ADP-Heptose

Synthetic ALPK1-TIFA inducer 

ADP-Heptose induced ALPK1-TIFA signaling

ADP-Heptose is an intermediary sugar in the biosynthesis of lipopolysaccharide (LPS), an essential component of the outer membrane of Gram‑negative bacteria. It is generated by a multi-step biosynthesis pathway, in which the final step is the interconversion between two isomers, ADP-D-glycero-β-D-manno-heptose and ADP-L-glycero-β-D-manno-heptose, catalyzed by an epimerase enzyme (e.g. HldD). Importantly, synthetic forms of ADP-Heptose D- and L-  isomers have been shown to trigger comparable NF-κB-dependent signaling in vitro [1-3]. InvivoGen readily provides ADP-D-glycero-β-D-manno-heptose (D‑isomer; ADP‑Heptose).

ADP-Heptose Background

ADP-Heptose has been identified as a potent pathogen-associated molecular pattern (PAMP) of Gram-negative bacteria (e.g. Helicobacter pylori and Shigella flexneri) that binds to the cytosolic pattern recognition receptor (PRR) ALPK1 [1-3]. By binding to ALPK1, ADP-Heptose triggers the oligomerization of TIFA (TRAF-interacting protein with FHA domain-containing protein A) and TRAF6 (TNF receptor-associated factor 6). This ultimately results in the activation of NF-κB and a strong pro-inflammatory response through the secretion of IL-8 [1].

ADP-Heptose is directly delivered to host cells (i.e. human embryonic kidney cells, intestinal epithelial cells, gastric cells, and cervical cancer cells) when bacteria are endocytosed. Additionally, it can be delivered via type III and type IV bacterial secretion systems (e.g. Yersinia pseudotuberculosis and H. pylori, respectively) [3,4]. Furthermore, extracellular ADP-Heptose can freely penetrate the host cell membrane and access the host cytoplasm. Thus, extracellular bacteria that do not encode these secretion systems (e.g. Neisseria meningitidis) are also able to activate the ALPK1-TIFA signaling axis [1, 3].

Features of ADP-Heptose:

• Potent LPS-intermediary metabolite produced by all Gram-negative bacteria
• Activates the cytosolic ALPK1-TIFA signaling pathway
• Easily penetrates the cell wall for delivery to the host cell cytoplasm

InvivoGen's ADP-Heptose is of the highest quality, guaranteed free of bacterial contamination, and has been functionally validated on HEK-Blue™ Null1-v cells. Additionally, to foster research into ADP-Heptose-dependent signaling, InvivoGen provides HEK-Blue™ KO-ALPK1 and HEK-Blue™ KO-TIFA cells.

References:

1. Pfannkuch, L. et al. 2019. ADP heptose, a novel pathogen-associated molecular pattern identified in Helicobacter pylori. FASEB J, fj201802555R.
2. Garcia-Weber, D. et al. 2018. ADP-heptose is a newly identified pathogen-associated molecular pattern of Shigella flexneri. EMBO Rep 19
3. Zhou, P. et al. 2018. Alpha-kinase 1 is a cytosolic innate immune receptor for bacterial ADP-heptose. Nature 561, 122-126.
4. Zimmermann, S. et al. 2017. ALPK1- and TIFA-Dependent Innate Immune Response Triggered by the Helicobacter pylori Type IV Secretion System. Cell Rep 20, 2384-2395.

Figures

ADP-Heptose induced NF-κB response. HEK-Blue™ Null1-v, HEK-Blue™ KO‑ALPK1, and HEK-Blue™ KO‑TIFA cells were incubated with increasing concentrations of ADP‑Heptose (0 - 30 µg/ml). After overnight incubation, the NF-κB response was assessed by measuring the activity of SEAP in the supernatant using QUANTI-Blue™ Solution, a SEAP detection reagent. Data are presented as optical density (OD) at 630 nm (mean ± SEM). The EC₅₀ value is indicated (± std error).

ADP-D-Heptose vs. ADP-L-Heptose NF-κB response. HEK-Blue™ Null1-v cells were incubated with increasing concentrations of either ADP‑D-Heptose or ADP-L-Heptose (0 - 10 µg/ml). After overnight incubation, the NF-κB response was assessed by measuring the activity of SEAP in the supernatant using QUANTI-Blue™ Solution, a SEAP detection reagent. Data are presented as optical density (OD) at 630 nm (mean ± SEM). The EC₅₀ value is indicated (± std error).

Specifications

Formula: C₁₇H₂₇N₅O₁₆P_{2 .} Et₃NH (stable form)

Molecular weight: 720.21 g/mol (stable form)

Solubility: 10 mg/ml in H₂O

Working concentration: 0.01 - 30 µg/ml

Quality Control

• Purity >95% (UHPLC)
• Activation of the ALPK1/TIFA signaling pathway has been confirmed using a cellular assay.
• Absence of bacterial contamination (i.e. endotoxins) has been confirmed using a kinetic chromogenic LAL assay, with an endotoxin level <1 EU/mg

Contents

• 250 µg ADP-Heptose (provided as a dried powder)

ADP-Heptose is shipped at room temperature.

Store at -20°C. Upon resuspension, prepare aliquots of ADP-Heptose and store at -20°C.

Resuspended product is stable 3 months at -20°C. 

Avoid repeated freeze-thaw cycles.