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Membrane recruitment of NOD2 in intestinal epithelial cells is essential for nuclear factor-{kappa}B activation in muramyl dipeptide recognition.

Barnich N, Aguirre JE, Reinecker HC, Xavier R, Podolsky DK - J. Cell Biol. (2005)

Bottom Line: To gain insight into the molecular mechanisms of NOD2 function, we performed a functional analysis of deletion and substitution NOD2 mutants.Membrane targeting and subsequent NF-kappaB activation are mediated by two leucine residues and a tryptophan-containing motif in the COOH-terminal domain of NOD2.The membrane targeting of NOD2 is required for NF-kappaB activation after the recognition of bacterial muramyl dipeptide in intestinal epithelial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA.

ABSTRACT
Nucleotide oligomerization domain (NOD) 2 functions as a mammalian cytosolic pathogen recognition molecule, and mutant forms have been genetically linked to Crohn's disease (CD). NOD2 associates with the caspase activation and recruitment domain of RIP-like interacting caspase-like apoptosis regulatory protein kinase (RICK)/RIP2 and activates nuclear factor (NF)-kappaB in epithelial cells and macrophages, whereas NOD2 mutant 3020insC, which is associated with CD, shows an impaired ability to activate NF-kappaB. To gain insight into the molecular mechanisms of NOD2 function, we performed a functional analysis of deletion and substitution NOD2 mutants. NOD2, but not NOD2 3020insC mutant, associated with cell surface membranes of intestinal epithelial cells. Membrane targeting and subsequent NF-kappaB activation are mediated by two leucine residues and a tryptophan-containing motif in the COOH-terminal domain of NOD2. The membrane targeting of NOD2 is required for NF-kappaB activation after the recognition of bacterial muramyl dipeptide in intestinal epithelial cells.

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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 3020insC mutant, and empty vector (pCMVtag2C) shows the membrane association of NOD2 (arrows) but not of the NOD2 mutant. NOD2 and 3020insC mutant were detected by using monoclonal anti-Flag antibody followed by fluorescein-conjugated anti–mouse IgG. (C) Western blot analysis using anti-Flag, anti–E-cadherin, or anti–lactate dehydrogenase antibodies. Caco-2 cells were transfected with Flag-NOD2 (WT) or Flag-NOD2 3020insC (mutant). Cytosolic (C) and membrane (M) fractions were separated as described in Materials and methods. Proteins were fractionated through 4–12% Tris-glycine SDS-PAGE and were subjected to Western blot analysis by using anti-Flag antibody to detect NOD2 expression. The ratio in the membrane and cytosolic fractions that were determined after the quantification of NOD2 3020insC mutant was compared with NOD2 wild type. The result is the mean of four separate experiments. Error bar represents SEM. *, P < 0.05. (D) Caco-2 cells were cotransfected with GFP-NOD2 and Flag-NOD2 or with GFP-NOD2 and Flag-NOD2 3020insC mutant and were detected using anti-Flag antibody for Flag-tagged constructs. Only the NOD2 wild type showed plasma membrane association (arrows). (E) Caco-2 cells that were transfected with GFP-NOD2 were stained with anti–E-cadherin antibody, a membrane marker, to confirm the plasma membrane association of the NOD2 wild type. Bars, 20 μm.
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fig2: 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 3020insC mutant, and empty vector (pCMVtag2C) shows the membrane association of NOD2 (arrows) but not of the NOD2 mutant. NOD2 and 3020insC mutant were detected by using monoclonal anti-Flag antibody followed by fluorescein-conjugated anti–mouse IgG. (C) Western blot analysis using anti-Flag, anti–E-cadherin, or anti–lactate dehydrogenase antibodies. Caco-2 cells were transfected with Flag-NOD2 (WT) or Flag-NOD2 3020insC (mutant). Cytosolic (C) and membrane (M) fractions were separated as described in Materials and methods. Proteins were fractionated through 4–12% Tris-glycine SDS-PAGE and were subjected to Western blot analysis by using anti-Flag antibody to detect NOD2 expression. The ratio in the membrane and cytosolic fractions that were determined after the quantification of NOD2 3020insC mutant was compared with NOD2 wild type. The result is the mean of four separate experiments. Error bar represents SEM. *, P < 0.05. (D) Caco-2 cells were cotransfected with GFP-NOD2 and Flag-NOD2 or with GFP-NOD2 and Flag-NOD2 3020insC mutant and were detected using anti-Flag antibody for Flag-tagged constructs. Only the NOD2 wild type showed plasma membrane association (arrows). (E) Caco-2 cells that were transfected with GFP-NOD2 were stained with anti–E-cadherin antibody, a membrane marker, to confirm the plasma membrane association of the NOD2 wild type. Bars, 20 μm.

Mentions: 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 (Fig. 2 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 (Fig. 2 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 (Fig. 2 C). GFP-tagged and Flag-tagged NOD2 colocalized in Caco-2 cells (Fig. 2 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 Fig. 2 E, the GFP-NOD2 wild type colocalized with E-cadherin in Caco-2 cells.


