ROLE OF THE DISULFIDE BOND IN SHIGA TOXIN A-CHAIN FOR TOXIN ENTRY INTO CELLS

Shiga toxin consists of an enzymatically active A-chain and a pentamer ic binding subunit. The A-chain has a trypsin-sensitive region, and up on cleavage two disulfide bonded fragments, A(1) and A(2), are generat ed. To study the role of the disulfide bond, it was eliminated by muta ting cysteine 242 to serine. In T47D cells this mutated toxin was more toxic than wild type toxin after a short incubation, whereas after lo nger incubation times wild type toxin was most toxic. Cells cleaved no t only wild type but also mutated A-chain into A(1) and A(2) fragments . The mutated A chain was more sensitive than wild type toxin to Prona se, and it was degraded at a higher rate in T47D cells. Subcellular fr actionation demonstrated transport of both wild type and mutated toxin to the Gels apparatus. Brefeldin A, which disrupts the Golgi apparatu s, protected not only against Shiga toxin but also against the mutated toxin, indicating involvement of the Golgi apparatus. After prebindin g of Shiga(C242S) toxin to wells coated with the Shiga toxin receptor, Gb(3), trypsin treatment induced dissociation of A(1) from the toxin- receptor complex demonstrating that in addition to stabilizing the A c hain, the disulfide bond prevents dissociation of the A(1) fragment fr om the toxin-receptor complex.