Peptide phage display library as source for inhibitors of clostridial neurotoxins.

Clostridial neurotoxins are the most powerful toxins known. There are no available antidotes to neutralize neurotoxins after they have been internalized by neuronal cells. Enzymatic domains of clostridial neurotoxins are zinc-endopeptidases specific for protein components of the neuroexocytosis apparatus. Thus, attempts were made to find such antidotes among molecules possessing chelating properties. Subsequently, it was proposed that the process of interaction between clostridial neurotoxins and their substrates might be more complex than viewed previously and may include several separate regions of interaction. Phage display technology is free from bias toward any particular model. This technology in combination with recombinantly produced light chains of botulinum neurotoxins serotypes A, B, and C was used to identify potential inhibitors of clostridial neurotoxins. Identified sequences did not show substantial similarity with substrate proteins of clostridial neurotoxins. Nevertheless, three peptides chosen for further analysis were able to inhibit enzymatic activity of all clostridial neurotoxins tested. This work demonstrates that at least one of these peptides could not be cleaved by clostridial neurotoxin. Attempts to delete amino acid residues from this peptide resulted in dramatic loss of its inhibitory activity. Finally, this work presents a novel approach to searching for inhibitors of clostridial neurotoxins.

Diphtheria toxin NAD affinity and ADP ribosyltransferase activity are reduced at tryptophan 153 substitutions for alanine or phenylalanine.

Previous studies on chemical modifications of diphtheria toxin (DT) fragment A have suggested that the Trp153 amino acid residue is essential for the ADP ribosylation of elongation factor 2. We verified this experimentally after replacing Trp153 by Phe or Ala residues through in vitro mutagenesis of a cloned toxin gene fragment. Each of the mutant fragment A forms were found to reveal a reduced ADP ribosyl transferase (ADPRT) activity as well as lower affinity for NAD. Both ADPRT activity and NAD affinity of DT fragment A were only partially destroyed by nearly synonymous Trp153 ==> Phe153 substitution, but dramatically destroyed by Ala153 substitution. At the same time, each of the mutant fragment A forms appeared to be thermostable, suggesting that the mutations do not dramatically destroy the structure of the protein. These results clearly demonstrate that Trp153 is not highly specific for DT fragment A structure maintenance, but is highly specific for the key toxin functions such as ADP ribosylation of elongation factor 2 and NAD binding. We suggest that the Trp153 role in DT functioning may be that of binding the ribose moiety of NAD, which is crucial for DT catalytic activity and hence for toxicity.

Simple and efficient method for heterologous expression of clostridial proteins.

Many clostridial proteins are poorly produced in Escherichia coli. It has been suggested that this phenomena is due to the fact that several types of codons common in clostridial coding sequences are rarely used in E. coli and the quantities of the corresponding tRNAs in E. coli are not sufficient to ensure efficient translation of the corresponding clostridial sequences. To address this issue, we amplified three E. coli genes, ileX, argU, and leuW, in E. coli; these genes encode tRNAs that are rarely used in E. coli (the tRNAs for the ATA, AGA, and CTA codons, respectively). Our data demonstrate that amplification of ileX dramatically increased the level of production of most of the clostridial proteins tested, while amplification of argU had a moderate effect and amplification of leuW had no effect. Thus, amplification of certain tRNA genes for rare codons in E. coli improves the expression of clostridial genes in E. coli, while amplification of other tRNAs for rare codons might not be needed for improved expression. We also show that amplification of a particular tRNA gene might have different effects on the level of protein production depending on the prevalence and relative positions of the corresponding codons in the coding sequence. Finally, we describe a novel approach for improving expression of recombinant clostridial proteins that are usually expressed at a very low level in E. coli.

Recombinant derivatives of clostridial neurotoxins as delivery vehicles for proteins and small organic molecules.

Clostridial neurotoxins are the most powerful toxins known. Nevertheless, derivatives of these toxins may find broad applications both in science and medicine because of their unique abilities to recognize neurons and deliver small and large molecules into them. In this paper we describe the construction of two types of such derivatives. Proteins belonging to the first class were designed to allow direct conjugation with one or few molecules of interest. Proteins belonging to the second class contain biotin residue and therefore could be easily connected to streptavidin loaded with multiple molecules of interest. Only C-terminal regions of neurotoxin heavy chains were incorporated in the structure of recombinant proteins. Nevertheless, recombinant proteins were found to be able to recognize specific neuronal receptors and target model molecules to rat synaptosomes and human neuroblastoma cells.

Ligand-toxin hybrids directed to the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein exhibit lower toxicity than native Pseudomonas exotoxin.

