IMPORTANCE OF GLYCOLIPID SYNTHESIS FOR BUTYRIC ACID-INDUCED SENSITIZATION TO SHIGA TOXIN AND INTRACELLULAR SORTING OF TOXIN IN A431 CELLS

The human epidermoid carcinoma cell line A431 becomes highly sensitive to Shiga toxin upon treatment with butyric acid. This strong sensitiz ation (>1000-fold) is accompanied by an increase in the fraction of ce ll-associated toxin transported to the Golgi apparatus and to the endo plasmic reticulum (ER). Furthermore, our previous work showed that the length of the fatty acyl chain of Gb3, the Shiga toxin receptor, also was changed (longer fatty acids). We have now investigated the import ance of this change by testing whether glycolipid synthesis is require d for the changed intracellular sorting and the toxin sensitivity. We demonstrate here that inhibition of glycosphingolipid synthesis by inh ibition of N-acyltransferase with fumonisin B-1, by inhibition of gluc osylceramide synthetase by PDMP or PPMP, or by inhibition of serine pa lmitoyl transferase by beta-fluoroalanine, inhibited the butyric acid- induced change in sensitivity and the increase in the fraction of cell -associated Shiga toxin transported to the Golgi apparatus and the ER. The block in butyric acid-induced sensitization caused by beta-fluoro alanine could be abolished by simultaneous addition of sphinganine or sphingosine. Thus, the data suggest that the fatty acyl chain length o f glycosphingolipids is important for intracellular sorting and transl ocation of Shiga toxin to the cytosol.