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 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.
Garred
O
O
Institute for Cancer Research at The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway.
Dubinina
E
E
Polesskaya
A
A
Olsnes
S
S
Kozlov
J
J
Sandvig
K
K
D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects / Q000235:genetics; D020126:Brefeldin A; D003517:Cyclopentanes / Q000494:pharmacology; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D003603:Cytotoxins / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005260:Female; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D012694:Serine / Q000235:genetics / Q000378:metabolism; D022603:Shiga Toxins; D018675:Toxicity Tests; D014407:Tumor Cells, Cultured
D001427:Bacterial Toxins; D003517:Cyclopentanes; D003603:Cytotoxins; D004220:Disulfides; D022603:Shiga Toxins; D020126:Brefeldin A; D012694:Serine; D003545:Cysteine