Creation of intercellular bonds by anchoring protein ligands to membranes using the diphtheria toxin T domain

We describe the creation of cell adhesion mediated by cell surface engineering. The Flt3-ligand was fused to a membrane anchor made of the diphtheria toxin translocation domain. The fusion protein was attached to the surface of a cell by an acid pulse. Contact with another cell expressing the receptor Flt3 lead to its activation. This activity involved direct cell-cell contact. A mean force of 20 nN was needed to separate functionalized cells after 5 min of contact. Overall, we showed that it is possible to promote specific cell-cell adhesion by attaching protein ligands at the surface of cells.     

Development of novel whole-cell immunoadsorbents by yeast surface display of the IgG-binding domain

The ZZ domain derived from Staphylococcus aureus, which binds to the Fc part of immunoglobulin G (IgG), was displayed on the cell surfaces of yeast Saccharomyces cerevisiae by cell-surface engineering using the C-terminal half of alpha-agglutinin under control of the 5'-upstream region of the isocitrate lyase gene from Candida tropicalis (UPR-ICL). Display of ZZ on the cell surface was confirmed by immunofluorescence microscopy. Enzyme-linked immunosorbent assay (ELISA) and sandwich ELISA using the S. cerevisiae cells displaying ZZ detected IgG and antigen (human serum albumin) down to a concentration of 1-10 ng/ml in both cases. The detection range covered by these assay systems was wide and could be varied by adjusting the amount of cells and reaction times with horseradish peroxidase (HRP) substrate. Moreover, yeast cells displaying ZZ were successfully used for repeated affinity purification of IgG from serum. These results indicate that S. cerevisiae displaying ZZ may constitute novel and genetically renewable whole-cell immunoadsorbents widely applicable to immunoassays and affinity purification.     

Receptor-mediated internalization and degradation of diphtheria toxin by monkey kidney cells.

The receptor-mediated internalization and degradation of radiolabeled diphtheria toxin by cultured monkey kidney cells was studied. The ability of a number of enzymes and chemicals to remove cell surface-bound toxin was tested; the combination of pronase and inositol hexaphosphate (PIHP) proved most effective. Using PIHP, the kinetics of toxin-cell association at 37 degrees C was resolved into two compounds: surface binding and internalization. The PIHP assay also allowed estimation of the half-time of toxin internalization (about 25 min). An assay involving precipitation of culture supernatants with trichloroacetic acid was developed and used to measure the rate of degradation and excretion of cell-associated toxin. Agents which markedly inhibited toxin internalization similarly prevented degradation, implying an intracellular location for the degradative process. The primary radioactive product excreted by Vero cells was monoiodotyrosine. The extent and rate of toxin degradation indicated lysosomal involvement. Finally, agents which blocked internalization or degradation, or both, (e.g. antibody and concanavalin A), protected cells from the cytotoxin action of diphtheria toxin, suggesting that these processes are necessary for expression of biological effect.     

Development of recombinant scFv-antibodies against diphtheria toxin using phage display system

Phage display technology is an effective approach to the development of the next generation of immunodiagnostic reagents. Naive murine phage display a library of single-chain variable antibodies (scFv) was used to isolate scFv recognizing the diphtheria toxin, an important diagnostic antigen of diphtheria. The diphtheria toxin B subunit-binding clone with affinity constant of 1.13 x 10(7) M(-1) was selected. scFv preserved activity on storage in the course of 8 months.     

Characterization of the diphtheria toxin receptor-binding domain

The carboxyl-terminal region of diphtheria toxin (DT) has been analysed in order to determine regions of receptor recognition. Biochemical cleavage of the toxin with hydroxylamine (HA) was used to generate the peptides HA9DT (residues 454–535), HA6DT (residues 482–535), and HA3DT (residues 454–461). Characterization of HA6DT demonstrated that the final 54 amino acids of DT are sufficient to constitute the receptor-binding domain of the toxin. Within HA9DT, the region encompassing HA3DT and containing the highly cationic polyphosphate-binding site did not contribute to the binding ability of HA6DT. Consistent with this observation, HA3DT itself did not compete for binding of radiolabelled DT to Vero cells. A 30-amino acid synthetic peptide composed of residues 506–535 did not block receptor binding of DT, indicating that residues toward the amino-terminus of HA6DT, or the entire HA6DT region, are required for receptor recognition.     

