Alteration of an amino acid residue outside the active site of the ricin A chain reduces its toxicity towards yeast ribosomes

Summary Yeast transformants containing integrated copies of a galactose-regulated, ricin toxin A chain (RTA) expression plasmid were constructed and used in an attempt to isolate RTA-resistant yeast mutants. Analysis of RNA from mutant strains demonstrated that approximately half contained ribosomes that had been partially modified by RTA, although all the strains analysed transcribed full-length RTA RNA. The mutant strains could have mutations in yeast genes giving rise to RTA-resistant ribosomes or they could contain alterations within the RTA-encoding DNA causing production of mutant toxin. Ribosomes isolated from mutant strains were shown to be susceptible to RTA modification in vitro suggesting that the strains contain alterations in RTA. This paper describes the detailed analysis of one mutant strain which has a point mutation that changes serine 203 to asparagine in RTA protein. Although serine 203 lies outside the proposed active site of RTA its alteration leads to the production of RTA protein with a greatly reduced level of ribosome modifying activity. This decrease in activity apparently allows yeast cells to survive expression of RTA as only a proportion of the ribosomes become modified. We demonstrate that the mutant RTA preferentially modifies 26S rRNA in free 60S subunits and has lower catalytic activity compared with native RTA when produced in Escherichia coli. Such mutations provide a valuable means of identifying residues important in RTA catalysis and of further understanding the precise mechanism of action of RTA.     

蓖麻毒素A链突变体(MRTA)的分子设计

利用同源模建的方法,借助分子力学优化,分子力学模拟退火设计构建了删除部分氨基酸序列的蓖麻毒素Ａ链突变体。采用泊松－玻尔兹曼方程对比分析了蓖麻毒素Ａ链与ＭＲＴＡ表现静电势分布,研究了ＲＴＡ与ＭＲＴＡ蛋白表面静电性质;     

蓖麻毒素A链突变体(MRTA)的分子设计

利用同源模建的方法,借助分子力学优化、分子动力学模拟退火设计构建了删除部分氨基酸序列的蓖麻毒素Ａ链突变体（ＭＲＴＡ）。采用泊松—玻尔兹曼方程对比分析了蓖麻毒素Ａ链（ＲＴＡ）与ＭＲＴＡ表观静电势分布,研究ＲＴＡ与ＭＲＴＡ蛋白表面静电性质;通过半经验量子化学ＡＭ１与分子力学结合方法探讨ＲＴＡ与ＭＲＴＡ功能域氨基酸前线分子轨道性质、能级分布,从理论上预测ＭＲＴＡ功能活性     

The isolation and characterization of temperature-dependent ricin A chain molecules in Saccharomyces cerevisiae

Ricin is a heterodimeric plant protein that is potently toxic to mammalian cells. Toxicity results from the catalytic depurination of eukaryotic ribosomes by ricin toxin A chain (RTA) that follows toxin endocytosis to, and translocation across, the endoplasmic reticulum membrane. To ultimately identify proteins required for these later steps in the entry process, it will be useful to express the catalytic subunit within the endoplasmic reticulum of yeast cells in a manner that initially permits cell growth. A subsequent switch in conditions to provoke innate toxin action would permit only those strains containing defects in genes normally essential for toxin retro-translocation, refolding or degradation to survive. As a route to such a screen, several RTA mutants with reduced catalytic activity have previously been isolated. Here we report the use of Saccharomyces cerevisiae to isolate temperature-dependent mutants of endoplasmic reticulum-targeted RTA. Two such toxin mutants with opposing phenotypes were isolated. One mutant RTA (RTAF108L/L151P) allowed the yeast cells that express it to grow at 37 degrees C, whereas the same cells did not grow at 23 degrees C. Both mutations were required for temperature-dependent growth. The second toxin mutant (RTAE177D) allowed cells to grow at 23 degrees C but not at 37 degrees C. Interestingly, RTAE177D has been previously reported to have reduced catalytic activity, but this is the first demonstration of a temperature-sensitive phenotype. To provide a more detailed characterization of these mutants we have investigated their N-glycosylation, stability, catalytic activity and, where appropriate, a three-dimensional structure. The potential utility of these mutants is discussed.     

