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.

Hamster diphtheria toxin receptor: a naturally occurring chimera of monkey and mouse HB-EGF precursors.

The sensitivity of mammalian cell lines to diphtheria toxin (DT) varies between species. Monkey (Mk) Vero cells are highly sensitive to DT, whereas rat and mouse (Ms) cells are resistant; hamster (Hm) cells display moderate DT sensitivity. The precursor of the Mk heparin-binding epidermal growth factor-like growth factor (proHB-EGF) functions as a DT receptor but the Ms proHB-EGF does not. In this study we have cloned, expressed, and characterized the Hm proHB-EGF/DT receptor. The expression of Hm proHB-EGF confers moderate DT sensitivity to normally DT-resistant mouse cells. The amino acid sequence of Hm preproHB-EGF shows that, overall, it more closely resembles the Ms preproHB-EGF sequence, except in the DT-binding region where it more closely resembles the Mk sequence. In the DT-binding region the Hm proHB-EGF sequence differs from the Mk proHB-EGF in only four amino acid residues (124, 126, 133, and 147); one of these residues, Ile133 in Mk proHB-EGF, has been previously reported to be important for DT binding and sensitivity. Analysis of Mk proHB-EGF mutants with residues substituted for Ile133 suggests that Asn133 in Hm proHB-EGF may be responsible for the moderate DT sensitivity of Hm proHB-EGF-expressing cells.

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.

Diphtheria toxin:receptor interaction: association, dissociation, and effect of pH.

Diphtheria toxin (DT) binds to a specific heparin-binding epidermal growth factor-like growth factor (HB-EGF) precursor that is expressed in DT-sensitive cells. DT binds to the cell-surface HB-EGF precursor with an apparent dissociation constant (KD) of approximately 1 x 10(-8) - 10(-9) M at 4 degreesC, a temperature at which toxin binds but is not internalized. The interaction of DT with the cell-surface receptor, however, may be influenced by other cell-surface components. We used a biosensor method to measure the binding of DT to immobilized recombinant human HB-EGF (hHB-EGF) at 25 degreesC with no other cellular components present. We observed that at pH 7.4, using this in vitro two component system, DT binds to hHB-EGF with an apparent KD of 2.7 x 10(-8) M. We also observed that the dissociation of DT from hHB-EGF at pH values that approach those of the endosome occurs at a faster rate as the pH is decreased. These results suggest that the low pH of the endosome is sufficient to allow DT to dissociate from the HB-EGF precursor, prior to the translocation of the enzymatically active fragment of DT into the cytosol.

Glutamic acid 141 of the diphtheria toxin receptor (HB-EGF precursor) is critical for toxin binding and toxin sensitivity.

The transmembrane precursor of the monkey heparin-binding EGF-like growth factor also functions as a diphtheria toxin receptor. The mouse precursor does not bind the toxin. Previously, the most important region for binding the toxin in the monkey precursor was narrowed down to residues 122-148 through the expression of chimeric mouse/monkey precursors and subsequent toxin-sensitivity assays. To define further the toxin binding domain of the monkey precursor, distinct monkey/mouse chimeric precursors were expressed and assayed. The region between monkey residues 136-148 was found to be absolutely necessary for the retention of toxin sensitivity. Within this region, the monkey and mouse precursors differ in only two residues (residues 141 and 147). A toxin-insensitive monkey/mouse chimera that contained monkey residues 1-136 was converted to a toxin-sensitive chimera by the mutation of a single residue (His141 to Glu141). Expression of a mutant monkey precursor in which a single monkey residue (Glu141) was converted to the mouse residue (His141) yielded a cell line that was approximately 100-fold less sensitive to the toxin and the mutant precursor bound the toxin approximately 12-fold less tightly than the wild-type monkey precursor. Taken together, these results indicate that Glu 141 plays a critical role in toxin binding and toxin sensitivity.

