Ricin is a heterodimeric protein produced in the seeds of the castor oil plant (Ricinus communis)... more Ricin is a heterodimeric protein produced in the seeds of the castor oil plant (Ricinus communis). It is exquisitely potent to mammalian cells, being able to fatally disrupt protein synthesis by attacking the Achilles heel of the ribosome. For this enzyme to reach its substrate, it must not only negotiate the endomembrane system but it must also cross an internal membrane and avoid complete degradation without compromising its activity in any way. Cell entry by ricin involves a series of steps: (i) binding, via the ricin B chain (RTB), to a range of cell surface glycolipids or glycoproteins having beta-1,4-linked galactose residues; (ii) uptake into the cell by endocytosis; (iii) entry of the toxin into early endosomes; (iv) transfer, by vesicular transport, of ricin from early endosomes to the trans-Golgi network; (v) retrograde vesicular transport through the Golgi complex to reach the endoplasmic reticulum; (vi) reduction of the disulphide bond connecting the ricin A chain (RTA) and the RTB; (vii) partial unfolding of the RTA to render it translocationally-competent to cross the endoplasmic reticulum (ER) membrane via the Sec61p translocon in a manner similar to that followed by misfolded ER proteins that, once recognised, are targeted to the ER-associated protein degradation (ERAD) machinery; (viii) avoiding, at least in part, ubiquitination that would lead to rapid degradation by cytosolic proteasomes immediately after membrane translocation when it is still partially unfolded; (ix) refolding into its protease-resistant, biologically active conformation; and (x) interaction with the ribosome to catalyse the depurination reaction. It is clear that ricin can take advantage of many target cell molecules, pathways and processes. It has been reported that a single molecule of ricin reaching the cytosol can kill that cell as a consequence of protein synthesis inhibition. The ready availability of ricin, coupled to its extreme potency when administered intravenously or if inhaled, has identified this protein toxin as a potential biological warfare agent. Therapeutically, its cytotoxicity has encouraged the use of ricin in 'magic…
Current Opinion in Drug Discovery Development, 2010
A significant number of therapeutic targets reside inside cells and intracellular organelles. The... more A significant number of therapeutic targets reside inside cells and intracellular organelles. Therapeutics therefore must be able to gain access to cellular compartments, and be able to interact specifically with a given molecule to exert a desired effect. Many naturally occurring toxins perform such targeting with apparent ease, making them excellent paradigms for the delivery of therapeutics to the cell interior. By studying the mechanisms of cell entry, trafficking and modes of toxicity of these model delivery vectors, researchers can decipher how cells transport both endogenous molecules and exogenously applied therapeutics inside cells. Perhaps more importantly, the exploitation of cell binding and trafficking motifs could allow a therapeutic to target specifically, traffic within and escape from cellular compartments; in addition, toxic domains can be used to disrupt cell function specifically for therapeutic purposes. This review provides an overview of recent developments in the understanding of toxin targeting and trafficking, and discusses how these developments could result in opportunities for the design of more specific and efficient systems for therapeutic targeting.
Some of the most important and interesting molecules in metazoan biology are glycoproteins. The i... more Some of the most important and interesting molecules in metazoan biology are glycoproteins. The importance of the carbohydrate component of these structures is often revealed by the disease phenotypes that manifest when the biosynthesis of particular glycoforms is disrupted. On the other hand, the presence of large amounts of carbohydrate can often hinder the structural and functional analysis of glycoproteins. There are often good reasons, therefore, for wanting to engineer and predefine the N-glycans present on glycoproteins, e.g., in order to characterize the functions of the glycans or facilitate their subsequent removal. Here, we describe in detail two distinct ways in which to usefully interfere with oligosaccharide processing, one involving the use of specific processing inhibitors, and the other the selection of cell lines mutated at gene loci that control oligosaccharide processing, using cytotoxic lectins. Both approaches have the capacity for controlled, radical alteration of oligosaccharide processing in eukaryotic cells used for heterologous protein expression, and have great utility in the structural analysis of glycoproteins.
