Role of Globotriaosyl Ceramide (Gb3 or CD77) in Apoptosis in Burkitt's Lymphoma

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Verotoxins (VT's, also called Shiga toxins) produced by E. coli serotype 0157:H7 and other serotypes have been implicated as causative agents of hemolytic uremic syndrome, hemorrhagic colitis, and thrombocytopenic purpura. The receptors for VT's are globotriaosyl ceramide (Gb3 or CD77) and possibly other glycosphingolipids which contain Gal alpha1-4 Gal residues. Gb3 is expressed at the germinal center stage of B lymphocyte development. Because of their phenotypic resemblance, Daudi cells and other Burkitt's lymphoma-derived cell lines serve as in vitro models of GC B lymphocytes. The Gb3-binding VT B subunits mimic the IFNAR-1 subunit of the interferon-alpha receptor, the B cell protein CD19, and MHC class II proteins in their amino acid sequences. Experimental evidence to date indicates that Gb3 and Gb3-binding proteins are essential components of a number of signal transduction pathways in Burkitt's lymphoma and GC B lymphocytes. These include a role for Gb3/IFNAR-1 in IFN-alpha induced growth inhibition and adhesion, Gb3/CD19 interaction in adhesion and Gb3/VT B-subunit interaction in the induction of apoptosis. However, there is relatively little data available concerning which signal transduction pathways require Gb3, or the precise role of Gb3 in the pathways leading to the observed responses. Our preliminary investigations indicate that Gb3 may play a major role in a number of apoptotic pathways in Gb3-positive B cells. We propose to further investigate the role of Gb3 in signal transduction relating to apoptosis using the Daudi cell line, which expresses high levels of Gb3, and the Daudi-derived Gb3-deficient VT500 cell line. The long term objectives of the proposed research are to determine the cellular functions of Gb3 and other Gal alpha 1-4 Galcontaining glycolipids and their endogenous protein ligands, and to determine how the targeting of Gb3-positive cells by VT affects immune responses and the pathogenesis of VT producing E. coli infection. Specific aims are to: 1) Identify and define apoptosis pathways which require Gb3 by comparing such pathways in Gb3+ and Gb3- Burkitt's lymphoma cell lines. 2) Determine differential gene expression in Gb3+ and Gb3- Burkitt's lymphoma cells prior to and following treatment with inducers of apoptosis by performing microarray analysis, RT-PCR and northern analysis. 3) Identify and model potential VT-like Gb3-binding sites on proteins involved in apoptosis pathways which require Gb3.