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PubMed1998

Toxin binding site of the diphtheria toxin receptor: loss and gain of diphtheria toxin binding of monkey and mouse heparin-binding, epidermal growth factor-like growth factor precursors by reciprocal site-directed mutagenesis.

The transmembrane precursor of the monkey (Mk) heparin-binding, epidermal growth factor-like growth factor (proHB-EGF) functions as a diphtheria toxin (DT) receptor, whereas the mouse (Ms) precursor does not. Previously, using chimeric Ms/Mk precursors, we have shown that DT resistance of cells bearing Ms proHB-EGF may be accounted for by several amino acid substitutions between residues 122 and 148 within the EGF-like domain and that Glu-141 is an important amino acid residue for DT binding. In this study, reciprocal site-directed mutagenesis was performed on the major non-conserved residues in the region of 122-148, alone or in combination, between Mk and Ms precursors to identify more precisely which amino acid residues are important for DT binding. Two approaches were used. The first, more traditional approach was to destroy DT sensitivity and binding of Mk proHB-EGF by substitution(s) with the corresponding Ms residue(s). From the single mutations, the greatest loss of DT sensitivity was observed with Mk/Glu-141His (approximately 4000-fold) and the next greatest with Mk/Ile-133Lys (approximately fourfold). The double mutations Mk/Leu-127Phe/Glu-141His, Mk/Ile-133Lys/Glu-141His and Mk/His-135Leu/Glu-141His resulted in complete toxin resistance (> 100000-fold). The second approach, both novel and complementary, was to gain DT binding and sensitivity of Ms proHB-EGF by substitution(s) with the corresponding Mk residue(s). Surprisingly, the single mutation Ms/His-141Glu resulted in the gain of moderate DT sensitivity (> 260-fold). The double mutation Ms/Lys-133Ile/His-141Glu and the triple mutation Ms/Lys-133Ile/Leu-135His/His-141Glu resulted in a progressive gain in toxin sensitivity (> 4700-fold and >16000-fold respectively) and affinity. This triple mutant cell line is essentially as sensitive (IC50 = 3.1 ng ml(-1)) as the highly toxin-sensitive monkey Vero cell line (IC50 = 4 ng ml(-1)), indicating that these three Mk residues enable the Ms proHB-EGF to act as a fully functional DT receptor. Taken together, these results indicate that Glu-141 plays the most critical role in DT binding and sensitivity and that two additional amino acid residues, Ile-133 and His-135, also play significant roles.

Amino Acid Sequence↗
PubMed1993

Hypersensitivity to diphtheria toxin by mouse cells expressing both diphtheria toxin receptor and CD9 antigen.

DTS-II is a highly diphtheria toxin (DT)-sensitive cell line previously isolated by transfection of wild-type DT-resistant mouse L-M(TK-) cells with the cDNA encoding a monkey Vero cell DT receptor. DTS-II cells are as toxin-sensitive as Vero cells, have approximately 3-fold more receptors than Vero cells, and have approximately 10-fold lower affinity for DT than Vero cells. We now cotransfected DTS-II cells with a plasmid containing the Vero cell cDNA coding for CD9 antigen (pCD9) and with a plasmid containing the gene for hygromycin resistance (pHyg). The stably transfected hygromycin-resistant colonies were screened for DT hypersensitivity employing a replica plate system. A DT-hypersensitive colony was isolated and purified. The purified DT-hypersensitive cells, DTS-III, (i) are approximately 10-fold more toxin-sensitive than DTS-II and Vero cells and (ii) bear approximately 10(6) DT receptors per cell (i.e., approximately 20-fold and approximately 60-fold more receptors than DTS-II and Vero cells, respectively), but their receptor affinity is still approximately 10-fold lower than that of Vero cells. Cross-linking experiments employing 125I-labeled DT demonstrated that DTS-II and DTS-III cells have essentially the same profile of DT-binding cell-surface protein(s), suggesting that CD9 antigen, although expressed on the cell surface of DTS-III cells, may not be in close proximity to the DT-binding domain of the receptor. CD9 may affect DT receptor expression by increasing receptor density at the cell surface. By employing DTS-III cells it should be possible to purify and characterize the DT cell-surface receptor protein(s).

Animals↗
PubMed1998

Diphtheria toxin translocation across endosome membranes. A novel cell permeabilization assay reveals new diphtheria toxin fragments in endocytic vesicles.

