Article25 March 2004free access Regulatory regions and critical residues of NOD2 involved in muramyl dipeptide recognition Tsuyoshi Tanabe Tsuyoshi Tanabe Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, MI, USA The Gene Discovery Research Center, The National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, 305-8562 Japan Search for more papers by this author Mathias Chamaillard Mathias Chamaillard Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Yasunori Ogura Yasunori Ogura Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Li Zhu Li Zhu Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Su Qiu Su Qiu Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Junya Masumoto Junya Masumoto Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Partho Ghosh Partho Ghosh Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Anthony Moran Anthony Moran Department of Microbiology, National University of Ireland, Galway, Ireland Search for more papers by this author Martina M Predergast Martina M Predergast Department of Microbiology, National University of Ireland, Galway, Ireland Search for more papers by this author Gerard Tromp Gerard Tromp Wayne State University School of Medicine, Detroit, MI, USA Search for more papers by this author Charlene J Williams Charlene J Williams Department of Medicine, Division of Rheumatology, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Naohiro Inohara Naohiro Inohara Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Gabriel Núñez Corresponding Author Gabriel Núñez Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Tsuyoshi Tanabe Tsuyoshi Tanabe Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, MI, USA The Gene Discovery Research Center, The National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, 305-8562 Japan Search for more papers by this author Mathias Chamaillard Mathias Chamaillard Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Yasunori Ogura Yasunori Ogura Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Li Zhu Li Zhu Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Su Qiu Su Qiu Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Junya Masumoto Junya Masumoto Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Partho Ghosh Partho Ghosh Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Anthony Moran Anthony Moran Department of Microbiology, National University of Ireland, Galway, Ireland Search for more papers by this author Martina M Predergast Martina M Predergast Department of Microbiology, National University of Ireland, Galway, Ireland Search for more papers by this author Gerard Tromp Gerard Tromp Wayne State University School of Medicine, Detroit, MI, USA Search for more papers by this author Charlene J Williams Charlene J Williams Department of Medicine, Division of Rheumatology, Thomas Jefferson University, Philadelphia, PA, USA Search for more papers by this author Naohiro Inohara Naohiro Inohara Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Gabriel Núñez Corresponding Author Gabriel Núñez Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Author Information Tsuyoshi Tanabe1,6, Mathias Chamaillard1, Yasunori Ogura1, Li Zhu1, Su Qiu1, Junya Masumoto1, Partho Ghosh2, Anthony Moran3, Martina M Predergast3, Gerard Tromp4, Charlene J Williams5, Naohiro Inohara1 and Gabriel Núñez 1 1Department of Pathology and Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, MI, USA 2Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA 3Department of Microbiology, National University of Ireland, Galway, Ireland 4Wayne State University School of Medicine, Detroit, MI, USA 5Department of Medicine, Division of Rheumatology, Thomas Jefferson University, Philadelphia, PA, USA 6The Gene Discovery Research Center, The National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, 305-8562 Japan *Corresponding author. Department of Pathology, University of Michigan Medical School, 4219 CCGC 0938, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0938, USA. Tel.: +1 734 764 8514; Fax: +1 734 647 9654; E-mail: [email protected] The EMBO Journal (2004)23:1587-1597https://doi.org/10.1038/sj.emboj.7600175 These authors share first authorship These authors share senior authorship PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Multiple genetic variants of CARD15/NOD2 have been associated with susceptibility to Crohn's disease and Blau syndrome. NOD2 recognizes muramyl dipeptide (MDP) derived from bacterial peptidoglycan (PGN), but the molecular basis of recognition remains elusive. We performed systematic mutational analysis to gain insights into the function of NOD2 