Investigations into the relationship between structure and function of diphtheria toxin.

Abstract

Studies on the structure-function relationship of diphtheria toxin are reported. New methods are described for the preparation of pure intact ("unnicked") molecule (or the A chain) to enter the cell (11). The complete amino acid sequence of the A chain has recently become available (12). A recent report by Iglewski and Rittenberg (13) indicated that protein synthesis in Ehrlich-Lettr6 ascites carcinoma cells is much more sensitive to the action of diphtheria toxin than that in normal mouse spleen cells, and that Ehrlich-Lettre tumors regressed in mice following an injection of the affected mice with diphtheria toxin. A difference in sensitivity to diphtheria toxin was also found in human breast carcinoma cells as compared to normal human cells. These observations suggest that diphtheria toxin may prove to be a useful anti-cancer agent, although more recent data indicate that the difference in sensitivity of tumor cells to toxin as compared to normal cells may not be as large as originally thought (14,15).It is therefore of considerable interest to us to investigate the mechanism of action of diphtheria toxin in greater detail. More specifically, we are trying to gain further information concerning the amino acid residues of the toxin that are essential for its activity, and to investigate the mechanism by which diphtheria toxin enters the cell. Some of the results obtained during this investigation are presented in this paper. MATERIALS AND METHODS Materials

Reconstitution of fully active diphtheria toxin from purified fragments A and B

“…The apparent discrepancy between our findings and [6] may well reflect the differences in the test systems employed. We feel that pre-incubation of cells with BSSOs affords less equivocal results than does incubation with a mixture of intact toxin and fragment B [5,6] as it demonstrates that interference with the toxic activity of diphtheria toxin requires the binding of fragment B to the cell surface and is The diphtheria toxin solution used in this experiment had an LD,, higher than that of fresh, native toxin ( fig.2) due to prolonged storage at 4°C.…”

“…The inhibition of protein synthesis observed [6] when HeI_a cells were treated for 3 h with diphtheria toxin, however, was unaffected by simultaneous treatment with BSSOs; from this it was deduced [6] that BSSQa does not bind to HeLa cells.…”

“…S-Sulphonated fragment B (BSSOa) was prepared from 50 mg diphtheria toxin as in [6], fractionated by Sephadex G-l 00 gel chromatography and shown to be pure by SDS-polyacrylamide gel electrophoresis [9]. The receptor binding activity of BSSOs was judged by its ability to bind to receptors on HeLa cells and so inhibit the subsequent binding and toxin effect of diphtheria toxin in the toxicity test system described below.…”

“…We report for the first time the purification of stable, biologically active diphtheria toxin fragment B by S-sulphonation [6] and its combination, by the ElsevierlNorth-Holland Biomedical Press asymmetric disulphide synthesis [8], with reduced fragment A to form a reconstituted toxin molecule possessing full activity.…”

Enhancement of cytotoxicities of ricin and Pseudomonas toxin in Chinese hamster ovary cells by nigericin.

“…Many of these toxins share common structural and functional features. They consist of two structural domains, A and B subunits which correspond to enzymatic and receptor-binding activities of the toxin molecules, respectively (1,3,12). Toxins may have similar enzymatic activities of their A subunits (or domains) but different B subunits (or domains) which are involved in the binding of toxins to the surface receptors of the susceptible cells.…”