Efficient Intracellular Transport Of Shiga Toxin, The Ligand Of Globotriaosylceramide(Gb3), Is Dependent On Its Structure

Shiga toxin (STx), is produced an important factor of Shigellosis by Shigella dysenteriae. Shiga toxin is composed of enzymatically active(A) and receptor binding(B) moieties. The receptor of STx is globotriaosylceramide(Gb3) which is enriched at the plasma membrane of mammalian cells. STx follows a complex intracellular pathway in order to kill susceptible cells. After binding to cell surface receptor, globotriaosylceramide, the toxin is internalized and trafficked in retrograde fashion to the endoplasmic reticulum (ER). From the ER lumen, the toxin gains access to the cytoplasm, where it enzymatically inactivates the 28S rRNA, inhibiting protein synthesis. The mechanism of its intracellular transport is still unclear.To study the effect of STxB structure on its intracellular transport, we modified the N-terminus of STxB by gene cloning.In this study, the sequence of STxB has been amplified by PCR. The products have been ligated into the pGEX-4T-1 vector which has a GST-tag. Thus, a GST-STxB expression plasmid GST-STxB-pGEX-4T-1 is constructed successfully. This plasmid was then used to transform a bacterium DH5α. The recombinant protein GST-STxB was expressed by IPTG induction and purified by glutathione Sepharose 4B gel affinity chromatography. The purity of protein was confirmed by SDS-PAGE and Western blot analysis. The oligomerization status of the protein was confirmed by HPLC using a Bio-sil SEC 250 column.The intracellular transport of the recombinant protein was investigated. Hela cells were first incubated with GST-STxB at 4℃for 30min or 37℃for 120min, and subsequently visualized by immuno-fluorescence microscopy. The intracellular transport of GST-STxB was inhibited, suggesting that the N-terminal structure of STxB is important to the intracellular transport. Further studies with flow cytometry indicated that the inhibition was caused by the reduction of GST-STxB proteins binding to their receptor Gb3 molecules.