Expression of fragment C of tetanus toxin fused to a carboxyl-terminal fragment of diphtheria toxin in Salmonella typhi CVD 908 vaccine strain.

We report the expression of fragment C of tetanus toxin (FC) fused to the eukaryotic cell binding domain (the carboxyl-terminus) of diphtheria toxin (FC-bDt fusion) in attenuated Salmonella typhi live vector vaccine strain CVD 908. The FC-bDt protein fusion was constructed using plasmid pTETnir15 which carries the gene encoding FC under control of the nirB promoter (nirBP). The open reading frame for FC was modified to incorporate an in-frame glycine-proline hinge region and a set of four restriction sites at the 3' end of the FC gene. A 482 bp DNA fragment encoding the eukaryotic cell binding domain of diphtheria toxin was then inserted at the 3' end of the modified FC gene to create an in-frame FC-bDt fusion gene. The resulting plasmid, pOG215, was able to express the FC-bDt fusion protein in both Escherichia coli DH5a and S. typhi CVD 908, as evidenced by Western immunoblots using anti-FC and anti-C-terminal diphtheria toxin monoclonal antibodies. Maximum expression of the FC-bDt fusion protein was achieved by growing CVD 908(pOG215) at the low oxidation-reduction potential of thioglycollate broth, i.e. in conditions that activate nirBP and drive transcription of the FC-bDt fusion gene. Whereas maximum expression of FC alone was also observed using thioglycollate broth, expression of bDt alone was unsuccessful using a variety of growth conditions. FC fusions constitute one strategy to "rescue" expression of proteins which are otherwise difficult to express.

The effect of receptor rapid-internalization signals on diphtheria toxin endocytosis and cell sensitivity.

Diphtheria toxin enters toxin-sensitive mammalian cells by receptor-mediated endocytosis employing the heparin-binding EGF-like growth factor precursor as its receptor. We reported previously (Almond and Eidels, 1994) that cytoplasmic domain mutants of the toxin receptor and cells expressing wild-type receptor internalize toxin slowly, the rate being approximately that of normal turnover of the plasma membrane. To determine whether it was possible to increase toxin sensitivity by increasing the rate of toxin internalization, we constructed diphtheria toxin cytoplasmic domain mutant cell lines containing rapid-internalization signals from either the low density lipoprotein receptor or from the lysosomal acid phosphatase precursor. Although cells transfected with mutant receptor genes internalized toxin at a faster rate than those expressing the wild-type receptor, they showed a decrease in toxin sensitivity. This decreased sensitivity may be accounted for by an observed decrease in the number of toxin-binding sites and by an increased rate of toxin internalization and degradation. These results suggest that the rate of toxin internalization may not be the rate-limiting step in the cytotoxic process.

Localization of a critical diphtheria toxin-binding domain to the C-terminus of the mature heparin-binding EGF-like growth factor region of the diphtheria toxin receptor.

The monkey and human heparin-binding EGF-like growth factor precursors are diphtheria toxin receptors. To investigate which portion of this growth factor precursor is responsible for binding the toxin, the monkey precursor was compared to the mouse precursor which does not bind the toxin. Three clusters of differing residues were identified. The human mature growth factor (residues 63-148), which contains two of three differing clusters, inhibited the binding of radiolabeled toxin to cell-surface receptors, thus narrowing the binding domain to 86 of the 208 residue precursor. To define further the binding domain, chimeric mouse/monkey precursors were expressed and assayed for toxin sensitivity. The third cluster, residues 122-135, was found to be the most important region for toxin binding. Total replacement of monkey residues with mouse residues N-terminal to this region yields a highly toxin-sensitive cell. Collectively, the results suggest that the most critical residues for toxin binding lie between residues 122-148.

The cytoplasmic domain of the diphtheria toxin receptor (HB-EGF precursor) is not required for receptor-mediated endocytosis.