By using cells overexpressing diphtheria toxin (DT) receptor and a novel method of permeabilizing the plasma membrane with a bacterial pore-forming toxin, specific translocation of fragment A to the cytosol was observed, whereas full-size DT and other minor species of DT-derived fragments were left in intracellular vesicles. The translocation competence of DT proteins with mutations in the transmembrane domain is consistent with their cytotoxicities. The charge-reversal mutants E349K and D352K do not translocate their fragment A to the cytosol, whereas D352N is partially competent. ADP-ribosyltransferase activity of fragment A is not required for translocation. Novel fragments of DT with apparent molecular masses of 28 and 35 kDa were detected in endocytic vesicles. The 28-kDa fragment consists of fragment A and an N-terminal piece of fragment B, whereas the 35-kDa fragment contains part of fragment B and may be complementary to the 28-kDa fragment. Time course studies show that the 28-kDa fragment appears in endocytic vesicles prior to translocation of fragment A to the cytosol, raising the possibility that the 28-kDa fragment is an intermediate in translocation. We present a model for translocation of fragment A that incorporates the observations made using the novel permeabilization method.

Animals↗
PubMed2000

Cell surface monkey CD9 antigen is a coreceptor that increases diphtheria toxin sensitivity and diphtheria toxin receptor affinity.

Monkey (Mk) CD9 antigen has been shown previously to increase the diphtheria toxin (DT) sensitivity of cells when co-expressed with Mk proHB-EGF (DT receptor). We have elucidated here the mechanism whereby Mk CD9 influences Mk proHB-EGF and present evidence that Mk CD9 is a coreceptor for DT. We observed that Mk CD9 not only increased the DT sensitivity but also increased the DT receptor affinity of cells. Furthermore, the higher the Mk CD9/Mk proHB-EGF ratio, the higher the affinity. In contrast, mouse (Ms) CD9 did not increase the toxin sensitivity or receptor affinity of cells when co-expressed with Mk proHB-EGF. Using Mk/Ms chimeric CD9 molecules, we determined that the second extracellular domain of Mk CD9 is responsible for both increased sensitivity and receptor affinity. This domain of Mk CD9 also interacts with Mk proHB-EGF in a yeast two-hybrid system. Our findings thus suggest that Mk CD9 has a direct physical interaction with Mk proHB-EGF to form a DT receptor complex and that this contact may change the conformation of the receptor to increase DT binding affinity and consequently increase toxin sensitivity. We thus propose that Mk CD9 is a coreceptor for DT.

Amino Acid Sequence↗
PubMed1988

Identification of diphtheria toxin receptor and a nonproteinous diphtheria toxin-binding molecule in Vero cell membrane.

Two substances possessing the ability to bind to diphtheria toxin (DT) were found to be present in a membrane fraction from DT-sensitive Vero cells. One of these substances was found on the basis of its ability to bind DT and inhibit its cytotoxic effect. This inhibitory substance competitively inhibited the binding of DT to Vero cells. However this inhibitor could not bind to CRM197, the product of a missense mutation in the DT gene, and did not inhibit the binding of CRM197 to Vero cells. Moreover, similar levels of the inhibitory activity were observed in membrane fractions from DT-insensitive mouse cells, suggesting the inhibitor is not the DT receptor which is specifically present in DT-sensitive cells. The second DT-binding substance was found in the same Vero cell membrane preparation by assaying the binding of 125I-labeled CRM197. Such DT-binding activity could not be observed in membrane preparation from mouse L cells. From competition studies using labeled DT and CRM proteins, we conclude that this binding activity is due to the surface receptor for DT. Treatment of these substances with several enzymes revealed that the inhibitor was sensitive to certain RNases but resistant to proteases, whereas the DT receptor was resistant to RNase but sensitive to proteases. The receptor was solubilized and partially purified by chromatography on CM-Sepharose column. Immunoprecipitation and Western blotting analysis of the partially purified receptor revealed that a 14.5-kD protein is the DT receptor, or at least a component of it.

Animals↗
PubMed1992

The 27-kD diphtheria toxin receptor-associated protein (DRAP27) from vero cells is the monkey homologue of human CD9 antigen: expression of DRAP27 elevates the number of diphtheria toxin receptors on toxin-sensitive cells.

