Search PubMed⌕ Search

Biomedical subjects

R J Youle

Publications and source records attributed to R J Youle.

At least 55 records · Page 3Linked to original sources
PubMed1996

X-ray crystallographic structure of recombinant eosinophil-derived neurotoxin at 1.83 A resolution.

The X-ray crystallographic structure of recombinant eosinophil-derived neurotoxin (rEDN) has been determined by molecular replacement methods and refined at 1.83 A resolution to a conventional R-factor ( = sigma magnitute of (magnitute of F(zero)-magnitude of Fc)/ sigma magnitude of F(zero) of 0.152 with excellent stereochemistry. The molecular model of rEDN contains all 1081 non-hydrogen protein atoms, two non-covalently bound sulfate anions and 121 ordered solvent molecules. The polypeptide fold of rEDN is related to those observed in the homologous structures of RNase A, Onconase and angiogenin. rEDN is one of the largest members of the pyrimidine-specific ribonuclease superfamily of vertebrates and has small insertions in four of its seven loop structures and a large insertion from Asp115 to Tyr123. The non-covalently bound SO4(A) and SO4(B) anions occupy phosphate-binding subsites of rEDN. The active site SO4(A) anion makes contacts in rEDN that are similar to those in RNase A and involve the side-chain atoms of Gln14, His15 and His129, and the NH group of Leu130. The SO4(B) anion makes contacts with the side-chain atoms of Arg36 and Asn39 and the main-chain atoms of Asn39 and Gln40. The equivalent residues of RNase A cannot make contacts similar to those observed in rEDN. The SO4(B) binding site of rEDN likely corresponds to the P-1 subsite and may be representative of how other homologous RNases bind the P-1 phosphate.

Amino Acid Sequence↗
PubMed1996

Role of the N terminus in RNase A homologues: differences in catalytic activity, ribonuclease inhibitor interaction and cytotoxicity.

A number of biochemical properties differ dramatically among homologues within the pancreatic ribonuclease superfamily. Human pancreatic ribonuclease (hRNase) has high enzyme activity, extreme sensitivity to ribonuclease inhibitor (RI) and is non-toxic, whereas a homologous RNase from frog eggs, called onconase, has much lower enzyme activity, is not sensitive to RI and is cytotoxic to cancer cell lines and animals. To explore the structural basis of these differences among members in the RNAse family we synthesized genes for onconase, hRNase, a mutant onconase (K9Q) and onconase-hRNase N-terminal hybrids and expressed the proteins in Escherichia coli with final yields of 10 to 50 mg per liter of culture after purification. A recombinant version of onconase with an N-terminal methionine instead of the native pyroglutamyl residue had decreased cytotoxicity and enzyme activity. Cleavage of the recombinant onconase Met-1 residue, and cyclization of the Gln1 residue to reform the pyroglutamyl N terminus, reconstituted cytotoxicity and enzyme activity. Thus a unique role of the pyroglutamyl residue in the active site of amphibian RNases is indicated. Replacement of one to nine residues of onconase with the homologous residues of hRNase increased the enzymatic activity against most of the substrates tested with a simultaneous shift in the enzyme specificity from high preference for poly(U) to slight preference for poly(C). Cytotoxicity of the chimera decreased, dissociating cytotoxicity from enzymatic activity. The molecular basis for the low binding affinity of onconase for RI has been examined experimentally with the recombinant RNases and by fitting onconase and RNase A structures to the coordinates from the recently published RNase A-RI complex.

Amino Acid Sequence↗
PubMed1996

Immunotoxins for central nervous system malignancy.

Current therapy for primary brain tumors, metastatic brain tumors and leptomeningeal carcinomatosus is inadequate. Monoclonal antibodies and certain biological ligands such as transferrin and epidermal growth factor bind selectively to tumors but in and of themselves have little therapeutic activity. Linking these binding moieties to protein toxins yields new molecules, called immunotoxins, that display enormous cell-type specific toxicity in vitro and could be valuable agents for treatment of CNS cancer. This review discusses our development of immunotoxins to treat brain tumors after systemic, intrathecal and intratumoral injection.

