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.     

Site-specific mutagenesis of mistletoe lectin: the role of RIP activity in apoptosis.

Recombinant mistletoe lectin (rML) belongs to the class of type II ribosome-inactivating proteins (RIP) composed of a catalytically active A-chain with rRNA N-glycosidase activity and a B-chain with carbohydrate binding properties. To investigate the contribution of the enzymatic activity of the rML A-chain to the observed cytotoxic and apoptotic effects, an rMLA E166Q R169Q molecule was developed by means of site-specific mutagenesis. Following heterologous expression, the activity of mutant rMLA was measured in a cell-free assay for rRNA-N-glycosidase activity. Moreover, after generation of heterodimer, the activities of mutant rML E166Q R169Q and rML wild type were determined in a cytotoxicity and apoptosis assay. Although the reduction of activity as measured in the cell-free RIP assay was more pronounced (factor 237) than in both cellular assays (factors 20-22), the data clearly indicate a close correlation between cytotoxicity, apoptosis, and the enzymatic activity of the rML A-chain. Thus, RIP activity is an essential feature of rML and therefore a prerequisite for its biological function as an anticancer agent.     

Complete structure determination of the A chain of mistletoe lectin III from Viscum album L. ssp. album.

The complete primary structure of the A chain of mistletoe lectin III (ML3A), a type II ribosome-inactivating protein, was determined using proteolytic digests of ML3A, HPLC separation of the peptides, Edman degration and MALDI-MS. Based on our results, ML3A consists of 254 amino acid residues, showing a high homology to the A chain of isolectin ML1 with only 24 amino acid residue exchanges. A striking important structural difference compared with ML1A is the lack of the single N-glycosylation site in ML3A due to an amino acid exchange at position 112 (ML1A: NL112GS ==> ML3A: T112GS). The alignment of ML3A with the A chains of ML1, isoabrins, ricin D, Ricinus communis agglutinin and three lectins, identified from the Korean mistletoe Viscum album ssp. coloratum, demonstrates the rigid conservation of all amino acid residues, responsible for the RNA-N-glycosidase activity as reported for ricin D. In addition, the fully determined primary structure of ML3A will give further information about the biological mechanism of mistletoe lectin therapy.     

Induction of apoptosis by mistletoe lectin I and its subunits. No evidence for cytotoxic effects caused by isolated A- and B-chains

Type II ribosome inactivating proteins (RIP II) are generally known to induce apoptosis in human cells by the inhibition of protein biosynthesis. Recent data from mistletoe RIP II proteins (eg. mistletoe lectin I; ML1) suggest an additional mode of apoptosis induction through the binding of their lectin part to certain cell surface receptors as is known for some human galectins. In order to clarify this possibility, we used highly sensitive flow cytometric apoptosis assays and mistletoe hololectin subunits of proven purity to show that neither human lymphocytes nor Molt-4 cells undergo apoptosis after treatment with isolated lectin-type B-chains. In contrast to earlier investigations, only the hololectin was able to induce apoptosis in these assays. We conclude that direct apoptosis induction by mistletoe lectins occurs only after uptake of the molecules into the cell due to the action of the ribosome inactivating A-chain.     

Mistletoe lectin dissociates into catalytic and binding subunits before translocation across the membrane to the cytoplasm.

Hybridomas producing monoclonal antibodies (mAbs) against the mistletoe lectin A-chain (MLA) were obtained to investigate the intracellular routing and translocation of ribosome-inactivating proteins. Anti-MLA mAb MNA5 did not bind the holotoxin but interacted with isolated MLA. This epitope was not recognized upon MLA denaturation or conjugation of MLA with the ricin binding subunit (RTB). Furthermore, the mAbs did not appreciably react with a panel of MLA synthetic octapeptides linked to the surface of polyethylene pins. A study of the cytotoxicity of mistletoe lectin, ricin, and chimeric toxin MLA/RTB for the hybridomas revealed that interchain disulfide bond reduction and subunit dissociation are required for cytotoxic activity of mistletoe lectin.     