Cytotoxicity of a recombinant ricin-A-chain fusion protein containing a proteolytically-cleavable spacer sequence

Chimeric proteins composed of ricin toxin A chain (RTA) and staphylococcal protein A (PA) have been produced in E. coli. Constructs consisting of N-terminal RTA and C-terminal PA (RTA-PA) or N-terminal PA and C-terminal (PA-RTA) were capable of binding to immunoglobulin G (via PA) and of specifically depurinating 28 S ribosomal RNA (via RTA). However, neither fusion protein was cytotoxic to antigen-bearing target cells in the presence of an appropriate monoclonal antibody presumably because the RTA could not be released from the PA within the cytosol where the ribosomal substrate of RTA is located. The overcome this, a short amino acid sequence from diphtheria toxin was engineered between the RTA and PA to produce a disulfide-linked loop containing a trypsin sensitive cleavage site. Cleavage of this fusion protein with trypsin converted the RTA-DT-PA to the two chain form consisting of RTA linked by a disulfide bond to PA. The cleaved fusion protein was highly toxic to Daudi cells coated with anti-immunoglobulin antibody suggesting that the RTA could be released from the PA by reduction within the cytosol.     

Preparation and characterization of recombinant proricin containing an alternative protease-sensitive linker sequence.

The aim of this study was to determine the feasibility of utilizing a factor Xa-specific cleavage site within a recombinant protein containing the ricin A chain (RTA) sequence. Release of RTA is believed to be an essential step during the intracellular phase of ricin intoxication. Failure to incorporate such cleavage sites in fusions containing RTA results in a loss of toxin action (O'Hare, M., et al. (1990) FEBS Lett. 273,200. Kim, J., and Weaver, R.F. (1988) Gene 68,315). In this report we describe the introduction of a factor Xa-specific site in the linker of proricin, which we use here as a model substrate. Upon purification of the recombinant mutant proricin after expression in Xenopus oocytes, we demonstrate that the protease does have access to the engineered recognition sequence (albeit at low efficiency) and that the presence of the latter does not interfere with disulfide bond formation or the lectin activity of the ricin B chain moiety. Upon cleavage and reduction, the RTA polypeptide displays ribosome-inactivating ability, indicating that the presence of the modified linker at its C-terminus does not interfere with its catalytic activity. The general applicability of using such a cleavage site in recombinant fusions with RTA is discussed.     

Modulation of toxin stability by 4-phenylbutyric acid and negatively charged phospholipids

AB toxins such as ricin and cholera toxin (CT) consist of an enzymatic A domain and a receptor-binding B domain. After endocytosis of the surface-bound toxin, both ricin and CT are transported by vesicle carriers to the endoplasmic reticulum (ER). The A subunit then dissociates from its holotoxin, unfolds, and crosses the ER membrane to reach its cytosolic target. Since protein unfolding at physiological temperature and neutral pH allows the dissociated A chain to attain a translocation-competent state for export to the cytosol, the underlying regulatory mechanisms of toxin unfolding are of paramount biological interest. Here we report a biophysical analysis of the effects of anionic phospholipid membranes and two chemical chaperones, 4-phenylbutyric acid (PBA) and glycerol, on the thermal stabilities and the toxic potencies of ricin toxin A chain (RTA) and CT A1 chain (CTA1). Phospholipid vesicles that mimic the ER membrane dramatically decreased the thermal stability of RTA but not CTA1. PBA and glycerol both inhibited the thermal disordering of RTA, but only glycerol could reverse the destabilizing effect of anionic phospholipids. In contrast, PBA was able to increase the thermal stability of CTA1 in the presence of anionic phospholipids. PBA inhibits cellular intoxication by CT but not ricin, which is explained by its ability to stabilize CTA1 and its inability to reverse the destabilizing effect of membranes on RTA. Our data highlight the toxin-specific intracellular events underlying ER-to-cytosol translocation of the toxin A chain and identify a potential means to supplement the long-term stabilization of toxin vaccines.     