Diphtheria toxin is believed to enter toxin-sensitive mammalian cells by receptor-mediated endocytosis employing the transmembrane cell surface precursor of heparin-binding epidermal growth factor-like growth factor as a receptor. To investigate the contribution of the receptor's cytoplasmic domain to the toxin internalization process, we constructed stable cell lines that express receptor molecules containing cytoplasmic domain mutations. Our results indicate that Tyr192 and surrounding amino acid residues are important for high toxin sensitivity. Cells expressing mutant receptors are less sensitive to toxin and have fewer toxin-specific binding sites but internalize toxin at rates similar to those of cells expressing the intact receptor. This rate of internalization is much slower (1-2%/min) than that of classical endocytic receptors (10-50%/min). Our results are consistent with a model in which the cytoplasmic domain of the toxin receptor lacks a signal for rapid internalization. We suggest that toxin-receptor complexes, nevertheless, are internalized by receptor-mediated endocytosis by entrapment in clathrin-coated pits as part of bulk phase turnover of cell surface proteins. Although the rate is slow, successful intoxication occurs because a single internalized enzymatically-active toxin molecule is sufficient to inhibit protein synthesis in the cell.

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).

Structure-function analyses of diphtheria toxin by use of monoclonal antibodies.

A large panel of hybridomas, secreting monoclonal antibodies (MAbs) specific for diphtheria toxin (DT) and prepared by immunization with either intact DT or its A or B fragment (DTA or DTB), have been isolated and characterized. The 213 MAbs were initially screened for reactivity to DT by enzyme-linked immunosorbent assay analyses and then were classified for their reactivity with DT, DTB, or DTA by solid-phase Western blot (immunoblot) analyses; 129 DTB-specific, 51 DTA-specific, and 33 non-fragment-assignable MAbs were obtained. Of the DTB MAbs, 118 recognize epitopes between residues 194 and 453, 10 recognize epitopes between residues 454 and 481, and 1 recognizes an epitope present in denatured toxin but not present in native DT located within the carboxyl-terminal receptor-binding region of DT (residues 482 to 535). Those MAbs that were the most protective in a cytotoxicity assay recognized native toxin in solution and inhibited binding of radiolabeled toxin to Vero cells to the greatest extent. A number of MAbs were able to detect epitopes that became more or less accessible when the toxin was preincubated at acidic (endosomal-mimicking) pH, suggesting that the epitopes they recognize may be important in the low-pH-induced insertion and/or translocation of DT across the endosomal membrane.

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 454-481). 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.

Expression cloning of a diphtheria toxin receptor: identity with a heparin-binding EGF-like growth factor precursor.

A monkey cDNA (pDTS) encoding a diphtheria toxin (DT) sensitivity determinant was isolated by expression cloning in mouse L-M cells. Mouse cells are naturally resistant to DT, because they lack functional cell surface receptors for the toxin. Unlike wild-type L-M cells, pDTS-transfected mouse cells are extremely toxin sensitive and specifically bind radioiodinated DT. Intoxication of the transfected cells requires receptor-mediated endocytosis of the bound toxin. The cDNA is predicted to encode an integral membrane protein that is identical to the precursor of a heparin-binding EGF-like growth factor. The DT sensitivity protein is thus a growth factor precursor that DT exploits as a receptor.

Expression of functional diphtheria toxin receptors on highly toxin-sensitive mouse cells that specifically bind radioiodinated toxin.