Cytotoxic activity of recombinant bFGF-rViscumin fusion proteins

A fusion protein (bFGF-rMLA), containing the mitogen basic fibroblast growth factor (bFGF) and the cytotoxic component of rViscumin (recombinant mistletoe lectin), the enzymatic A-chain (rMLA), was expressed in Escherichia coli, purified, and functionally characterized. bFGF-rMLA is cytotoxic for mouse B16 melanoma cells expressing the FGF receptor with an IC(50) value of approximately 1 nM. rMLA shows no significant effect on the viability of the B16 cells up to a concentration of 141 nM. Additionally, bFGF-rMLA was associated with the rViscumin B-chain (rMLB) in an in vitro folding procedure. The IC(50) value of bFGF-rMLA/rMLB to B16 cells in the presence of lactose-to block rMLB lectin activity-was 134 pM. Thus, it was possible to enhance the efficacy of a rViscumin A-chain mitotoxin through addition of rMLB. We conclude that rViscumin fusion proteins may be generally applicable for the receptor-specific inactivation of target cells and point out their potential in drug development.     

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.     

Preferential binding of the anticancer drug rViscumin (recombinant mistletoe lectin) to terminally alpha2-6-sialylated neolacto-series gangliosides

Production of biochemically defined recombinant mistletoe lectin was achieved by cloning and separate expression of the single catalytically active A-chain and the B-chain with carbohydrate binding properties in Escherichia coli, yielding an active heterodimeric protein named rViscumin (Eck et al. [1999] Eur. J. Biochem., 265, 788-797). Employing solid phase binding assays, rViscumin was shown to preferentially bind to terminally alpha2-6-sialylated neolacto-series gangliosides IV(6)Neu5Ac-nLc4Cer, VI(6)Neu5Ac-nLc6Cer, and VIII(6)Neu5Ac-nLc8Cer isolated from human granulocytes. Only marginal binding of rViscumin to galactose-terminated neutral GSLs was determined, whereas reinvestigation of ricin specificity demonstrated this lectin as a galactose-binding protein. Human promyelotic HL-60 cells exhibited an IC(50) value (half maximum cytotoxicity) of 1.16 pM and human bladder carcinoma 5637 cells of 12.1 pM rViscumin; CHO-K1 cells were resistant to rViscumin treatment up to a concentration of 5.26 nM tested. Quantification of the predominant receptor ganglioside IV(6)Neu5Ac-nLc4Cer by means of a specific anti-Neu5Acalpha2-6Galbeta1-4GlcNAc-R antibody revealed 3.68 x 10(6) and 1.54 x 10(6) receptor molecules per HL-60 and 5637 cell, respectively; CHO-K1 cells were negative, lacking alpha2-6-sialylated gangliosides. The data imply a direct correlation of rViscumin cytotoxicity and the expression of receptor ganglioside. Moreover, CHO-K1 cells were rendered susceptible toward rViscumin cytotoxicity after exogenous application of human granulocyte gangliosides. Thus, (1) rViscumin has to be considered as a sialic acid-specific rather than a galactose-specific type II ribosome-inactivating protein, and (2) neolacto-series gangliosides with Neu5Acalpha2-6Galbeta1-4GlcNAc-terminus are true functional and physiologically relevant rViscumin receptors.     

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.     

Crystal structure of mistletoe lectin I from Viscum album

The crystal structure of the ribosome-inactivating protein (RIP) mistletoe lectin I (ML-I) from Viscum album has been solved by molecular replacement techniques. The structure has been refined to a crystallographic R-factor of 24.5% using X-ray diffraction data to 2.8 A resolution. The heterodimeric 63-kDa protein consists of a toxic A subunit which exhibits RNA-glycosidase activity and a galactose-specific lectin B subunit. The overall protein fold is similar to that of ricin from Ricinus communis; however, unlike ricin, ML-I is already medically applied as a component of a commercially available misteltoe extract with immunostimulating potency and for the treatment of human cancer. The three-dimensional structure reported here revealed structural details of this pharmaceutically important protein. The comparison to the structure of ricin gives more insights into the functional mechanism of this protein, provides structural details for further protein engineering studies, and may lead to the development of more effective therapeutic RIPs.     

