Screening and Identification of Novel cGAS Homologues Using a Combination of in Vitro and In Vivo Protein Synthesis

Abstract

The cyclic GMP-AMP synthase (cGAS) catalyzes the synthesis of the multifunctional second messenger, cGAMP, in metazoans. Although numerous cGAS homologues are predicted in protein databases, the catalytic activity towards cGAMP synthesis has been proven for only four of them. Therefore, we selected five novel and yet uncharacterized cGAS homologues, which cover a broad range in the field of vertebrates. Cell-free protein synthesis (CFPS) was used for a pre-screening to investigate if the cGAS genes originating from higher organisms can be efficiently expressed in a bacterial expression system. As all tested cGAS variants were expressible, enzymes were synthesized in vivo to supply higher amounts for a subsequent in vitro activity assay. The assays were carried out with purified enzymes and revealed vast differences in the activity of the homologues. For the first time, the cGAS homologues from the Przewalski's horse, naked mole-rat, bald eagle, and zebrafish were proven to catalyze the synthesis of cGAMP. The extension of the list of described cGAS variants enables the acquisition of further knowledge about the structural and molecular mechanism of cGAS, potentially leading to functional improvement of the enzyme.The chemical synthesis of 2 3 -cGAMP contains eight reaction steps and suffers from low yields [8,9], whereas the enzymatic reaction reaches nearly full conversion within a few hours [6]. In recent studies, human cGAS was investigated predominantly with regards to the identification of key residues [10,11] and the kinetic mechanism [12]. Although several homologous enzymes are known, only murine cGAS [6,7], porcine cGAS [10,13], and chicken cGAS [14] received more attention. For the murine and porcine homologue, crystal structures are also available and, in parts, crucial amino acids with relevance for the catalytic function are known. Alignments of amino acid sequences of identified cGAS homologues revealed a low-sequence homology in regions without determined function [10,15]. This high level of variation and the markedly reduced activity of human cGAS in comparison to murine cGAS [11] demonstrate the possibility that other cGAS homologues might be available with enhanced properties or different characteristics and functions. For characterization, cGAS enzymes were synthesized with eukaryotic cell lines or expressed recombinantly in Escherichia coli. Eukaryotic genes tend to be difficult to express in bacterial systems, though fusion-tags, such as maltose-binding protein (MBP, 42.5 kDa) [16], glutathione S-transferase (GST, 26 kDa) [2], and small ubiquitin-like modifier (SUMO, 12 kDa) [1], were used for better solubility and functional expression of cGAS. Challenges remain to find suitable expression systems for eukaryotic protein production with good yield and purity, and under conditions conducive to functional protein studies. To realize higher throughput in heterologous protein synthesis, other methods than traditional recombinant expression can be considered.

A Multi-Enzyme Cascade Reaction for the Production of 2′3′-cGAMP

“…As shown in Table 2 , the reaction rate of 2′3′-cGAMP synthesis was the highest in the first experiment with a specific activity of 0.052 ± 0.014 U mg −1 , although higher thscGAS concentrations were used in the subsequent experiments. This value is in the typical range for the intrinsic specific activity of thscGAS, demonstrating that the thscGAS-catalyzed reaction was running close to its optimum in these enzyme cascade conditions [ 25 ]. The specific thscGAS activity with changed enzyme ratios (50:5:50:120) was only 0.012 ± 0.004 U mg −1 , which indicates that not enough ATP was provided by the cascade to fully exploit the activity of thscGAS.…”

“…In order to provide sufficient amounts of intermediates for the subsequent reaction step, the synthesis of 2′3′-cGAMP was started after 35 min by adding thscGAS, 0.5 mM GTP as the second substrate and 0.1 mg mL −1 HT DNA, required for thscGAS activation. A thscGAS concentration of 40 mg L −1 was chosen, as this concentration was commonly used in previously established assays [ 24 , 25 ]. The results of the reaction progress of the first thscGAS coupled cascade experiment are shown in Figure 2 .…”

“…The specific 2′3′-cGAMP yield related to GTP in this experiment was 0.40 ± 0.01 mol 2′3′-cGAMP per mol GTP. In screening studies with thscGAS, Rolf et al achieved twice as high specific 2′3′-cGAMP yields related to GTP of 0.79 ± 0.24 mol 2′3′-cGAMP per mol GTP, which indicates the need for further optimization [ 25 ]. One possibility would be to re-phosphorylate the dephosphorylated GTP by suitable kinases to provide it again to the thscGAS-catalyzed reaction.…”

“…This demonstrates the need to provide excess ATP for an efficient 2′3′-cGAMP synthesis. Alternatively, the 2′3′-cGAMP synthesis rate could be increased by using another cGAS homologue as it is already known that the murine cGAS homolog has an almost fourfold higher activity of 0.279 U mg −1 and could thus lead to higher synthesis rates in this multi-enzyme cascade reaction [ 25 ].…”

Cytosolic Sensors for Pathogenic Viral and Bacterial Nucleic Acids in Fish

“…Currently, specific activities of cGAS variants were determined using in vitro assay and RP-HPLC measurements of cGAMP production. Although, the zebrafish cGAS homologue has low amino acid identity to human cGAS (less than 35%), their similar activities were determined [ 179 ]. Liu and co-workers [ 160 ] reported that overexpression of zebrafish cGASa/b in HEK293T cells and zebrafish embryos significantly activated NF-κB and type I IFN signaling pathways in a STING-dependent manner, and that cGASa, but not cGASb, was involved in immunoglobulin Z-mediated mucosal immunity in gill-associated lymphoid tissue, suggesting differential functions between the two DrcGASs [ 160 ].…”