Ricin A-chain Research Articles

A BRIEF COMMUNICATION

Saponins are amphiphilic substances consisting of a hydrophobic backbone with one or two hydrophilic sugar units. Recently it was shown that saponinum album (SA) from Gypsophila paniculata L. enhanced cytotoxicity of a saporin-based chimeric toxin (up to 385,000-fold) as well as the toxicity of saporin (up to 100,000-fold) with N-glycosidase activity. Previously we have shown that toxicity of other N-glycosidases such as ricin A-chain, nigrin b, and toxins such as diphtheria toxin or microcystin-LR was not enhanced by SA. This points to a specific SA-dependent mechanism of toxicity enhancement on saporin and saporin-based toxins. Although the cytotoxicity enhancing effect was observed in up to 10 different cell lines, nothing is known about the kinetic of SA under cell culture conditions. Therefore SA was titrated, and the uptake respective liberation profile of SA was investigated in ECV-304 cells. Treatment of cells with [3H]-SA leads to an immediate uptake of saponin molecules. After cells were saturated with [3H]-SA, a first equilibrium (first eq.) was reached. The first eq. was disturbed by washing until a second equilibrium was reached between the activity observed within cells and that seen in the supernatant. After a further extensive washing, a small portion of saponin molecules remained durable associated with the cell. This portion was sufficient to induce a drastic toxicity enhancement on saporin indicating a long-lasting sensitization of cells against the toxin.

1504: Preparation of Ricin A-Chain Carrying Microbubble and its Anti-Tumor Effects in Vitro

1504: Preparation of Ricin A-Chain Carrying Microbubble and its Anti-Tumor Effects in Vitro

Anti‐endoglin monoclonal antibodies are effective for suppressing metastasis and the primary tumors by targeting tumor vasculature

Anti-metastatic activity of an antitumor agent is exceedingly important because metastasis is the primary cause of death for most solid cancer patients. In this report, we show that 3 anti-endoglin (ENG) monoclonal antibodies (mAbs) SN6a, SN6j and SN6k which define individually distinct epitopes of ENG of tumor vasculature are capable of suppressing tumor metastases in the multiple metastasis models. The metastasis models were generated by i.v., s.c. (into flank) or mammary gland fat pad injection of 4T1 murine mammary carcinoma cells and splenic injection of two types of colon26 murine colorectal carcinoma cells. Individual mAbs were injected i.v. via the tail vein of mice. SN6a and SN6j effectively suppressed the formation of metastatic colonies of 4T1 in the lung in all of the three 4T1 metastatic models. In addition, these mAbs were effective for suppressing the primary tumors of 4T1 in the skin and mammary fat pad. These mAbs effectively suppressed microvessel density and angiogenesis in tumors as measured by the Matrigel plug assay in mice. No significant side effects of the administered mAbs were detected. Furthermore, SN6a and SN6j extended survival of the tumor-bearing mice. SN6j, SN6k and their immunoconjugates with deglycosylated ricin A-chain were all effective for suppressing hepatic metastasis of colon26. The findings in the present study are clinically relevant in view of the ongoing clinical trial of a humanized (chimerized) form of SN6j.

Size Limitation in Translocation of Fibroblast Growth Factor 1 Fusion Proteins across the Endosomal Membrane

After binding to its receptor on the surface of mammalian cells and subsequent endocytosis, FGF1 is translocated across the membrane into the cytosol. The growth factor is then further transported into the nucleus. In order to characterize more closely the translocation mechanism utilized by FGF1, we introduced additional amino acids into FGF1 to test the size dependence of the translocated substrate. We constructed mutants containing an increasing number of copies of the myc tag (1-13 copies) in a surface loop of the FGF1 molecule. All of the constructs bound to specific FGF receptors and to heparin and were taken up by endocytosis. However, only FGF1 mutants harboring up to three myc tags (53 amino acids) were translocated while mutants with five myc tags (77 amino acids) or more were not translocated through the membrane. We further showed that insertion of other, unrelated polypeptides into FGF1, i.e., 3xFLAG tag (22 amino acids) and streptavidin binding peptide (50 amino acids), was also translocated. Larger insertions into FGF1, like the CBP-SBP tag (82 amino acids) or ricin A-chain (272 amino acids), resulted in fusion proteins that failed to translocate. The presented data imply that it is possible to employ FGF1 to import various polypeptides into the cytosol and nucleus of cells. Furthermore, the strict size dependence of FGF1 fusion proteins in membrane translocation argues against simple leakage of FGF1 from ruptured endosomal membranes but rather points to a specific translocation apparatus involving a proteinaceous pore.

