The Effect of Shiga Toxin Binding to Globotriaosylceramide in Rafts of Human Kidney Cells and Burkitt's Lymphoma Ramos Cells

Abstract

Shiga toxin, an enterotoxin produced from Shigella dysenteriae serotype and enterohemorrhagic Escherichia coli binds specifically to globotriaosylceramide, Gb3, on the cell surface and causes cell death. Stx is shown to induce apoptosis in several cell lines, including human renal tubular cell line ACHN and Burkitt's lymphoma Ramos cells. In order to study the early signal transduction after Stx addition, Gb3-enriched microdomains were prepared from these cells by sucrose density gradient centrifugation of Triton X-100 lysate as buoyant, detergent-insoluble microdomain, rafts. Gb3 was only recovered in rafts and associated with Src family kinase Yes or Lyn. Tyrosine residues of raft proteins were hyperphosphorylated in 10min and turned to resting level in 30min after Stx addition. Yes was thought to be responsible for hyperphosphorylation observed in raft proteins, because Yes activity increased in 3-10min after Stx addition. Unexpectedly, however, all of the Yes activity was obtained in high-density, detergent-soluble fraction, but not in rafts. Stx was known to be internalized as complex with Gb3 via retrograde endocytosis. Therefore, Yes was also thought to enter into intracellular space with Stx/Gb3 complex, where Yes was activated and increased solubility for Triton X-100. Since Stx B subunit could alone induce Yes activation, the binding of Stx to Gb3 might cause temporal activation of Yes in DS in the process of apoptosis.