Antibodies, Monoclonal↗
PubMed1995

A study of the intracellular routing of cytotoxic ribonucleases.

Several ribonucleases serve as cytotoxic agents in host defense and in physiological cell death pathways. Although certain members of the pancreatic ribonuclease A superfamily can be toxic when applied to the outside of cells, they become thousands of times more toxic when artificially introduced into the cytosol, indicating that internalization is the rate-limiting step for cytotoxicity. We have used three agents that disrupt the Golgi apparatus by distinct mechanisms, retinoic acid, brefeldin A, and monensin, to probe the intracellular pathways ribonucleases take to reach the cytosol. Retinoic acid and monensin potentiate the cytotoxicity of bovine seminal RNase, Onconase, angiogenin, and human ribonuclease A 100 times or more. Retinoic acid-mediated potentiation of ribonucleases is completely blocked by brefeldin A. Ribonucleases appear to route more efficiently into the cytosol through the Golgi apparatus disrupted by monensin or retinoic acid. Intracellular RNA degradation by BS-RNase increased more than 100 times in the presence of retinoic acid confirming that the RNase reaches the cytosol and indicating that degradation of RNA is the intracellular lesion causing toxicity. As retinoic acid alone and Onconase are in clinical trials for cancer therapy, combinations of RNases and retinoic acid in vivo may offer new clinical utility.

Amino Acid Sequence↗
PubMed1995

The role of free radicals and p53 in neuron apoptosis in vivo.

Apoptosis is a mechanism of cell death operative in the normal development and regulation of vertebrate tissues and organ cellularity. During the postnatal development of the mouse cerebellum, extensive granule neuron apoptosis occurs that may regulate the final granule cell to Purkinje cell stoichiometry observed in the adult. Cerebellar granule cells are highly sensitive to genotoxic agents such as gamma-irradiation and methylazoxymethanol during the first 2 weeks of postnatal development. We demonstrate that ionizing radiation induces extensive cerebellar granule cell death via apoptosis in vivo. In p53 null mice, however, the cerebellar granule cells do not undergo apoptosis in response to gamma-irradiation. In mice heterozygous for the p53 allele, the granule cells apoptosis is delayed, indicating an intermediate response. The developmental apoptosis of cerebellar granule cells, however, occurs similarly in wild-type and p53 null mice. Therefore, neurons undergo p53-dependent and p53-independent apoptosis, depending upon the initiating stimulus that triggers DNA fragmentation. In contrast to x-ray damage, the extensive death of cerebellar granule cells induced by methylazoxymethanol was found to be independent of the DNA fragmentation characteristic of apoptosis, and was also independent of expression of p53. Ablation of neuron progenitor cells with genotoxic agents may occur by p53-dependent apoptosis or by p53-independent mechanisms not associated with DNA fragmentation.

Animals↗
PubMed1994

Apoptosis and free radicals.

Necrotic cell death is usually a consequence of extensive insult to the cell, leading to release of intracellular contents and an inflammatory response. Apoptosis, however, is a physiological response to damaging influences that requires sufficient maintenance of homeostasis to allow execution of the pathway. Apoptosis circumvents the induction of an inflammatory response, which can be disadvantageous and, therefore, would be more beneficial than necrosis under many circumstances. The apoptotic response appears complicated and involves many factors, including the mitotic rate, the stage of differentiation, the type and strength of the initiating stimulus, and exogenous factors. Recent evidence, however, implicates free radicals as a causal agent in some types of apoptosis, both physiologically and pathologically.

Animals↗
PubMed1994

Expression and characterization of recombinant human eosinophil-derived neurotoxin and eosinophil-derived neurotoxin-anti-transferrin receptor sFv.