Ricin accumulation and degradation during castor seed development and late germination

The castor plant ( Ricinus communis L.) is a euphorb, commonly grown for its high seed oil content. The seed also contains a deadly toxin known as ricin (RCA 60) and the less toxic Ricinus communis agglutinin (RCA 120) in addition to potentially harmful allergens. Using Western blotting, an antibody screening technique, ricin content was evaluated in various plant parts with a ricin A-chain specific antibody. Seed, cotyledon, root, hypocotyl, and leaf tissues were tested. Western blot results indicated that ricin is only present in the seed. In order to determine when ricin accumulates in the seed, reproductive tissue samples were harvested during seed development and upon germination. Developing seed were collected from inflorescences in 4-day intervals beginning with the day of pollination (day 0) until the seed had fully matured (day 44). Ricin was absent from the developing seed throughout development until the 28th day post-pollination. Ricin content quickly increased as the seed matured and the capsule dehydrated. Mature seed were also germinated and collected at 2-day intervals once radicle emergence began, until the 12th day, when seedlings were fully expanded. From the sixth day forward, germinating seedlings were dissected into root, hypocotyl, and cotyledonary tissue. Western blot analysis indicated that after the 6th day, ricin is degraded below levels of detection. Ricin is only detectable in the seed tissue from day 0 (ungerminated seed) through the sixth day after radicle emergence and is not detected in any of the root, hypocotyl, or cotyledonary tissue. These data suggest that ricin is generated and stored in the seed for a specific purpose; most likely the protection of the seed from predation in its quiescent state.

Detecting Ricin: Sensitive Luminescent Assay for Ricin A-Chain Ribosome Depurination Kinetics

Ricin is a family member of the lethal ribosome-inactivating proteins (RIP) found in plants. Ricin toxin A-chain (RTA) from castor beans catalyzes the hydrolytic depurination of a single base from a GAGA tetraloop of eukaryotic rRNA to release a single adenine from the sarcin-ricin loop (SRL). Protein synthesis is inhibited by loss of the elongation factor binding site resulting in cell death. We report a sensitive coupled assay for the measurement of adenine released from ribosomes or small stem-loop RNAs by RTA catalysis. Adenine phosphoribosyl transferase (APRTase) and pyruvate orthophosphate dikinase (PPDK) convert adenine to ATP for quantitation by firefly luciferase. The resulting AMP is cycled to ATP to give sustained luminescence proportional to adenine concentration. Subpicomole adenine quantitation permits the action of RTA on eukaryotic ribosomes to be followed in continuous, high-throughput assays. Facile analysis of RIP catalytic activity will have applications in plant toxin detection, inhibitor screens, mechanistic analysis of depurinating agents on oligonucleotides and intact ribosomes, and in cancer immunochemotherapy. Kinetic analysis of the catalytic action of RTA on rabbit reticulocyte 80S ribosomes establishes a catalytic efficiency of 2.6 x 10(8) M(-1) s(-1), a diffusion limited reaction indicating catalytic perfection even with large reactants.

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 alpha-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 alpha-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 DeltaP1 and DeltaP2 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.

