Nod2-Nodosome in a Cell-Free System: Implications in Pathogenesis and Drug Discovery for Blau Syndrome and Early-Onset Sarcoidosis

Nucleotide-binding oligomerization domain-containing protein (Nod) 2 is an intracellular pattern recognition receptor, which recognizes muramyl dipeptide (N-Acetylmuramyl-L-Alanyl-D-Isoglutamine: MDP), a bacterial peptidoglycan component, and makes a NF-κB-activating complex called nodosome with adaptor protein RICK (RIP2/RIPK2). Nod2 mutants are associated with the autoinflammatory diseases, Blau syndrome (BS)/early-onset sarcoidosis (EOS). For drug discovery of BS/EOS, we tried to develop Nod2-nodosome in a cell-free system. FLAG-tagged RICK, biotinylated-Nod2, and BS/EOS-associated Nod2 mutants were synthesized, and proximity signals between FLAG-tagged and biotinylated proteins were detected by amplified luminescent proximity homogeneous assay (ALPHA). Upon incubation with MDP, the ALPHA signal of interaction between Nod2-WT and RICK was increased in a dose-dependent manner. The ALPHA signal of interaction between RICK and the BS/EOS-associated Nod2 mutants was more significantly increased than Nod2-WT. Notably, the ALPHA signal between Nod2-WT and RICK was increased upon incubation with MDP, but not when incubated with the same concentrations, L-alanine, D-isoglutamic acid, or the MDP-D-isoform. Thus, we successfully developed Nod2-nodosome in a cell-free system reflecting its function in vivo, and it can be useful for screening Nod2-nodosome-targeted therapeutic molecules for BS/EOS and granulomatous inflammatory diseases.

It was also discovered that an autoinflammatory disease, Blau syndrome (BS)/early-onset sarcoidosis (EOS), was caused by a point mutation of Nod2, encoding a constitutively active form, resulting in NF-B activation [9][10][11]. BS/EOS is a systemic granulomatous disease, and patients with BS/EOS have nonnecrotizing granulomas of the skin, eyes, and joints [12]. Other granulomatous inflammatory diseases were similarly reported to be associated with Nod2 [13,14]. Therefore, Nod2 has been thought to be involved in granulomatous disorders [15] and may be an attractive drug target for treatment of these chronic inflammatory granulomatous diseases.
Thus, we aimed to develop a reconstituted protein-protein interaction assay system between wild-type Nod2 and the BS/EOS-associated mutants of Nod2 and RICK in a cell-free system, further called the reconstituted Nod2-nodosome in a cell-free system [16].

Western Blotting Analysis.
A total of 1.5 g of synthetic protein was subjected to SDS-PAGE followed by Western blotting analysis. Protein detection on the blotting membranes was performed using anti-FLAG mAb M2 (Sigma-Aldrich, St. Louis, MO, USA), followed by peroxidaseconjugated affinity-purified F(ab ) 2 fragment of goat antimouse IgG, F(ab ) 2 fragment-specific (Jackson ImmunoResearch, West Grove, PA, USA) for FLAG-tagged proteins, or HRP-conjugated streptavidin (Nacalai, Kyoto, Japan) for biotinylated proteins.

Statistics.
Results are presented as the mean and standard deviation. Levels of significance were evaluated using Student's -test. A value < 0.05 was considered statistically significant.

MDP-Induced Interaction between Nod2 and RICK in Nodosome in a Cell-Free
System. Nod2-WT-Btn was coprecipitated with FLAG-RICK-WT when incubated with 5.33 mg/mL MDP, but not without MDP in pull-down assay (Figure 2(a)). The baseline ALPHA signal of Nod2-WT-Btn and FLAG-RICK-WT interaction was approximately 2000 counts, with no stimulation (Figure 2(b)). The ALPHA signal of interaction between Nod2-WT-Btn and FLAG-RICK-WT increased upon incubation with 5.33 mg/mL MDP (Sigma-Aldrich, St. Louis, MO, USA) ( < 0.01), whereas no increase was detected upon incubation with the same amount (5.33 mg/mL) of N-Acetylmuramyl-D-Alanyl-D-Isoglutamine (MDP-D-isomer) (Invivogen, San Diego, CA, USA) (Figure 2(b)). The ALPHA signal of interaction between Nod2-CARDs-Btn and FLAG-RICK-WT increased without MDP ( < 0.01). Furthermore, the ALPHA signal of interaction between Nod2-CARDs-Btn and FLAG-RICK-CARD without MDP was significantly increased ( < 0.01), whereas the ALPHA signal of interaction between Nod2-WT-Btn and FLAG-RICK-CARD was not increased and was similar to baseline levels (Figure 2(b)).