The gene for the human recombinant eosinophil-derived neurotoxin (rEDN) was synthesized and fused to the gene encoding a single chain antibody (sFv) to the human transferrin receptor (EDNsFv). Both rEDN and EDNsFv were expressed as insoluble proteins in inclusion bodies in Escherichia coli BL21(DE3). Following denaturation and renaturation, EDN and EDNsFv were partially purified by chromatography on heparin-Sepharose. Final purification of EDN was achieved by Sephadex G-100, whereas EDNsFv which contained a 6-histidyl residue carboxyl terminus was highly purified using the metal chelate resin, Ni(2+)-nitriloacetic acid. Whereas the recombinant EDN had ribonuclease activity that was similar to the native protein, the fusion protein had enzymatic activity that was 6-13% that of native EDN. The fusion protein was able to bind to the human transferrin receptor. In contrast to rEDN that had no inherent cytotoxicity to human tumor cells, the EDNsFv fusion protein was cytotoxic to human leukemia cells that express the human transferrin receptor with an IC50, 0.2-1 nM. At 1.3 nM EDNsFv, no cytotoxicity was observed on cells that lack the human transferrin receptor. Free antibody to the human transferrin receptor, E6, inhibited the cytotoxicity of the EDNsFv. Human enzymes may be engineered to acquire cytotoxic properties by fusing them to antibodies. Thus, they may be candidates for the construction of immunofusion proteins that may be less immunogenic than immunotoxins containing bacterial- or plant-derived toxin moieties.

3T3 Cells↗
PubMed1994

Characterization of the mechanism of cellular and cell free protein synthesis inhibition by an anti-tumor ribonuclease.

Onconase, a protein with anti-tumor activity, causes potent inhibition of protein synthesis in the rabbit reticulocyte lysate (IC50 10(-11) M) and when microinjected into Xenopus oocytes (IC50 10(-10) M). Onconase is a member of the RNase A superfamily; however, unlike RNase A, the mechanism of protein synthesis inhibition does not involve apparent degradation of lysate or cellular ribosomal RNAs. Rather, reticulocyte and oocyte tRNA is hydrolyzed after Onconase treatment. Furthermore, re-addition of tRNA to Onconase pretreated lysates or oocytes restores the translational capacity of the system. Taken together these results suggest that Onconase causes potent protein synthesis inhibition by a mechanism involving inactivation of cellular tRNA.

Animals↗
PubMed1994

RNase inhibition of human immunodeficiency virus infection of H9 cells.

Onconase and bovine seminal RNase, two members of the RNase A superfamily, inhibit human immunodeficiency virus type 1 replication in H9 leukemia cells 90-99.9% over a 4-day incubation at concentrations not toxic to uninfected H9 cells. Two other members of the same protein family, bovine pancreatic RNase A and human eosinophil-derived neurotoxin, have no detectable antiviral activity, demonstrating a strikingly selective antiviral activity among homologous ribonucleases. The antiviral RNases do not appear to affect viral particles directly but inhibit replication in host cell cultures. Onconase, already in clinical trials for cancer therapy, and bovine seminal RNase have potential as antiviral therapeutics.

Animals↗
PubMed1994

Retinoic acid disrupts the Golgi apparatus and increases the cytosolic routing of specific protein toxins.

All-trans retinoic acid can specifically increase receptor mediated intoxication of ricin A chain immunotoxins more than 10,000 times, whereas fluid phase endocytosis of ricin A chain alone or ricin A chain immunotoxins was not influenced by retinoic acid. The immunotoxin activation by retinoic acid does not require RNA or protein synthesis and is not a consequence of increased receptor binding of the immunotoxin. Vitamin D3 and thyroid hormone T3, that activate retinoic acid receptor (RAR) cognates, forming heterodimers with retinoid X receptor (RXR), do not affect the potency of immunotoxins. Among other retinoids tested, 13-cis retinoic acid, which binds neither RAR nor RXR, also increases the potency of the ricin A chain immunotoxin. Therefore, retinoic acid receptor activation does not appear to be necessary for immunotoxin activity. Retinoic acid potentiation of immunotoxins is prevented by brefeldin A (BFA) indicating that in the presence of retinoic acid, the immunotoxin is efficiently routed through the Golgi apparatus en route to the cytoplasm. Directly examining cells with a monoclonal antibody (Mab) against mannosidase II, a Golgi apparatus marker enzyme, demonstrates that the Golgi apparatus changes upon treatment with retinoic acid from a perinuclear network to a diffuse aggregate. Within 60 min after removal of retinoic acid the cell reassembles the perinuclear Golgi network indistinguishable with that of normal control cells. C6-NBD-ceramide, a vital stain for the Golgi apparatus, shows that retinoic acid prevents the fluorescent staining of the Golgi apparatus and eliminates fluorescence of C6-NBD-ceramide prestained Golgi apparatus. Electron microscopy of retinoic acid-treated cells demonstrates the specific absence of any normal looking Golgi apparatus and a perinuclear vacuolar structure very similar to that seen in monensin-treated cells. This vacuolization disappears after removal of the retinoic acid and a perinuclear Golgi stacking reappears. These results indicate that retinoic acid alters intracellular routing, probably through the Golgi apparatus, potentiating immunotoxin activity indepedently of new gene expression. Retinoic acid appears to be a new reagent to manipulate the Golgi apparatus and intracellular traffic. As retinoic acid and immunotoxins are both in clinical trials for cancer therapy, their combined activity in vivo would be interesting to examine.