Comparative analysis of depurination catalyzed by ricin A-chain on synthetic 32mer and 25mer oligoribonucleotides mimicking the sarcin/ricin domain of the rat 28S rRNA and E. coli 23S rRNA

Ricin A-chain can inactivate eukaryotic ribosomes, but exhibits no N-glycosidase activity on intact E. coli ribosomes. In the present research, in order to avoid using radiolabeled oligoribonucleotides, two kinds of synthetic 5′-FAM fluorescence-labeled oligoribonucleotide substrates were used to mimic the sarcin/ricin domains of rat 28S rRNA and E. coli 23S rRNA (32mer and 25mer, named as Rat FAM-SRD and E. coli FAM-SRD, respectively). Ricin A-chain was able to specifically release adenine from the first adenosine of the GAGA tetraloop and exhibited specific N-glycosidase activity under neutral and weak acidic conditions with both substrates. However, under more acidic conditions, ricin A-chain was able to release purines from other sites on eukaryotic substrates, but it retained specific depurination activity on prokaryotic substrates. At pH 5.0, the Michaelis constant ( K m) for the reaction with Rat FAM-SRD (4.57 ± 0.28 μM) corresponded to that with E. coli FAM-SRD (4.64 ± 0.26 μM). However, the maximum velocity ( V max) for ricin A-chain with Rat FAM-SRD was 0.5 ± 0.024 μM/min, which is higher than that with E. coli FAM-SRD (0.32 ± 0.011 μM/min).

A dynamics study of the A-chain of ricin by terahertz vibrational calculation and normal modes analysis

We studied the terahertz (THz) spectroscopy and low frequency normal modes of both apo- and holo- (adenosine monophosphate (AMP)-bound) ricin-A-chain (RTA) as a means to understand the dynamical changes that RTA undergoes upon substrate binding. The calculated THz spectra of apo- and holo-RTAs demonstrated a general intensity suppression upon substrate binding, which is attributed to the reduced number of collective motion in THz region. In normal mode analysis of RTA we find a shearing motion that is shared by both the apo- and holo-RTAs, whereas a breathing motion, and an upward hinge rising and an α-G bending characteristic motion are dampened significantly upon AMP binding, suggesting these motions are involved in the necessary flexibility of the active site. In contrast, we find a normal mode motion that separates domains I and II of RTA at the interface that is more common in the holo-protein. We hypothesized that the flexibility of the entrance of RTA can facilitate the entry of rRNA and allow the substrate to adjust its conformation and orientation prior to depurination. This process suggests an rRNA binding pathway which is supplemental the current RTA depurination mechanism.

Calculation of Absolute Ligand Binding Free Energy to a Ribosome-Targeting Protein as a Function of Solvent Model

A comparative analysis is provided of the effect of different solvent models on the calculation of a potential of mean force (PMF) for determining the absolute binding affinity of the small molecule inhibitor pteroic acid bound to ricin toxin A-chain (RTA). Solvent models include the distance-dependent dielectric constant, several different generalized Born (GB) approximations, and a hybrid explicit/GB-based implicit solvent model. We found that the simpler approximation of dielectric screening and a GB model, with Born radii fitted to a switching-window dielectric-boundary surface Poisson solvent model, severely overpredicted the binding affinity as compared to the experimental value, estimated to range from -4.4 to -6.0 kcal/mol. In contrast, GB models that are parametrized to fit the Lee-Richards molecular surface performed much better, predicting binding free energy within 1-3 kcal/mol of experimental estimates. However, the predicted free-energy profiles of these GB models displayed alternative binding modes not observed in the crystal structure. Finally, the most rigorous and computationally costly approach in this work, which used a hybrid explicit/implicit solvent model, correctly determined a binding funnel in the PMF near the crystallographic bound state and predicted an absolute binding affinity that was 2 kcal/mol more favorable than the estimated experimental binding affinity.

