leading to rapid shutdown of protein synthesis. They are homologous in sequence and are probably evolutionarily related. We have determined the complete primary amino acid sequence of alpha-trichosanthin and have found it to be homologous, as expected, to that of abrin A-chain and ricin A-chain. A crystal structure for ricin, which includes ricin A-chain and ricin B-chain, has been determined from x-ray diffraction data. Based on the sequence homologies of these proteins, we fit the primary sequences of abrin A-chain and alpha-trichosanthin to the backbone structure for ricin A-chain and have generated energy-minimized molecular models for them. These models should prove useful in studying the structural-functional relationships of these proteins in particular and of the class in general.     

Identification of the ricin lipase site and implication in cytotoxicity

Ricin is a heterodimeric plant toxin and the prototype of type II ribosome-inactivating proteins. Its B-chain is a lectin that enables cell binding. After endocytosis, the A-chain translocates through the membrane of intracellular compartments to reach the cytosol where its N-glycosidase activity inactivates ribosomes, thereby arresting protein synthesis. We here show that ricin possesses a functional lipase active site at the interface between the two subunits. It involves residues from both chains. Mutation to alanine of catalytic serine 221 on the A-chain abolished ricin lipase activity. Moreover, this mutation slowed down the A-chain translocation rate and inhibited toxicity by 35%. Lipase activity is therefore required for efficient ricin A-chain translocation and cytotoxicity. This conclusion was further supported by structural examination of type II ribosome-inactivating proteins that showed that this lipase site is present in toxic (ricin and abrin) but is altered in nontoxic (ebulin 1 and mistletoe lectin I) members of this family.     

Structural and functional studies of cinnamomin, a new type II ribosome-inactivating protein isolated from the seeds of the camphor tree.

Cinnamomin is a new type II ribosome-inactivating protein (RIP). Its A-chain exhibits RNA N-glycosidase activity to inactivate the ribosome and thus inhibit protein synthesis, whereas the glycosylated B-chain is a lectin. The primary structure of cinnamomin, which exhibits approximately 55% identity with those of ricin and abrin, was deduced from the nucleotide sequences of cDNAs of cinnamomin A- and B-chains. It is composed of a total of 549 amino-acid residues: 271 residues in the A-chain, a 14-residue linker and 264 residues in the B-chain. To explore its biological function, the cinnamomin A-chain was expressed in Escherichia coli with a yield of 100 mg per L of culture, and purified through two-step column chromatography. After renaturation, the recovery of the enzyme activity of the expressed A-chain was 80% of that of native A-chain. Based on the modeling of the three-dimensional structure of the A-chain, the functional roles of five amino acids and the only cysteine residues were investigated by site-directed mutagenesis or chemical modification. The conserved single mutation of the five amino-acid residues led to 8-50-fold losses of enzymatic activity, suggesting that these residues were crucial for maintaining the RNA N-glycosidase activity of the A-chain. Most interestingly, the strong electric charge introduced at the position of the single cysteine in A-chain seemed to play a role in enzyme/substrate binding.     

The rice tapetum-specific gene RA39 encodes a type I ribosome-inactivating protein

A tapetum-specific cDNA encoded by a rice gene, RA39, was isolated by cDNA subtractive hybridization, differential screening and rapid amplification of cDNA ends. RA39 is a single-copy gene in the rice genome. mRNA in situ hybridization indicates that this gene is a tapetum-specific gene, and highly expressed in the tapetal cells at the meiosis and tetrad stages. The RA39 cDNA is 1,013 bp in length with an open reading frame encoding 298 amino acid residues. This cDNA sequence does not show significant homology to any known sequences in GenBank databases, but its deduced amino acid sequence (RA39) has between 19 and 34% sequence identity to ribosome-inactivating proteins (RIPs). Optimal alignment reveals that the five amino acid residues constituting the active site of the ricin A-chain (Tyr⁸⁰, Tyr¹²³, Glu¹⁷⁷, Arg¹⁸⁰ and Trp²¹¹), which are invariant among all RIPs published to date, are conserved in RA39. Recombinant RA39 protein expressed in Escherichia coli was purified to homogeneity. The purified protein exhibits the RNA N-glycosidase activity of RIPs. This demonstrates that RIPs occur in the reproductive organs of rice. The possible function of RA39 in anther development is discussed.     