Diphtheria toxin (DT), a bacterial protein exotoxin, inactivates mammalian cell elongation factor 2 after toxin internalization by receptor-mediated endocytosis. To isolate the DT receptor, we cotransfected DT-resistant wild-type mouse L-M cells with a cDNA library constructed from RNA of highly toxin-sensitive monkey Vero cells and with a neomycin-resistance gene. Stably transfected G418-resistant L-M colonies were screened for DT sensitivity in a replica plate assay. After screening of 8000 colonies, one DT-sensitive (DTS) colony was isolated. The purified DTS mouse cells are highly toxin-sensitive; they are at least 1000-fold more sensitive than wild-type L-M cells and only approximately 10-fold less sensitive than Vero cells. Incubation of the DTS mouse cells with CRM 197, a nontoxic form of DT that competitively inhibits the binding of native DT to the toxin receptor, protected them from DT-mediated toxicity. More important, these DTS mouse cells express receptors on their cell surface that bind radioiodinated DT in a specific fashion, a property hitherto readily demonstrable only with highly toxin-sensitive cells of monkey origin. Furthermore, HA6DT, a DT fragment comprising the Mr 6000 carboxyl-terminal receptor-binding domain, inhibited the binding of radioiodinated toxin to these DTS mouse cells to the same extent as unlabeled DT. With these DTS mouse cells as a source of monkey cDNA, it should be possible to clone the gene encoding the DT receptor.

Isolation of diphtheria toxin-sensitive mouse cells from a toxin-resistant population transfected with monkey DNA.

Diphtheria toxin (DTX)-sensitive mouse cells were isolated from a toxin-resistant thymidine kinase (TK)-negative L-M(TK-) mouse cell population that was transfected with DNA from highly toxin-sensitive monkey Vero cells. Sensitivity to DTX was screened by using a replica plate assay. The purified toxin-sensitive mouse cells were characterized with respect to their ability to bind, internalize, and translocate DTX into the cytosol. In contrast to the L-M(TK-) cells, these DTX-sensitive mouse cells were able to bind and internalize radioiodinated toxin into intracellular vesicles at 37 degrees C. Specific binding of radioiodinated toxin to their cell surface (at 4 degrees C) could not be demonstrated. However, the following evidence for functional receptors capable of binding DTX was obtained: (i) when the toxin-sensitive mouse cells were first allowed to bind DTX at 4 degrees C, followed by washing the cells and shifting the temperature to 37 degrees C (allowing cell surface-bound toxin to enter the cells), the cells were killed; (ii) when cells with surface-bound DTX were exposed briefly to an acidic medium (allowing the toxin to penetrate the plasma membrane directly), protein synthesis was inhibited; and (iii) when cells were incubated with DTX in the presence of the CRM 197, a nontoxic form of DTX with binding properties similar to native DTX, the cytotoxic effect of DTX was markedly decreased. The results demonstrate that the toxin-sensitive mouse cells are killed by a mechanism similar to that observed in naturally occurring toxin-sensitive cell lines. The data further suggest that the transfected mouse cells express functional receptors for DTX.

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.

Anti-idiotypic antibodies that protect cells against the action of diphtheria toxin.

An anti-idiotypic serum prepared against the combining site (idiotype) of specific anti-diphtheria toxoid antibodies was characterized with respect to its interaction with highly diphtheria toxin-sensitive Vero cells. Although the anti-idiotypic serum protected Vero cells against the cytotoxic action of diphtheria toxin, it did not prevent the binding of 125I-labeled diphtheria toxin to the cells but did inhibit the internalization and degradation of 125I-labeled toxin. This anti-idiotypic serum immunoprecipitated a cell-surface protein from radiolabeled Vero cells with an apparent Mr of approximately 15,000. These results are consistent with the hypothesis that the anti-idiotypic serum contains antibodies that carry an internal image of an internalization site on the toxin and that a cell-surface protein involved in toxin internalization possesses a complementary site recognized by both the toxin and the anti-idiotypic antibodies.

Specific cleavage of diphtheria toxin by human urokinase.

Diphtheria toxin must undergo a specific cleavage reaction and subsequent reduction to express the enzymatic ADP-ribosyltransferase activity that is responsible for its toxicity. In an effort to identify potential cellular enzymes that might be involved in this process we have found that a human urinary plasminogen activator, urokinase, is capable of specifically cleaving diphtheria toxin to yield an enzymatically active A fragment (more homogeneous than that produced by trypsin cleavage) and a B fragment (with an identical amino-terminal sequence to that produced by trypsin cleavage). The results raise the possibility that urokinase or urokinase-like enzymes play a role in diphtheria toxin-mediated intoxication.