and molecular mechanisms of disease susceptibility. Using an archive of 519 mutations covering ∼50% of the amino-acid residues of NOD2, the essential regulatory domains and specific residues of NOD2 involved in recognition of MDP were identified. The analysis revealed distinct roles for N-terminal and C-terminal leucine-rich repeats (LRRs) in the modulation of NOD2 activation and bacterial recognition. Within the C-terminal LRRs, variable residues predicted to form the β-strand/βturn structure were found to be essential for the response to MDP. In addition, we analyzed NOD1, a NOD2-related protein, revealing conserved and nonconserved amino-acid residues involved in PGN recognition. These results provide new insights into the molecular function and regulation of NOD2 and related NOD family proteins. Introduction The susceptibility to genetic diseases is largely dependent on the physiological consequence of genetic alterations. In certain disorders including neurofibromatosis and Crohn's disease (CD), the genetic alteration involves highly mutated disease-associated proteins (Rasmussen and Friedman, 2000; Hugot et al, 2001). In CD, a common chronic inflammatory disorder of the intestinal tract, nearly 40 variants of the CARD15 gene have been detected (Hugot et al, 2001). NOD2, the product of CARD15, is a member of a growing family of proteins, called NODs (for nucleotide-binding oligomerization domains), that have been implicated in the regulation of immune responses and cell death in animals and plants (Girardin et al, 2002; Inohara and Nunez, 2003). NOD2 acts as a bacterial peptidoglycan (PGN) recognition molecule through specific detection of the conserved muramyl dipeptide (MDP) structure (Bonen et al, 2003; Chamaillard et al, 2003c; Girardin et al, 2003a; Inohara et al, 2003). Three major CD-associated NOD2 mutations, R702W, G908R and L1007fsinsC (Hugot et al, 2001; Ogura et al, 2001a), and multiple rare variants (Chamaillard et al, 2003c) have been found to be deficient in their ability to sense PGN and/or synthetic MDP (Girardin et al, 2003a; Inohara et al, 2003). Homozygosity and compound heterozygosity increase up to ∼40-fold the genotype relative risk for CD compared to simple heterozygosity (∼2–4-fold) (Hampe et al, 2001; Hugot et al, 2001; Ogura et al, 2001a). The human NOD protein family contains approximately 20 members including NOD1, NOD2, Cryopyrin, Apaf-1 and CIITA (Inohara and Nunez, 2003). The great majority of NODs contain an N-terminal effector domain, a centrally located NOD and C-terminal leucine-rich repeats (LRRs) (Inohara and Nunez, 2003). NOD1 and NOD2 have one and two caspase-recruitment domains (CARDs), respectively, as N-terminal effector domains and activate NF-κB through interaction with the downstream factor RICK (also called RIP2 and CARDIAK) (Bertin et al, 1999; Inohara et al, 1999; Ogura et al, 2001b). Oligomerization of NOD1, and presumably of NOD2, promotes the proximity of RICK molecules and of the I-κB kinase (IKK) subunits, leading to IKK and NF-κB activation (Chamaillard et al, 2003a; Inohara and Nunez, 2003). In addition to CD, recurrent missense mutations (i.e. R334Q-W and L469F) in the NOD domain of NOD2 have been associated with Blau syndrome (BS), a monogenic, dominantly inherited disease characterized by early-onset granulomatous arthritis (Miceli-Richard et al, 2001). The BS-causing mutations exhibit enhanced basal NF-κB activity compared to wild-type NOD2 (Chamaillard et al, 2003c). Three other closely related autosomal-dominant diseases, familial cold autoinflammatory syndrome (FCAS), Muckle–Wells syndrome (MWS), and chronic infantile neurological cutaneous and articular syndrome (CINCA), are associated with missense mutations that localize to the NOD region of Cryopyrin (Chamaillard et al, 2003a; Inohara and Nunez, 2003). Moreover, mutations in the LRRs of CIITA are associated with type II bare lymphocyte syndrome, indicating an essential role for the LRRs in CIITA function (Chamaillard et al, 2003a; Inohara and Nunez, 2003). Similarly, plant NOD proteins are a class of disease-resistance (R) proteins that recognize pathogens through their LRRs and induce a signalling response against pathogens (Dangl and Jones, 2001; Meyers et al, 2003). In the present study, we generated a comprehensive library of NOD2 and NOD1 variants to study the molecular basis of bacteria-induced NF-κB stimulation. In this study, 519 amino-acid residues of NOD2 were mutated. Functional analyses revealed mechanisms of NOD2 regulation as well as conserved and nonconserved mechanisms whereby NOD1 and NOD2 mediate the