Cell-mediated reduction of the interfragment disulfide in nicked diphtheria toxin. A new system to study toxin entry at low pH.

J.O Moskaug,K Sandvig,S Olsnes

doi:10.1016/s0021-9258(18)61118-4

Abstract

When 125I-labeled nicked diphtheria toxin bound to Vero cells was exposed to pH less than 5.0, a small fraction was reduced to yield A- and B-fragments. The pH required for reduction correlates well with that required to induce intoxication, and the amount of A-fragment released was of the same order as that required to intoxicate the cells. Conditions that protect cells against intoxication, such as acidification of the cytosol, treatment with anion transport inhibitors, or treatment with anti-diphtheria toxin antibodies, prevented the reduction of the interfragment disulfide in cell-bound toxin. In vitro, thioredoxin reduced nicked diphtheria toxin only at pH 5.0 and lower, and the reduction was inhibited by anti-toxin antibodies. This indicates that a conformational change in the toxin, necessary for reduction by the thioredoxin system, is prevented by the antibodies. Reduction by glutathione and cysteine was most efficient at neutral pH and was not inhibited by anti-toxin. The results are consistent with the possibility that cell-mediated reduction of the interfragment disulfide is a measure of the entry of fragment A into the cytosol.

Highlights

From the Institute for Cancer Research ut the Norwegian Radium Hospitaland the Norwegian Cancer Society, Montebello, Oslo, Norway
Required for reduction correlates well with that required to induce intoxication, and the amount of Afragment released was of the same order as that required to intoxicate the cells. conditions that protect cells against intoxication, such as acidification of the cytosol, treatment with anion transport inhibitors, or treatment with anti-diphtheria toxin antibodies, prevented the reduction of the interfragment disulfide in cell-bound toxin
Thioredoxin reduced nicked diphtheria toxin only at pH 5.0 and lower, and the reduction was inhibited by anti-toxin antibodies

Summary

LOW pH*

When ‘251-labelednicked diphtheria toxin bound to nicked toxin can release its A-fragment into the cytosol [6]. The Na+/H+ ionophore monensinw, hich prevents the formation of a proton gradient across the endosomal membrane, blocks the entry of diphtheria toxin into the cytosol It is, possible to induce entry of toxinintomonensintreated cells by exposing cellswith surface-bound toxin tlow pH [12, 14]. In the present work we have exposed cells with surface-bound ‘251-labelednicked diphtheria toxin(’*‘I-nDT)’ to low pH and measured the extent of disulfide reduction. The cells were pH 7.2; lane 2, cells with prebound toxin were incubated at pH 4.5; incubated with Hepes medium, pH 7.2, containing 10 pM monensin and 1 mM NEM for 5 min a t room temperature. The same total amount of radioactivity was applied in each wellof the gel

RESULTS

Glutathione and cysteine may be the sulfhydryls involved

Findings

DISCUSSION