Disruption of an Internal Membrane-Spanning Region in Shiga Toxin 1 Reduces Cytotoxicity

Abstract

Shiga toxin type 1 (Stx1) belongs to the Shiga family of bipartite AB toxins that inactivate eukaryotic 60S ribosomes. The A subunit of Stxs are N-glycosidases that share structural and functional features in their catalytic center and in an internal hydrophobic region that shows strong transmembrane propensity. Both features are conserved in ricin and other ribosomal inactivating proteins. During eukaryotic cell intoxication, holotoxin likely moves retrograde from the Golgi apparatus to the endoplasmic reticulum. The hydrophobic region, spanning residues I224 through N241 in the Stx1 A subunit (Stx1A), was hypothesized to participate in toxin translocation across internal target cell membranes. The TMpred computer program was used to design a series of site-specific mutations in this hydrophobic region that disrupt transmembrane propensity to various degrees. Mutations were synthesized by PCR overlap extension and confirmed by DNA sequencing. Mutants StxAF226Y, A231D, G234E, and A231D-G234E and wild-type Stx1A were expressed in Escherichia coli SY327 and purified by dye-ligand affinity chromatography. All of the mutant toxins were similar to wild-type Stx1A in enzymatic activity, as determined by inhibition of cell-free protein synthesis, and in susceptibility to trypsin digestion. Purified mutant or wild-type Stx1A combined with Stx1B subunits in vitro to form a holotoxin, as determined by native polyacrylamide gel electrophoresis immunoblotting. StxA mutant A231D-G234E, predicted to abolish transmembrane propensity, was 225-fold less cytotoxic to cultured Vero cells than were the wild-type toxin and the other mutant toxins which retained some transmembrane potential. Furthermore, compared to wild-type Stx1A, A231D-G234E Stx1A was less able to interact with synthetic lipid vesicles, as determined by analysis of tryptophan fluorescence for each toxin in the presence of increasing concentrations of lipid membrane vesicles. These results provide evidence that this conserved internal hydrophobic motif contributes to Stx1 translocation in eukaryotic cells.

The C‐terminus of pokeweed antiviral protein has distinct roles in transport to the cytosol, ribosome depurination and cytotoxicity

“…, 1993) and has been suggested to become exposed to solvent upon separation of the A and the B chains (Deeks et al. , 2002; Suhan and Hovde, 1998). A point mutation, which changed a conserved proline‐250 to alanine at the C‐terminus of ricin A chain reduced the cytotoxicity of the holotoxin (Simpson et al.…”

Immuno‐informatics: Mining genomes for vaccine components

“…One means of identifying such proteins has been to identify them by cell fraction separation methods, 2‐D gel electrophoresis, and highly focused sequencing methodologies (such as electron spray mass spectrometry) 35 . Alternatively, such proteins can be identified by scanning genomic sequences with computer programs that predict secretory signal peptides (SignalP and SPScan), 36 transmembrane domains (Tmpred) 37 and lipoprotein attachment sites (Prosite Scan) 38 …”

A simple method for expression and purification of Shiga toxin 1 (Stx1) with biological activities by using a single‐promoter vector and native signal peptide

“…Only one pair of primers (P1 and P2) was used to amplify the whole stx1 gene and indirectly used E. coli O157:H7 as a template to amplify the entire stx1 gene. This is done rather than amplifying the genes of the subunits or initially purifying from a toxin‐converting phage, then using the toxin‐converting phage as a PCR template, which is much easier than the method of previous investigators . Furthermore, we could get final yield of the purified rStx1 ranged from 2 to 3 mg/L by one‐step nickel affinity gel column chromatography, which could be comparable yield from some previous affinity purification methods of Stx1 using globotriaosylceramide (Gb3) or egg white glycoprotein‐immobilized resins [17, 18].…”

“…Suhan et al. successfully made holotoxin in vitro using a combination of purified subunits . Nishikawa et al.…”