Role of the Disulfide Bond in Shiga Toxin A-chain for Toxin Entry into Cells

Abstract

Shiga toxin consists of an enzymatically activeA-chain and a pentameric binding subunit. The A-chain has a trypsin-sensitive region, and upon cleavage two disulfide bonded fragments, A 1 and A 2 , 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 A 1 and A 2 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, Gb 3 , trypsin treatment induced dissociation of A 1 from the toxin-receptor complex demonstrating that in addition to stabilizing the A-chain, the disulfide bond prevents dissociation of the A 1 fragment from the toxinreceptor complex.

The Intracellular Journey of Shiga Toxins

“…For full catalytic activity, the A 1 fragment must presumably dissociate from A 2 , since residues 258-262 of A 2 lie adjacent to the active site cleft, and the side chain of Met 260 protrudes into the active site itself [109]. Removal of the disulfide bridge is sufficient for the A 1 fragment to dissociate from the A 2 -StxB-Gb3 complex [5]. How the disulfide bridge is reduced is not known, but in analogy with cholera toxin, whose active A 1 fragment also needs to be cleaved from its disulfide-linked A 2 fragment, it is possible that the reduction also of StxA is carried out by the ER-resident enzyme protein disulfide isomerase [110,111].…”

“…The Shiga toxins are AB 5 -toxins consisting of a pentameric binding moiety (StxB) and an enzymatically active A-moiety (StxA) (Fig. 1).…”

“…Furin cycles between the TGN and the cell surface, and is involved in cleavage and activation of not only Shiga toxin, but also diphtheria toxin and Pseudomonas exotoxin A [4]. The pH-optimum for toxin cleavage varies with the substrate, and for Shiga toxin, furin-induced cleavage has a low pH-optimum [5], indicating that efficient cleavage can occur shortly after endocytosis. After cleavage, the A 1 fragment remains attached to the A 2 part via the internal disulfide bond [5], and the cleaved toxin is transported further via the Golgi complex to the endoplasmic reticulum (ER).…”

“…The pH-optimum for toxin cleavage varies with the substrate, and for Shiga toxin, furin-induced cleavage has a low pH-optimum [5], indicating that efficient cleavage can occur shortly after endocytosis. After cleavage, the A 1 fragment remains attached to the A 2 part via the internal disulfide bond [5], and the cleaved toxin is transported further via the Golgi complex to the endoplasmic reticulum (ER). From the ER, the A 1 -fragment is translocated to the cytosol and functions as a highly specific N-glycosidase that removes adenine from one particular adenosine residue in the 28S RNA of the 60S ribosomal subunit [6].…”

The Rediscovery of Shiga Toxin and Its Role in Clinical Disease

“…Toxin activity is increased dramatically by proteolytic nicking, and this appears to take place primarily within an intracellular compartment, and there is good evidence that this event is mediated by the intravesicular protease, furin (47). Removal of the interchain loop by site-directed mutagenesis of one cysteine results in an initial enhancement of toxin activity, perhaps by another proteolytic event, but much less total activity at later times of incubation, apparently due to enhanced degradation of the toxin when the disulfide loop is not present (48). Purification of Stx toxins and subunits in large scale quantity have permitted their crystallization and 3-dimensional constructions of the relationship of the A and B pentamer subunits (51,52).…”