4-Chloro-7-nitrobenzofurazan↗
PubMed1994

Efficacy of direct intratumoral therapy with targeted protein toxins for solid human gliomas in nude mice.

Targeted protein toxins are a new class of reagents with the potential for great tumor selectivity and cytotoxic potency. Two such compounds were studied: 1) Tf-CRM107, a conjugate of human transferrin (Tf) and diphtheria toxin with a point mutation (CRM107); and 2) 454A12-rRA, a conjugate of a monoclonal antibody (454A12) to the human Tf receptor and recombinant ricin A chain (rRA). Both compounds are potent and specific in killing human glioblastoma cell lines in vitro. The authors investigated the activity of these reagents administered intratumorally against solid U251 MG human gliomas in vivo. Nude mice with established U251 MG flank tumors (0.5 to 1.0 cm in diameter) were randomly assigned to be treated with 100-microliters intratumoral injections of Tf-CRM107 (10 micrograms) or 454A12-rRA (10 micrograms), equimolar doses of CRM107 (4.3 micrograms), 454A12 antibody (7.5 micrograms), or rRA (1.5 micrograms), or phosphate-buffered saline (PBS) every 2 days for a total of four doses. Tumor volume and animal weight were assessed by a blinded observer before each treatment and biweekly for 30 days after initiating therapy. With Tf-CRM107 administration, tumor regression of greater than 95% occurred by Day 14 (p < 0.01) and tumors did not recur by Day 30. Treatment with 454A12-rRA caused a 30% decrease in tumor volume by Day 14 (p < 0.01). Treatment with equimolar doses of the unconjugated targeted protein toxin components CRM107, 454A12, or rRA caused significant U251 MG tumor growth inhibition, but the effects were less potent than the antitumor effects of the conjugates. This study also characterized the dose-response effect of Tf-CRM107 on tumor growth and tumor weight on Day 30. Nude mice with established U251 MG flank tumors (0.5 to 1.0 cm in diameter) were treated with 100-microliters intratumoral injections of 10, 1.0, or 0.1 microgram of Tf-CRM107 or PBS every 2 days for a total of four doses. All three doses of Tf-CRM107 significantly inhibited tumor growth by Day 14 (p < 0.01) and at Day 30 (p < 0.05), with a significant dose-response relationship. This study demonstrated in vivo efficacy of the targeted toxins Tf-CRM107 and 454A12-rRA against a human glioma. With intratumoral administration, the effect of Tf-CRM107 was tumor-specific and in some animals curative. Regional therapy with these potent tumor-specific agents using direct intratumoral infusion should limit systemic toxicity and may be efficacious against brain tumors.

Animals↗
PubMed1994

Toxicity of an antitumor ribonuclease to Purkinje neurons.

