Intracellular targeting of the endoplasmic reticulum/nuclear envelope by retrograde transport may determine cell hypersensitivity to verotoxin via globotriaosyl ceramide fatty acid isoform traffic

Abstract

The pentameric B subunit of verotoxin (VT) mediates the attachment to cell surface globotriaosyl ceramide (Gb3) to facilitate receptor-mediated endocytosis of the toxin. In highly toxin-sensitive tumor cells, the holotoxin and VT1 B subunit is targeted intracellularly to elements of the endoplasmic reticulum (ER)/nuclear membrane. In less sensitive cells, the toxin is targeted to components of the Golgi apparatus. We have studied two cell systems: the induced VT hypersensitivity of human astrocytoma cell lines cultured in the presence of sodium butyrate (compared to sodium propionate and capronate) and the increased VT sensitivity of multiple drug-resistant mutants as compared to parental human ovarian carcinoma cells. In both cases, a difference in the intracellular retrograde transport of the receptor-bound internalized toxin to the ER/nuclear envelope, as opposed to the Golgi, correlated with a >1,000-fold increase in cell sensitivity to VT. This change in intracellular routing may be due to sorting of Gb3 fatty acid isoforms, since nuclear targeting was found in turn to correlate with the preferential synthesis of Gb3 containing shorter chain (primarily C16) fatty acid species. We propose that the isoform-dependent traffic of Gb3 from the cell surface to the ER/nuclear membrane provides a new signal transduction pathway for Gb3 binding proteins.

Bacterial protein toxins and lipids: role in toxin targeting and activity

“…Thus these cells become more sensitive to ST (Lauvrak et al, 2004). In addition, phospholipid composition in the surrounding membrane and interaction of Gb3-bound toxin with surface proteins are probably involved in the location of ST into membrane microdomains and subsequent sorting of endocytosis trafficking (Arab and Lingwood, 1996;Arab et al, 1998;Shimizu et al, 2003). Then, ST retrograde transport from the Golgi to ER uses a COP-Iindependent and Rab6-dependent pathway, the signalling molecules of which remain to be defined White et al, 1999).…”

Membrane cytosolic translocation of verotoxin A1 subunit in target cells

“…To avoid the cytotoxic effects associated with VTA, most VT1 transport studies have utilized VTB (Arab & Lingwood, 1998;Johannes et al, 1997). Although some studies have monitored VT1 as a whole (Sandvig & van Deurs, 1996), no studies have monitored both VTA and VTB.…”

Human microvascular endothelial cells resist Shiga toxins by IFN-γ treatment in vitro

“…On the other hand, the intracellular toxin in the Stx-resistant phenotype was present at 2?6 fmol per 10 5 cells and was more than 100-fold greater than in the native ones, even if the native cells incorporated the total toxin input (0?011 fmol). There still remained a possibility that Stxs were transferred to lysozomes and degraded, instead of travelling through the Golgi apparatus to the cytoplasm in an active form (Arab & Lingwood, 1998). However, a significant proportion of the intracellular Stx2 was recovered from the cytoplasmic fraction without any apparent degradation, where the intracellular toxin inactivated ribosomes by N-glycosidase activity (Endo et al, 1988).…”

“…The endothelial cells express the specific receptor for Stxs, globotriaosyl ceramide (Gb3), and its content is enhanced by various proinflammatory cytokines, such as TNF-a (Molostvov et al, 2001;Eisenhauer et al, 2001). After endocytosis, Stxs travel through the Golgi apparatus (Arab & Lingwood, 1998), are processed by furin (Garred et al, 1995) and then, finally, reach their cellular target, the 60S ribosome. The A-subunit N-glycosidase activity inactivates the ribosome, resulting in protein-synthesis inhibition (Endo et al, 1988).…”