Evidence for glycosphingolipid modification of the type 1 IFN receptor

P1-determinant glycolipids that include two membrane glycosphingolipids, globotriaosylceramide (Gal alpha 1-4Gal beta 1-4Glc-ceramide, Gb3) and galabiosylceramide (Gal alpha 1-4Gal-ceramide, Gb2) are receptors for an Escherichia coli-derived subunit toxin, verotoxin (VT-1). Studies with Daudi cells and glycosphingolipid-deficient Daudi mutants and U937 cells identified that the presence of Gb2/Gb3 correlates with IFN-alpha sensitivity. Comparison of amino acid sequences between VT-1 and the extracellular region of the 63-kDa IFN-alpha beta receptor (IFNAR) peptide reveals regions of identity, specifically in those domains in the VT-1 B subunit, that have been implicated as Gb2/Gb3 binding sites. In direct ligand binding studies, we show that membrane Gb2/Gb3 content affects the binding capacity of cells for IFN-alpha, although IFNAR cell surface expression is unaffected. Binding of IFN-alpha to the receptor leads to kinase-associated phosphorylation of the latent transcription factor, ISGF3, which activates transcription by binding to IFN-stimulated regulatory elements in IFN-sensitive genes. Electrophoretic mobility band shift assays indicated that U-937 and Daudi mutant cells, deficient in Gb2 and Gb3, exhibited reduced nuclear factor binding to the human 2-5A synthetase IFN-stimulated regulatory element when compared with wild-type Daudi cells, after exposure to IFN-alpha. Moreover, when Daudi cells were treated with a ceramide analogue, 1-phenyl-2-(decanoylamino)-3-morpholino-1-propanol, Gb2 and Gb3 synthesis was inhibited and a concomitant reduction in IFN-induced ISGF3 activation was noted. IFNAR cell surface expression was unaffected by 1-phenyl-2-(decanoylamino)-3-morpholino-1-propanol treatment. By using a fusion protein of the extracellular domain of IFNAR linked at the carboxyl terminus to the Fc portion of IgG1, we demonstrate that IFNAR is able to bind preferentially to Gb2. These results suggest that an association of IFNAR with membrane Gal alpha 1-4Gal containing glycolipids facilitates receptor-mediated signaling.