Purkinje cell toxicity is one of the characteristic features of the Gordon phenomenon, a syndrome manifested by ataxia, muscular rigidity, paralysis, and tremor that may lead to death (Gordon, 1933). Two members of the RNase superfamily found in humans, EDN (eosinophil-derived neurotoxin) and ECP (eosinophil cationic protein), cause the Gordon phenomenon when injected intraventricularly into guinea pigs or rabbits. We have found that another member of the RNase superfamily, an antitumor protein called onconase, isolated from Rana pipiens oocytes and early embryos, will also cause the Gordon phenomenon when injected into the cerebrospinal fluid of guinea pigs at a dose similar to that of EDN (LD50, 3-4 micrograms). Neurologic abnormalities of onconase-treated animals were indistinguishable from those of EDN-treated animals, and histology showed dramatic Purkinje cell loss in the brains of onconase-treated animals. The neurotoxic activity of onconase correlates with ribonuclease activity. Onconase modified by iodoacetic acid to eliminate 70% and 98% of the ribonuclease activity of the native enzyme displays a similar decrease in ability to cause the Gordon phenomenon. In contrast, the homologous bovine pancreatic RNase A injected intraventricularly at a dose 5000 times greater than the LD50 dose of EDN or onconase is not toxic and does not cause the Gordon phenomenon. A comparison of the RNase activities of EDN, onconase, and bovine pancreatic RNase A using three pancreatic RNA substrates demonstrates that onconase is orders of magnitude less active enzymatically than EDN and RNase A. Thus, another member of the RNase superfamily in addition to EDN and ECP can cause the Gordon phenomenon.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Pharmacokinetics and toxicology of immunotoxins administered into the subarachnoid space in nonhuman primates and rodents.

Immunotoxins have been suggested as possible therapeutic agents in patients with leptomeningeal carcinomatosis. The pharmacokinetics, stability, and toxicity of immunotoxins injected into the i.t. space were examined in rats and rhesus monkeys. Monoclonal antibodies specific for the human (454A12 and J1) and rat (OX26) transferrin receptors were coupled to recombinant ricin A chain. In monkeys, the maximally tolerated dose of the anti-human transferrin receptor immunotoxin (454A12-rRA) was a dose that yielded a nominal cerebrospinal fluid (CSF) concentration of approximately 1.2 x 10(-7) M. In rats, the 10% lethal dose (LD10) of the anti-human transferrin receptor immunotoxin was a dose yielding a nominal CSF concentration of 8.8 x 10(-7) M whereas the LD10 of the anti-rat transferrin receptor immunotoxin (OX26-rRA) was a dose yielding a nominal CSF concentration of 1.2 x 10(-7) M. Thus, the species-relevant antibody resulted in toxicity at a concentration one-seventh that of the immunotoxin with the irrelevant antibody. A comparison of the area under the concentration curve at the LD10 for rats with the area under the concentration curve at the maximally tolerated dose in monkeys and humans shows that the species-relevant immunotoxin was a better predictor of the toxic dose of the anti-transferrin receptor immunotoxin in humans than the irrelevant immunotoxin. The pharmacokinetics of the 454A12-rRA immunotoxin within the CSF of monkeys showed a biphasic clearance with an early-phase half-life of 1.4 h and a late phase half-life of 10.9 h. The clearance was 4.4 ml/h or approximately twice the estimated clearance due to bulk flow of CSF. Loss by degradation was ruled out because immunoblot analysis showed that the immunotoxin was stable for up to 24 h after administration. Possible losses in addition to sampling include diffusion into brain tissue and transcapillary permeation. The apparent volume of distribution was 10.1 ml or approximately three-fourths the total CSF volume of the monkey. Dose limiting toxicity corresponded with the selective elimination of Purkinje cells in both rats and monkeys and was manifested clinically as ataxia and lack of coordination. The onset of ataxia in monkeys occurred within 5 days and, in the more mild form, was reversible with time. There was evidence of only minimal inflammation within the CSF, and there were no signs of systemic toxicity. Immunotoxins injected into the subarachnoid space may have potential for treatment of leptomeningeal carcinomatosis.

Animals↗
PubMed1993

A cytotoxic ribonuclease. Study of the mechanism of onconase cytotoxicity.