recognition of PGN. Results Construction of a library of NOD2 mutants To define amino-acid residues that are important for NOD2 function and disease susceptibility, we designed an approach to introduce systematically point mutations in the entire coding region of CARD15/NOD2 at a controlled rate by polymerase chain reactions (Shafikhani et al, 1997). The approach combines random mutagenesis and systematic functional analysis of each generated mutant clones. Because NOD2 is composed of 1040 amino acids, the mutants were constructed with four different cassettes separated by unique restriction sites (Figure 1A). Under our experimental conditions, the mutant library for each cassette contained an average of three nucleotide substitutions per 1000 base pairs (range 1–7) resulting in ∼2 amino-acid substitutions per cassette (data not shown). In total, 806 nucleotide substitutions in the coding region were analyzed resulting in 519 amino-acid changes (∼50% of the NOD2 residues; Figure 1B). Our present study involved 338 independent clones of which 148 contained single amino-acid substitutions and 190 contained multiple (range 2–5) amino-acid replacements in NOD2. A summary of all NOD2 mutant clones including the type of mutation, location of the mutation and in vitro functional activity is shown in Figure 1B. The ability of each mutant to induce MDP-dependent and -independent activation of NF-κB was determined using a luciferase reporter NF-κB assay (Inohara et al, 2001; Ogura et al, 2001b). Briefly, mutant and wild-type NOD2 clones were transiently expressed in HEK293T cells in the presence or absence of MDP. The analysis was primarily based on the 127 clones with single missense mutations and the 59 clones with nonsense and frameshift mutations. However, the results of all generated clones are provided in Supplementary Tables 1, 2 and 3. Figure 1.Strategy for NOD2 mutagenesis and summary of studied NOD2 mutations. (A) Schematic representation of NOD2 including mutagenized cassettes (I–IV). The location of CARDs, NOD domain and leucine-rich repeat domain (LRD) are shown. Numbers represent the position of amino-acid residues. Restriction enzyme sites flanking each cassette are shown. (B) Summary of NOD2 mutations generated and analyzed. WT, wild-type; ND, not determined. Download figure Download PowerPoint CARDs of NOD2 in RICK-dependent NF-κB activation We mutated ∼57% (127/220) of the amino acids in the CARDs of NOD2 (Figure 1B and Supplementary Table 1). The analysis included 14 mutants with only one amino-acid substitution in the CARDs of NOD2, of which seven (Q31H, E69K, T91I, A106V, L145P, P177S and R180K) exhibited complete loss and/or greatly reduced activity in response to MDP (Supplementary Table 1 and Figure 2B), indicating that these CARD residues are essential for NF-κB activation. Representative results from a mutant clone with wild-type phenotype (M152L) and loss-of-function mutants with single amino-acid replacements in CARD1 (Q31H, E69K and A106V) or CARD2 (L145P and R180K) are shown in Figure 2B and/or in Supplementary Table 1. Immunoblotting analysis revealed that most of the loss-of-function mutants were expressed at levels similar to those of the wild-type protein (Figure 2C and Supplementary Table 1). However, some of the CARD point mutants including F161I, P177S and R180K were not expressed, suggesting that these mutations are associated with reduced protein stability (Figure 2C and Supplementary Table 1). Figure 2.Mutational analyses of the CARD of NOD2. (A) Schematic representation of the N-terminus region of NOD2. Numbers indicate the position of amino-acid residues. Loss-of-function mutations are shown in black. Clones with wild-type activity are depicted in gray and mutations found in complex mutant clones (i.e. more than one amino-acid substitution) with wild-type activity are shown in gray italic. Mutations associated with reduced expression are labelled with the symbol a. Mutations found in Crohn's patients are labelled with the symbol b. (B) NF-κB activity of representative CARD mutants. E778K and ΔLRR are shown as controls. Values represent the mean of normalized data±s.d. of triplicate cultures. (C) Interaction of NOD2 mutants with RICK. Extracts from HEK293T cells expressing indicated mutants were immunoprecipitated with anti-Myc antibody and immunoblotted with anti-NOD2 antibody. IP, immunoprecipitation; total, immunoblotting of total lysates. A black dot denotes the mobility shift of the phosphorylated form of RICK. (D) Inhibition of RICK-induced NF-κB activation by CARD mutants and PEA15 control plasmid. Values