Immunity | Jiang Zhengfan's team reveals that mucopolysaccharides (sulfated glycosaminoglycans) are the cause of STING co-ligand and STING translocation to Golgi activation

Responsible editor | Xi cGAS is a DNA receptor in the cell.
After binding to DNA and activating, it uses ATP and GTP as substrates to synthesize the second messenger 2'3'-cGAMP [1,2], which activates STING (MITA or ERIS) 【3-5】, induce I-type interferon and anti-viral/tumor immune response.

Manganese ions (Mn2+) released from organelles after virus infection are essential to resist DNA virus infection [6]; Mn2+ (rather than Mg2+) in cells is preferentially used by cGAS for 2'3'-cGAMP synthesis, and the catalytic efficiency is higher.
high.

Therefore, Mn2+ is the second cGAS agonist in the cell [7,8].

DNA from any source in the cytoplasm can activate cGAS, and various stresses on cells, such as oxidation, metabolism, and DNA damage, can cause the release of organelles Mn2+ and promote cGAS activation.

Therefore, cGAS is not only a DNA sensor, but also a sensor of cell pressure and genome instability, and plays an important role in autoimmune diseases, cell damage/senescence, and tumor immune surveillance [9-13].

The "manganese immunotherapy" combined with Mn2+ and PD-1 antibody can significantly enhance the tumor treatment effect of PD-1 antibody [13].

After the 2'3'-cGAMP produced by cGAS is combined with STING, STING needs to accumulate from the endoplasmic reticulum via the Golgi apparatus in the pernuclear vesicles and recruit and activate TBK1, which is the so-called transport of STING.

There are many important discoveries about the regulatory proteins involved in STING transport.
For example, the laboratory of Professor Shu Hongbing of Wuhan University found that iRhom2 is essential for STING transport and function [14].

But why the activation of STING must be translocated from the endoplasmic reticulum to the Golgi apparatus has been an unresolved scientific question.

Existing studies have shown that the translocation and activation of STING are tightly coupled: BFA, an inhibitor of membrane vesicle transport, can completely block the activation of STING, while self-activating mutants of STING will spontaneously localize to the Golgi apparatus.

More interesting findings come from recent studies on autoimmune diseases of COPA syndrome [15-18].

COPA is a key subunit of the COPI vesicle (responsible for the recovery of protein from the Golgi apparatus to the endoplasmic reticulum) complex.
Its mutation will cause the STING that escapes to the Golgi apparatus to be unable to be recycled to the endoplasmic reticulum, thereby causing STING to accumulate in the Golgi apparatus.
And self-activation, causing autoimmune diseases.

The activation of STING in cells carrying COPA mutations does not depend on cGAMP at all, indicating that STING localized in the Golgi can be activated spontaneously, which suggests that there may be unknown ligands that activate STING in the Golgi.

There is an extracellular matrix composed of proteins and polysaccharides secreted by cells.
The extracellular matrix not only provides a scaffold for cells and tissues, but also plays an important role in regulating cell survival, development, migration, proliferation and other biological functions.

Sulfated glycosaminoglycans (sGAGs, also known as mucopolysaccharides) are an important member of the extracellular matrix and the main component of bone matrix and connective tissue.

Mucopolysaccharides are a class of linear linear polymers composed of disaccharide repeating units synthesized in the Golgi apparatus, and are also called aminoglycans (one of the disaccharide units is aminohexose).

Based on the different composition of disaccharide units, the family can be divided into four categories: ①dermatin sulfate (DS); ②chondroitin sulfate (CS); ③keratan sulfate (KS) ; ④heparan sulfate (HS) and heparin (heparin, Hp).

Sulfated glycosaminoglycans are widely distributed in the extracellular matrix, cell surface or intracellular membrane vesicles (Golgi apparatus, endosomes and secretory vesicles).
Due to the large number of negatively charged sulfated modifications, sulfated glycosaminoglycans can interact with The combination of positively charged amino acids or polar amino acid residues on the target protein affects the interaction of the target protein with other proteins or induces the oligomerization of the target protein [19].

In recent years, as the research of mucopolysaccharides in biomedicine continues to deepen, they have been found to have various pharmacological activities such as anticoagulation, hypolipidemic, antiviral, antitumor and antiradiation.

Mucopolysaccharide is one of the active ingredients of animal medicine, commonly found in skin (ejiao, sea cucumber, cicada slough, snake slough, etc.
), bones (tiger bones, dog bones, etc.
), horns (rhino horns, antlers, etc.
), scales (pangolins, tortoise shells, etc.
) , Turtle shell, hawksbill turtle, etc.
) and mucus (snails, loach, etc.
) and other medicinal materials, these medicinal materials are the treasures of thousands of years of traditional Chinese medicine culture.

Existing studies have shown that the extracellular matrix or sulfated glycosaminoglycans on the cell surface play an important role in regulating many physiological functions outside the cell, but little is known about the physiological functions of the intracellular sulfated glycosaminoglycans.
[19,20], their function of participating in the innate immune response has never been reported.