Onconase, or P-30, is a protein initially purified from extracts of Rana pipiens oocytes and early embryos based upon its anticancer activity both in vitro and in vivo. It is a basic single-chain protein with an apparent molecular mass of 12,000 daltons and is homologous to RNase A. In cultured 9L glioma cells, onconase inhibits protein synthesis with an IC50 of about 10(-7) M. The inhibition of protein synthesis correlates with cell death determined by clonogenic assays. 125I-Labeled onconase binds to specific sites on cultured 9L glioma cells. Scatchard analysis of the binding data shows that onconase appears to bind to cells with two different affinities, one with a Kd of 6.2 x 10(-8) and another of 2.5 x 10(-7) M. Each cell could bind about 3 x 10(5) molecules of onconase at each of the two affinity sites. The low affinity Kd is similar to the IC50 for onconase toxicity. Onconase also demonstrates a saturability of cytotoxicity at a concentration that would saturate the low affinity binding site. Incubation at 4 degrees C increased the binding of onconase to cells relative to 37 degrees C binding and also increased the sensitivity of cells to onconase toxicity, indicating that receptor binding may be an initial step in cell toxicity. Onconase cytotoxicity can be blocked by metabolic inhibitors, NaN3 and 2-deoxyglucose, and cytotoxicity is potentiated 10-fold by monensin. Ribonuclease activity appears necessary for onconase toxicity because alkylated onconase, which only retains 2% of the ribonuclease activity, was at least 100-fold less potent in inhibiting protein synthesis in cells. Onconase inhibition of protein synthesis in 9L cells coincides with the degradation of cellular 28 S and 18 S rRNA. In contrast to RNase A, onconase is resistant to two RNase inhibitors, placental ribonuclease inhibitor and Inhibit-Ace. Northern hybridization with placental ribonuclease inhibitor cDNA probe indicates that 9L glioma cells contain endogenous placental ribonuclease inhibitor mRNA. Based on these results, we propose that onconase toxicity results from onconase binding to cell surface receptors, internalization to the cell cytosol where it degrades ribosomal RNA, inhibiting protein synthesis and causing cell death.

Animals↗
PubMed1993

The spatial distribution of immunotoxins in solid tumors: assessment by quantitative autoradiography.

The spatial distribution of i.v. administered immunotoxins in s.c. human rhabdomyosarcoma RD2 xenografts was studied. The toxin and immunotoxins were: (a) diphtheria toxin (DT); (b) a binding-deficient form of DT (CRM107) linked to a monoclonal IgG1 antibody (454A12) directed against the human transferrin receptor (454A12-107); (c) the binding-deficient form of DT linked to the Fab' fragment of 454A12 (Fab'-107); and (d) the binding-deficient form of DT coupled to MOPC21, a monoclonal IgG1 with no significant binding to RD2 cells. DT and the immunotoxins were radiolabeled with 125I and injected via the tail vein into tumor-bearing athymic mice (median tumor weight, 0.25 g). Tumors were removed 2, 6, and 24 h after injection of DT or immunotoxin. Film images of 20-microns frozen sections were digitized by video microscopy, and gray levels were converted to tissue concentrations based upon the film response to radioactivity standards and the specific activity of the radiolabeled toxins. Images of the tumors were characterized quantitatively by the kurtosis and the area above threshold; the kurtosis is a measure of the spatial heterogeneity of the radiolabeled immunotoxins, and the area above threshold is defined here as the fractional tumor area that reaches or exceeds 1.5% of the initial plasma concentration. The spatial distribution of DT in the tumors was extremely uniform, characterized by low kurtosis values. In contrast, the autoradiograms of 454A12-107 were punctate in appearance and were characterized by very high kurtosis values. Fab'-107, which has approximately one-half the molecular weight of the intact immunotoxin and binds only monovalently, also produced punctate images with kurtosis values similar to those for 454A12-107. The nonbinding immunotoxin distributed somewhat less uniformly than DT but much more homogeneously than either of the binding immunotoxins. DT, 454A12-107, and Fab'-107 have similar affinities for their respective receptors, but the concentration of binding sites for DT on RD2 cells (<3,000 receptors/cell) is much lower than the concentration of transferrin receptor (60,000 receptors/cell). Thus, the heterogeneous distribution of 454A12-107 and Fab'-107 probably reflects retarded penetration due to binding to the tumor cells. The area above threshold was greatest for DT and lowest for 454A12-107; the fragment and nonbinding immunotoxins had intermediate values. The lower area above threshold for the nonbinding immunotoxin as compared with DT may be due to the considerably large molecular weight and hence the lower capillary permeability and diffusion coefficient of the immunotoxin.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Characterization of single-chain antibody (sFv)-toxin fusion proteins produced in vitro in rabbit reticulocyte lysate.