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.     

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.     

Complete structure determination of N‐acetyl‐D‐galactosamine‐binding mistletoe lectin‐3 from Viscum album L. album

The primary structure of the B chain of the N‐acetyl‐D ‐galactosamine‐recognizing mistletoe lectin‐3 (ML‐3B) has been deduced from proteolytic digest peptides of the purified glycoprotein, their HPLC‐separation and Edman degradation and confirmation of the peptide sequences by MALDI‐MS. ML‐3B consists of 262 amino acid residues including 10 cysteine moieties. The structure and linkage of the carbohydrate side chains, connected to two N‐glycosylation sites at positions Asn⁹⁵ and Asn¹³⁵ of the lectin, were determined by a combination of glycosidase treatment and MALDI‐MS of corresponding glycopeptide fragments. The sequence alignment reveals a high homology with other B chains of type‐II RIPs, although there are remarkable differences in the D ‐galactose‐specific mistletoe lectin‐1B chain. The recently published primary structure of the mistletoe lectin‐3A chain 1 and the now available primary sequence of the 3B chain allowed the construction of a preliminary homology model of ML‐3. The model demonstrates, unequivocally, that ML‐3 is a member of the type‐II RIP family with rigid conservation of the enzymatic active site of the A chain and an identical overall protein fold. Specific amino acid residue exchanges and the different glycosylation pattern in comparison with ML‐1 are discussed and related to the properties of the two glycoproteins. The knowledge of the complete primary structure of mistletoe lectin‐3 is a major contribution towards more insight into the mechanism of the biological activity of commercial mistletoe preparations. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

Comparative study of interaction of two type II ribosome-inactivating proteins and their A-chains with model membrane

Both cinnamomin and ricin are type II ribosome-inactivating proteins. Cinnamomin is less cytotoxic compared with ricin. In order to clarify the mechanism of their different cytotoxicities, the interaction of cinnamomin and its A-chain with model membrane was investigated and compared with that of ricin and its A-chain. It was revealed that cinnamomin is less effective than ricin in interacting with model membrane. Cinnamomin A-chain interacts with model membrane much less violently than ricin A-chain. The differences in the interaction of cinnamomin, ricin or their A-chains with model membrane might at least in part indicate the different cytotoxicity between cinnamomin and ricin.     

Molecular characterization of the recombinant A-chain of a type II ribosome-inactivating protein (RIP) from Viscum album coloratum and structural basis on its ribosome-inactivating activity and the sugar-binding properties of the B-chain

Mistletoe (Viscum album) lectins, which are classified as a type II ribosome-inactivating protein (RIP) due to their unique biological function and the potential medical and therapeutic application in cancer cells, receive a rising attention. The heterodimeric glycoproteins contain the Achain with catalytic activity and the B-chain with sugar binding properties. In recent years, studies involving the lectins from the white berry European mistletoe (Viscum album) and the yellow berry Korean mistletoe (Viscum album coloratum) have been described. However, the detailed mechanism in exerting unique cytotoxic effect on cancer cells still remains unclear. Here, we aim to understand and define the molecular basis and biological effects of the type II RIPs, through the studies of the recombinant Korean mistletoe lectin. To this end, we expressed, purified the recombinant Korean mistletoe lectin (rKML), and investigated its molecular characteristics in vitro, its cytotoxicity and ability to induce apoptotic cell death in cancer cells. To gain structural basis for its catalytic activity and sugar binding properties, we performed homology modeling studies based on the high degree of sequence identity and conserved secondary structure prediction between Korean and European, Himalayan mistletoe lectins, and Ricin.