On April 14, 2021, the Laboratory of Jiang Zhengfan, School of Life Sciences, Peking University, published an online research article on Immunity on the mechanism of Golgi transport required for STING "Golgi-synthesized sulfated glycosaminoglycans mediate polymerization and activation of the cGAMP sensor STING" , Revealing that mucopolysaccharide (sulfated glycosaminoglycan) is the cause of STING co-ligand and STING translocation to Golgi activation.

In this study, Dr.
Fang Run and Jiang Qifei from the laboratory of Jiang Zhengfan cleverly used the self-activating mutant of STING to induce cell death, and performed a CRISPR-Cas9-mediated genome-wide screening in diploid cells HT1080 , Found that a number of key genes involved in the synthesis of sulfated glycosaminoglycans (mucopolysaccharides) are highly enriched (Figure 1). Knockout of these key genes for the synthesis of mucopolysaccharides makes the cells lose their responsiveness to STING.

Subsequently, they constructed gene knockout cell lines in different cells to further prove that the lack of mucopolysaccharide synthesis in the Golgi can severely weaken the activation of the cGAS-STING pathway.

In order to study whether sulfated glycosaminoglycans act directly on STING, they selected the core protein SRGN loaded with sulfated glycosaminoglycans as the indicator of sulfated glycosaminoglycans, and found that STING and SRGN had DNA virus-induced co-localization and Interaction (Figure 2A).

In vitro pull-down experiments with magnetic beads carrying heparin also showed that STING and heparin have a direct interaction, and STING that binds cGAMP has stronger heparin binding ability (Figure 2B).

Sulfated glycosaminoglycans are loaded with a large number of negatively charged sulfated modifications, so they function mainly by binding to positively charged amino acid residues on the target protein through electrostatic interactions.

Since the intracellular sulfated glycosaminoglycans are encapsulated in the Golgi lumen, they speculated that the three positively charged amino acid residues (H42, R45, H50) located between the first and second transmembrane regions of STING may be involved in the binding of sulfated Glycosaminoglycans (Figure 2C).

Mutation of these three positively charged amino acids will significantly weaken the activation of STING (Figure 2D).

Further mutation of the polar amino acids located in the first and second transmembrane regions and the third and fourth transmembrane regions of STING will gradually reduce the activity of STING until it disappears completely.

Interestingly, the restoration of any one of the three positively charged amino acids H42, R45, and H50 can restore part of the activity of the completely inactive mutant.

Reverting position 42 or position 50 to lysine or arginine with stronger electrical properties, the activity of STING will gradually increase as the positive charge of the restored amino acid increases.

Interestingly, the introduction of positively charged amino acids (P110R) in the third and fourth transmembrane regions that do not contain positively charged amino acids will also partially revert the activity of STING.

Co-immunoprecipitation experiments found that the interaction strength of various STING mutants with SRGN is very consistent with the activity of these mutants; more importantly, by introducing additional positively charged arginine (Y46R/H50R, H50R/P110R, etc.
) will make STING constitutively bind to sulfated glycosaminoglycans and self-activate (Figure 2E).

These results indicate that the binding ability of sulfated glycosaminoglycans is one of the determinants of whether STING can be activated and the intensity of activation.

In order to further study how sulfated glycosaminoglycans mediate the activation of STING, they found through in vitro experiments that a variety of sulfated glycosaminoglycans and purified full-length STING protein and TBK1 protein can induce STING to different degrees when co-incubated.
The formation of aggregates and phosphorylation of TBK1, of which heparin sulfate (HS) and heparin (Hp) have the best activation effects (Figure 2E).

Further experiments found that the activation of STING by sulfated glycosaminoglycans is sugar chain length dependent.
The shortest sugar chain formed by four monosaccharides has the ability to activate STING, and this activation depends on the O-sulfation of the sugar chain.
Retouch.

More importantly, through the analysis of STING from four sources of human, mouse, bat and chicken, it is found that the mechanism of sulfated glycosaminoglycan-mediated STING activation is highly conserved in evolution.

Finally, they demonstrated through mouse experiments that the synthesis of sulfated glycosaminoglycans is critical for mice to resist infection by the DNA virus VACV.

In summary, the study found that the sulfated glycosaminoglycans synthesized in the Golgi are essential for the activation of STING-TBK1 and are very conservative: the sGAGs in the Golgi directly bind to the translocated STING, which triggers the increase in STING like a hinge.
Polymerization, which in turn recruits and promotes TBK1 autophosphorylation and downstream pathway activation (Figure 3).

This discovery reveals why STING needs to leave the intranet and translocate to the Golgi body to activate this puzzle that has troubled for many years.
It is expected to open up the "last mile" of STING activation. In addition, this research fills the gap in the study of the intracellular physiological functions of sulfated glycosaminoglycans, reveals the key immunological functions of biopolysaccharides in cells, and provides new ideas for future biopolysaccharides in antiviral and antitumor clinical practice.
clue.

The postdoctoral fellow of Peking University's Academy of Sciences and 2015 doctoral student Jiang Qifei are the co-first authors of this article, and Professor Jiang Zhengfan from the Academy of Sciences/Peking University-Tsinghua Life Sciences Joint Center is the corresponding author.

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Original link: https://doi.
org/10.
1016/j.
celrep.
2020.
108053 Platemaker: Qi Jiang