Chimeric proteins consisting of a fusion between binding-deficient mutants of diphtheria toxin (DT) or Pseudomonas exotoxin A (PE) and a single-chain antibody (E6 sFv) against the human transferrin receptor (TfnR) were expressed in a rabbit reticulocyte lysate system. Molecules utilizing PE40 (the carboxyl terminus 40 kDa of PE, lacking the binding domain) exhibited significant E6 sFv-mediated, cell type-specific cytotoxicity (IC50 1 x 10(-10) M) against a human erythroleukemia-derived cell line, K562. In contrast, a fusion protein between the same sFv and a DT mutant, DTM1 (containing two amino acid substitutions in the binding domain [S(508)F, S(525)F]) was not significantly cytotoxic, despite being enzymatically active. A tripartite protein in the form NH2-DTM1-E6 sFv-PE40-COOH exhibited cytotoxicity comparable to that of the PE40-sFv fusion (IC50 1 x 10(-10) M), suggesting that the deficit in activity of DTM1-sFv is not a function of misfolding of the sFv moiety or of a reduced ability to bind TfnR. In contrast to DTM1-E6 sFv, a fusion protein between a second DT mutant, CRM 107 [S(525)F], and the E6 sFv was specifically cytotoxic (IC50 1 x 10(-9) M), and toxicity could be blocked by addition of excess E6 antibody. The cell-free in vitro expression system we describe is rapid and may be used to express functional toxin-sFv fusion proteins. No protein refolding procedures are required, and the technique may be used to express proteins which, due to restrictions imposed on manipulation of toxin-encoding genes in Escherichia coli, could not be produced by more conventional methods.

ADP Ribose Transferases↗
PubMed1993

The role of proline 345 in diphtheria toxin translocation.

Diphtheria toxin (DT) can translocate across endosomal membranes in response to low pH. Buried hydrophobic domains localized in the 37-kDa toxin B chain become exposed in response to acidic conditions and are thought to participate in the membrane translocation process. The crystal structure of DT has revealed a structurally distinct translocation domain composed of nine alpha-helices with their interconnecting loops (Choe, S., Bennett, M., Fujii, G., Curmi, P., Kantardjieff, K., Collier, R., and Eisenberg, D. (1992) Nature 357, 216-222). Two of these alpha-helices, TH8 and TH9, are unusually apolar and constitute the central core of the translocation domain. It has been proposed that these domains and the highly charged interconnecting loop undergo a conformation change under acidic conditions producing a dagger-like structure capable of inserting into the membrane thus initiating the translocation process. Proline 345 occupies a strategic location at the end of the TH8 alpha-helix. Proline residues have the ability to undergo a cis-trans isomerization reaction and because of this have been proposed to play a role in the conformational change that is a prerequisite for toxin translocation. The role of the proline at position 345 in membrane translocation was investigated. Pro was mutagenized to Glu and to Gly using a two-step recombinant polymerase chain reaction procedure, and the mutant proteins were expressed in vitro. Glu, an alpha-helix former, and Gly, an alpha-helix breaker, were selected for mutagenesis to distinguish between a structural role for Pro as an alpha-helix breaker and alternative roles, perhaps involving cis-trans isomerization-related conformational changes. Replacing Pro at position 345 with Glu or Gly resulted in a 99% reduction in toxicity to Vero cells. The enzymatic and binding activity of the toxin were not altered by the mutations. Instead, the reduction in toxicity is due to decreased translocation ability, suggesting that the Pro at position 345 plays a specific role in toxin membrane translocation.

Amino Acid Sequence↗