cGAS produces a 2′-5′-linked cyclic dinucleotide second messenger that activates STING

Abstract

Detection of cytoplasmic DNA represents one of the most fundamental mechanisms of the innate immune system to sense the presence of microbial pathogens1. Moreover, erroneous detection of endogenous DNA by the same sensing mechanisms has an important pathophysiological role in certain sterile inflammatory conditions2,3. The endoplasmic-reticulum-resident protein STING is critically required for the initiation of type I interferon signalling upon detection of cytosolic DNA of both exogenous and endogenous origin4,5,6,7,8. Next to its pivotal role in DNA sensing, STING also serves as a direct receptor for the detection of cyclic dinucleotides, which function as second messenger molecules in bacteria9,10,11,12,13. DNA recognition, however, is triggered in an indirect fashion that depends on a recently characterized cytoplasmic nucleotidyl transferase, termed cGAMP synthase (cGAS), which upon interaction with DNA synthesizes a dinucleotide molecule that in turn binds to and activates STING14,15. We here show in vivo and in vitro that the cGAS-catalysed reaction product is distinct from previously characterized cyclic dinucleotides. Using a combinatorial approach based on mass spectrometry, enzymatic digestion, NMR analysis and chemical synthesis we demonstrate that cGAS produces a cyclic GMP-AMP dinucleotide, which comprises a 2′-5′ and a 3′-5′ phosphodiester linkage >Gp(2′-5′)Ap(3′-5′)>. We found that the presence of this 2′-5′ linkage was required to exert potent activation of human STING. Moreover, we show that cGAS first catalyses the synthesis of a linear 2′-5′-linked dinucleotide, which is then subject to cGAS-dependent cyclization in a second step through a 3′-5′ phosphodiester linkage. This 13-membered ring structure defines a novel class of second messenger molecules, extending the family of 2′-5′-linked antiviral biomolecules.

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Figure 1: The R231A STING mutant uncouples cyclic di-GMP sensing from cGAS-induced activation.
Figure 2: The cGAS reaction product is distinct from cGAMP(3′-5′).
Figure 3: The second messenger produced by cGAS is >Gp(2′-5′)Ap(3′-5′)>.
Figure 4: cGAMP(2′-5′) is a potent activator of human and murine STING.
Figure 5: >Gp(2′-5′)Ap(3′-5′)> is synthesized in a two-step process.

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Acknowledgements

We thank M. Pelegrin for providing us with LL171 cells. K.-P.H. is supported by the National Institutes of Health (U19AI083025), the European Research Council Advanced Grant 322869, and the Center for Integrated Protein Science Munich (CIPSM). A.A. and V.H. are members of the excellence cluster ImmunoSensation. V.H. is supported by grants from the German Research Foundation (SFB670) and the European Research Council (ERC 243046).

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A.A., M.G., T.C., G.W., T.D., I.R., J.L., K.-P.H. and V.H. designed experiments and analysed the data. A.A., M.G., T.C., G.W., T.D. and I.R. performed experiments. A.A. and V.H. wrote the manuscript. V.H. supervised the project.

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Correspondence to Andrea Ablasser or Veit Hornung.

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Ablasser, A., Goldeck, M., Cavlar, T. et al. cGAS produces a 2′-5′-linked cyclic dinucleotide second messenger that activates STING. Nature 498, 380–384 (2013). https://doi.org/10.1038/nature12306

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