Diphtheria toxin receptor. Identification of specific diphtheria toxin-binding proteins on the surface of Vero and BS-C-1 cells.

The biochemical characteristics of specific receptor molecules for diphtheria toxin on the surface of two toxin-sensitive cell lines (Vero and BS-C-1) were examined. Diphtheria toxin was found to bind to a number of different proteins in Nonidet P-40 solubilized extracts of 125I-labeled cells. In contrast, permitting diphtheria toxin to bind first to labeled intact cells, which were subsequently solubilized and subjected to immunoprecipitation with anti-diphtheria toxin, resulted in a far more restricted profile of diphtheria toxin-binding proteins that possessed Mrs in the range of 10,000-20,000. Direct chemical cross-linking of radioiodinated diphtheria toxin to cell surface proteins resulted in the appearance of several predominant bands possessing Mrs of approximately 80,000. The Mr approximately 80,000 complexes were shown to be composed of radiolabeled diphtheria toxin (Mr 60,000) and unlabeled Mr approximately 20,000 cellular proteins. These complexes were judged to be a result of specific binding in that their appearance could be preferentially inhibited by the addition of a 100-fold excess of unlabeled diphtheria toxin. The formation of the Mr approximately 80,000 complexes was sensitive to prior trypsin treatment of the cells and to known inhibitors of diphtheria toxin binding. Furthermore, prior incubation of the cells with diphtheria toxin at 37 degrees C ("down regulation") markedly and specifically reduced the subsequent formation of the Mr approximately 80,000 cross-linked complexes, and these down-regulated cells were less sensitive to diphtheria toxin in cytotoxicity assays. Further incubation of down-regulated cells at 37 degrees C restored their ability to form Mr approximately 80,000 complexes; this regeneration requires protein synthesis and restores the cells' sensitivity to diphtheria toxin-mediated cytotoxicity. These results strongly suggest that a Mr 10,000-20,000 cell surface protein is, or constitutes a portion of, the functional diphtheria toxin receptor.

Role of glycosylation in expression of functional diphtheria toxin receptors.

We have previously demonstrated, by using a detergent-solubilized system, the existence of specific diphtheria toxin-binding glycoproteins on the surface of toxin-sensitive cells. We have now tested the effect of tunicamycin treatment on the sensitivity of cells in culture to diphtheria toxin and have investigated the toxin sensitivity of mutant cells with known defects in glycosylation of asparagine-linked glycoproteins. Treatment of CHO-K1 cells with tunicamycin, which blocks the synthesis of both high-mannose-type and complex-type oligosaccharide chains of asparagine-linked glycoproteins, resulted in a 50- to 100-fold decrease in sensitivity to diphtheria toxin. In contrast, CHO-K1 mutants, defective in the synthesis of either high-mannose-type or complex-type oligosaccharides, showed no difference in toxin sensitivity compared with that of their parental cell lines. When we used an acid shock system, which is believed to result in receptor-dependent direct toxin penetration at the cell surface, the toxin sensitivity of tunicamycin-treated cells was not restored to that of untreated cells, suggesting that tunicamycin treatment results in a decrease in functional toxin receptors. Direct binding studies with 125I-labeled toxin demonstrated that this decrease in functional receptors is due to a decrease in the affinity of the receptors rather than to a change in the number of receptors. Taken together, these data are consistent with the interpretation that the diphtheria toxin receptor is a glycoprotein and suggest that the toxin binds neither to carbohydrate residues unique to the high-mannose-type oligosaccharides nor to those unique to the complex-type oligosaccharides. Furthermore, these data are consistent with the hypothesis that diphtheria toxin binds to the peptide backbone of the glycoprotein receptor.