Induction and increase of HLA-DR antigen expression by immune interferon on ML-3 cell line enhances the anti-HLA-DR immunotoxin activity

In order to evaluate the impact of induction and increase target antigen expression on immunotoxin potency, we measured the potentiating effect of recombinant immune interferon-gamma (rIFN-gamma) on the cytotoxicity of an anti HLA-DR ricin A-chain immunotoxin (2G5 RTA-IT) on the myeloid cell line ML-3. After 48 h of incubation with rIFN-gamma (500 U/ml) the percentage of 2G5-positive cells increased from 40% to 79%, and the 2G5 mean density was enhanced by 10-fold (11,000 versus 110,000 molecules/cell). Concurrently, rIFN-gamma pretreatment induced a dramatic improvement of 2G5 RTA-IT dose-effect cytotoxicity, as well as immunotoxin cytotoxicity kinetics. When 2G5 RTA-IT was used at the optimal dose of 10(-8)M (the maximum dose which avoided non-specific ricin A-chain cytotoxicity), the immunotoxin-induced cell kill increased with the percentage of DR-positive ML-3 cells according to a similar linear-logarithmic function of rIFN-gamma concentration. Moreover, in the same range of rIFN-gamma concentrations, the killing values and the percentage of DR-positive ML-3 cells were similar if not identical. These findings imply that the enhancement of 2G5 RTA-IT cytotoxicity by rIFN-gamma is mainly related to the rIFN-gamma 2G5 antigen induction on HLA-DR negative cells when immunotoxin was used at 10(-8) M. Furthermore, 2G5 RTA-IT dose-effect cytotoxicity on DR-expressing ML-3 cells, when used at lower concentrations, was also increased by rIFN-gamma in a dose-dependent manner. This result suggests that for immunotoxin concentrations close to the limiting membrane saturation dose (10(-10)M), rIFN-gamma may not solely act by inducing HLA-DR expression on DR-negative ML-3 subpopulation but also by increasing individual cellular DR density on DR expressing ML-3 cells. Finally, our study showed that immunotoxin potency on malignant cell populations which display an heterogeneous antigen expression, could be greatly improved by the use of rIFN-gamma.

Steered unfolding of ricin A and B chains

Highly toxic, heterodimeric protein ricin binds itself to the cell surface glycolipids or glycoproteins via its B-chain. The toxic A-chain halts protein synthesis by inactivating the ribosomes, leading to cell death. The translocation step requires partial unfolding of the protein. In this work mechanical unfolding of intact ricin as well as the individual A- and B-chains has been studied. A total of 110 ns simulation run has been performed to observe the unfolding of ricin dimer using steered molecular dynamics simulation. A gradual unfolding against a constant pulling velocity is observed for the ricin A-chain leaving the B-chain in its native-like structure. The breakage of the disulfide linkage connecting the two chains and reversal of the pulling ends of B-chain surprisingly reversed the picture as the B-chain starts to unfold from its N-terminal end. Due to the unfolding of B-chain from N-terminal end, the A-chain appears structurally rigid, which comes from the strong interfacial interactions (hydrophobic, hydrogen bonding, salt bridge). Mechanical unfolding of the individual monomers has also been performed to compare their stabilities in the monomeric and dimeric forms.

Detection of an abasic site in RNA with stem-loop DNA beacons: Application to an activity assay for Ricin Toxin A-Chain

The catalytic ability of Ricin Toxin A-Chain (RTA) to create an abasic site in a 14-mer stem-tetraloop RNA is exploited for its detection. RTA catalyzes the hydrolysis of the N-glycosidic bond of a specific adenosine in the G A GA tetraloop of stem-loop RNA. Thus, a 14-mer stem-loop RNA substrate containing an intact “G A GA” sequence can be discriminated from the product containing an abasic “G ab GA” sequence by hybridization with a 14-mer DNA stem-loop probe sequence and following the fluorescent response of the heteroduplexes. Three DNA beacon probe designs are described. Beacon 1 probe is a stem-loop structure and has a fluorophore and a quencher covalently linked to the 5′- and 3′-ends. In this format the probe–substrate heteroduplex gives a fluorescent signal while the probe–product one remains quenched. Beacon 2 is a modified version of 1 and incorporates a pyrene deoxynucleoside for recognition of the abasic site. In this format both the substrate and product heteroduplexes give a fluorescent response. Beacon 3 utilizes a design where the fluorophore is on the substrate RNA sequence at its 5′-end while the quencher is on the probe DNA sequence at its 3′-end. In this format the fluorescence of the substrate–probe heteroduplex is quenched while that of the product–probe one is enhanced. The lower limit of detection with beacons is 14 ng/mL of RTA.

