Abstract
Shiga toxin consists of an enzymatically active A-chain and a pentameric binding subunit. The A-chain has a trypsin-sensitive region, and upon cleavage two disulfide bonded fragments, A1 and A2, are generated. To study the role of the disulfide bond, it was eliminated by mutating cysteine 242 to serine. In T47D cells this mutated toxin was more toxic than wild type toxin after a short incubation, whereas after longer incubation times wild type toxin was most toxic. Cells cleaved not only wild type but also mutated A-chain into A1 and A2 fragments. The mutated A-chain was more sensitive than wild type toxin to Pronase, and it was degraded at a higher rate in T47D cells. Subcellular fractionation demonstrated transport of both wild type and mutated toxin to the Golgi apparatus. Brefeldin A, which disrupts the Golgi apparatus, protected not only against Shiga toxin but also against the mutated toxin, indicating involvement of the Golgi apparatus. After prebinding of Shiga(C242S) toxin to wells coated with the Shiga toxin receptor, Gb3, trypsin treatment induced dissociation of A1 from the toxin-receptor complex demonstrating that in addition to stabilizing the A-chain, the disulfide bond prevents dissociation of the A1 fragment from the toxin-receptor complex.
Highlights
Shiga toxin is produced by Shigella dysenteriae type 1 and consists of an enzymatically active A-chain in noncovalent association with a pentamer of B-chains responsible for binding to Gb3, the receptor for Shiga toxin [1,2,3,4,5,6]
In the present article we demonstrate that the disulfide bond in the A-chain of Shiga toxin is not required for rapid intoxication of cells but seems to be important for toxicity after a long incubation time
To test whether the disulfide bond is required for intoxication of cells, we measured the ability of Shiga(C242S) toxin to intoxicate T47D cells, which are highly sensitive to Shiga toxin
Summary
Materials—Brefeldin A (BFA) was purchased from Epicentre Technologies (Madison, WI). Pronase, trypsin, Hepes, Gb3, N-ethylmaleimide, and PMSF were obtained from Sigma. In other experiments 125I-labeled toxin was prebound at 0 °C for 20 min, the cells were washed in Hepes medium, and the incubation was continued for the indicated period. The supernatant was transferred to an Eppendorf tube on ice containing a final concentration of 5 mM PMSF and sample buffer; the wells were placed on ice and washed once with Hepes medium, and sample buffer with 2-mercaptoethanol and 5 mM PMSF was added. Pronase Sensitivity of Wild Type Shiga Toxin and Shiga(C242S) Toxin—125I-Labeled toxin at a concentration of 800 ng/ml in a reaction volume of 25 l of Hepes medium was incubated with increasing concentration of Pronase (0, 1, 3, 10, 30, and 100 g/ml) for 10 min at room temperature. 5% trichloroacetic acid, dissolved in sample buffer containing 2-mercaptoethanol, and analyzed by SDS-PAGE and autoradiography
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