Interaction of the membrane-inserted diphtheria toxin T domain with peptides and its possible implications for chaperone-like T domain behavior

The T domain of diphtheria toxin is believed to aid the low-pH-triggered translocation of the partly unfolded A chain (C domain) through cell membranes. Recent experiments have suggested the possibility that the T domain aids translocation by acting as a membrane-inserted chaperone [Ren, J., et al. (1999) Science 284, 955-957]. One prediction of this model is that the membrane-inserted T domain should be able to interact with sequences that mimic unfolded proteins. To understand the basis of interaction of the membrane-inserted T domain with unfolded polypeptides, its interaction with water-soluble peptides having different sequences was studied. The membrane-inserted T domain was able to recognize helix-forming 23-residue Ala-rich peptides. In the presence of such peptides, hydrophobic helix 9 of the T domain underwent the previously characterized conformational change from a state exhibiting shallow membrane insertion to one exhibiting deep insertion. This conformational change was more readily induced by the more hydrophobic peptides that were tested. It did not occur at all in the presence a hydrophilic peptide in which alternating Ser and Gly replaced Ala or in the presence of unfolded hydrophilic peptides derived from the A chain of the toxin. Interestingly, a peptide with a complex sequence (RKE(3)KE(2)LMEW(2)KM(2)SETLNF) also interacted with the T domain very strongly. We conclude that the membrane-inserted T domain cannot recognize every unfolded amino acid sequence. However, it does not exhibit strong sequence specificity, instead having the ability to recognize and interact with a variety of amino acid sequences having moderate hydrophobicity. This recognition was not strictly correlated with the strength of peptide binding to the lipid, suggesting that more than just hydrophobicity is involved. Although it does not prove that the T domain functions as a chaperone, T domain recognition of hydrophobic sequences is consistent with it having polypeptide recognition properties that are chaperone-like.     

Concerted protonation of key histidines triggers membrane interaction of the diphtheria toxin T domain

The translocation domain (T domain) of the diphtheria toxin contributes to the transfer of the catalytic domain from the cell endosome to the cytosol, where it blocks protein synthesis. Translocation is initiated when endosome acidification induces the interaction of the T domain with the membrane of the compartment. We found that the protonation of histidine side chains triggers the conformational changes required for membrane interaction. All histidines are involved in a concerted manner, but none is indispensable. However, the preponderance of each histidine varies according to the transition observed. The pair His(223)-His(257) and His(251) are the most sensitive triggers for the formation of the molten globule state in solution, whereas His(322)-His(323) and His(251) are the most sensitive triggers for membrane binding. Interestingly, the histidines are located at key positions throughout the structure of the protein, in hinges and at the interface between each of the three layers of helices forming the domain. Their protonation induces local destabilizations, disrupting the tertiary structure and favoring membrane interaction. We propose that the selection of histidine residues as triggers of membrane interaction enables the T domain to initiate translocation at the rather mild pH found in the endosome, contributing to toxin efficacy.     

Binding properties of diphtheria toxin to cells are altered by mutation in the fragment A domain

CRM197, CRM176, and CRM228 are products of single or multiple missense mutations in the diphtheria toxin gene. CRM197 differs from wild-type toxin in 1 amino acid residue of the fragment A region, and also CRM176 and CRM228 have amino acid substitution(s) in fragment A. We compared the binding properties of CRM197 to toxin-sensitive Vero cells with those of diphtheria toxin and other CRMs. Nicked CRM197 is about 50 times more effective than intact CRM197 in inhibiting the action of diphtheria toxin on sensitive cells, as shown by inhibition of diphtheria toxin cytotoxicity or inhibition of binding of 125I-diphtheria toxin. The binding of native toxin or other CRMs was not significantly affected by nicking. Moreover, the binding of CRM197 to cells was unaffected by ATP, although ATP clearly inhibits binding of diphtheria toxin, CRM176, and CRM228. Two kinds of hybrid protein were formed using fragment B of CRM197: one with fragment A of diphtheria toxin and one with fragment A of CRM228. ATP inhibited the binding of these hybrid proteins. Furthermore, the affinities of these hybrid proteins for diphtheria toxin-sensitive cells were the same as that of native toxin. Thus, it was concluded that the altered binding properties of CRM197 were due to alteration of fragment A and what the interaction of diphtheria toxin with ATP involves both fragments. The results also suggest that fragment A plays a role in diphtheria toxin-receptor interaction.     