Current topics in microbiology and immunology, 2012
A number of protein toxins bind at the surface of mammalian cells and after endocytosis traffic t... more A number of protein toxins bind at the surface of mammalian cells and after endocytosis traffic to the endoplasmic reticulum, where the toxic A chains are liberated from the holotoxin. The free A chains are then dislocated, or retrotranslocated, across the ER membrane into the cytosol. Here, in contrast to ER substrates destined for proteasomal destruction, they undergo folding to a catalytic conformation and subsequently inactivate their cytosolic targets. These toxins therefore provide toxic probes for testing the molecular requirements for retrograde trafficking, the ER processes that prepare the toxic A chains for transmembrane transport, the dislocation step itself and for the post-dislocation folding that results in catalytic activity. We describe here the dislocation of ricin A chain and Shiga toxin A chain, but also consider cholera toxin which bears a superficial structural resemblance to Shiga toxin. Recent studies not only describe how these proteins breach the ER membran...
The murine monoclonal antibody H17E2 recognises placental alkaline phosphatase (PLAP), an antigen... more The murine monoclonal antibody H17E2 recognises placental alkaline phosphatase (PLAP), an antigen present in the human term placenta and also expressed by many tumours. The antibody is of value in both immunoscintigraphy and radioimmunotherapy in testicular and ovarian cancer. The small size of genetically engineered single chain antibodies (SCAs) should give diagnostic and therapeutic advantages of improved tumour penetration and increased blood clearance compared to IgG. Employing recombinant DNA techniques a SCA based on H17E2 has been expressed in Escherichia coli and has been shown to bind placental alkaline phosphatase specifically. When administered to nude mice bearing human tumour xenografts, the H17E2 SCA effectively localised to tumour whilst a co-administered non-specific SCA did not. H17E2 SCA achieves tumour: blood ratios that are superior to those achieved with whole IgG, probably owing to its rapid blood clearance. We conclude that the H17E2 SCA is suitable for furth...
We report bacterial expression of a single-chain antibody (ScFv) reactive against the haptens 4-h... more We report bacterial expression of a single-chain antibody (ScFv) reactive against the haptens 4-hydroxy-3 nitrophenylacetic acid (NP) and 4-hydroxy-3-iodo-5-nitrophenylacetic acid (NIP) that is suitable for targeting to mammalian cells in vitro in a novel two-step targeting strategy. Hapten-derivatized primary antibodies of known specificity, bound to target cells, can capture the ScFv. Specificity resides in the interaction of the primary targeting antibody with the target and the interaction of the ScFv for NP/NIP, since the ScFv does not bind cells and nonderivatized antibodies bound at cells cannot capture the ScFv. The ScFv described here can therefore be considered as a universal agent for delivery of drugs, toxins, or radionuclides to any cell type for which a previously characterized antibody exists.
We screened a panel of compounds derived from Exo2 - a drug that perturbs post-Golgi compartments... more We screened a panel of compounds derived from Exo2 - a drug that perturbs post-Golgi compartments and trafficking in mammalian cells - for their effect on the secretory pathway in Arabidopsis root epidermal cells. While Exo2 and most related compounds had no significant effect, one Exo2 derivative, named LG8, induced severe morphological alterations in both the Golgi (at high concentrations) and the endoplasmic reticulum (ER). LG8 causes the ER to form foci of interconnecting tubules, which at the ultrastructural level appear similar to those previously reported in Arabidopsis roots after treatment with the herbicide oryzalin. In cotyledonary leaves, LG8 causes redistribution of a trans Golgi network (TGN) marker to the vacuole. LG8 affects the anterograde secretory pathway by inducing secretion of vacuolar cargo and preventing the brassinosteroid receptor BRI1 from reaching the plasma membrane. Uptake and arrival at the TGN of the endocytic marker FM4-64 is not affected. Unlike the...
... Ion Niculescu-Duvaz, Robert Spooner, Richard Marais, and Caroline J. Springer*. CRC Centre ... more ... Ion Niculescu-Duvaz, Robert Spooner, Richard Marais, and Caroline J. Springer*. CRC Centre for Cancer Therapeutics at the Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, United Kingdom, and ...
There is a vast number of bacterial and plant toxins that affect cytosolic targets in mammalian c... more There is a vast number of bacterial and plant toxins that affect cytosolic targets in mammalian cells, and whether the purpose of the toxin is to act as a defence mechanism against predators, or to cause deliberate cell death in order to form an environment more suitable for bacterial growth, each of these toxins must cross a lipid membrane barrier in order to exert their effect. This review looks at the methods employed by bacterial and plant toxins in order to reach their target. We examine the trafficking methods involved in toxin transport throughout the cell, highlighting the proteins necessary for the toxins movement, and noting how many of the toxins hijack the cells own trafficking and protein processing machinery in order to reach their goals. Studying the trafficking of toxins has led to a greater understanding of retrograde transport, a process which has key relevance to the correct intracellular delivery of pharmacological agents.