The MDP Degradation Products Did Not Induce Interaction between Nod2 and RICK in the Cell-Free System.
Using the Nod2-nodosome in a cell-free system established above, we assessed whether MDP degradation products, such as L-alanine, D-isoglutamic acid, and N-acetylglucosamine (GlcNAc), could induce interaction between Nod2 and RICK in the cell-free system. No increased signals were observed upon incubation with a negative control (−), MDP-D-isomer, L-alanine, D-isoglutamic acid, and GlcNAc (Figure 2(c)).   Figure 2: Construction of Nod2-nodosome containing the BS/EOS-associated mutation in a cell-free system. Synthetic protein-protein interactions were detected by pull-down assay and amplified luminescent proximity homogeneous assay (ALPHA). (a) Biotinylated-Nod2-WT (Nod2-WT-Btn) and 1 g FLAG-tagged RICK-WT (FLAG-RICK-WT) lysed in 300 L NP-40 buffer were precipitated with 20 L streptavidin-conjugated agarose beads with or without MDP. The precipitations were subjected to SDS-PAGE and immunoblotting. Detection on the blotting membranes was performed using anti-FLAG mAb M2 or anti-Nod2 mAb. (b) A total of 100 ng of each protein indicated was incubated with 5 g/mL anti-FLAG mAb M2, 16.67 g/mL protein-A-conjugated ALPHA acceptor beads, and 16.67 g/mL streptavidinconjugated ALPHA donor beads for 24 hours, with or without 5.33 mg/mL MDP or 5 mg/mL N-Acetylmuramyl-D-Alanyl-D-Isoglutamine (MDP-D-isomer). Responses (counts) were measured using the EnSpire6 Multimode Plate Reader. (c) Activation of Nod2-nodosome in a cell-free system by MDP degradation components. Interactions between Nod2-WT-Btn and FLAG-RICK-WT were detected by ALPHA. A total of 100 ng of Nod2-WT-Btn and FLAG-RICK-WT were incubated with 5 g/mL anti-FLAG mAb M2, 16.67 g/mL protein-A-conjugated ALPHA acceptor beads and 16.67 g/mL streptavidin-conjugated ALPHA donor beads for 24 hours, without or with 5.33 mg/mL MDP, 5.33 mg/mL MDP (D-isomer), 5.33 mg/mL N-acetylglucosamine (GlcNAc), 5.33 mg/mL L-alanine (L-Ala), or 5.33 mg/mL D-isoglutamine (D-isoGlu). Responses (counts) were measured using the EnSpire Multimode Plate Reader. The results are representative of three independent experiments and given as means ± standard deviation from triplicate wells. (d) A total of 100 ng of Nod2-WT-Btn, or Nod2-R334W-Btn, or Nod2-N670K-Btn with FLAG-RICK-WT was incubated with 5 g/mL anti-FLAG mAb M2, 16.67 g/mL protein-A-conjugated ALPHA acceptor beads, and 16.67 g/mL streptavidin-conjugated ALPHA donor beads for 24 hours, with or without 5.33 mg/mL or 13.33 mg/mL MDP or 13.33 mg/mL MDP-D-isomer. The results are representative of three independent experiments and given as means ± standard deviation from triplicate wells. CARD, caspase recruitment domain; MDP, muramyl dipeptide; WT, wild-type; P, precipitation; WB, western blot. * value < 0.05 and * * value < 0.01 were considered statistically significant using Student's -test. 6 The Scientific World Journal in a cell-free system with or without BS/EOS-associated mutations, we assessed the interaction between FLAG-RICK-WT and Nod2-WT-Btn, Nod2-R334W-Btn, or Nod2-N670K-Btn, in a cell-free system. The ALPHA signal between FLAG-RICK-WT and Nod2-WT-Btn increased upon incubation with 5.33 mg/mL and 13.33 mg/mL MDP, in a dose-dependent manner (Figure 2(d)). The baseline ALPHA signal between the BS/EOS-associated Nod2 mutants, such as Nod2-R334W-Btn and Nod2-N670K-Btn, and FLAG-RICK-WT with no stimulation was higher than that between Nod2-WT-Btn and FLAG-RICK-WT ( < 0.01, < 0.01, resp.) (Figure 2(d), left columns). Upon incubation with 5.33 mg/mL, the ALPHA signal between Nod2-WT-Btn and FLAG-RICK-WT increased in a dose-dependent manner, and signals between Nod2-R334W-Btn or Nod2-N670K-Btn and FLAG-RICK-WT were more significantly increased ( < 0.05, < 0.01, resp.) (Figure 2(d), center columns). Upon incubation with 13.33 mg/mL MDP, the ALPHA signal between Nod2-WT-Btn and FLAG-RICK-WT increased in a dosedependent manner, and signals between Nod2-R334W-Btn or Nod2-N670K-Btn and FLAG-RICK-WT were more significantly increased ( < 0.05, < 0.01, resp.) (Figure 2(d), right columns).