An antibody that inhibits the binding of diphtheria toxin to cells revealed the association of a 27-kDa membrane protein with the diphtheria toxin receptor

A monoclonal antibody that blocks the binding of diphtheria toxin to Vero cells was isolated by immunizing mice with Vero cell membrane. The antibody inhibits the binding of diphtheria toxin and also CRM197, a mutant form of diphtheria toxin, to Vero cells, and consequently inhibits the cytotoxicity of diphtheria toxin. This antibody does not directly react with the receptor molecule of diphtheria toxin (DTR14.5). Immunoprecipitation and immunoblotting studies revealed that this antibody binds to a novel membrane protein of 27 kDa (DRAP27). When diphtheria toxin receptor was passed through an affinity column made with this antibody, the receptor was trapped only in the presence of DRAP27. These results indicate that DRAP27 and DTR14.5 closely associate in Vero cell membrane and that the inhibition of the binding of diphtheria toxin to the receptor is due to the binding of the antibody to the DRAP27 molecule. Binding studies using 125I-labeled antibody showed that there are many more molecules of DRAP27 on the cell surface than diphtheria toxin-binding sites. However, there is a correlation between the sensitivity of a cell line to diphtheria toxin and the number of DRAP27 molecules on the cell surface, suggesting that DRAP27 is involved in the entry of diphtheria toxin into the target cell.     

Fate of tetanus toxin bound to the surface of primary neurons in culture: evidence for rapid internalization

We examined the nature of the tetanus toxin receptor in primary cultures of mouse spinal cord by ligand blotting techniques. Membrane components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose sheets, which were overlaid with 125I-labeled tetanus toxin. The toxin bound only to material at or near the dye front, which was lost when the cells were delipidated before electrophoresis. Gangliosides purified from the lipid extract were separated by thin-layer chromatography and the chromatogram was overlaid with 125I-toxin. The toxin bound to gangliosides corresponding to GD1b and GT1b. Similar results were obtained with brain membranes; thus, gangliosides rather than glycoproteins appear to be the toxin receptors both in vivo and in neuronal cell cultures. To follow the fate of tetanus toxin bound to cultured neurons, we developed an assay to measure cell-surface and internalized toxin. Cells were incubated with tetanus toxin at 0 degree C, washed, and sequentially exposed to antitoxin and 125I-labeled protein A. Using this assay, we found that much of the toxin initially bound to cell surface disappeared rapidly when the temperature was raised to 37 degrees C but not when the cells were kept at 0 degree C. Some of the toxin was internalized and could only be detected by our treating the cells with Triton X-100 before adding anti-toxin. Experiments with 125I-tetanus toxin showed that a substantial amount of the toxin bound at 0 degree C dissociated into the medium upon warming of the cells. Using immunofluorescence, we confirmed that some of the bound toxin was internalized within 15 min and accumulated in discrete structures. These structures did not appear to be lysosomes, as the cell-associated toxin had a long half-life and 90% of the radioactivity released into the medium was precipitated by trichloroacetic acid. The rapid internalization of tetanus toxin into a subcellular compartment where it escapes degradation may be important for its mechanism of action.     

The number of subunits comprising the channel formed by the T domain of diphtheria toxin.

In the presence of a low pH environment, the channel-forming T domain of diphtheria toxin undergoes a conformational change that allows for both its own insertion into planar lipid bilayers and the translocation of the toxin's catalytic domain across them. Given that the T domain contributes only three transmembrane segments, and the channel is permeable to ions as large as glucosamine(+) and NAD(-), it would appear that the channel must be a multimer. Yet, there is substantial circumstantial evidence that the channel may be formed from a single subunit. To test the hypothesis that the channel formed by the T domain of diphtheria toxin is monomeric, we made mixtures of two T domain constructs whose voltage-gating characteristics differ, and then observed the gating behavior of the mixture's single channels in planar lipid bilayers. One of these constructs contained an NH(2)-terminal hexahistidine (H6) tag that blocks the channel at negative voltages; the other contained a COOH-terminal H6 tag that blocks the channel at positive voltages. If the channel is constructed from multiple T domain subunits, one expects to see a population of single channels from this mixture that are blocked at both positive and negative voltages. The observed single channels were blocked at either negative or positive voltages, but never both. Therefore, we conclude that the T domain channel is monomeric.     