新丰毒蛋白——一种新的具有活性小A链的核糖体失活蛋白

辛纳毒蛋白是从香樟种子中分离的一种Ⅱ核糖体失活蛋白.最近,从香樟种子中还分离到另一种微型双链核糖体失活蛋白,命名为新丰毒蛋白.还原的新丰毒蛋白表现出与还原的辛纳毒蛋白同样的RNA N-糖苷酶和体外对抑制蛋白质翻译的活力.新丰毒蛋白的B链与辛纳毒蛋白的B链具有同样的分子质量和相同的N端10个氨基酸序列.它的A链N端10个氨基酸序列也与辛纳毒蛋白的A链完全一致,并且C端与辛纳毒蛋白的A链一样具有半胱氨酸,但是它的分子质量却只有辛纳毒蛋白A链的一半.RT-PCR和RNA印迹结果表明体内不存在新丰毒蛋白的mRNA.推测新丰毒蛋白是从辛纳毒蛋白通过蛋白质剪接而产生的,是一种研究蛋白质剪接的好材料.     

Formation of γ-BTC in Flooded Rice Field Soils Treated with γ-BHC

The amino acids of the B-chains of two abrins (designated as abrin-a and abrin-b) from the seeds of Abrus precatorius have been sequenced. The sequence of the B-chain of abrin-a was solved by analysis of peptides derived by enzymatic digestions with trypsin, Iysylendopeptidase, and chymotrypsin, as well as by chemical cleavage with cyanogen bromide. The sequence of the B-chain of abrin-b was analyzed by sequence analysis of tryptic peptides and comparing these sequences with those of corresponding peptides of the B-chain of abrin-a. The B-chains of abrin-a and abrin-b consist of 268 amino acid residues and share 256 identical residues. Comparison of their sequences with that of the ricin B-chain shows that 60% of the residues of both abrin B-chains are identical to those of the ricin B-chain and that two saccharide-binding sites in ricin B-chain identified by a crystallographic study are highly conserved in both abrin B-chains.     

Identification of the tryptophan residue located at the low-affinity saccharide binding site of ricin D

The saccharide binding ability of the low affinity (LA-) binding site of ricin D was abrogated by N-bromosuccinimide (NBS)-oxidation, while in the presence of lactose the number of tryptophan residues eventually oxidized decreased by 1 mol/mol and the saccharide binding ability was retained (Hatakeyama et al., (1986) J. Biochem. 99, 1049-1056). Based on these findings, the tryptophan residue located at the LA-binding site of ricin D was identified. Two derivatives of ricin D which were modified with NBS in the presence and absence of lactose were separated into their constituent polypeptide chains (A- and B-chains), respectively. The modified tryptophan residue or residues was/were found to be contained in the B-chain, but not in the A-chain. From lysylendopeptidase and chymotryptic digests, peptides containing oxidized tryptophan residues were isolated by gel filtration on Bio-Gel P-30 and HPLC. Analysis of the peptides containing oxidized tryptophan revealed that three tryptophan residues at positions 37, 93, and 160 on the B-chain were oxidized in the inactive derivative of ricin D, in which the saccharide binding ability of the LA-binding site was abrogated by NBS-oxidation. On the other hand, the modified residues were determined to be tryptophans at positions 93 and 160 in the active derivative of ricin D which was modified in the presence of lactose, indicating that upon binding with lactose, the tryptophan residue at position 37 of the B-chain was protected from NBS-oxidation. From these results, it is suggested that tryptophan at position 37 on the B-chain is the essential residue for saccharide binding at the LA-binding site of ricin D.     

