Fig. 1
Identification of a bioactive mammalian disaccharide Manβ1-4GlcNAc. a Size exclusion fractionation of Trex1−/− MEFs fOS pool and bioactivity of each fraction. Top panel, FACE analysis of each fraction. Bottom panel, quantitative RT-PCR analysis of mCxcl10 mRNA in RAW264.7 cells (permeabilized by digitonin, same below) stimulated for 24 h with each fraction. b Two-dimensional HPLC analysis of fOS enriched in wild-type (WT), Trex1−/− MEFs and Trex1−/− fOS treated with α-mannosidases (see Methods). Quantitation and structure of top five enriched fOSs, identified by the second reverse-phase HPLC, are shown in Supplementary Fig. 2. c FACE analysis of Trex1−/− MEFs fOS pool, key fractions and synthetic standards (as shown on top). d Quantitative RT-PCR analysis of mCxcl10 mRNA in RAW264.7 cells that were stimulated with increasing amounts (1, 10, and 100 μM) of the synthetic Man2GlcNAc1 and ManGlcNAc1. e, f FACE analysis (e) and bioactivity (f) of untreated or α- or β-mannosidase digested Trex1−/− MEFs fOS pool or the synthetic ManGlcNAc disaccharide. Bioactivity of each fOS sample was measured by quantitative RT-PCR analysis of mCxcl10 mRNA in RAW264.7 cells stimulated for 24 h with indicated fOS samples. (g) FACE analysis of Trex1−/− MEFs fOS pool, and synthetic Manβ1-4GlcNAc, Manα1-4GlcNAc, Man9GlcNAc2, Man5GlcNAc2. h Quantitative RT-PCR analysis of mCxcl10 and mIfit1 mRNA in RAW264.7 cells that were stimulated with increasing amounts (1, 10, and 100 μM) of the synthetic Manβ1-4GlcNAc and Manα1-4GlcNAc for 24 h. i Quantitative RT-PCR analysis of mCxcl10 and mIfit1 mRNA in RAW264.7 cells that were stimulated with 10 μM of Glcβ1-4GlcNAc, Galβ1-4GlcNAc, and Manβ1-4GlcNAc. j Quantitative RT-PCR analysis of mCxcl10 mRNA in RAW264. 7 cells that were stimulated with increasing amounts (10, 100, and 1000 μM) of Mannose, GlcNAc, Manα1-CH3, Manβ1-CH3, and Manβ1-4GlcNAc. Data are representative of at least three independent experiments. Error bars indicate SEM. Unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001,****P < 0.0001, ns not significant (same throughout)
