title: Role of processing and intracellular transport for optimal toxicity of Shiga toxin and toxin mutants.
abstract: Cleavage of Shiga toxin A-fragment at a highly trypsin-sensitive site increases its enzymatic activity. To investigate the role of this cleavage site in intoxication of cells, we studied the routing, cleavage, and toxicity of mutant toxin where the trypsin-sensitive site had been eliminated. Ultrastructural analysis of toxin tagged with horseradish peroxidase demonstrated that wild-type and mutant toxins were transported from endosomes to the trans-Golgi network and further through the Golgi cisterns to the endoplasmic reticulum. Wild-type toxin was much more efficient than the mutants in provoking rapid intoxication, but after prolonged incubation time also mutants were highly toxic. The cells were able to cleave both wild-type Shiga toxin and the mutants, but the cellular location for cleavage appears to differ. Wild-type toxin was cleaved in the presence of brefeldin A, which disrupts the Golgi cisterns. This indicates that the cleavage occurs in the endosomes or in the trans-Golgi network. In contrast, the mutant Shiga-His (R248H/R251H) was not cleaved in the presence of brefeldin A, indicating that the cleavage can occur only after the toxin has left the trans-Golgi network. In vitro experiments showed that the cytosolic enzyme calpain is able to cleave Shiga-His, and results from in vivo experiments are consistent with the possibility that cleavage is carried out by calpain after the mutant A-fragment has reached the cytosol.
authors.affiliation: Institute for Cancer Research, Norwegian Radium Hospital, Montebello, Oslo.
authors.forename: O
authors.initials: O
authors.lastname: Garred
authors.forename: E
authors.initials: E
authors.lastname: Dubinina
authors.forename: P K
authors.initials: PK
authors.lastname: Holm
authors.forename: S
authors.initials: S
authors.lastname: Olsnes
authors.forename: B
authors.initials: B
authors.lastname: van Deurs
authors.forename: J V
authors.initials: JV
authors.lastname: Kozlov
authors.forename: K
authors.initials: K
authors.lastname: Sandvig
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000302:isolation & purification / Q000378:metabolism* / Q000633:toxicity; D001483:Base Sequence; D001692:Biological Transport; D002470:Cell Survival / Q000187:drug effects; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D017931:DNA Primers; D010450:Endopeptidases / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D004768:Enterotoxins / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D006735:Horseradish Peroxidase / Q000378:metabolism; D007700:Kinetics; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000633:toxicity; D015183:Restriction Mapping; D017384:Sequence Deletion; D022603:Shiga Toxins; D012760:Shigella; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism*; D014709:Vero Cells
chemical_list: D001427:Bacterial Toxins; D017931:DNA Primers; D004768:Enterotoxins; D011993:Recombinant Fusion Proteins; D022603:Shiga Toxins; D006735:Horseradish Peroxidase; D010450:Endopeptidases; D014357:Trypsin