Localization of the diphtheria toxin receptor-binding domain to the carboxyl-terminal Mr approximately 6000 region of the toxin

The carboxyl-terminal Mr = 5982 peptide of diphtheria toxin was prepared by specific cleavage of the toxin with hydroxylamine and purified by fast performance liquid chromatography. The identity of the peptide was established by a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, reactivity with specific monoclonal antibodies, and amino-terminal sequence analysis. The Mr = 5982 peptide was shown to protect highly toxin-sensitive Vero cells from the lethal action of diphtheria toxin. This protection was shown to be due to inhibition of the initial step in the cytotoxic process, the binding of toxin to its receptor. These results strongly suggest that the Mr = 5982 carboxyl-terminal region (amino acid residues 482-535) is, or contains, the receptor-binding domain of diphtheria toxin.     

Isolation of the fifth IgG-binding domain from Staphylococcus aureus protein A

Using affinity chromatography on IgG-Sepharose at pH 5.0, a new fragment capable of binding to IgG (domain E) was isolated from trypsin hydrolysate of protein A. Trypsinolysis of protein A was performed at low temperatures. Thus, the intact structure of protein A was found to include six domains, of which five interact with IgG.     

Functional display of foreign protein on surface of Escherichia coli using N-terminal domain of ice nucleation protein

We investigated the ability of the N-terminal domain of InaK, an ice nucleation protein from Pseudomonas syringae KCTC1832, to act as an anchoring motif for the display of foreign proteins on the Escherichia coli cell surface. Total expression level and surface display efficiency of green fluorescent protein (GFP) was compared following their fusion with either the N-terminal domain of InaK (InaK-N), or with the known truncated InaK containing both N- and C-terminal domains (InaK-NC). We report that the InaK-N/GFP fusion protein showed a similar cell surface display efficiency ( approximately 50%) as InaK-NC/GFP, demonstrating that the InaK N-terminal region alone can direct translocation of foreign proteins to the cell surface and can be employed as a potential cell surface display motif. Moreover, InaK-N/GFP showed the highest levels of total expression and surface display based on unit cell density. InaK-N was also successful in directing cell surface display of organophosphorus hydrolase (OPH), confirming its ability to act as a display motif.     

Cell surface display of CD8 ecto domain on Escherichia coli using ice nucleation protein

A new system for cell surface display of recombinant proteins on Escherichia coli was tested for expression of the ecto domain of CD8, which is the surface protein of human T cytotoxic lymphocytes. Immunofluorescence microscopy, ELISA, and immunodot blotting confirmed successful expression of the CD8 ecto domain fused to ice nucleation protein (INP), an outer membrane protein of Pseudomonas syringae. © Rapid Science Ltd. 1998     

Behavior of the N-terminal helices of the diphtheria toxin T domain during the successive steps of membrane interaction

During intoxication of a cell, the translocation (T) domain of the diphtheria toxin helps the passage of the catalytic domain across the membrane of the endosome into the cytoplasm. We have investigated the behavior of the N-terminal region of the T domain during the successive steps of its interaction with membranes at acidic pH using tryptophan fluorescence, its quenching by brominated lipids, and trypsin digestion. The change in the environment of this region was monitored using mutant W281F carrying a single native tryptophan at position 206 at the tip of helix TH1. The intrinsic propensity to interact with the membrane of each helix of the N-terminus of the T domain, TH1, TH2, TH3, and TH4, was also studied using synthetic peptides. We showed the N-terminal region of the T domain was not involved in the binding of the domain to the membrane, which occurred at pH 6 mainly through hydrophobic effects. At that stage of the interaction, the N-terminal region remained strongly solvated. Further acidification eliminated repulsive electrostatic interactions between this region and the membrane, allowing its penetration into the membrane by attractive electrostatic interactions and hydrophobic effects. The peptide study indicated the nature of forces contributing to membrane penetration. Overall, the data suggested that the acidic pH found in the endosome not only triggers the formation of the molten globule state of the T domain required for membrane interaction but also governs a progressive penetration of the N-terminal part of the T domain in the membrane. We propose that these physicochemical properties are necessary for the translocation of the catalytic domain.     

