Fig. 3: NAGK phosphorylates MDP to render it NOD2-agonistic.
From: Phosphorylation of muramyl peptides by NAGK is required for NOD2 activation
a, Left, GlcNAc conversion to GlcNAc-6-phosphate in a NAGK-dependent manner. Right, MDP. Sugar moieties are indicated in blue. The arrow indicates the phosphorylatable OH group in the C6 position of MDP. b, In vitro kinase reactions with indicated ligands were analysed by TLC. The dashed line indicates where the image has been cropped to remove superfluous lanes. The image represents three biological replicates. c, Wild-type KBM-7-IL-1BmScarlet cells were treated with indicated doses MDP (l-d isomer), MDP (l-l isomer) or 6-amino MDP for 16 h. Data are mean ± s.e.m of n = 4 independent biological samples. Two-way ANOVA with Dunnett’s multiple-comparisons test. Comparisons were made with untreated controls, which are depicted three times for comparability. d, Extracted ion chromatogram of the MDP in vitro kinase reaction without (left) or with (middle) recombinant NAGK (rNAGK). Chromatographic peaks indicate either MDP (black) or 6-phospho-MDP (purple). Right, corresponding mass spectra from these peaks, with theoretical and detected mass. e, Schematic of the cellular L18-MDP, MDP and 6-phospho-MDP content analysis by LC–MS. f, L18-MDP, MDP and 6-phospho-MDP analysis in wild-type and NAGK-knockout KBM-7 cells treated with L18-MDP. Tukey-style box plots of n = 6 independent biological samples. Two-way ANOVA with Šídák’s multiple-comparisons test. Stim, stimulated. g, IL-1B–mScarlet expression in wild-type, NAGK-knockout or NOD2-knockout KBM-7-IL-1BmScarlet cells that were untreated or stimulated with MDP, pMDP or TNF in digitonin buffer for 16 h. Data are mean ± s.e.m of n = 3 independent biological samples. Two-way ANOVA (MDP and pMDP) or one-way ANOVA (TNF) with Dunnett’s multiple-comparisons test. Comparisons to untreated controls (MDP or pMDP) or wild-type cells (TNF). h,i, NF-kB reporter activity in wild-type or NAGK-deficient NOD2-expressing HEK cells stimulated with MDP or pMDP in digitonin buffer as indicated for 16 h. Data are mean ± s.e.m of n = 3 independent biological samples. A four-parameter dose-response curve was fitted to calculate half-maximal effective concentration (EC50).