Prediction of a conserved, neutralizing epitope in ribosome-inactivating proteins

The secondary structures, side-chain solvent accessibilities, and superpositioned crystal structures of the A-chain of ricin and four other plant rRNA N-glycosidases (ribosome-inactivating proteins, RIPs) were examined. Previously, a 26-residue fragment from the A-chain of ricin was determined to bind to a neutralizing monoclonal antibody. The region in the native ricin A-chain, to which this peptide corresponds, is solvent-exposed and contains a negatively charged residue that has been hypothesized to participate in the toxin's function, namely, rRNA binding and/or enzymatic activity. This region appears to be conserved in all of the structurally defined plant RIPs examined. Moreover, other plant RIPs, whose tertiary structures are, as yet, unknown, were predicted to have an analogous, solvent-exposed region containing a conserved, negatively charged residue. By analogy, these conserved structural and functional features lead to the suggestion that this exposed region represents a logical starting point for experiments designed to locate neutralizing epitopes in these RIPs. In contrast, the tertiary structure of the analogous region in a bacteria-derived RIP (Shiga toxin) is a less solvent-exposed, truncated loop and is a structure that is not as likely to be a neutralizing epitope. Because most of the amino acid residues are not conserved within this exposed region, these RIPs are predicted to be antigenically distinct.     

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.     

Features of the structure of catalytic subunits of toxins, inhibiting protein synthesis. I. The effect of pH and interaction with the B-chain of ricin

A comparative study of gelonin and A-chains of ricin, mistletoe lectin I and diphtheria toxin was undertaken. The effect of pH was studied on: a) the conformation of the proteins under study using intrinsic fluorescence; b) interaction of these proteins with ricin B-chain using gel-filtration. Structural stability of the proteins was assessed according to denaturing action of guanidine hydrochloride and temperature, and localization of tryptophan residues was determined using fluorescence quenching by I-, Cs+ and acrylamide. All investigated proteins were shown to undergo the conformational changes when a environment became acidic. In comparison with an intact protein--gelonin, the A-chains of ricin, a mistletoe lectin and a diphtheria toxin are less stable. At pH less than 5.0 tryptophan residues became more accessible to quencher and a positive charge of the surrounding area increases (in the case of gelonin it is negatively charged). No reliable interaction of a ricin B-chain with both gelonin and A-chain of diphtheria toxin was observed. The interaction of a ricin B-chain with a A-chain of mistletoe lectin I is weaker than that with ricin A-chain and is practically pH-independent.     

Interaction of Cibacron blue F3GA and polynucleotides with ricin A-chain, 60 S ribosomal subunit-inactivating protein

Cibacron blue F3GA, a sulfonated polyaromatic blue dye, inhibited the ability of ricin A-chain to inactivate ribosomes. Difference-spectroscopic study revealed that the dye bound to the A-chain (Kd = 0.72 microM), producing a difference spectrum with a single maximum at 688 nm and two minima at 585 and 628 nm. Such a significant difference spectrum was not observed in the presence of ricin B-chain or intact ricin, neither of which can inactivate ribosomes. Modification of arginine residues in the A-chain with phenylglyoxal showed a correlation between the loss of inhibitory activity on protein synthesis and the loss of difference absorbance produced by the dye-A-chain interaction. Both losses occurred significantly at an early stage of the modification. Furthermore, the dye protected the A-chain against a loss of its inhibitory activity resulting from the modification of arginine residues. These results suggest that the same arginine residues participate both in the interaction with the dye and in the inactivation of ribosomes. Based on these data, the dye appears to interact with the active site of the A-chain. Addition of several polynucleotides, namely rRNA, tRNA, poly(U) and DNA, to the dye-A-chain complex resulted in a marked displacement of the dye, whereas mono- and dinucleotides had little or no effect on the dye-A-chain interaction. These findings indicate the possible existence of a polynucleotide binding site in the active site of the A-chain. A combination of these and other results suggests that the A-chain recognizes and acts on some part of RNA of the 60 S ribosomal subunit.     