Laboratory investigation; a journal of technical methods and pathology, 1996
Goodpasture's disease is defined by the presence of autoantibodies to the glomerular basement... more Goodpasture's disease is defined by the presence of autoantibodies to the glomerular basement membrane and characterized clinically by rapidly progressive glomerulonephritis and pulmonary hemorrhage. P1, a murine monoclonal antibody to the Goodpasture antigen (the noncollagenous domain of the alpha 3 chain of type IV collagen, alpha 3(IV)NC1), has been a valuable reagent in investigating the pathogenesis of this disorder. The purpose of this study was to generate and characterize a recombinant form of P1 as a single-chain Fv (scFv). First strand cDNA was made from RNA extracted from the P1 hybridoma cell line, and DNA encoding the antibody light and heavy chain variable domains was amplified by polymerase chain reaction, using universal oligonucleotides. The purified products were ligated sequentially into an expression plasmid separated by a sequence encoding a 15 amino acid flexible oligopeptide linker. The resulting scFv was expressed in E. coli. Functional scFv, designated H...
The plant cytotoxin ricin enters mammalian cells by receptor-mediated endocytosis, undergoing ret... more The plant cytotoxin ricin enters mammalian cells by receptor-mediated endocytosis, undergoing retrograde transport to the ER (endoplasmic reticulum) where its catalytic A chain (RTA) is reductively separated from the holotoxin to enter the cytosol and inactivate ribosomes. The currently accepted model is that the bulk of ER-dislocated RTA is degraded by proteasomes. We show in the present study that the proteasome has a more complex role in ricin intoxication than previously recognized, that the previously reported increase in sensitivity of mammalian cells to ricin in the presence of proteasome inhibitors simply reflects toxicity of the inhibitors themselves, and that RTA is a very poor substrate for proteasomal degradation. Denatured RTA and casein compete for a binding site on the regulatory particle of the 26S proteasome, but their fates differ. Casein is degraded, but the mammalian 26S proteasome AAA (ATPase associated with various cellular activities)-ATPase subunit RPT5 acts ...
Ricin is a heterodimeric protein produced in the seeds of the castor oil plant (Ricinus communis)... more Ricin is a heterodimeric protein produced in the seeds of the castor oil plant (Ricinus communis). It is exquisitely potent to mammalian cells, being able to fatally disrupt protein synthesis by attacking the Achilles heel of the ribosome. For this enzyme to reach its substrate, it must not only negotiate the endomembrane system but it must also cross an internal membrane and avoid complete degradation without compromising its activity in any way. Cell entry by ricin involves a series of steps: (i) binding, via the ricin B chain (RTB), to a range of cell surface glycolipids or glycoproteins having beta-1,4-linked galactose residues; (ii) uptake into the cell by endocytosis; (iii) entry of the toxin into early endosomes; (iv) transfer, by vesicular transport, of ricin from early endosomes to the trans-Golgi network; (v) retrograde vesicular transport through the Golgi complex to reach the endoplasmic reticulum; (vi) reduction of the disulphide bond connecting the ricin A chain (RTA) and the RTB; (vii) partial unfolding of the RTA to render it translocationally-competent to cross the endoplasmic reticulum (ER) membrane via the Sec61p translocon in a manner similar to that followed by misfolded ER proteins that, once recognised, are targeted to the ER-associated protein degradation (ERAD) machinery; (viii) avoiding, at least in part, ubiquitination that would lead to rapid degradation by cytosolic proteasomes immediately after membrane translocation when it is still partially unfolded; (ix) refolding into its protease-resistant, biologically active conformation; and (x) interaction with the ribosome to catalyse the depurination reaction. It is clear that ricin can take advantage of many target cell molecules, pathways and processes. It has been reported that a single molecule of ricin reaching the cytosol can kill that cell as a consequence of protein synthesis inhibition. The ready availability of ricin, coupled to its extreme potency when administered intravenously or if inhaled, has identified this protein toxin as a potential biological warfare agent. Therapeutically, its cytotoxicity has encouraged the use of ricin in 'magic…
Current Opinion in Drug Discovery Development, 2010
A significant number of therapeutic targets reside inside cells and intracellular organelles. The... more A significant number of therapeutic targets reside inside cells and intracellular organelles. Therapeutics therefore must be able to gain access to cellular compartments, and be able to interact specifically with a given molecule to exert a desired effect. Many naturally occurring toxins perform such targeting with apparent ease, making them excellent paradigms for the delivery of therapeutics to the cell interior. By studying the mechanisms of cell entry, trafficking and modes of toxicity of these model delivery vectors, researchers can decipher how cells transport both endogenous molecules and exogenously applied therapeutics inside cells. Perhaps more importantly, the exploitation of cell binding and trafficking motifs could allow a therapeutic to target specifically, traffic within and escape from cellular compartments; in addition, toxic domains can be used to disrupt cell function specifically for therapeutic purposes. This review provides an overview of recent developments in the understanding of toxin targeting and trafficking, and discusses how these developments could result in opportunities for the design of more specific and efficient systems for therapeutic targeting.
