ADP-Heptose (L-isomer)

Synthetic ALPK1-TIFA inducers  

ADP-Heptose (D- & L-isomer) induced ALPK1-TIFA signaling

Bacterial 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 the D- and L- isomers of ADP-Heptose have been shown to trigger comparable NF-κB-dependent signaling in vitro [1-3].

InvivoGen has synthesized and provides the L-isomer, ADP‑L-Heptose– ADP-L-glycero-β-D-manno-heptose.

Both isomers of ADP-Heptose have been identified as potent PAMPs from Gram-negative bacteria that bind to the cytosolic receptor, ALPK1 [1-3]. By binding to ALPK1, ADP-Heptose triggers the oligomerization of TIFA and the recruitment of TRAF6. Ultimately, this results in the activation of NF-κB and a strong pro-inflammatory response [1].

Key features:

• 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-L-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.

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)

Isomer: L isomer

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.
• The 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 of ADP-L-Heptose (provided as a dried powder)
• 1.5 ml of endotoxin-free water

 Product is shipped at room temperature.

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

 The resuspended product is stable for 3 months at -20°C. 

Avoid repeated freeze-thaw cycles.