Diphtheria toxin entry into cells is facilitated by low pH.

Sandvig, Kirsten; Olsnes, Sjur

doi:10.1083/jcb.87.3.828

Cited by 454 publications

(273 citation statements)

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“…Whether or not the phenomenon by itself is involved in the translocation process has not been elucidated. Diphtheria toxin, which requires an acidic environment for internalization [14,15], was found to induce fusion of DPPC vesicles only at acidic pH [ 161. Recently, from several experimental results [5,7,14], it' was proposed that the acidic environment might not be required for ricin internalization.…”

Section: Effect Of the A-chain On The Membrane Mixing Of Dmpc And Dppmentioning

confidence: 99%

Ricin A‐chain induces fusion of small unilamellar vesicles at neutral pH

1989

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The interaction of ricin and its constituent polypeptides, the A-and B-chain, with small unilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC) or dimyristoylphosphatidylcholine (DMPC) was investigated by means of differential scanning calorimetry measurements. The A-chain, at neutral pH, entirely shifted the endothermic peak of small unilamellar vesicles of DPPC from 37°C to 41°C at low protein/lipid ratios. The potency of either ricin or the B-chain to induce the shift of endothermic peak was much less than that of the A-chain. The A-chain was also found to cause mixing of endothermic peaks of DMPC vesicles and DPPC vesicles. These data strongly suggest that the A-chain has the ability to induce fusion of phospholipid vesicles.

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Section: Effect Of the A-chain On The Membrane Mixing Of Dmpc And Dppmentioning

confidence: 99%

Ricin A‐chain induces fusion of small unilamellar vesicles at neutral pH

1989

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“…The DT receptor, a 185-residue integral membrane protein, has been cloned from monkey kidney cells (Naglich et al, 1992) and has 97% sequence identity with human heparinbinding epidermal growth factor precursor (Higashiyama et al, 1992). Second, DT is triggered by low pH to undergo a conformational change and insert into the endosomal membrane, an event that has been unraveled by mimicking the process at the plasma membrane by exposing cells to low pH (Sandvig & Olsnes, 1980). In vitro, DT undergoes a cooperative conformational transition at pH 5.0, characterized by increased hydrophobicity, exposure of buried tryptophan residues, and altered susceptibility to proteases (Blewitt et al, 1985;Dumont & Richards, 1988).…”

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Refined structure of dimeric diphtheria toxin at 2.0 Å resolution

1994

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The refined structure of dimeric diphtheria toxin (DT) at 2.0 A resolution, based on 37,727 unique reflections ( F > l o ( F ) ) , yields a final R factor of 19.5% with a model obeying standard geometry. The refined model consists of 523 amino acid residues, 1 molecule of the bound dinucleotide inhibitor adenylyl 3'-5' uridine 3' monophosphate (ApUp), and 405 well-ordered water molecules. The 2.0-A refined model reveals that the binding motif for ApUp includes residues in the catalytic and receptor-binding domains and is different from the Rossmann dinucleotide-binding fold. ApUp is bound in part by a long loop (residues 34-52) that crosses the active site. Several residues in the active site were previously identified as NAD-binding residues. Glu 148, previously identified as playing a catalytic role in ADP-ribosylation of elongation factor 2 by DT, is about 5 A from uracil in ApUp.The trigger for insertion of the transmembrane domain of DT into the endosomal membrane at low pH may involve 3 intradomain and 4 interdomain salt bridges that will be weakened at low pH by protonation of their acidic residues. The refined model also reveals that each molecule in dimeric DT has an "open" structure unlike most globular proteins, which we call an open monomer. Two open monomers interact by "domain swapping" to form a compact, globular dimeric DT structure. The possibility that the open monomer resembles a membrane insertion intermediate is discussed.Keywords: ADP-ribosyltransferase; diphtheria; membrane insertion Diphtheria toxin (DT) is secreted from toxic strains of the bacterium Corynebacterium diphtheriae. Toxigenic conversion of C. diphtheriae occurs by lysogenization with corynephage 0 (Freeman, 1951), which carries the DT gene encoding a 535-residue protein (M, 58,342) (Uchida et al., 1971; Greenfield et al., 1983). DT kills eukaryotic cells by inactivating an essential component of the protein translation machinery, elongation factor 2 (EF-2) (Collier, 1975).DT is produced as a protomer of 2 fragments connected by a loop containing a proteolysis site and a disulfide bond. Limited proteolysis with trypsin and reduction of the disulfide separates the 2 fragments, which are denoted fragment A and fragment B (Drazin et al., 1971). Fragment A consists of an independent folding domain, the catalytic (C) domain (residues 1-190) (Choe et al., 1992). Fragment B consists of 2 folding domains, the transmembrane (T) domain (residues 191-378) and receptor-binding (R) domain (residues 379-535) (Choe et al., 1992).

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“…The toxins bound to cell surface are then internalized by endocytosis (33), and the toxins, or at least their A fragments, enter the cytoplasm to exert its effects. Like in the case of Semliki Forest virus (SFV) (11), intravesicular low pH is required for the penetration of the toxin into cytoplasm (6,17,20,27,36).Some information has been obtained on the biochemical properties of DT receptor. The treatment of DT-sensitive cells with some proteases or phospholipase C reduces the sensitivity to DT (32), whereas treatment with neuraminidase increases the sensitivity (26).…”

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

Mekada

Okada

Uchida

1988

The Journal of Cell Biology

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Abstract. 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 Veto 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 ~25I-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.D PHTHERIA toxin (DT) ~ is a cytotoxic protein which inhibits cellular protein synthesis (4, 40) in eukaryotes by catalyzing the ADP-ribosylation of EF-2, which results in its inactivation (9, 12). The first step of intoxication by DT is binding of the toxin to a susceptible cell. A specific receptor for DT is believed to be involved in this step (13,43). Cells from a number of mammals including humans and monkeys are sensitive to DT, but mouse and rat cells are insensitive (29). Several lines of evidence show that the difference in sensitivity to DT between species is primarily determined by the presence or absence of a cell surface receptor (18,28,31,48). However, this receptor has not been isolated. The toxins bound to cell surface are then internalized by endocytosis (33), and the toxins, or at least their A fragments, enter the cytoplasm to exert its effects. Like in the case of Semliki Forest virus (SFV) (11), intravesicular low pH is required for the penetration of the toxin into cytoplasm (6,17,20,27,36).Some information has been obtained on the biochemical properties of DT receptor. The treatment of DT-sensitive cells with some proteases or phospholipase C reduces the sensitivity to DT (32), whereas treatment with neuraminidase increases the sensitivity (26). Although these studies 1. Abbreviations used in this paper: CRMs, cross reacting materials related to diphtheria toxin; DT, diphtheria toxin.give a clue to the chemical nature of DT receptor, the possibility that ...

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Diphtheria toxin entry into cells is facilitated by low pH.

Cited by 454 publications

References 20 publications

Ricin A‐chain induces fusion of small unilamellar vesicles at neutral pH

Ricin A‐chain induces fusion of small unilamellar vesicles at neutral pH

Refined structure of dimeric diphtheria toxin at 2.0 Å resolution

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

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