Membrane recruitment of NOD2 in intestinal epithelial cells is essential for nuclear factor-{kappa}B activation in muramyl dipeptide recognition.

Barnich N, Aguirre JE, Reinecker HC, Xavier R, Podolsky DK - J. Cell Biol. (2005)

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 3020insC mutant, and empty vector (pCMVtag2C) shows the membrane association of NOD2 (arrows) but not of the NOD2 mutant. NOD2 and 3020insC mutant were detected by using monoclonal anti-Flag antibody followed by fluorescein-conjugated anti–mouse IgG. (C) Western blot analysis using anti-Flag, anti–E-cadherin, or anti–lactate dehydrogenase antibodies. Caco-2 cells were transfected with Flag-NOD2 (WT) or Flag-NOD2 3020insC (mutant). Cytosolic (C) and membrane (M) fractions were separated as described in Materials and methods. Proteins were fractionated through 4–12% Tris-glycine SDS-PAGE and were subjected to Western blot analysis by using anti-Flag antibody to detect NOD2 expression. The ratio in the membrane and cytosolic fractions that were determined after the quantification of NOD2 3020insC mutant was compared with NOD2 wild type. The result is the mean of four separate experiments. Error bar represents SEM. *, P < 0.05. (D) Caco-2 cells were cotransfected with GFP-NOD2 and Flag-NOD2 or with GFP-NOD2 and Flag-NOD2 3020insC mutant and were detected using anti-Flag antibody for Flag-tagged constructs. Only the NOD2 wild type showed plasma membrane association (arrows). (E) Caco-2 cells that were transfected with GFP-NOD2 were stained with anti–E-cadherin antibody, a membrane marker, to confirm the plasma membrane association of the NOD2 wild type. Bars, 20 μm.
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fig2: 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 3020insC mutant, and empty vector (pCMVtag2C) shows the membrane association of NOD2 (arrows) but not of the NOD2 mutant. NOD2 and 3020insC mutant were detected by using monoclonal anti-Flag antibody followed by fluorescein-conjugated anti–mouse IgG. (C) Western blot analysis using anti-Flag, anti–E-cadherin, or anti–lactate dehydrogenase antibodies. Caco-2 cells were transfected with Flag-NOD2 (WT) or Flag-NOD2 3020insC (mutant). Cytosolic (C) and membrane (M) fractions were separated as described in Materials and methods. Proteins were fractionated through 4–12% Tris-glycine SDS-PAGE and were subjected to Western blot analysis by using anti-Flag antibody to detect NOD2 expression. The ratio in the membrane and cytosolic fractions that were determined after the quantification of NOD2 3020insC mutant was compared with NOD2 wild type. The result is the mean of four separate experiments. Error bar represents SEM. *, P < 0.05. (D) Caco-2 cells were cotransfected with GFP-NOD2 and Flag-NOD2 or with GFP-NOD2 and Flag-NOD2 3020insC mutant and were detected using anti-Flag antibody for Flag-tagged constructs. Only the NOD2 wild type showed plasma membrane association (arrows). (E) Caco-2 cells that were transfected with GFP-NOD2 were stained with anti–E-cadherin antibody, a membrane marker, to confirm the plasma membrane association of the NOD2 wild type. Bars, 20 μm.
Mentions: 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 (Fig. 2 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 (Fig. 2 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 (Fig. 2 C). GFP-tagged and Flag-tagged NOD2 colocalized in Caco-2 cells (Fig. 2 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 Fig. 2 E, the GFP-NOD2 wild type colocalized with E-cadherin in Caco-2 cells.

Bottom Line: To gain insight into the molecular mechanisms of NOD2 function, we performed a functional analysis of deletion and substitution NOD2 mutants.Membrane targeting and subsequent NF-kappaB activation are mediated by two leucine residues and a tryptophan-containing motif in the COOH-terminal domain of NOD2.The membrane targeting of NOD2 is required for NF-kappaB activation after the recognition of bacterial muramyl dipeptide in intestinal epithelial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA.

ABSTRACT
Nucleotide oligomerization domain (NOD) 2 functions as a mammalian cytosolic pathogen recognition molecule, and mutant forms have been genetically linked to Crohn's disease (CD). NOD2 associates with the caspase activation and recruitment domain of RIP-like interacting caspase-like apoptosis regulatory protein kinase (RICK)/RIP2 and activates nuclear factor (NF)-kappaB in epithelial cells and macrophages, whereas NOD2 mutant 3020insC, which is associated with CD, shows an impaired ability to activate NF-kappaB. To gain insight into the molecular mechanisms of NOD2 function, we performed a functional analysis of deletion and substitution NOD2 mutants. NOD2, but not NOD2 3020insC mutant, associated with cell surface membranes of intestinal epithelial cells. Membrane targeting and subsequent NF-kappaB activation are mediated by two leucine residues and a tryptophan-containing motif in the COOH-terminal domain of NOD2. The membrane targeting of NOD2 is required for NF-kappaB activation after the recognition of bacterial muramyl dipeptide in intestinal epithelial cells.

Show MeSH
Related in: MedlinePlus