Some of the most important and interesting molecules in metazoan biology are glycoproteins. The i... more Some of the most important and interesting molecules in metazoan biology are glycoproteins. The importance of the carbohydrate component of these structures is often revealed by the disease phenotypes that manifest when the biosynthesis of particular glycoforms is disrupted. On the other hand, the presence of large amounts of carbohydrate can often hinder the structural and functional analysis of glycoproteins. There are often good reasons, therefore, for wanting to engineer and predefine the N-glycans present on glycoproteins, e.g., in order to characterize the functions of the glycans or facilitate their subsequent removal. Here, we describe in detail two distinct ways in which to usefully interfere with oligosaccharide processing, one involving the use of specific processing inhibitors, and the other the selection of cell lines mutated at gene loci that control oligosaccharide processing, using cytotoxic lectins. Both approaches have the capacity for controlled, radical alteration of oligosaccharide processing in eukaryotic cells used for heterologous protein expression, and have great utility in the structural analysis of glycoproteins.
Current topics in microbiology and immunology, 2012
A number of protein toxins bind at the surface of mammalian cells and after endocytosis traffic t... more A number of protein toxins bind at the surface of mammalian cells and after endocytosis traffic to the endoplasmic reticulum, where the toxic A chains are liberated from the holotoxin. The free A chains are then dislocated, or retrotranslocated, across the ER membrane into the cytosol. Here, in contrast to ER substrates destined for proteasomal destruction, they undergo folding to a catalytic conformation and subsequently inactivate their cytosolic targets. These toxins therefore provide toxic probes for testing the molecular requirements for retrograde trafficking, the ER processes that prepare the toxic A chains for transmembrane transport, the dislocation step itself and for the post-dislocation folding that results in catalytic activity. We describe here the dislocation of ricin A chain and Shiga toxin A chain, but also consider cholera toxin which bears a superficial structural resemblance to Shiga toxin. Recent studies not only describe how these proteins breach the ER membran...
The murine monoclonal antibody H17E2 recognises placental alkaline phosphatase (PLAP), an antigen... more The murine monoclonal antibody H17E2 recognises placental alkaline phosphatase (PLAP), an antigen present in the human term placenta and also expressed by many tumours. The antibody is of value in both immunoscintigraphy and radioimmunotherapy in testicular and ovarian cancer. The small size of genetically engineered single chain antibodies (SCAs) should give diagnostic and therapeutic advantages of improved tumour penetration and increased blood clearance compared to IgG. Employing recombinant DNA techniques a SCA based on H17E2 has been expressed in Escherichia coli and has been shown to bind placental alkaline phosphatase specifically. When administered to nude mice bearing human tumour xenografts, the H17E2 SCA effectively localised to tumour whilst a co-administered non-specific SCA did not. H17E2 SCA achieves tumour: blood ratios that are superior to those achieved with whole IgG, probably owing to its rapid blood clearance. We conclude that the H17E2 SCA is suitable for furth...
We report bacterial expression of a single-chain antibody (ScFv) reactive against the haptens 4-h... more We report bacterial expression of a single-chain antibody (ScFv) reactive against the haptens 4-hydroxy-3 nitrophenylacetic acid (NP) and 4-hydroxy-3-iodo-5-nitrophenylacetic acid (NIP) that is suitable for targeting to mammalian cells in vitro in a novel two-step targeting strategy. Hapten-derivatized primary antibodies of known specificity, bound to target cells, can capture the ScFv. Specificity resides in the interaction of the primary targeting antibody with the target and the interaction of the ScFv for NP/NIP, since the ScFv does not bind cells and nonderivatized antibodies bound at cells cannot capture the ScFv. The ScFv described here can therefore be considered as a universal agent for delivery of drugs, toxins, or radionuclides to any cell type for which a previously characterized antibody exists.