Pseudomonas exotoxin (PE) binds the heavy chain of the alpha2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP). To understand the significance of this interaction, novel toxin-derived gene fusions were constructed with two ligands that also bind this receptor. A 39-kDa cellular protein, termed RAP, binds LRP with high affinity and often co-purifies with it. Two RAP toxins were constructed, one with PE and one with diphtheria toxin (DT). RAP, which replaced the toxins binding domains, was combined with each of the corresponding translocating and ADP-ribosylating domains. Both RAP-toxins bound LRP with an apparent higher affinity than native PE. Despite this, RAP-PE and DT-RAP were less toxic than native PE. Apparently, RAP-toxin molecules bound and entered cells but used a pathway that afforded only low efficiency of toxin transport to the cytosol. This was evident because co-internalization with adenovirus increased the toxicity of RAP-toxins by 10-fold. We speculate that the high affinity of RAP binding may not allow the toxin's translocating and ADP-ribosylating domains to reach the cytosol but rather causes the toxin to take another pathway, possibly one that leads to lysosomes. To test this hypothesis, additional RAP-PE fusions were constructed. N-terminal or C-terminal fragments of RAP were joined to PE to produce two novel fusion proteins which were likely to have reduced affinity for LRP. Both of these shorter fusion proteins exhibited greater toxicity than full-length RAP-PE. A second ligand-toxin gene fusion was constructed between plasminogen activator inhibitor type 1 and DT. DT-plasminogen activator inhibitor type 1 formed a complex with tissue-type plasminogen activator and inhibited its proteolytic activity. However, like the RAP-toxins, this hybrid was less toxic for cells than native PE.

Construction and expression in Escherichia coli of hybrid genes composed of sequences encoding diphtheria toxin and human CD4 receptor.

Derivatives of natural toxins possessing substituted receptor-recognition domains of different specificities can be used as instruments for the selective elimination of target cells. We have constructed two different types of hybrid genes that encode proteins composed of diphtheria toxin (DT) lacking the C-terminal residues that mediate toxin binding fused with the N-terminal region of human CD4 (Lys10 to Glu152). One of these hybrids encodes a protein with CD4 at the N terminus, while the other encodes a protein with CD4 at the C terminus. The stability of these two proteins was dramatically different. We could not detect a full-size product when the first construct was expressed in Escherichia coli. In contrast, proteins encoded by the second construct were more stable. In the latter case, the amount of full-size hybrid protein was 1-2% of the total cell protein. We speculate on the involvement of the region that resembles the processing site of Pseudomonas aeruginosa exotoxin A in the proteolytic degradation of the product encoded by the first type of hybrid.

[Cytotoxic properties of a recombinant hybrid of the A protein of Staphylococcus aureus with a fragment of exotoxin A of Pseudomonas aeruginosa]. / Tsitotoksicheskie svoistva rekombinantnogo gibrida A-belka Staphylococcus aureus s fragmentom ékzotoksina A Pseudomonas aeruginosa.

By gene-engineering technique a chimeric protein made up of fragments of Staphylococcus aureus protein A and . Pseudomonas aeruginosa exotoxin A has been constructed. The chimeric protein was shown to preserve features characteristic of its both constituents--it ADP-ribosylates elongation factor 2 and binds to Ig. Cytotoxic properties of the chimeric protein were studied in two model systems. Treatment of target cells in both systems was performed successively with antibodies against corresponding antigens and after washing--with recombinant chimeric toxin which bound to antibodies on the surface of target cells. In the first model system human B-lymphoma cells (Daudi line) carrying Ig molecules on their surface were treated with polyclonal antibodies against human Ig L-chains. In the other system, human T-lymphoma cells (Jurkat line) were treated successively with monoclonal antibodies against cell surface CD5 antigen and further on--with polyclonal antibodies against mouse Ig. In both systems, only a slight inhibition of the target cells' growth was registered. The probable reasons of low cytotoxic activity of the chimeric protein and prospects of increasing it are discussed.

[Construction of recombinant genes coding for products containing the fragment of Staphylococcus aureus protein A and the fragment of Pseudomonas aeruginosa exotoxin A]. / Konstruirovanie rekombinantnykh genov, produkty kotorykh soderzhat v svoem sostave fragment A belka Staphylococcus aureus i fragment ékzotoksina A Pseudomonas aeruginosa.

The genes coding for Pseudomonas aeruginosa exotoxin-A and Staphylococcus aureus A-protein have been cloned. Different fragments of the genes were subcloned on the plasmid vestors. The plasmids pAPA4 and pAPA42 have been constructed. The plasmids are coding for the hybrid protein which consists of the immunoglobulin binding A-protein domain at its NH2-terminus and the catalytically active fragment of exotoxin-A at its COON-terminus. The hybrid gene is expressed in Escherichia coli cells under the control of lambda cro-gene expression elements (pAPA42) or lac-operon expression elements (pAPA4). The latter proved to be most productive.

[Structure and function of diphtheria toxin]. / Struktura i funktsii difteriinogo toksina.

The survey of the literature on the problem of structural and functional relationship of different parts of diphtheria toxin (i.e. in the binding of toxin to eucaryotic cells receptors, intracellular transport of a-fragment of diphtheria toxin and toxin-mediated ADP-ribosylation of EF2) is presented. Some data concerning structural similarities of A-fragment of diphtheria toxin and C-terminal part of Pseudomonas aeruginosa exotoxin A are presented.

[Construction of plasmids coding for diphtheria toxin fragments]. / Konstruirovanie plazmid, kodiruiushchikh fragmenty difteriinogo toksina.

Plasmids coding different nontoxic derivatives (toxoids) of the diphtheria toxin were constructed. A secretion of toxoids that carry a signal sequence was found in the periplasmic space of E. coli and Erwinia carotovora. Toxoids without a signal sequence appear in the cytoplasm. We believe that the toxoids secreted in E. coli and E. carotovora cells undergo a limited proteolysis. According to the molecular weights of the fragments there are three targets for proteolysis. One of them being just between A- and B-fragments of the diphtheria toxin. The others are localised in the B-fragment. The role of E. coli signal peptidase in the specific cutting is discussed.