Diphtheria toxin-binding glycoproteins on hamster cells: candidates for diphtheria toxin receptors

Proia, Richard L.; Eidels, Leon; Hart, David A.

doi:10.1128/iai.25.3.786-791.1979

Cited by 24 publications

(16 citation statements)

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“…42:1809, 1983; manuscript in preparation) have been identified and characterized. It has been suggested that these DT-binding cell surface glycoproteins are likely candidates for the DT receptor (27)(28)(29)(30). These glycoproteins have a high molecular weight (ca.…”

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“…These glycoproteins have a high molecular weight (ca. 150,000 and 70,000) and show anomalous behavior on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating the presence of a significant degree of carbohydrate (27). The binding of DT to these cell surface glycoproteins has been shown to be specific for the B fragment of the toxin (27,28,30).…”

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“…This binding is inhibited by nucleotides, polyphosphates, and polycations (28, 29), compounds which are known to protect cells by interfering with toxin-receptor interactions (8,22). The binding is saturable with respect to both toxin and receptor (27,30) and is reversible (L. L. Ross and L. Eidels, unpublished data).In this investigation we have attempted to define further the glycoprotein nature of the DT receptor and the possible role of carbohydrates in the toxin binding process. To this end we have investigated the toxin sensitivity of tunicamycintreated cells and the toxin sensitivity of CHO-Ki mutants with known alterations in glycoprotein biosynthesis.…”

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“…In our laboratory, specific DT-binding cell surface glycoproteins from guinea pig lymph node cells (30), hamster lymph node and thymus cells (27), and more recently from monkey kidney Vero cells and Chinese hamster ovary (CHO-Kl) cells (C. P. Robles, D. A. Hart, and L. Eidels, Fed. Proc.…”

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“…150,000 and 70,000) and show anomalous behavior on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating the presence of a significant degree of carbohydrate (27). The binding of DT to these cell surface glycoproteins has been shown to be specific for the B fragment of the toxin (27,28,30). This binding is inhibited by nucleotides, polyphosphates, and polycations (28,29), compounds which are known to protect cells by interfering with toxin-receptor interactions (8,22).…”

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Role of glycosylation in expression of functional diphtheria toxin receptors

Hranitzky¹,

Durham²,

Hart³

et al. 1985

Infect Immun

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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-Kl cells with tunicamycin, which blocks the synthesis of both high-mannose-type and complex-type oligosaccharide chains of asparagine-linked glycoproteins, resulted in a 50to 100-fold decrease in sensitivity to diphtheria toxin. In contrast, CHO-Kl 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.Diphtheria toxin (DT) is synthesized as a single polypeptide chain (molecular weight, 58,342) with two disulfide bridges and no free sulfhydryl groups. Limited proteolysis yields an amino-terminal fragment, the A fragment (molecular weight, 21,167), and a carboxy-terminal fragment, the B fragment (molecular weight, 37,195), which remain associated by an interchain disulfide bond (1, 2, 26). The A fragment inhibits protein synthesis through the NADdependent ADP ribosylation of elongation factor 2 present in the cytosol of eucaryotic cells (2, 26). The B fragment is responsible for interaction of the toxin with specific receptors on toxin-sensitive cells (23,47). It is widely held that the DT receptor possesses a protein component, since treatment of toxin-sensitive cells with such proteases as trypsin, pronase (21, 24), or bromelain (K. Hranitzky, D. A. Hart, and L. Eidels, Fed. Proc. 43:1956, 1984; manuscript in preparation) results in a significant decrease in sensitivity to the toxin. In contrast, treatment of toxin-sensitive cells with neuraminidase has been reported to result in a slight increase in sensitivity to DT (21,35), suggesting that carbohydrate structures that become exposed on removal of sialic...

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