We screened a panel of compounds derived from Exo2 - a drug that perturbs post-Golgi compartments... more We screened a panel of compounds derived from Exo2 - a drug that perturbs post-Golgi compartments and trafficking in mammalian cells - for their effect on the secretory pathway in Arabidopsis root epidermal cells. While Exo2 and most related compounds had no significant effect, one Exo2 derivative, named LG8, induced severe morphological alterations in both the Golgi (at high concentrations) and the endoplasmic reticulum (ER). LG8 causes the ER to form foci of interconnecting tubules, which at the ultrastructural level appear similar to those previously reported in Arabidopsis roots after treatment with the herbicide oryzalin. In cotyledonary leaves, LG8 causes redistribution of a trans Golgi network (TGN) marker to the vacuole. LG8 affects the anterograde secretory pathway by inducing secretion of vacuolar cargo and preventing the brassinosteroid receptor BRI1 from reaching the plasma membrane. Uptake and arrival at the TGN of the endocytic marker FM4-64 is not affected. Unlike the...
... Ion Niculescu-Duvaz, Robert Spooner, Richard Marais, and Caroline J. Springer*. CRC Centre ... more ... Ion Niculescu-Duvaz, Robert Spooner, Richard Marais, and Caroline J. Springer*. CRC Centre for Cancer Therapeutics at the Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, United Kingdom, and ...
There is a vast number of bacterial and plant toxins that affect cytosolic targets in mammalian c... more There is a vast number of bacterial and plant toxins that affect cytosolic targets in mammalian cells, and whether the purpose of the toxin is to act as a defence mechanism against predators, or to cause deliberate cell death in order to form an environment more suitable for bacterial growth, each of these toxins must cross a lipid membrane barrier in order to exert their effect. This review looks at the methods employed by bacterial and plant toxins in order to reach their target. We examine the trafficking methods involved in toxin transport throughout the cell, highlighting the proteins necessary for the toxins movement, and noting how many of the toxins hijack the cells own trafficking and protein processing machinery in order to reach their goals. Studying the trafficking of toxins has led to a greater understanding of retrograde transport, a process which has key relevance to the correct intracellular delivery of pharmacological agents.
Laboratory investigation; a journal of technical methods and pathology, 1996
Goodpasture's disease is defined by the presence of autoantibodies to the glomerular basement... more Goodpasture's disease is defined by the presence of autoantibodies to the glomerular basement membrane and characterized clinically by rapidly progressive glomerulonephritis and pulmonary hemorrhage. P1, a murine monoclonal antibody to the Goodpasture antigen (the noncollagenous domain of the alpha 3 chain of type IV collagen, alpha 3(IV)NC1), has been a valuable reagent in investigating the pathogenesis of this disorder. The purpose of this study was to generate and characterize a recombinant form of P1 as a single-chain Fv (scFv). First strand cDNA was made from RNA extracted from the P1 hybridoma cell line, and DNA encoding the antibody light and heavy chain variable domains was amplified by polymerase chain reaction, using universal oligonucleotides. The purified products were ligated sequentially into an expression plasmid separated by a sequence encoding a 15 amino acid flexible oligopeptide linker. The resulting scFv was expressed in E. coli. Functional scFv, designated H...
The plant cytotoxin ricin enters mammalian cells by receptor-mediated endocytosis, undergoing ret... more The plant cytotoxin ricin enters mammalian cells by receptor-mediated endocytosis, undergoing retrograde transport to the ER (endoplasmic reticulum) where its catalytic A chain (RTA) is reductively separated from the holotoxin to enter the cytosol and inactivate ribosomes. The currently accepted model is that the bulk of ER-dislocated RTA is degraded by proteasomes. We show in the present study that the proteasome has a more complex role in ricin intoxication than previously recognized, that the previously reported increase in sensitivity of mammalian cells to ricin in the presence of proteasome inhibitors simply reflects toxicity of the inhibitors themselves, and that RTA is a very poor substrate for proteasomal degradation. Denatured RTA and casein compete for a binding site on the regulatory particle of the 26S proteasome, but their fates differ. Casein is degraded, but the mammalian 26S proteasome AAA (ATPase associated with various cellular activities)-ATPase subunit RPT5 acts ...
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