DOI: 10.5061/dryad.rr4xgxdgs
title: Data from: The β-D-manno-heptoses are immune agonists across kingdoms

description: Bacterial small molecule metabolites such as
 adenosine-diphosphate-D-glycero-β-D-manno-heptose (ADP-heptose) and their
 derivatives act as effective innate immune agonists in mammals. We showed
 that functional nucleotide-diphosphate-heptose biosynthetic enzymes (HBEs)
 are distributed widely in bacteria, archaea, eukaryotes, and viruses. We
 identified a conserved STTR5 motif as a hallmark of heptose
 nucleotidyltransferases that can synthesize not only ADP-heptose but also
 cytidine-diphosphate (CDP)- and uridine-diphosphate
 (UDP)-heptose. Both CDP- and UDP-heptoses are agonists that trigger
 stronger alpha-protein kinase 1 (ALPK1)-dependent immune responses than
 ADP-heptose in human and mouse cells and mice. We also produced
 ADP-heptose in archaea and verified its innate immune agonist functions.
 Hence, the β-D-manno-heptoses are cross-kingdom small molecule
 pathogen-associated molecular patterns that activate the ALPK1-dependent
 innate immune signaling cascade.
description: Paper: **The β-D-*manno*-heptoses are immune agonists across kingdoms**
 \[Access this dataset on Dryad](
 [https://doi.org/10.5061/dryad.rr4xgxdgs](https://doi.org/10.5061/dryad.rr4xgxdgs)) **Descriptions** This folder contains the enzymatic assay data of NDP-heptose biosynthetic enzymes (HBEs) data, phylogenesis of HBEs performing nucleotidyltransferase activities (HENases) beyond bacteria, source data of cytokines in mice, qPCR source data, and dual luciferase assay data. **Enzymatic assay data of HBEs** NDP-heptoses generated by HENases combined with appropriate HBEs in four-step reactions (Fig. 2G). Synthesis of NDP-heptoses by different HBE combinations at a simulated physiological condition (pH 7.2, S7P 0.1 mM, ATP 3.5 mM, CTP 0.3 mM, and UTP 0.7 mM) (Fig. 3A). **Phylogenesis** Three .fasta files and three .nwk files were contained. The “Viral HENase aligments.fas”, “Archaeal HENase aligments.fas”, “Eukaryotic HENase aligments.fas” contain the aligned sequences of representative HENases from viruses, archaea, and eukaryota, respectively. Three phylogenetic trees were generated by MEGA 6 using the aligned sequences and formed the following files: “Tree of viral HENase.nwk”, “Tree of archaeal HENase.nwk”, “Tree of eukaryotic HENase.nwk”. **Source data of cytokines in mice** Cytokine concentrations in the serum of wild-type (WT) and *Alpk1*−/− mice injected with ADP-, CDP-, or UDP-heptose intravenously (Fig. 4F and Fig. S13 ). Cytokine concentrations in the lungs of WT or *Alpk1*−/− mice infected with *Burkholderia multivorans* ATCC BAA-247 intranasally (Fig. S14). **qPCR source data** qPCR source data of *IP-10* expression in BMDM cells upon the incubation of ADP-, CDP-, or UDP-heptose (Fig. S9). **Source data of NF-kB luciferase assays** ADP-, CDP-, or UDP-heptose triggered NF-κB activation in 293T cell or its *ALPK1*−/− mutant (Fig.4E). Electroporation of ADP-, CDP-, or UDP-heptose into 293T cells (Fig. S10B). NF-κB activation triggered by the cell lysates of *Methanococcus maripaludis* 01 in 293T cell or its *ALPK1*−/− mutant (Fig. S11D). Assessments of possible interactive effects among ADP-, CDP-, and UDP-heptoses via NF-κB-driven luciferase assays (Fig. S12). Assessments of ADP-heptose triggered NF-κB activation in 293T *ALPK1*−/−cells expressing varied ALPK1 homologues (Fig.S16C).