Protective effects of anti-ricin A-chain RNA aptamer against ricin toxicity

To investigate the therapeutic potential of an RNA ligand (aptamer) specific for the catalytic ricin A-chain (RTA), the protective effects of a 31-nucleotide RNA aptamer (31RA), which formed a high affinity complex with RTA, against ricin-induced toxicity in cell-based luciferase translation and cell cytotoxicity assays were evaluated. To test the therapeutic potential of anti-RTA aptamers in Chinese hamster ovary (CHO) AA8 cells stably transfected with a tetracycline regulatable promoter, ricin ribotoxicity was measured using luciferase and ricin-induced cytotoxicity was ascertained by MTS cell proliferation assay with tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]. Inhibition of protein synthesis by ricin in CHO AA8 cells resulted in diminished luciferase activity and treatment with polyclonal antibody against deglycosylated RTA (dgA) neutralized the inhibitory effects of ricin on luciferase activity and protected against ricin-induced cytotoxicity as measured by MTS assay. The 31RA anti-RTA aptamer inhibited the translation of luciferase mRNA in cell-free reticulocyte translation assay. 31RA aptamer also partially neutralized the inhibitory effects of ricin on luciferase activity and partially protected against ricin-induced cytotoxicity in CHO AA8 cells. We have shown that anti-RTA RNA aptamer can protect against ricin ribotoxicity in cell-based luciferase and cell cytotoxicity assays. Hence, RNA aptamer that inhibits RTA enzymatic activity represents a novel class of nucleic acid inhibitor that has the potential to be developed as a therapeutic agent for the treatment of ricin intoxication.

Coarse-grained lattice model simulations of sequence-structure fitness of a ribosome-inactivating protein

Many realistic protein-engineering design problems extend beyond the computational limits of what is considered practical when applying all-atom molecular-dynamics simulation methods. Lattice models provide computationally robust alternatives, yet most are regarded as too simplistic to accurately capture the details of complex designs. We revisit a coarse-grained lattice simulation model and demonstrate that a multiresolution modeling approach of reconstructing all-atom structures from lattice chains is of sufficient accuracy to resolve the comparability of sequence-structure modifications of the ricin A-chain (RTA) protein fold. For a modeled structure, the unfolding-folding transition temperature was calculated from the heat capacity using either the potential energy from the lattice model or the all-atom CHARMM19 force-field plus a generalized Born solvent approximation. We found, that despite the low-resolution modeling of conformational states, the potential energy functions were capable of detecting the relative change in the thermodynamic transition temperature that distinguishes between a protein design and the native RTA fold in excellent accord with reported experimental studies of thermal denaturation. A discussion is provided of different sequences fitted to the RTA fold and a possible unfolding model.

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.

Ricin A Chain from Ricinus sanguineus: DNA Sequence, Structure and Toxicity

The gene coding for the ricin A-chain from Ricinus sanguineus (RsTA) was isolated and sequenced (GB: DQ661048). Comparison of RsTA with the ricin A-chain from Ricinus communis (RTA) revealed the presence of five differences in the gene sequence. At the protein level only two differences were noticed, the replacement of Asn136 by Ser (N136S) and Ile173 by Val (I174 V). From the RsTA structure model (PMDB: PM0074652), the N136S mutation was predicted to have no effect on RsTA structure. The I174V mutation is believed to have no effect on the RsTA structure or on toxicity since this replacement was found in the Ricinus agglutinin's A-chain and the latter has a comparable toxicity to RTA. The Ser221 of the putative lipase active site was found. Toxicity experiments showed that RsTA and RTA have similar toxicities. This finding proves that the N136S and I174 V mutations have no effect on RsTA toxicity.

