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Hydrolysis of 2'3'-cGAMP by ENPP1 and design of nonhydrolyzable analogs.

Li L, Yin Q, Kuss P, Maliga Z, Millán JL, Wu H, Mitchison TJ - Nat. Chem. Biol. (2014)

Bottom Line: We report activity-guided partial purification and identification of ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP1) to be the dominant 2'3'-cGAMP hydrolyzing activity in cultured cells.We synthesized a hydrolysis-resistant bisphosphothioate analog of 2'3'-cGAMP (2'3'-cG(s)A(s)MP) that has similar affinity for hSTING in vitro and is ten times more potent at inducing IFN-β secretion from human THP1 monocytes.Studies in mouse Enpp1(-/-) lung fibroblasts indicate that resistance to hydrolysis contributes substantially to its higher potency. 2'3'-cG(s)A(s)MP is therefore improved over natural 2'3'-cGAMP as a model agonist and has potential as a vaccine adjuvant and cancer therapeutic.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
Agonists of mouse STING (TMEM173) shrink and even cure solid tumors by activating innate immunity; human STING (hSTING) agonists are needed to test this therapeutic hypothesis in humans. The endogenous STING agonist is 2'3'-cGAMP, a second messenger that signals the presence of cytosolic double-stranded DNA. We report activity-guided partial purification and identification of ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP1) to be the dominant 2'3'-cGAMP hydrolyzing activity in cultured cells. The hydrolysis activity of ENPP1 was confirmed using recombinant protein and was depleted in tissue extracts and plasma from Enpp1(-/-) mice. We synthesized a hydrolysis-resistant bisphosphothioate analog of 2'3'-cGAMP (2'3'-cG(s)A(s)MP) that has similar affinity for hSTING in vitro and is ten times more potent at inducing IFN-β secretion from human THP1 monocytes. Studies in mouse Enpp1(-/-) lung fibroblasts indicate that resistance to hydrolysis contributes substantially to its higher potency. 2'3'-cG(s)A(s)MP is therefore improved over natural 2'3'-cGAMP as a model agonist and has potential as a vaccine adjuvant and cancer therapeutic.

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ENPP1 is an efficient hydrolase for 2′3′-cGAMP(a) Ion dependency and (b) pH preference of the dominant hydrolase activity in MDA-MB231 cells. (c) Activity of recombinant ENPP1 alone or (d) coupled to alkaline phosphatase in buffer condition: 0.2% NP-40, 20 mM Tris-HCl, pH 9.0, 2 mM Ca2+, 200 μM Zn2+. (e) Kinetics of 2′3′-cGAMP and ATP hydrolysis by recombinant ENPP1. 1 nM ENPP1 was tested in the same buffer condition as (f) and (g). Data are presented as mean and standard error.
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Figure 2: ENPP1 is an efficient hydrolase for 2′3′-cGAMP(a) Ion dependency and (b) pH preference of the dominant hydrolase activity in MDA-MB231 cells. (c) Activity of recombinant ENPP1 alone or (d) coupled to alkaline phosphatase in buffer condition: 0.2% NP-40, 20 mM Tris-HCl, pH 9.0, 2 mM Ca2+, 200 μM Zn2+. (e) Kinetics of 2′3′-cGAMP and ATP hydrolysis by recombinant ENPP1. 1 nM ENPP1 was tested in the same buffer condition as (f) and (g). Data are presented as mean and standard error.

Mentions: We next sought a bulk tissue source to purify and identify the hydrolase. Liver is a classic source for organelle and enzyme purification. We detected high activity in mouse livers, again was in the 7000 G pellet (Supplementary Fig. 3a). Because a calf liver provides more starting material, we performed differential centrifugation followed by detergent solubilization, anion exchange fractionation, and size exclusion fractionation on calf liver extract (Supplementary Fig. 3b-h). Fraction 26 from the last purification step has low protein concentration undetectable by SDS-PAGE gel, but high hydrolytic activity. Mass spectrometry analysis of this fraction revealed 377 proteins and a top ranking protein annotated as ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP1) appeared to be the most plausible candidate (Supplementary Dataset)26,27. ENPP1 is a plasma membrane and ER lumen protein28, which agrees with our cell fractionation results. In addition, the structure of ENPP1 revealed that it has a Ca2+ binding domain and chelates two Zn2+ ions in the catalytic site29. Consistent with the ion dependency of ENPP1, addition of Ca2+and Zn2+ both boosted the hydrolase activity in the active fractions (Fig. 2a and Supplementary Fig. 4a). Moreover, we found the optimal pH for the liver hydrolase activity to be 9.0, which agrees with that of ENPP1 (Fig. 2b and Supplementary Fig. 4b). We noticed that 2′3′-cGAMP migrates differently at pH 6.5 and 7.0 compared to other pH conditions. This is due to changed polarity of 2′3′-cGAMP at these conditions rather than its hydrolysis state because the same shift can be observed without lysate (Supplementary Fig. 5a). Nevertheless, 2′3′-cGAMP and the hydrolysis product at the baseline are well separated in all conditions. Finally, recombinant ENPP1 at nano-molar concentrations efficiently degraded 2′3′-cGAMP (Fig. 2c). The degradation product co-migrates with AMP on TLC. Addition of alkaline phosphatase converted the product to inorganic phosphate, showing that no phosphodiester bonds remained after ENPP1 action (Fig. 2d). Together, our results suggest that ENPP1 accounts for the observed hydrolase activity.


