Interleukin-6 (IL-6) induces the activation of the Src family kinase Hck, which is associated with the IL-6 receptor ␤-chain, gp130. Here we describe the identification of an "acidic" domain comprising amino acids 771 to 811 of gp130 as a binding region for Hck, which mediates proliferative signaling. The deletion of this region of gp130 (i.e., in deletion mutant d771-811) resulted in a significant reduction of Hck kinase activity and cell proliferation upon stimulation of gp130 compared to wild-type gp130. In addition, d771-811 disrupted the growth factor-stimulated activation of Erk and the dephosphorylation of Pyk2. Based on these findings, we propose a novel, acidic domain of gp130, which is responsible for the activation of Hck, Erk, and Pyk2 and signals cell proliferation upon growth factor stimulation.To exert its biological effects, interleukin-6 (IL-6) must bind to the IL-6 receptor (IL-6R), composed of two ␣-chains (IL-6R␣, 80 kDa) and two ␤-chains (IL-6R␤ or gp130, 130 kDa). Two moieties of IL-6 and two pairs of these receptor chains form a functional hexameric IL-6R complex (42,43,55). The subsequent intracellular signaling events are activated via gp130, which is the common ␤-chain of the receptors for cardiotrophin 1, ciliary neurotrophic factor, oncostatin M, leukemia inhibitory factor, . Activation of the IL-6R stimulates at least two major signaling pathways, the Src homology 2 (SH2) domain containing protein tyrosine phosphatase 2 (Shp-2)/mitogen-activated protein kinase (MAPK) signaling cascade (8,26,31,32,41,46) and the Janus kinase (Jak)/signal transducer and activator of transcription (STAT) pathway (6,18,25,45). It was shown in recent in vivo studies that gp130-mediated signals were regulated by a balance between these two pathways (33). However, the signaling cascades mediating IL-6-induced cell growth are not fully defined. It was shown that Jak and STAT proteins are activated by IL-6 in multiple myeloma (MM) cells independently of the proliferative response. In contrast, MAPK was activated only in cells showing a proliferative response to IL-6 (32). Moreover, the physical separation of gp130 and Shp-2 reduced cell proliferation (26).We have shown previously that at least three members of the Src family of tyrosine kinases, i.e., Fyn, Hck, and Lyn, coprecipitate with gp130 in lysates of MM cells (20). Stimulation of cells with IL-6 increased the activity of these kinases. The association of Hck kinase with gp130 appeared to be stronger than either of the other two kinases. Therefore, we decided to focus on the Hck kinase to elucidate the mechanism(s) and biological significance of the IL-6-mediated Src kinase activation. To identify the gp130 binding domain for Hck, several mutants of gp130 were constructed. These mutants were based on a chimeric receptor consisting of the extracellular part of the erythropoietin receptor (EPOR) and the intracellular part of human gp130 (23). These EPOR/gp130 receptor chimeras (Eg) allowed study of the activation of gp130 by erythropoietin (EPO) after tr...

The human Gb3/CD77 synthase, encoded by the A4GALT gene, is an unusually promiscuous glycosyltransferase. It synthesizes the Galα1→4Gal linkage on two different glycosphingolipids (GSLs), producing globotriaosylceramide (Gb3, CD77, P k ) and the P1 antigen. Gb3 is the major receptor for Shiga toxins (Stxs) produced by enterohemorrhagic Escherichia coli . A single amino acid substitution (p.Q211E) ramps up the enzyme’s promiscuity, rendering it able to attach Gal both to another Gal residue and to GalNAc, giving rise to NOR1 and NOR2 GSLs. Human Gb3/CD77 synthase was long believed to transfer Gal only to GSL acceptors, therefore its GSL products were, by default, considered the only human Stx receptors. Here, using soluble, recombinant human Gb3/CD77 synthase and p.Q211E mutein, we demonstrate that both enzymes can synthesize the P1 glycotope (terminal Galα1→4Galβ1→4GlcNAc-R) on a complex type N-glycan and a synthetic N-glycoprotein (saposin D). Moreover, by transfection of CHO-Lec2 cells with vectors encoding human Gb3/CD77 synthase and its p.Q211E mutein, we demonstrate that both enzymes produce P1 glycotopes on N-glycoproteins, with the mutein exhibiting elevated activity. These P1-terminated N-glycoproteins are recognized by Stx1 but not Stx2 B subunits. Finally, cytotoxicity assays show that Stx1 can use P1 N-glycoproteins produced in CHO-Lec2 cells as functional receptors. We conclude that Stx1 can recognize and use P1 N-glycoproteins in addition to its canonical GSL receptors to enter and kill the cells, while Stx2 can use GSLs only. Collectively, these results may have important implications for our understanding of the Shiga toxin pathology.

No abstract

Recombinant human erythropoietin (EPO) is the main therapeutic glycoprotein for the treatment of anemia in cancer and kidney patients.T he in-vivo activity of EPO is carbohydrate-dependent with the number of sialic acid residues regulating its circulatory half-life.E PO carries three Nglycans and thus obtaining pure glycoforms provides am ajor challenge.W eh ave developed ar obust and reproducible chemoenzymatic approach to glycoforms of EPO with and without sialic acids.E PO was assembled by sequential native chemical ligation of two peptide and three glycopeptide segments.The glycopeptides were obtained by pseudoprolineassisted Lansbury aspartylation. Enzymatic introduction of the sialic acids was readily accomplished at the level of the glycopeptide segments but even more efficiently on the refolded glycoprotein. Biological recognition of the synthetic EPOs was shown by formation of 1:1c omplexes with recombinant EPO receptor.