represent the mean of normalized data±s.d. of triplicate cultures. (E) Specific inhibition of MDP-induced NOD2-mediated NF-κB by the L145P mutant. An LPS preparation containing MDP-like activity (closed bars) was used in this assay. (F) Inhibition of MDP-induced NF-κB activation by the L145P mutant and dominant-negative form of IKKγ in HEK293T cells. DN denotes dominant-negative form. Download figure Download PowerPoint NOD2 associates with its downstream effector RICK through a homophilic CARD–CARD interaction to activate NF-κB (Ogura et al, 2001b). To determine whether the CARD mutants interact with RICK, we carried out immunoprecipitation experiments in which NOD2 and RICK proteins were transiently coexpressed in HEK293T cells. Immunoprecipitation analyses revealed that RICK interacted with T91I and Q204R, a control mutant with wild-type phenotype, as effectively as with wild-type NOD2 molecule (Figure 2E). In contrast, the loss-of-function NOD2 mutants, Q31H, A106V and L145P, exhibited reduced association with RICK (Figure 2C). As expected, the F161I mutant, which displayed greatly reduced expression, did not immunoprecipitate with RICK (Figure 2C). RICK has been found to be phosphorylated when overexpressed in mammalian cells (Ogura et al, 2001b), although the physiological role of phosphorylated RICK remains unclear. Notably, the NOD2 mutants with impaired ability to interact with RICK did not induce the mobility shift associated with RICK phosphorylation (Figure 2C), indicating that RICK phosphorylation correlates with the ability of NOD2 to interact with RICK and with the activation of NF-κB. A dominant-negative mutant of NOD2 signalling The interaction between NOD2 and RICK appears essential for NF-κB activation (Ogura et al, 2001b). Therefore, we tested if the loss-of-function CARD mutants could inhibit the activity induced through the NOD2 signalling pathway. The analysis revealed that L145P, a CARD2 mutant, but not A106V, Q31H nor F161I, inhibited the ability of RICK to activate NF-κB (Figure 2D). Moreover, L145P, but not mutants lacking one or two CARDs, inhibited the ability of wild-type NOD2 to activate NF-κB in the absence or presence of bacterial components (Figure 2E). HEK293T cells express a low level of endogenous NOD2 and activate NF-κB in response to a high concentration of MDP (5 μg/ml) (Supplementary Table 2). To determine whether the L145P mutant could inhibit the activity of endogenous NOD2, we transfected HEK293T cells with constructs expressing the L145P mutant or dominant-negative forms of TBK1 and MyD88 (as negative controls) or IKKγ as a positive control and stimulated the cells with MDP. We found that the L145P mutant, like a dominant-negative form of IKKγ, exhibited a dominant-negative effect on NOD2-mediated signalling, whereas interfering mutants of TBK or MyD88 did not (Figure 2F). These results indicate that the dominant-negative effect of the mutation L145P is specific. Critical residues in the NOD domain required for bacterial recognition This work included missense mutants with substitutions in 249 amino-acid residues of the NOD domain (Figures 1B and 3A). The analysis of the generated mutant clones was primarily restricted to the 59 mutants with a single amino-acid substitution. Initial functional screening of these mutants using synthetic MDP revealed 21 residues in the NOD domain that are critical for NOD2 function (Figure 3A). These included mutation of the conserved Asp residue at position 379, D379A, in the Walker's B box (Figure 3B), which is essential for binding to the Mg2+-substrate and nucleotide hydrolysis (Walker et al, 1982). The D379A NOD2 mutant did not respond to MDP (Supplementary Table 1), suggesting that nucleotide hydrolysis is essential for MDP-dependent NF-κB activation. The equivalent mutation of D284A in NOD1 as well as the K208R mutation in the A box of NOD1 resulted in loss of ligand-dependent NF-κB activation (Supplementary Table 2). Thus, the requirement of the B box for the response to bacterial components is conserved between NOD1 and NOD2. In addition, mutation of amino-acid residues A232, V295, F327, C333, S344, S396, T401, F408, A429, V492, L626, P639, K655, G680, L690, A691, S714 and H734, located within the NOD domain, resulted in loss of MDP-dependent NF-κB activation (Figure 3B). Similarly to D284A, most of these NOD mutants retained their ability to activate NF-κB when overexpressed in HEK293T cells (Figure 3B and Supplementary Table 1). Immunoblotting analysis showed appropriate expression of the mutants except for C333Y and A232P whose expression was undetectable (Figure 3B), suggesting that the latter residues are important for protein stability. Figure 3.Mutational analyses of the NOD domain of NOD2. (A) Schematic representation of the NOD domain. The Walker's A and B boxes are depicted by solid dots. Numbers indicate the position of amino-acid residues. Symbols and color code are described in Figure 2. The gain-of-function mutations are labelled with the symbol c. (B) NF-κB activity of representative NOD domain mutants in the presence or absence of MDP. E778K and ΔLRR are shown as controls. Expression of NOD2 proteins is shown on top. (C) Interaction of NOD2 mutants with RICK. Extracts from HEK293T cells expressing indicated mutants were co-immunoprecipitated with anti-Myc antibody and immunoblotted with anti-NOD2 antibody. IP, immunoprecipitation; total, immunoblotting of total lysates. A black dot denotes phosphorylated form of RICK. (D) Evidence of intramolecular interaction between the C-terminal region of the NOD domain and proximal LRRs by functional complementation. NF-κB activity in the presence or absence of MDP is shown for NOD2 mutant and wild-type clones. Values represent the mean of normalized data±s.d. of triplicate cultures. Download figure Download PowerPoint Distinct functional alteration of NOD2 variants associated with BS and CD Several missense NOD2 mutants in NOD were identified that are found at a low frequency (i.e. less than 1%) in Western European populations (Hugot et al, 2001; Lesage et al, 2002). Seven of these natural mutations, A140T, R373C, S431L, V793M, N853S, V955I and G978E, were generated through random mutagenesis of NOD2. In addition, we engineered R334W and R334Q, two recurrent BS-causing mutations. Consistent with a recent report (Chamaillard et al, 2003c), both R334W and R334Q exhibited increased basal NF-κB activity, which was comparable to that observed with NOD2 lacking the LRRs (Figure 3B). Unlike the ΔLRR NOD2 mutant, the NF-κB activity of R334W and R334Q was further enhanced by addition of MDP (Figure 3B). Thus, the BS-associated R334W and R334Q variants function as hyper-responsive mutations. To determine whether the two BS-causing mutations (R334W and R334Q) and loss-of function mutants (D379A and E778K) could exhibit abnormal interaction with RICK, we coexpressed the NOD2 mutants or V295M (a mutant with wild-type phenotype) and Myc-tagged RICK and immunoprecipitated the cell lysates with anti-Myc antibody. Immunoprecipitation analyses showed that the activity of the mutants cannot be explained by an altered interaction with RICK (Figure 3C). A novel regulatory region of NOD2 Multiple mutations (i.e. frameshift and nonsense mutations) resulted in premature C-terminal truncation of NOD2 (Figure 4). Truncation at the first or second CARD of NOD2 resulted in mutations that were unable to activate NF-κB (Figure 4). This is consistent with the observation that both CARDs of NOD2 are required for association with RICK, an essential downstream factor for NOD2 activity (Ogura et al, 2001b; Kobayashi et al, 2002). Truncation at the most C-terminal LRRs (residues 855-1040) of NOD2 resulted in mutants that were able to activate NF-κB at a level comparable to that of the wild-type protein, but were unresponsive to bacterial components (Figure 4 and Supplementary Table 1). Notably, sequential truncation from residue 664 to 854 resulted in NOD2 mutants with elevated basal NF-κB activity (Figures 4 and 5A and Supplementary Table 1). Additional truncation N-terminal to residue 664 or C-terminal to residue 855 (located in the fifth LRR) resulted in loss of the constitutive ability of NOD2 to activate NF-κB (Figures 4 and 5A). Immunoblotting analysis revealed that the expression levels of the truncated NOD2 mutants could not explain the observed differences in NF-κB activity (Figure 5B). These results indicate that the borders of the regulatory region are located between residues 664 and 854. Furthermore, the region from residue 855 to the C-terminus, which expands from the distal part of the fifth LRR to the 11th LRR, is sufficient to suppress the constitutive activation of NOD2. To test whether the mechanism of activation is conserved between NOD1 and NOD2, we generated a panel of NOD1 mutants and tested their ability to induce NF-κB in the presence or absence of a commercial lipopolysaccharide (LPS) preparation from Escherichia coli O55:B5, with traces of PGN-derived γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP), the moiety recognized by NOD1 (Chamaillard et al, 2003b; Girardin et al, 2003b). These experiments revealed that truncation of NOD1 from residues