Single Nucleotide Polymorphisms of Human STING Can Affect Innate Immune Response to Cyclic Dinucleotides

The STING (stimulator of interferon genes) protein can bind cyclic dinucleotides to activate the production of type I interferons and inflammatory cytokines. The cyclic dinucleotides can be bacterial second messengers c-di-GMP and c-di-AMP, 3’5’-3’5’ cyclic GMP-AMP (3’3’ cGAMP) produced by Vibrio cholerae and metazoan second messenger 2’5’-3’5’ Cyclic GMP-AMP (2’3’ cGAMP). Analysis of single nucleotide polymorphism (SNP) data from the 1000 Genome Project revealed that R71H-G230A-R293Q (HAQ) occurs in 20.4%, R232H in 13.7%, G230A-R293Q (AQ) in 5.2%, and R293Q in 1.5% of human population. In the absence of exogenous ligands, the R232H, R293Q and AQ SNPs had only modest effect on the stimulation of IFN-β and NF-κB promoter activities in HEK293T cells, while HAQ had significantly lower intrinsic activity. The decrease was primarily due to the R71H substitution. The SNPs also affected the response to the cyclic dinucleotides. In the presence of c-di-GMP, the R232H variant partially decreased the ability to activate IFN-βsignaling, while it was defective for the response to c-di-AMP and 3’3’ cGAMP. The R293Q dramatically decreased the stimulatory response to all bacterial ligands. Surprisingly, the AQ and HAQ variants maintained partial abilities to activate the IFN-β signaling in the presence of ligands due primarily to the G230A substitution. Biochemical analysis revealed that the recombinant G230A protein could affect the conformation of the C-terminal domain of STING and the binding to c-di-GMP. Comparison of G230A structure with that of WT revealed that the conformation of the lid region that clamps onto the c-di-GMP was significantly altered. These results suggest that hSTING variation can affect innate immune signaling and that the common HAQ haplotype expresses a STING protein with reduced intrinsic signaling activity but retained the ability to response to bacterial cyclic dinucleotides.干扰素基因刺激因子（stimulator of interferon genes，STING）蛋白可结合环状二核苷酸（cyclic dinucleotides），以激活I型干扰素与炎性细胞因子的产生。此类环状二核苷酸可涵盖细菌来源的第二信使c-di-GMP与c-di-AMP、霍乱弧菌（Vibrio cholerae）产生的3'5'-3'5'环状GMP-AMP（3'3' cGAMP），以及后生动物来源的第二信使2'5'-3'5'环状GMP-AMP（2'3' cGAMP）。对千人基因组计划（1000 Genome Project）的单核苷酸多态性（single nucleotide polymorphism，SNP）数据进行分析后发现，人群中R71H-G230A-R293Q（HAQ）、R232H、G230A-R293Q（AQ）与R293Q的携带率分别为20.4%、13.7%、5.2%与1.5%。在无外源配体的条件下，R232H、R293Q与AQ型SNP对HEK293T细胞中干扰素-β（IFN-β）与核因子-κB（NF-κB）启动子活性的刺激作用仅为轻度影响，而HAQ型的固有活性则显著降低，该活性下降主要源于R71H突变。上述SNP亦会影响细胞对环状二核苷酸的应答反应。在c-di-GMP存在的情况下，R232H突变体激活IFN-β信号通路的能力部分下降，而其对c-di-AMP与3'3' cGAMP的应答则存在缺陷。R293Q突变体则会显著降低对所有细菌配体的刺激应答反应。令人意外的是，在配体存在的条件下，AQ与HAQ突变体仍保留部分激活IFN-β信号通路的能力，该特性主要源于G230A突变。生化分析结果显示，重组G230A突变体蛋白可改变STING的C端结构域构象及其与c-di-GMP的结合能力。将G230A突变体的蛋白结构与野生型（wild type，WT）结构进行比对后发现，用于钳合c-di-GMP的盖状区域构象发生了显著改变。上述结果表明，人源STING（hSTING）的遗传变异可影响固有免疫信号通路，且常见的HAQ单倍型所编码的STING蛋白固有信号活性降低，但仍保留对细菌环状二核苷酸的应答能力。