As fertilized eggs begin to develop, they engulf materials from the yolk sac including GDP-fucose analogs and other nutrients. selected inhibitory analogs were converted to GDP-fucose analogs within mammalian cells. Fucose analogs incorporated by Pofut1 into Notch EGF repeats disrupted CI 972 Delta-, but not Jagged-induced Notch signaling. Our data further suggest that fucose analog incorporation caused steric clashes with Delta ligands, but not with Jag1, and was responsible for inhibition of Notch signaling. Finally, these inhibitory fucose analogs were used to block Notch dependent T-cell differentiation. Fucose analogs thus represent a novel tool for the inhibition of Notch signaling. Results Fucose analogs inhibit Notch signaling in Zebrafish We generated a panel of GDP-fucose derivatives (Compounds 1C8, Figure 1a) and corresponding peracetylated fucose analogs (Compounds 9C16, Figure 1b; Supplementary Results, Supplementary Figure 1a) with different substituents at the 6-carbon position of L-fucose (Supplementary Figure 1b). To screen for fucose analogs with an inhibitory effect on Notch signaling, we utilized transgenic Zebrafish Tg(Tp1bglob:eGFP)um14 embryos expressing a Notch reporter transgene (GFP under the control of elements responsive to NICD)23. GFP fluorescence induced by activation of the Notch reporter serves as a sensitive and specific reflection of Notch signaling intensity and was used to monitor Notch signaling at 48 hours post fertilization, a developmental period when activation by Delta ligands predominates in Zebrafish. GDP-fucose analogs were injected into the yolk sac of embryos at the one cell stage, bypassing the fucose salvage pathway. As fertilized eggs begin to develop, they engulf materials from the yolk sac including GDP-fucose analogs and other nutrients. The analogs in our panel had a range of effects on Notch signaling. As expected, untreated and natural GDP-fucose (1) treated embryos expressed relatively high levels of GFP indicating CI 972 robust Notch signaling (Figure 1c). Inhibition of GDP-fucose biosynthesis by knocking down GDP-mannose-4,6-dehydratase (gmds MO)24 served as a positive control for Notch signaling inhibition due to reduced fucose on Notch (Figure 1c, bottom left panel). GDP-fucose analogs 2 and 5 did not cause any substantial reduction in Notch signaling compared to negative controls. By contrast, compounds 7 and 8 caused a partial reduction in GFP levels, whereas compounds 3, 4 and 6 with the C-6 ethynyl, ethenyl or OH substituents respectively, had the greatest inhibitory effect, almost entirely eliminating the GFP Notch reporter signal (Figure 1c). Open in a separate window Figure 1 Effects of fucose analogs on Notch signaling in Zebrafish embryos(a) Numbered structures of GDP-fucose analogs screened as potential inhibitors of Notch signaling in Zebrafish. (b) Peracetylated fucose analogs selected for further analysis in cell-based assays. See Supplementary Figure 1a for structures of other peracetylated fucose CI 972 analogs. (c) Transgenic Zebrafish embryos expressing a GFP fluorescent Notch signaling reporter showed that some injected GDP-fucose analogs, indicated in each panel, reduced Notch signaling. Knock down of GDP-mannose-4,6-dehydratase (MO) to inhibit endogenous GDP-fucose biosynthesis was used as a positive control for the effect of eliminating Notch the analogs are transferred by Pofut1, and when incorporated into EGF repeats, interfere with Notch signaling. To address this question, we incubated HEK293T cells expressing EGF1C18 of Notch1 with peracetylated versions of the fucose analogs (Figure 1b, Supplementary Figure 1a), which are more readily taken up Rabbit Polyclonal to MEF2C by cells than the GDP-fucose analogs in cell culture19,20. The success of this approach requires that a peracetylated analog be taken up by cells, efficiently converted to the corresponding GDP derivative, and transported into the endoplasmic reticulum (Supplementary Figure 1c) for utilization by Pofut119,20. In Zebrafish embryos (Figure 1c), we injected GDP-fucose analogs directly into the yolk, thereby bypassing the need for conversion of the analogs to their corresponding GDP derivatives. Using mass spectral glycoproteomic methods, we confirmed that HEK293T cells treated with compounds 10 and 11 (the peracetylated versions of 3 and 4, respectively) did CI 972 not act as inhibitors of Pofut1, but were transferred by Pofut1 CI 972 onto Notch1 EGF repeats. Extracted ion chromatograms (EICs) were generated to compare the relative amounts of ions corresponding to the fucose analog and fucose-modified glycoforms of a peptide from Notch1 EGF6 that contains an the fluoro group in compounds 5 or 13) did not alter Notch activity in either Zebrafish or mammalian cell systems, even though this analog was efficiently incorporated into Notch 1 EGF repeats by Pofut1 (Supplementary Figure 3). Larger groups (i.e. compounds 6, 7 and 8) also inhibited Notch activation in the Zebrafish system as would be expected (Figure 1c), but the peracetylated versions of these compounds (compounds 14, 15 and 16) were not incorporated into Notch1 EGF repeats (Supplementary Figure 3), so could not.