Discussion
Autoinflammatory syndromes are known to stem from aberrant innate immune complex disorders, some of which are known to be due to the excessive innate immune activation by mutations of the pathogen-recognizing receptors [17]. BS/EOS is one of the autoinflammatory syndromes, which is characterized by systemic chronic granulomatous inflammation, resulting in nonnecrotizing granuloma of the skin, eyes, and joints [12]. The susceptibility gene product of BS/EOS has been identified to be Nod2/CARD15 [11,18].
In the present study, we developed Nod2-nodosomes containing wild-type and the BS/EOS-associated mutants R334W and N670K in a cell-free system, assessed by ALPHA. In general, recombinant protein synthesis has been used for bacterial or viral systems, such as Escherichia coli, for structural and functional studies. However, synthetic proteins sometimes are unable to be investigated, because many proteins are expressed in an insoluble form [19]. Therefore, we employed the wheat germ cell-free protein synthesis system, and Nod2-WT-Btn, Nod2-R334W-Btn, Nod2-N670K-Btn, Nod2-CARDs, FLAG-RICK-WT, and FLAG-RICK-CARD were successfully synthesized (Figure 1). To date, we reported the generation of AIM2-inflammasome in a cell-free system, which is another intracellular pattern recognition receptor with adaptor ASC oligomerization, which was suitable for wheat germ cell-free protein synthesis [20]. These results suggested that the wheat germ cell-free protein synthesis system has an advantage for reconstituting intracellular pattern recognition receptor complexes for resolving innate immune complex-mediated autoinflammatory diseases.
First, we confirmed whether the Nod2-nodosome could recognize the bacterial cell-wall component MDP, which is a previously reported Nod2-ligand [6,7]. As shown in Figure 2(a), Nod2-WT-Btn was coprecipitated with FLAG-RICK-WT when incubated with MDP, but not without MDP in pull-down assay (Figure 2(a)). That was consistent with ALPHA in Figure 2(b). As shown in Figure 2(b), the ALPHA signal of interaction between Nod2-WT-Btn and FLAG-RICK-WT was increased upon incubation with MDP, but not without a ligand or with the MDP-D-isomer or MDP-degraded components (Figures 2(b) and 2(c)). These results indicated that Nod2-nodosome in a cell-free system is capable of detecting for its specific ligand, MDP.
Next, we tested whether the BS/EOS-associated Nod2 mutations induced increased activity. As shown in Figure 2(d), the ALPHA signal of interaction between FLAG-RICK-WT and Nod2-R334W-Btn or Nod2-N670K-Btn was much higher than when with Nod2-WT-Btn, with or without MDP stimulation (Figure 2(d)). These results are consistent with previous studies on the clinical manifestations of BS/EOS [21], suggesting that Nod2-nodosome in a cell-free system may suitably reflect the characteristics of endogenous Nod2-nodosome.
It is noted that there are some limitations of nodosome in a cell-free system. Only the initial event of Nod2-RICK interaction is detected. Upstream, downstream, and regulatory events in the activation of Nod2 cannot be measured with the nodosome in a cell-free system, as presented.
In conclusion, we developed Nod2-nodosome in a cellfree system, assessed by ALPHA. Nod2-nodosomes containing the BS/EOS-associated mutations Nod2-R334W and Nod2-N670K were more sensitive to MDP than Nod2-WT. Therefore, the Nod2-nodosomes in a cell-free system developed in the present study can be a useful tool for further investigation of pathogenesis of BS/EOS and discovery of its therapeutics.

Disclosure
The funders had no role in the study design, data collection, and analysis, decision to publish, or preparation of the paper.