Hydrolysis of 2'3'-cGAMP by ENPP1 and design of nonhydrolyzable analogs.

Li L, Yin Q, Kuss P, Maliga Z, Millán JL, Wu H, Mitchison TJ - Nat. Chem. Biol. (2014)

ENPP1 is an efficient hydrolase for 2′3′-cGAMP(a) Ion dependency and (b) pH preference of the dominant hydrolase activity in MDA-MB231 cells. (c) Activity of recombinant ENPP1 alone or (d) coupled to alkaline phosphatase in buffer condition: 0.2% NP-40, 20 mM Tris-HCl, pH 9.0, 2 mM Ca2+, 200 μM Zn2+. (e) Kinetics of 2′3′-cGAMP and ATP hydrolysis by recombinant ENPP1. 1 nM ENPP1 was tested in the same buffer condition as (f) and (g). Data are presented as mean and standard error.
© Copyright Policy
Related In: Results  -  Collection

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Figure 2: ENPP1 is an efficient hydrolase for 2′3′-cGAMP(a) Ion dependency and (b) pH preference of the dominant hydrolase activity in MDA-MB231 cells. (c) Activity of recombinant ENPP1 alone or (d) coupled to alkaline phosphatase in buffer condition: 0.2% NP-40, 20 mM Tris-HCl, pH 9.0, 2 mM Ca2+, 200 μM Zn2+. (e) Kinetics of 2′3′-cGAMP and ATP hydrolysis by recombinant ENPP1. 1 nM ENPP1 was tested in the same buffer condition as (f) and (g). Data are presented as mean and standard error.
Mentions: We next sought a bulk tissue source to purify and identify the hydrolase. Liver is a classic source for organelle and enzyme purification. We detected high activity in mouse livers, again was in the 7000 G pellet (Supplementary Fig. 3a). Because a calf liver provides more starting material, we performed differential centrifugation followed by detergent solubilization, anion exchange fractionation, and size exclusion fractionation on calf liver extract (Supplementary Fig. 3b-h). Fraction 26 from the last purification step has low protein concentration undetectable by SDS-PAGE gel, but high hydrolytic activity. Mass spectrometry analysis of this fraction revealed 377 proteins and a top ranking protein annotated as ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP1) appeared to be the most plausible candidate (Supplementary Dataset)26,27. ENPP1 is a plasma membrane and ER lumen protein28, which agrees with our cell fractionation results. In addition, the structure of ENPP1 revealed that it has a Ca2+ binding domain and chelates two Zn2+ ions in the catalytic site29. Consistent with the ion dependency of ENPP1, addition of Ca2+and Zn2+ both boosted the hydrolase activity in the active fractions (Fig. 2a and Supplementary Fig. 4a). Moreover, we found the optimal pH for the liver hydrolase activity to be 9.0, which agrees with that of ENPP1 (Fig. 2b and Supplementary Fig. 4b). We noticed that 2′3′-cGAMP migrates differently at pH 6.5 and 7.0 compared to other pH conditions. This is due to changed polarity of 2′3′-cGAMP at these conditions rather than its hydrolysis state because the same shift can be observed without lysate (Supplementary Fig. 5a). Nevertheless, 2′3′-cGAMP and the hydrolysis product at the baseline are well separated in all conditions. Finally, recombinant ENPP1 at nano-molar concentrations efficiently degraded 2′3′-cGAMP (Fig. 2c). The degradation product co-migrates with AMP on TLC. Addition of alkaline phosphatase converted the product to inorganic phosphate, showing that no phosphodiester bonds remained after ENPP1 action (Fig. 2d). Together, our results suggest that ENPP1 accounts for the observed hydrolase activity.

Bottom Line: We report activity-guided partial purification and identification of ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP1) to be the dominant 2'3'-cGAMP hydrolyzing activity in cultured cells.We synthesized a hydrolysis-resistant bisphosphothioate analog of 2'3'-cGAMP (2'3'-cG(s)A(s)MP) that has similar affinity for hSTING in vitro and is ten times more potent at inducing IFN-β secretion from human THP1 monocytes.Studies in mouse Enpp1(-/-) lung fibroblasts indicate that resistance to hydrolysis contributes substantially to its higher potency. 2'3'-cG(s)A(s)MP is therefore improved over natural 2'3'-cGAMP as a model agonist and has potential as a vaccine adjuvant and cancer therapeutic.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
Agonists of mouse STING (TMEM173) shrink and even cure solid tumors by activating innate immunity; human STING (hSTING) agonists are needed to test this therapeutic hypothesis in humans. The endogenous STING agonist is 2'3'-cGAMP, a second messenger that signals the presence of cytosolic double-stranded DNA. We report activity-guided partial purification and identification of ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP1) to be the dominant 2'3'-cGAMP hydrolyzing activity in cultured cells. The hydrolysis activity of ENPP1 was confirmed using recombinant protein and was depleted in tissue extracts and plasma from Enpp1(-/-) mice. We synthesized a hydrolysis-resistant bisphosphothioate analog of 2'3'-cGAMP (2'3'-cG(s)A(s)MP) that has similar affinity for hSTING in vitro and is ten times more potent at inducing IFN-β secretion from human THP1 monocytes. Studies in mouse Enpp1(-/-) lung fibroblasts indicate that resistance to hydrolysis contributes substantially to its higher potency. 2'3'-cG(s)A(s)MP is therefore improved over natural 2'3'-cGAMP as a model agonist and has potential as a vaccine adjuvant and cancer therapeutic.

Show MeSH
Related in: MedlinePlus