A single point mutation in ricin A-chain increases toxin degradation and inhibits EDEM1-dependent ER retrotranslocation

Ricin is a potent plant cytotoxin composed of an A-chain [RTA (ricin A-chain)] connected by a disulfide bond to a cell binding lectin B-chain [RTB (ricin B-chain)]. After endocytic uptake, the toxin is transported retrogradely to the ER (endoplasmic reticulum) from where enzymatically active RTA is translocated to the cytosol. This transport is promoted by the EDEM1 (ER degradation-enhancing α-mannosidase I-like protein 1), which is also responsible for directing aberrant proteins for ERAD (ER-associated protein degradation). RTA contains a 12-residue hydrophobic C-terminal region that becomes exposed after reduction of ricin in the ER. This region, especially Pro250, plays a crucial role in ricin cytotoxicity. In the present study, we introduced a point mutation [P250A (substitution of Pro250 with alanine)] in the hydrophobic region of RTA to study the intracellular transport of the modified toxin. The introduced mutation alters the secondary structure of RTA into a more helical structure. Mutation P250A increases endosomal-lysosomal degradation of the toxin, as well as reducing its transport from the ER to the cytosol. Transport of modified RTA to the cytosol, in contrast to wild-type RTA, appears to be EDEM1-independent. Importantly, the interaction between EDEM1 and RTA(P250A) is reduced. This is the first reported evidence that EDEM1 protein recognition might be determined by the structure of the ERAD substrate.     

HeLa cell mutants resistant to epidermal growth factor ricin A-chain conjugate

Epidermal growth factor (EGF) was linked to the toxic A chain of ricin toxin (RTA) to produce an EGF-receptor-specific cytotoxic agent, EGF-RTA. Three EGF-RTA-resistant mutants of the human HeLa cell line were selected. These mutant cell lines are 10-fold to more than 100-fold more resistant to EGF-RTA when compared to HeLa cells. The EGF-RTA-resistant mutants have at least as many EGF receptors as parent cells; the basis for the EGF-RTA-resistant phenotype must be distal to EGF binding. The EGF-RTA-resistant cells are not cross-ressitant to ricin or to diphtheria toxin; their mutant phenotype appears to be EGF specific. The EGF-RTA-resistant mutants are able to internalize and degrade EGF. However, the mutants have altered EGF receptor down-regulation and phorbol 12-tetradecanoate 13-acetate modulation properties. EGF-RTA/ammonium chloride and EGF-RTA/adenovirus co-treatment data suggest that the mutant defect(s) which confers EGF-RTA resistance is either in the endosome or at a step(s) in the intracellular EGF processing pathway between the endosome and the lysosome.     

Killing of cultured hepatocytes by conjugates of asialofetuin and EGF linked to the A chains of ricin or diphtheria toxin

A disulfide-linked conjugate between asialofetuin (ASF) and the toxic A chain (RTA) of ricin is as potent a toxin for cultured rat hepatocytes as our previously described conjugate between ASF and fragment A of diphtheria toxin (DTA). An RTA conjugate of epidermal growth factor (EGF) was a potent toxin for 3T3 cells. In contrast, EGF-DTA was essentially nontoxic for 3T3 cells. We have now examined the toxicity of EGF-RTA and EGF-DTA on cultured hepatocytes. The EGF-DTA conjugate, nontoxic to 3T3 cells, is also a potent toxin for hepatocytes. We also observed a decrease with time of culture in the sensitivity of hepatocytes to the ASF and EGF conjugates. This decrease is not a result of a decrease in EGF or asialoglycoprotein receptors.     

