title: 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 pathogens. Moreover, erroneous detection of endogenous DNA by the same sensing mechanisms has an important pathophysiological role in certain sterile inflammatory conditions. 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 origin. 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 bacteria. 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 STING. 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.
authors.affiliation: Institute for Clinical Chemistry and Clinical Pharmacology, University Hospital, University of Bonn, 53127 Bonn, Germany. andrea.ablasser@uni-bonn.de
authors.forename: Andrea
authors.initials: A
authors.lastname: Ablasser
authors.forename: Marion
authors.initials: M
authors.lastname: Goldeck
authors.forename: Taner
authors.initials: T
authors.lastname: Cavlar
authors.forename: Tobias
authors.initials: T
authors.lastname: Deimling
authors.forename: Gregor
authors.initials: G
authors.lastname: Witte
authors.forename: Ingo
authors.initials: I
authors.lastname: Röhl
authors.forename: Karl-Peter
authors.initials: KP
authors.lastname: Hopfner
authors.forename: Janos
authors.initials: J
authors.lastname: Ludwig
authors.forename: Veit
authors.initials: V
authors.lastname: Hornung
mesh_terms: D000249:Adenosine Monophosphate / Q000737:chemistry; D000818:Animals; D055162:Biocatalysis; D002460:Cell Line; D006152:Cyclic GMP / Q000737:chemistry; D003500:Cyclization; D057809:HEK293 Cells; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D015394:Molecular Structure; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D009843:Oligoribonucleotides / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D015290:Second Messenger Systems / Q000502:physiology*; D000099197:STING Protein; D000099235:Cyclic Guanosine Monophosphate-Adenosine Monophosphate Synthase
chemical_list: D000249:Adenosine Monophosphate; D006152:Cyclic GMP; D008565:Membrane Proteins; D009713:Nucleotidyltransferases; D009843:Oligoribonucleotides; C582108:Gp(2'-5')Ap(3'-5') dinucleotide; C532857:STING1 protein, human; D000099197:STING Protein; C530855:Sting1 protein, mouse; C578391:cGAS protein, mouse; D000099235:Cyclic Guanosine Monophosphate-Adenosine Monophosphate Synthase