Vinyldeoxyadenosine in a Sarcin−Ricin RNA Loop and Its Binding to Ricin Toxin A-Chain

8-Vinyl-2'-deoxyadenosine (8vdA) is a fluorophore with a quantum yield comparable to that of 2-aminopurine nucleoside. 8vdA was incorporated into a 10-mer stem-tetraloop RNA (8vdA-10) structure for characterization of the properties of the base, 8-vinyladenine (8-vA), with respect to adenine as a substrate or inhibitor for ribosome-inactivating proteins. Ricin toxin A-chain (RTA) and pokeweed antiviral protein (PAP) catalyze the release of adenine from a specific adenosine on a stem-tetraloop (GAGA) sequence at the elongation factor (eEF2) binding site of the 28S subunit of eukaryotic ribosomes, thereby arresting translation. RTA does not catalyze the release of 8-vinyladenine from 8vdA-10. Molecular dynamics simulations implicate a role for Arg180 in oxacarbenium ion destabilization and the lack of catalysis. However, 8vdA-10 is an active site analogue and inhibits RTA with a Ki value of 2.4 microM. Adenine is also released from the second adenosine in the modified tetraloop, demonstrating an alternative mode for the binding of this motif in the RTA active site. The 8vdA analogue defines the specificities of RTA for the two adenylate depurination sites in a RNA substrate with a GAGA tetraloop. The rate of nonenzymatic acid-catalyzed solvolysis of 8-vinyladenine from the stem-loop RNA is described. Unlike RTA, PAP catalyzes the slow release of 8-vinyladenine from 8vdA-10. The isolation of 8-vA and its physicochemical characterization is described.

Circular DNA and DNA/RNA Hybrid Molecules as Scaffolds for Ricin Inhibitor Design

Ricin Toxin A-chain (RTA) catalyzes the hydrolytic depurination of A4324, the first adenosine of the GAGA tetra-loop portion of 28S eukaryotic ribosomal RNA. Truncated stem-loop versions of the 28S rRNA are RTA substrates. Here, we investigate circular DNA and DNA/RNA hybrid GAGA sequence oligonucleotides as minimal substrates and inhibitor scaffolds for RTA catalysis. Closing the 5'- and 3'-ends of a d(GAGA) tetraloop creates a substrate with 92-fold more activity with RTA (kcat/Km) than that for the d(GAGA) linear form. Circular substrates have catalytic rates (kcat) comparable to and exceeding those of RNA and DNA stem-loop substrates, respectively. RTA inhibition into the nanomolar range has been achieved by introducing an N-benzyl-hydroxypyrrolidine (N-Bn) transition state analogue at the RTA depurination site in a circular GAGA motif. The RNA/DNA hybrid oligonucleotide cyclic GdAGA provides a new scaffold for RTA inhibitor design, and cyclic G(N-Bn)GA is the smallest tight-binding RTA inhibitor (Ki = 70 nM). The design of such molecules that lack the base-paired stem-loop architecture opens new chemical synthetic approaches to RTA inhibition.

Improved formulation of a recombinant ricin A-chain vaccine increases its stability and effective antigenicity

Ricin is a potent toxin associated with bioterrorism for which no vaccine or specific countermeasures are currently available. A stable, non-toxic and immunogenic recombinant ricin A-chain vaccine (RTA 1-33/44-198) has been developed by protein engineering. We identified optimal formulation conditions for this vaccine under which it remained stable and potent in storage for up to 18 months, and resisted multiple rounds of freeze–thawing without stabilizing co-solvents. Reformulation from phosphate buffer to succinate buffer increased adherence of the protein to aluminum hydroxide adjuvant from 15 to 91%, with a concomitant increase of nearly threefold in effective antigenicity in a mouse model. Using Fourier-transform infrared spectroscopy, we examined the secondary structure of the protein while it was adhered to aluminum hydroxide. Adjuvant adsorption produced only a small apparent change in secondary structure, while significantly stabilizing the protein to thermal denaturation. The vaccine therefore may be safely stored in the presence of adjuvant. Our results suggest that optimization of adherence of a protein antigen to aluminum adjuvant can be a useful route to increasing both stability and effectiveness, and support a role for a “depot effect” of adjuvant.