Immunotoxins constructed with chimeric,short-lived anti-CD22 monoclonal antibodies induce less vascular leak without loss of cytotoxicity

An immunotoxin (IT) constructed with RFB4, a murine anti-CD22 monoclonal antibody, and the “deglycosylated” A chain of ricin has shown activity at safe doses in patients with non-Hodgkin lymphoma and in children with acute lymphoblastic leukemia. The dose limiting toxicity is vascular leak syndrome (VLS), which appears to be due to a unique amino acid motif in the ricin toxin A (RTA) chain that damages vascular endothelial cells. We mutated recombinant (r) RTA to disable this site, but await testing of the IT prepared with this mutant RTA in humans. Another possible approach to reducing IT-induced VLS is to shorten the half-life of the IT in vivo. We previously constructed a mouse-human chimeric RFB4 by grafting the variable genes of RFB4 onto the human IgG1k constant regions. Here, we report the expansion of our panel of mutant chimeric RFB4s (mcRFB4s) that lack the ability to bind to the neonatal Fc receptor (FcRn). In comparison with cRFB4, which had a T1/2 of 263 h, the mcRFB4s had T1/2s ranging from 39 to 106 h. ITs were constructed with these mcRFB4s and rRTA. The mcRFB4-RTA ITs retained their cytotoxicity in vitro and had shorter half lives than the parental cRFB4-RTA IT. In addition, the mcRFB4 IT with the shortest T1/2 induced less pulmonary vascular leak in mice, which we have postulated is a surrogate marker for VLS in humans.     

Identification of an Htm1 (EDEM)-dependent,Mns1-independent Endoplasmic Reticulum-associated Degradation (ERAD) Pathway in Saccharomyces cerevisiae: APPLICATION OF A NOVEL ASSAY FOR GLYCOPROTEIN ERAD*

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a quality control system for newly synthesized proteins in the ER; nonfunctional proteins, which fail to form their correct folding state, are then degraded. The cytoplasmic peptide:N-glycanase is a deglycosylating enzyme that is involved in the ERAD and releases N-glycans from misfolded glycoproteins/glycopeptides. We have previously identified a mutant plant toxin protein, RTA (ricin A-chain nontoxic mutant), as the first in vivo Png1 (the cytoplasmic peptide:N-glycanase in Saccharomyces cerevisiae)-dependent ERAD substrate. Here, we report a new genetic device to assay the Png1-dependent ERAD pathway using the new model protein designated RTL (RTA-transmembrane-Leu2). Our extensive studies using different yeast mutants identified various factors involved in RTL degradation. The degradation of RTA/RTL was independent of functional Sec61 but was dependent on Der1. Interestingly, ER-mannosidase Mns1 was not involved in RTA degradation, but it was dependent on Htm1 (ERAD-related α-mannosidase in yeast) and Yos9 (a putative degradation lectin), indicating that mannose trimming by Mns1 is not essential for efficient ERAD of RTA/RTL. The newly established RTL assay will allow us to gain further insight into the mechanisms involved in the Png1-dependent ERAD-L pathway.     

Using homology modeling to interrogate binding affinity in neutralization of ricin toxin by a family of single domain antibodies

In this report we investigated, within a group of closely related single domain camelid antibodies (V_HHs), the relationship between binding affinity and neutralizing activity as it pertains to ricin, a fast‐acting toxin and biothreat agent. The V1C7‐like V_HHs (V1C7, V2B9, V2E8, and V5C1) are similar in amino acid sequence, but differ in their binding affinities and toxin‐neutralizing activities. Using the X‐ray crystal structure of V1C7 in complex with ricin's enzymatic subunit (RTA) as a template, Rosetta‐based homology modeling coupled with energetic decomposition led us to predict that a single pairwise interaction between Arg29 on V5C1 and Glu67 on RTA was responsible for the difference in ricin toxin binding affinity between V1C7, a weak neutralizer, and V5C1, a moderate neutralizer. This prediction was borne out experimentally: substitution of Arg for Gly at position 29 enhanced V1C7's binding affinity for ricin, whereas the reverse (ie, Gly for Arg at position 29) diminished V5C1's binding affinity by >10 fold. As expected, the V5C1_R29G mutant was largely devoid of toxin‐neutralizing activity (TNA). However, the TNA of the V1C7_G29R mutant was not correspondingly improved, indicating that in the V1C7 family binding affinity alone does not account for differences in antibody function. V1C7 and V5C1, as well as their respective point mutants, recognized indistinguishable epitopes on RTA, at least at the level of sensitivity afforded by hydrogen‐deuterium mass spectrometry. The results of this study have implications for engineering therapeutic antibodies because they demonstrate that even subtle differences in epitope specificity can account for important differences in antibody function.     

