Cellular localization of endogenous NOD2/CARD15 in intestinal epithelial cells
Expression of NOD2/CARD15 was assessed by Western blot analysis in several intestinal epithelial cell lines as well as in COS7 and HEK293 cells. Colo205, SW480, HT29, and LS174 exhibited a strong expression of endogenous NOD2. In contrast, T84 and Caco-2 expressed little or no NOD2 protein. No endogenous expression was observed in COS7 or HEK293 cells ( A). NOD2 that was expressed in HT29 did not contain any of the three common mutations that are associated with CD, and stimulation by MDP-LD induced a 6.2-fold increase in NF-κB activity, indicating functional NOD2 in this cell line ( B).
Figure 1. Expression and cellular localization of endogenous NOD2. (A) Western blot analysis using rabbit NOD2 antiserum HM2559 and anti–β-actin. 10 μg of total protein from different intestinal epithelial cells (IEC), COS7, and HEK293 cell (more ...)
Immunostaining of HT29 cells with a rabbit NOD2 antiserum revealed cytoplasmic and vesicular staining and membrane association. No membrane staining was observed with preimmune serum. In addition, no staining was observed in Caco-2 cells lacking endogenous NOD2 ( C). In colonic epithelial cells, MDP-LD may be taken up by the hPepT1 transporter (Vavricka et al., 2004
). The expression of this transporter is increased in chronically inflamed intestinal mucosa. If MDP-LD is indeed taken up via the hPepT1 transporter, the membrane targeting of NOD2 could play a crucial role in the activation of NF-κB, allowing MDP to interact with NOD2.
The COOH-terminal domain of NOD2 facilitates membrane targeting
Given the fact that the NOD2 3020insC mutant, which is associated with CD, did not respond to MDP-LD stimulation, the membrane association that was observed for NOD2 led us to examine the subcellular localization of the NOD2 3020insC mutant. Confocal microscopic examination of Caco-2 and COS7 cells that were transfected with Flag or GFP-tagged NOD2 was performed to achieve finer delineation of the spatial distribution of NOD2. Confocal analysis showed that the expressed protein Flag-NOD2 is located in the cytosol and also appears enriched close to the plasma membrane, recapitulating the distribution of endogenous NOD2 ( B). In contrast to wild-type NOD2, Flag-NOD2 3020insC mutant was only present in the cytosol and vesicular compartment. The loss of membrane targeting of the NOD2 3020insC mutant was confirmed by Western blot analysis after separation of cytosolic and membrane fractions. The relative purity of cytosolic and membrane fractions was confirmed by Western blot analysis using antibody against E-cadherin as a membrane marker and antibody anti–lactate dehydrogenase as a cytosolic marker ( C). The ratio of the Flag-tagged NOD2 3020insC protein between membrane and cytosolic fractions was significantly decreased compared with that of the NOD2 wild-type protein, taken as one ( C). GFP-tagged and Flag-tagged NOD2 colocalized in Caco-2 cells ( D), indicating that the nature of the NH2-terminal tag did not affect cellular localization. However, GFP-tagged NOD2 and Flag-tagged NOD2 3020insC mutant did not colocalize near the plasma membrane, confirming the specific membrane association of the NOD2 wild type. This membrane association was confirmed by using anti–E-cadherin antibody, a membrane marker. As shown in E, the GFP-NOD2 wild type colocalized with E-cadherin in Caco-2 cells.
Figure 2. Membrane association of expressed NOD2. (A) Amino acid sequences of the COOH-terminal domain of NOD2 and the NOD2 3020insC mutant, which is associated with CD. (B) Confocal microscopy examination of Caco-2 cells that are transfected with Flag-NOD2, Flag-NOD2 (more ...)
The COOH-terminal protein domain directs the membrane targeting of NOD2
The difference between NOD2 wild type and NOD2 3020insC mutant is the deletion of the final 33 COOH-terminal amino acid residues, indicating that this region can be responsible for membrane targeting. The domain of NOD2 that is responsible for membrane targeting was more specifically identified by serial deletion mutants ( A). Mutants 1 and 2 failed to localize to the cell membranes in Caco-2 cells ( B), indicating that the terminal six amino acid residues of NOD2 are required for membrane targeting. Mutants 3 and 4 were constructed by adding selected COOH-terminal amino acids (last 6 and 17 amino acid residues of NOD2) to the NOD2 3020insC mutant to determine whether these amino acids were sufficient to confer NOD2 membrane association. No membrane targeting in Caco-2 cells that were transfected with these two mutant forms was observed. Instead, these NOD2 mutants were targeted to an intracellular vesicle compartment ( B). Therefore, the last six amino acid residues of NOD2 are necessary but not sufficient to confer membrane targeting. Finally, the deletion of LERNDILE (mutant 5) and VWLRGNTF (mutant 6), which are located in the COOH-terminal domain of NOD2, also resulted in the loss of membrane targeting in transfected Caco-2 cells ( B). The identical distribution of all of these mutant forms of NOD2 was observed when transfected into COS7 cells, including the concomitant loss of membrane targeting (unpublished data) compared with wild-type NOD2, which indicates that the final 33 amino acids play a crucial role for NOD2 membrane recruitment.