Diphtheria toxin (DT) receptor associates with a 27-kD membrane protein (DRAP27) in monkey Vero cells. A cDNA encoding DRAP27 was isolated, and its nucleotide sequence was determined. The deduced amino acid sequence revealed that DRAP27 is the monkey homologue of human CD9 antigen. DRAP27 is recognized by CD9 antibodies. A human-mouse hybrid cell line (3279-10) possessing human chromosome 5, sensitive to DT, but not expressing CD9 antigen, was used for transfection experiments with DRAP27. When the cloned cDNA encoding DRAP27 was transiently expressed in 3279-10 cells, the total DT binding capacity was three to four times higher than that of untransfected controls. Transfectants stably expressing DRAP27 have an increased number of DT binding sites on the cell surface. Furthermore, the transfectants are 3-25 times more sensitive to DT than untransfected cells, and the sensitivity of these cells to DT is correlated with the number of DRAP27 molecules on the surface. However, when the cloned cDNA was introduced into mouse cell lines that do not express DT receptors, neither an increased DT binding nor enhancement of DT sensitivity was observed. Hence, we conclude that DRAP27 itself does not bind DT, but serves to increase DT binding and consequently enhances DT sensitivity of cells that have DT receptors. 12 proteins related to DRAP27/CD9 antigen were found through homology search analysis. These proteins appear to belong to a new family of transmembrane proteins.

Amino Acid Sequence↗
PubMed2003

Protection against group A streptococcus by immunization with J8-diphtheria toxoid: contribution of J8- and diphtheria toxoid-specific antibodies to protection.

A conformationally constrained, minimally conserved peptide from the M protein of group A streptococcus (GAS) has been defined. It consists of 12 amino acids from the C-repeat region within a non-M protein helix-forming sequence and is referred to as "J8." Here, we investigate the immunogenicity of a J8-diphtheria toxoid (DT) conjugate adjuvanted with the human-compatible adjuvants, SBAS2 and alum, and demonstrate that it is capable of inducing opsonic antibodies and can protect outbred mice from virulent challenge. In a range of experiments, protection correlated with the titer of J8-specific antibodies and not with the induction of J8-specific T cells. However, DT-specific antibodies (as well as J8-specific antibodies) were shown to stain the surface of fixed GAS and to be capable of opsonizing live organisms. DT may be an ideal carrier protein for J8 and other GAS peptides for GAS vaccines.

Adjuvants, Immunologic↗
PubMed1991

Diphtheria toxin receptor-binding domain substitution with interleukin 6: genetic construction and interleukin 6 receptor-specific action of a diphtheria toxin-related interleukin 6 fusion protein.

We have genetically replaced that portion of the diphtheria toxin structural gene which encodes the native receptor-binding domain with a synthetic gene encoding the cytokine interleukin 6 (IL-6/IFN-beta 2/BSF-2). The resulting gene fusion encodes the chimeric toxin DAB389-IL-6. Following expression and purification, we demonstrate that DAB389-IL-6 is selectively cytotoxic for eukaryotic cells bearing the interleukin 6 receptor. In addition, the cytotoxic action of DAB389-IL-6 is shown to require binding to the IL-6 receptor, internalization by receptor-mediated endocytosis and passage through an acidic compartment. Following the delivery of the catalytically active fragment A to the cytosol of target cells, cellular protein synthesis is inhibited by the ADP-ribosylation of elongation factor 2. While eukaryotic cells which are devoid of the IL-6 receptor are uniformly resistant to the action of this fusion toxin, the data presented suggest that a minimal number of IL-6 receptors may be necessary to mediate the internalization of sufficient levels of DAB389-IL-6 to result in the intoxication of target cells.

Animals↗
PubMed1997

Characterization of lipoprotein IRP1 from Corynebacterium diphtheriae, which is regulated by the diphtheria toxin repressor (DtxR) and iron.

The Corynebacterium diphtheriae irp1 gene is negatively regulated by DtxR and iron. The nucleotide sequence of irp1 revealed that it has homology with genes involved in iron acquisition. Expression of the irp1 gene showed that it encodes a lipoprotein (IRP1) with a predicted size of 38 kDa. Northern blot experiments indicated that transcription from the irp1 promoter is repressed in high-iron medium and suggested that irp1 is part of an iron-regulated operon.

Amino Acid Sequence↗