Structural analysis of nested neutralizing and non‐neutralizing B cell epitopes on ricin toxin's enzymatic subunit

In this report, we describe the X‐ray crystal structures of two single domain camelid antibodies (V_HH), F5 and F8, each in complex with ricin toxin's enzymatic subunit (RTA). F5 has potent toxin‐neutralizing activity, while F8 has weak neutralizing activity. F5 buried a total of 1760 Ų in complex with RTA and made contact with three prominent secondary structural elements: α‐helix B (Residues 98–106), β‐strand h (Residues 113–117), and the C‐terminus of α‐helix D (Residues 154–156). F8 buried 1103 Ų in complex with RTA that was centered primarily on β‐strand h. As such, the structural epitope of F8 is essentially nested within that of F5. All three of the F5 complementarity determining regions CDRs were involved in RTA contact, whereas F8 interactions were almost entirely mediated by CDR3, which essentially formed a seventh β‐strand within RTA's centrally located β‐sheet. A comparison of the two structures reported here to several previously reported (RTA‐V_HH) structures identifies putative contact sites on RTA, particularly α‐helix B, associated with potent toxin‐neutralizing activity. This information has implications for rational design of RTA‐based subunit vaccines for biodefense. Proteins 2016; 84:1162–1172. © 2016 Wiley Periodicals, Inc.     

Mutations affecting the activity of the Shiga-like toxin I A-chain.

Like ricin, Escherichia coli Shiga-like toxin I (SLT-I) inactivates eukaryotic ribosomes by catalytically depurinating adenosine 4324 in 28S rRNA. Although the primary structure of the enzymatic portion of the molecule (Slt-IA) is known to contain regions of significant homology to the ricin A chain (RTA), and although certain residues have been implicated in catalysis, the crystal structure of Slt-IA has not been solved nor has the geometry of its active site been well defined. In order to derive a more complete understanding of the nature of the Slt-IA active site, we placed the slt-IA gene under control of an inducible promoter in Saccharomyces cerevisiae. Induction of the cloned element was lethal to the host. This lethality was the basis for selection of an attenuated mutant of Slt-IA changed at tyrosine 77, a locus not previously linked to the active site. As well, it permitted evaluation of the toxicity of a number of mutant Slt-IA cassettes that we constructed in vitro. Putative active-site residues implicated in this fashion and in other studies were mapped to an energy-minimized computer model of Slt-IA that had been generated on the basis of the known crystal structure of RTA. A cleft was identified on one face of the protein in which all implicated residues clustered, irrespective of their distances from one another in the primary structure of the molecule. Many of the chemical features anticipated in the active site of an RNA N-glycosidase are indeed present on the amino acid side chains occupying the cleft.     

A relatively low level of ribosome depurination by mutant forms of ricin toxin A chain can trigger protein synthesis inhibition,cell signaling and apoptosis in mammalian cells

The A chain of the plant toxin ricin (RTA) is an N-glycosidase that inhibits protein synthesis by removing a specific adenine from the 28S rRNA. RTA also induces ribotoxic stress, which activates stress-induced cell signaling cascades and apoptosis. However, the mechanistic relationship between depurination, protein synthesis inhibition and apoptosis remains an open question. We previously identified two RTA mutants that suggested partial independence of these processes in a yeast model. The goals of this study were to establish an endogenous RTA expression system in mammalian cells and utilize RTA mutants to examine the relationship between depurination, protein synthesis inhibition, cell signaling and apoptosis in mammalian cells. The non-transformed epithelial cell line MAC-T was transiently transfected with plasmid vectors encoding precursor (pre) or mature forms of wild-type (WT) RTA or mutants. PreRTA was glycosylated indicating that the native signal peptide targeted RTA to the ER in mammalian cells. Mature RTA was not glycosylated and thus served as a control to detect changes in catalytic activity. Both pre- and mature WT RTA induced ribosome depurination, protein synthesis inhibition, activation of cell signaling and apoptosis. Analysis of RTA mutants showed for the first time that depurination can be reduced by 40% in mammalian cells with minimal effects on inhibition of protein synthesis, activation of cell signaling and apoptosis. We further show that protein synthesis inhibition by RTA correlates more linearly with apoptosis than ribosome depurination.     