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.     

Cytotoxic ribosome-inactivating lectins from plants

A class of heterodimeric plant proteins consisting of a carbohydrate-binding B-chain and an enzymatic A-chain which act on ribosomes to inhibit protein synthesis are amongst the most toxic substances known. The best known example of such a toxic lectin is ricin, produced by the seeds of the castor oil plant, Ricinnus communis. For ricin to reach its substrate in the cytosol, it must be endocytosed, transported through the endomembrane system to reach the compartment from which it is translocated into the cytosol, and there avoid degradation making it possible for a few molecules to inactivate a large proportion of the ribosomes and hence kill the cell. Cell entry by ricin involves the following steps: (i) binding to cell-surface glycolipids and glycoproteins bearing beta-1,4-linked galactose residues through the lectin activity of the B-chain (RTB); (ii) uptake by endocytosis and entry into early endosomes; (iii) transfer by vesicular transport to the trans-Golgi network; (iv) retrograde vesicular transport through the Golgi complex and into the endoplasmic reticulum (ER); (v) reduction of the disulfide bond connecting the A- and B-chains; (vi) a partial unfolding of the A-chain (RTA) to enable it to translocate across the ER membrane via the Sec61p translocon using the pathway normally followed by misfolded ER proteins for targeting to the ER-associated degradation (ERAD) machinery; (vi) refolding in the cytosol into a protease-resistant, enzymatically active structure; (vii) interaction with the sarcin-ricin domain (SRD) of the large ribosome subunit RNA followed by cleavage of a single N-glycosidic bond in the RNA to generate a depurinated, inactive ribosome. In addition to the highly specific action on ribosomes, ricin and related ribosome-inactivating proteins (RIPs) have a less specific action in vitro on DNA and RNA substrates releasing multiple adenine, and in some instances, guanine residues. This polynucleotide:adenosine glycosidase activity has been implicated in the general antiviral, and specifically, the anti HIV-1 activity of several single-chain RIPs which are homologous to the A-chains of the heterodimeric lectins. However, in the absence of clear cause and effect evidence in vivo, such claims should be regarded with caution.     

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.     

Secretion of human insulin by a transformed yeast cell

A yeast expression plasmid encoding a mini-proinsulin molecule was constructed and transformed into Saccharomyces cerevisiae. The plasmid encoded the sequence: B-Arg-Arg-Leu-Gln-Lys-Arg-A in which B represents the B-chain (30 amino acid residues) and A represents the A-chain (21 amino acid residues) of human insulin. The secreted peptides were shown to be a mixture of human insulin and des(B-30)human insulin. Thus, correct disulphide bridges can be established in proinsulin-like molecules devoid of a normal C-peptide region. Furthermore, the specificity of the yeast processing enzymes is so similar to the proinsulin converting enzymes in the human pancreatic beta-cell that it allows the processing of the mini-proinsulin to insulin.     

Carbohydrate-specifically polyethylene glycol-modified ricin A-chain with improved therapeutic potential

Ricin A-chain, which exhibits excellent cytotoxicity to tumor cells, has been widely used as an immunotoxin source. However, it has the fatal shortcoming of poor pharmacokinetics due to the tremendous liver uptake via carbohydrate-mediated recognition. Modification of proteins with polyethylene glycol, PEGylation, has the advantages of shielding the specific sites and prolonging the biological half-life. In this study, the carbohydrate-specific PEGylation of ricin A-chain was considered to be a novel approach to overcome this limitation. The carbohydrate group of ricin A-chain was oxidized by sodium m-periodate and further specifically conjugated with hydrazide-derivatized PEG. For a comparative study, the PEGylated ricin A-chain at amino groups was prepared using the hydroxysuccinimide ester-derivatized PEG. The carbohydrate-specifically PEGylated ricin A-chain showed a markedly lower liver uptake and systemic clearance compared with the amine-directly PEGylated ricin A-chain as well as the unmodified ricin A-chain. Furthermore, carbohydrate-specifically PEGylated ricin A-chain showed a significantly higher in vitro ribosome-inactivating activity than the amine-directly PEGylated ricin A-chain. These findings clearly demonstrate that the carbohydrate-specificity as well as PEGylation plays an important role in improving the in vivo pharmacokinetic properties and in vitro bioactivity. Therefore, these results suggest that the therapeutic use of immunotoxins constructed using this carbohydrate-specifically PEGylated ricin A-chain has potential as a cancer therapy.     

