Sulfated Glycosaminoglycans Activate STING By Inducing Its Polymerization

The cGAS-STING pathway is responsible for the detection of abnormally located cytoplasmic DNA that serves as a danger signal of pathogen invasion or tissue damage.The cytosolic DNA sensor cGAS(cyclicGMP-AMP[cGAMP]synthase)is activated by DNA binding or Mn²⁺to produce the second messenger 2'3'-cGAMP,which binds to and activates the endoplasmic reticulum(ER)-membrane adaptor protein STING(also known as MITA,ERIS,or MPYS).Once activated,STING translocates from ER membrane to the Golgi apparatus where it gets polymerized to recruit and activate the kinase TBK1 and subsequently the transcriptional factor IRF3,ultimately leading to the production of various cytokines including type I-IFNs.Though the structures of cGAMP-bond STING tetramer and cGAMP-bond STING dimer in complex with TBK1 reveal the requirement of STING polymerization for efficient signal activation,it remains unknown how STING polymers are formed inside cells and why STING needs its translocation from ER to the Golgi apparatus for activation.In this work,using a genome-wide CRISPR–Cas9-mediated screen and biochemical analysis,we discover that sulfated glycosaminoglycans(s GAGs)in Golgi or s GAGs-containing vesicles drive STING polymerization and activation.Multiple key genes that are critically involved in s GAG biosynthesis encoding PAPs transporter(SLC35B2)and enzymes(XYLT2,B4GALT7,B3GALT6,B3GAT3,UXS1,UGDH,and FAM20B)were identified by the screen and confirmed in the corresponding gene-deficient cells showing that s GAGs are essential for STING polymerization and activation.STING translocated to s GAG-containing vesicles upon VACV infection,for STING binds to s GAGs through its luminal positively charged and polar residues,which represent an evolutionarily conserved determinants for STING activation,where the binding strength determined the activation level of STING.Critically,purified or chemically synthesized s GAGs potently induce STING polymerization and TBK1 activation in vitro,with heparin(Hp)and heparan sulfate(HS)displaying the highest activity.Analysis of Hp-derived oligosaccharides reveals the dependence of chain-length and O-linked sulfation of Hp for STING activation.Consistently,s GAGs are required for the host defense against DNA virus infection in vivo.Thus,s GAGs-binding is both necessary and sufficient for STING polymerization,which provide a mechanistic explanation for Golgi translocation-dependent STING activation.