A trans-Golgi network retention signal YQRL fused to ricin A chain significantly enhances its cytotoxicity

Ricin enters the cells by receptor-mediated endocytosis, followed by translocation across the membranes of intracellular organelles. A trans-Golgi retention peptide signal YQRL was fused to the C-terminus of ricin A chain (RTA) by polymerase chain reaction. The recombinant RTA and RTA-YQRL were expressed in Escherichia coli using plasmid pKK223.3 under the control of a tac promoter. The recombinant proteins were purified by affinity chromatography on a Blue-Sepharose 6B column. The cytotoxicities of RTA and the fusion toxin RTA-YQRL were measured by the MTT assay in HeLa, SKOV-3, and WISH cells following fluid-phase endocytosis. The rRTA-YQRL was 2-, 10-, and 40-fold more cytotoxic than rRTA itself in the three cell lines, respectively. The results indicate that addition of a TGN retention signal YQRL to the C-terminus of RTA can markedly increase its cytotoxicity, suggesting TGN may play an important role in the intracellular routing and translocation of RTA.     

Immunotoxins constructed with chimeric,short-lived anti-CD22 monoclonal antibodies induce less vascular leak without loss of cytotoxicity

An immunotoxin (IT) constructed with RFB4, a murine anti-CD22 monoclonal antibody, and the “deglycosylated” A chain of ricin has shown activity at safe doses in patients with non-Hodgkin lymphoma and in children with acute lymphoblastic leukemia. The dose limiting toxicity is vascular leak syndrome (VLS), which appears to be due to a unique amino acid motif in the ricin toxin A (RTA) chain that damages vascular endothelial cells. We mutated recombinant (r) RTA to disable this site, but await testing of the IT prepared with this mutant RTA in humans. Another possible approach to reducing IT-induced VLS is to shorten the half-life of the IT in vivo. We previously constructed a mouse-human chimeric RFB4 by grafting the variable genes of RFB4 onto the human IgG1k constant regions. Here, we report the expansion of our panel of mutant chimeric RFB4s (mcRFB4s) that lack the ability to bind to the neonatal Fc receptor (FcRn). In comparison with cRFB4, which had a T_1/2 of 263 h, the mcRFB4s had T_1/2s ranging from 39–106 h. ITs were constructed with these mcRFB4s and rRTA. The mcRFB4-RTA ITs retained their cytotoxicity in vitro and had shorter half lives than the parental cRFB4-RTA IT. In addition, the mcRFB4 IT with the shortest T_1/2 induced less pulmonary vascular leak in mice, which we have postulated is a surrogate marker for VLS in humans.Key words: chimeric, anti-CD22, monoclonal antibody, Fc mutations, ricin A chain, immunotoxins     

The X-ray structure of ricin A chain with a novel inhibitor

Ricin is a potent heterodimeric cytotoxin; the B chain binds eucaryotic cell surfaces aiding uptake and the A chain, RTA, reaches the cytoplasm where it enzymatically depurinates a key ribosomal adenine, inhibiting protein synthesis. Ricin is known to be an agent in bioterrorist repertoires and there is great interest in finding, or creating, efficacious inhibitors of the toxin as potential antidotes. We have previously identified two families of bicyclic RTA inhibitors, pterins and purines. Both classes have poor solubility which impairs inhibitor development. Here we report the use of 2-amino-4,6-dihydroxy-pyrimidines as RTA inhibitors. Unlike previously observed single ring inhibitor platforms, these displace Tyr 80 and bind deep in the RTA specificity pocket. These compounds are at least 10 times more soluble than pterin-based inhibitors and appear to be useful new class of ricin inhibitors.