Solution and membrane-bound chaperone activity of the diphtheria toxin translocation domain towards the catalytic domain

During cell intoxication by diphtheria toxin, endosome acidification triggers the translocation of the catalytic (C) domain into the cytoplasm. This event is mediated by the translocation (T) domain of the toxin. Previous work suggested that the T domain acts as a chaperone for the C domain during membrane penetration of the toxin. Using partitioning experiments with lipid vesicles, fluorescence spectroscopy, and a lipid vesicle leakage assay, we characterized the dominant behavior of the T domain over the C domain during the successive steps by which these domains interact with a membrane upon acidification: partial unfolding in solution and during membrane binding, and then structural rearrangement during penetration into the membrane. To this end, we compared, for each domain, isolated or linked together in a CT protein (the toxin lacking the receptor-binding domain), each of these steps. The behavior of the T domain is marginally modified by the presence or absence of the C domain, whereas that of the C domain is greatly affected by the presence of the T domain . All of the steps leading to membrane penetration of the C domain are triggered at higher pH by the T domain , by 0.5-1.6 pH units. The T domain stabilizes the partially folded states of the C domain corresponding to each step of the process. The results unambiguously demonstrate that the T domain acts as a specialized pH-dependent chaperone for the C domain. Interestingly, this chaperone activity acts on very different states of the protein: in solution, membrane-bound, and membrane-inserted.     

Single mutation in the A domain of diphtheria toxin results in a protein with altered membrane insertion behavior

The insertion of the A domain of diphtheria toxin into model membranes has been shown to be both pH- and temperature-dependent (Hu and Holmes (1984) J. Biol. Chem. 259, 12226-12233). In this report, the insertion behavior of two mutant proteins of diphtheria toxin, CRM197 and CRM9, was studied and compared to that of wild-type toxin. Results indicated that both CRM197 and CRM9 resembled toxin with respect to the pH-dependence of binding to negatively-charged liposomes at room temperature. However, CRM197 differed from toxin with respect to both the pH- and temperature-dependence of fragment A insertion; fragment A197 inserts more readily into the bilayer at 0 degrees C and low pH or at neutral pH and room temperature than does wild type fragment A under these same conditions. This result indicates that the single amino acid substitution in the A domain of CRM197 facilitates entry of fragment A197 into the membrane, suggesting that CRM197 may be conformationally distinct from native toxin. In fact, the fluorescence spectra of CRM197 and wild-type toxin as well as their respective tryptic peptide patterns indicate that, at pH 7, CRM197 more closely resembles the acid form of wild-type toxin than the native form of toxin. These data suggest that CRM197 may be naturally in a more 'insertion-competent' conformation. In contrast, the mutation in the B domain of CRM9 which results in a 1000-fold decrease in binding affinity for plasma membrane receptors apparently does not cause a change in either the insertion of fragment A9 or the lipid-binding properties of CRM9 relative to toxin.     

Modification of the cell surface with neuraminidase increases the sensitivities of cells to diphtheria toxin and Pseudomonas aeruginosa exotoxin.

When cell lines that are susceptible to diphtheria toxin, such as human FL cells, were treated with C. perfringens neuraminidase their sensitivities to the toxin were increased. The sensitivities of the cells to the toxin were also increased by treatment with neuraminidase from Arthrobacter ureafaciens or HVJ (Sendai virus). Neuraminidase did not have this effect on a toxin-resistant cell line. It also did not increase the cytotoxic effect of a large concentration of fragment A of diphtheria toxin, which lacks the moiety of the toxin molecule that binds to the cell membrane. Neuraminidase from C. perfringens or HVJ also increased the sensitivity of cells to ricin toxin. Furthermore, neuraminidase from C. perfringens or A. ureafaciens increased the sensitivity of cells to Pseudomonas aeruginosa exotoxin (PA toxin), but in this case neuraminidase from HVJ did not have a similar effect.