at positions 648 and 668 yielded mutants with enhanced basal NF-κB activity but unresponsive to the LPS preparation (Figure 5C and Supplementary Table 2). These results indicate that the distal LRRs of both NOD1 and NOD2 are essential for suppression of the constitutive activation of these proteins as well as for recognition of bacterial components (Figure 4 and below). Figure 4.Analysis of truncated NOD2 mutants. The location of CARDs, NOD domain and LRD are shown. The Walker's A and B boxes in the NOD domain are depicted by solid dots. The basal NF-κB activity (BNA) and NF-κB response to bacterial components (BR) are shown. +, response similar to that obtained with the wild-type protein; −, no response above control plasmid; +++, response at least three-fold greater than that obtained with wild-type NOD2. Representative results are shown in Figure 3. Numbers represent the position of amino-acid residues. The names of individual mutations are shown on the left. WT, wild-type clone. Download figure Download PowerPoint Figure 5.Analyses of truncated mutants reveal an inhibitory domain in NOD1 and NOD2. (A) NOD2 activity in the absence (open bars) and presence of LPS preparation (closed bars). A schematic diagram of part of NOD and LRD of NOD2 is shown. Numbers indicate the position (amino-acid residue) of C-terminal truncations. WT, wild-type. E778K represents a control loss-of-function mutant. (−) represents results obtained with control plasmid. (B) Immunoblotting analysis of NOD2 mutants. Analysis was performed with cell extracts from HEK293T cells transfected with indicated mutant or wild-type NOD2 plasmid or control plasmid (−) and immunoblotted with anti-FLAG antibody. (C) NF-κB activity of NOD domain gain-of-function mutants in the presence or absence of MDP. Wild-type (WT) and mock cells (−) are shown as controls. Values represent the mean of normalized data±s.d. of triplicate cultures. (D) NOD1 activity in the absence (open bars) and presence of LPS preparation (closed bars). A schematic diagram of part of NOD and LRD of NOD1 is shown. Numbers indicate the position (amino-acid residue) of C-terminal truncation. WT, wild-type. (E) Immunoblotting analysis of NOD1 proteins. The analysis was performed with cell extracts from HEK293T cells transfected with indicated mutant and wild-type NOD1 plasmids or control plasmid (−) and immunoblotted with anti-NOD1 antibody. Download figure Download PowerPoint The analysis uncovered three loss-of-function mutants within the regulatory region that could be rescued in cis by mutations within the proximal LRRs. For example, G680W was a loss-of function mutant in that it failed to respond to MDP (Figure 3D). Yet, the double G680W/G761S, as the single G761S, was fully competent to be activated by MDP (Figure 3D). Similarly, the point mutant G775D functioned as a loss-of-function mutant, whereas the A725T/A726T and G775D/A725T/A726T mutants responded normally to MDP (Supplementary Table 2). Similar complementation was observed with two additional loss-of-function mutants G680R and C779Y in that the triple G680R/C779Y/N872K exhibited a normal response to MDP (Supplementary Table 1). These results reveal functional interactions between residues located in the C-terminal region of the NOD domain and the proximal LRRs. Missense mutations in the C-terminal region of the NOD domain can exhibit constitutive NF-κB activity We identified several missense mutations within the inhibitory domain described above (P668H, I673F, G680R/C710Y, F719I/K731R and N637I/A725V) that exhibited constitutive NF-κB activity (Figure 5E and Supplementary Table 1). Unlike the BS-associated mutations, the increased basal activity of these mutants was not further enhanced by MDP (Figure 5C). The G680R mutation by itself is a loss-of-function mutation, but the G680R/C710Y mutant clone is a gain-of-function mutant, suggesting intramolecular interactions within the regulatory region (Supplementary Table 1). P668H and I673F represent mutants with single amino-acid substitutions. Significantly, we identified a point mutant, L620Q, in the corresponding region of NOD1 that also exhibited constitutive NF-κB activity (Supplementary Table 2), suggesting a conserved mechanism of NF-κB activation regulation. Amino-acid alignment of NOD2, Cryopyrin and related NOD family members revealed that I673 in NOD2 corresponds to amino-acid residue F573 in Cryopyrin (Supplementary Figure 1). Notably, an F573S mutation in Cryopyrin causes the autoinflammatory disorder CINCA (Hull et al, 2003). Thus, F573S may represent a constitutively active mutation of Cry