Figure 3. NOD2 membrane association is COOH-terminal dependent. (A) Sequences of Flag-NOD2 COOH-terminal deletion and substitution mutants. (B) Caco-2 cells were transfected with Flag-NOD2 mutant constructs. Mutants 1–12 were detected by confocal microscopy (more ...)
Several substitution mutations were also introduced in the COOH-terminal domain of NOD2 ( A). Mutant 7—with substitution of amino acids GG for LL at the end of NOD2—and mutant 11—with substitution WLR1017GGG—lost their membrane targeting, whereas the other substitution mutants still showed membrane association ( B). For mutants that failed to colocalize with the plasma membrane, as well as for the NOD2 3020insC mutant, some vesicular staining was observed. By using calnexin, an ER marker, we found that some of these vesicles are within the ER compartment ( C). These results indicate that the two last leucine residues and the WLR motif are required for membrane targeting.
NF-κB activation by MDP-LD is dependent on the membrane targeting of NOD2
NOD2 is known to activate the nuclear transcription factor NF-κB after MDP-LD stimulation, and the NOD2 3020insC mutant's ability to activate NF-κB is impaired (Girardin et al., 2003
). The inability of the NOD2 mutant to respond to MDP-LD and activate NF-κB in CD is paradoxical, considering that NF-κB is responsible for the induction of a large number of inflammatory mediators (O'Neill, 2004
). To investigate whether the membrane targeting of NOD2 is required for MDP response, we transfected HEK293 cells with NOD2 wild type and different COOH-terminal mutants without tag. NOD2 expression was confirmed by Western blot analysis using antiserum HM2563 against NOD2 ( A). As shown in B, NOD2 wild type induced a 47-fold increase in NF-κB activation, whereas mutants 1–7 and 11 induced less than a 3-fold increase in NF-κB. However, mutants 8–10 and 12, which are still membrane associated, activated NF-κB. No NF-κB activation was observed after stimulation with an MDP L-Ala, L-Glx (MDP-LL)–inactive form (unpublished data). The release of IL-8 by HEK293 cells transfected with NOD2 and selected mutants after MDP-LD stimulation was determined by ELISA to confirm the NF-κB activation. The amount of IL-8 found in the supernatant of HEK293 cells that were transfected with NOD2 and stimulated with MDP-LD (10.55 pg/ml) was significantly increased compared with untransfected cells (0.47 pg/ml). The amount of IL-8 released in the supernatant of HEK293 cells that were transfected with the selected NOD2 mutants correlated with NF-κB activation ( C). These results demonstrate that the membrane targeting of NOD2 in intestinal epithelial cells is required for NF-κB activation upon the recognition of MDP. NOD2 binds to RIP2, a cytoplasmic protein, via a CARD–CARD interaction (Kobayashi et al., 2002
), suggesting that signaling of NOD2 at the membrane implies either NOD2 redistribution to the cytoplasm after MDP-LD stimulation or recruitment of RIP2 to the membrane.
Figure 4. Ligand-induced NF-κB activation and IL-8 release are dependent on NOD2 membrane association. (A) Expression of NOD2 and mutants without tag that were transfected into HEK293 cells was determined by Western blot analysis using NOD2 antiserum HM2563. (more ...)
Role of WLR1017, R702, and G908 in MDP-LD recognition
To determine which amino acid residues in the WLR motif are required for MDP recognition, we constructed three additional mutants ( A). NF-κB activation showed that both W1017G (mutant 13) and L1018G (mutant 14) play vital roles in MDP-LD recognition because NF-κB activation is significantly decreased compared with wild-type NOD2. However, R1019G substitution (mutant 15) had no effect on NOD2 MDP-LD recognition. These results were confirmed by determining the amounts of IL-8 released in the supernatant of HEK293 cells that were transfected with these mutants (unpublished data). Substitutions W1017G and L1018G led only to a twofold decrease of NF-κB, whereas WLR1017GGG substitutions had impaired NF-κB activation, indicating that both W and L amino acid residues are important but not sufficient to completely abrogate NOD2 function. However, the two other NOD2 mutants that are associated with CD—NOD2 R702W and NOD2 G908R—still showed membrane association in transfected COS7 cells, whereas these two mutants showed decreased levels of NF-κB activation after MDP-LD stimulation. This suggested that these two amino acids are required for MDP-LD recognition but did not play any role in the membrane targeting of NOD2 ( B).
Figure 5. Ligand-induced NF-κB activation are dependent on a three–amino acid motif that is required for membrane association. (A, 1) Amino acid sequences of Flag-NOD2 COOH-terminal substitution mutants. (2) Expression of these mutants was determined (more ...)
In summary, the subcellular localization of NOD2 is important for ligand binding because cell membrane localization in intestinal epithelial cells was required for NOD2 to recognize MDP-LD after its transport across the cell membrane. However, in contrast to Toll-like receptors that recognize bacterial components outside the cell, NOD2 does not contain a transmembrane domain and does not act as a membrane receptor. Furthermore, the observed membrane association of NOD2 could be an interaction with the inner side of the plasma membrane but not with cell surface expression. This study suggested that NOD2 membrane association is necessary for NOD2 function as an innate immune receptor, which is impaired for the most common NOD2 mutant associated with CD.