A new class of enzyme acting on damaged ribosomes: ribosomal RNA apurinic site specific lyase found in wheat germ

A new enzyme, which we named ribosomal RNA apurinic site specific lyase (RALyase), is described. The protein was found in wheat embryos and has a molecular weight of 50 625 Da. The enzyme specifically cleaves the phosphodiester bond at the 3' side of the apurinic site introduced by ribosome-inactivating proteins into the sarcin/ricin domain of 28S rRNA. The 3' and 5' ends of wheat 28S rRNA at the cleavage site are 5'-GUACG-alpha-hydroxy-alpha, beta-unsaturated aldehyde and pGAGGA-3', demonstrating that the enzyme catalyzes a beta-elimination reaction. The substrate specificity of the enzyme is extremely high: it acts only at the apurinic site in the sarcin/ricin domain of intact ribosomes, not on deproteinized rRNA or DNA containing apurinic sites. The amino acid sequences of five endopeptidase LysC-liberated peptides from the purified enzyme were determined and used to obtain a cDNA sequence. The open reading frame encodes a protein of 456 amino acids, and a homology search revealed a related rice protein. Similar enzyme activities were also found in other plants that express ribosome-inactivating proteins. We believe that RALyase is part of a complex self-defense mechanism.     

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.     

Nucleotide sequence of cDNA coding for saporin-6, a type-1 ribosome-inactivating protein from Saponaria officinalis

We have isolated and sequenced partial cDNA clones that encode SO-6, a ribosome-inactivating protein from Saponaria officinalis. A cDNA library was constructed from the leaves of this plant and screened with synthetic oligonucleotide probes representing various portions of the protein. The deduced amino acid sequence shows the signal peptide and a coding region virtually accounting for the entire amino acid sequence of SO-6. The sequence reveals regions of similarity to other ribosome-inactivating proteins, especially in a region of the molecule where critical amino acid residues might participate in the active site.     

Adenine nucleotide N-glycosidase activity of the A-chain of cinnamomin characterized by 1H-nuclear magnetic resonance

Plant ribosome-inactivating proteins specifically cleave an N-glycosidic bond of a unique adenosine in the largest ribosomal RNA, releasing an adenine from ribosomes of different sources. Here, 1H-nuclear magnetic resonance is used to analyze the enzymatic products of the A-chain of cinnamomin, a type-II ribosome-inactivating protein (RIP) acting on the nucleotides in situ. The enzymatic activities of the RIP on nine nucleotides are compared. Cinnamomin A-chain can cleave the N-glycosidic bond and release an adenine base from adenine nucleotides except 5'-ATP; however, it cannot act on 5'-GMP, 5'-CMP, and 5'-UMP. The A-chain in the mixture of cinnamomin A- and B-chain exhibits higher activity toward adenine nucleotides than the A-chain alone does, suggesting that the B-chain can conformationally stabilize the A-chain. Intact cinnamomin also exhibits lower activity toward adenine nucleotides. However, cinnamomin B-chain and heat-denatured intact cinnamomin cannot hydrolyze all the tested nucleotides. We conclude that hydrolysis of the N-C glycosidic bond of nucleotide compounds by cinnamomin A-chain has a base preference, and the negatively charged phosphate group(s) reduces the recognition ability of the A-chain to adenine nucleotide.