Nucleotide oligomerization domain-2 interacts with 2'-5'-oligoadenylate synthetase type 2 and enhances RNase-L function in THP-1 cells

Abstract

Nucleotide-binding and oligomerization domain-2 (NOD2) is an intracellular protein involved in innate immunity and linked to chronic inflammatory diseases in humans. Further characterization of the full spectrum of proteins capable of binding to NOD2 may provide new insights into its normal functioning as well as the mechanisms by which mutated forms cause disease. Using a proteomics approach to study human THP-1 cells, we have identified 2'-5'-oligoadenylate synthetase type 2 (OAS2), a dsRNA binding protein involved in the pathway that activates RNase-L, as a new binding partner for NOD2. The interaction was confirmed using over-expression of OAS2 and NOD2 in HEK cells. Further confirmation was obtained by detecting NOD2 in immunoprecipitates of endogenous OAS2 in THP-1 cells. Finally, over-expression of NOD2 in THP-1 cells led to enhanced RNase-L activity in cells treated with poly(I:C), a mimic of double-stranded RNA virus infection. These data indicate connectivity in pathways involved in innate immunity to bacteria and viruses and suggest a regulatory role whereby NOD2 enhances the function of RNase-L.

Figure 1

Transduced THP-1 cells over-express NOD2. Lysates from THP-1 cells stably transduced with CBP-SBP vector control (lane 1), CBP-SBP-NOD2 (lane 2) and CBP-SBP-NOD2-R334Q) (lane 3) analyzed by immunoblotting with anti-NOD2 antisera.

Figure 2

OAS2 is detected in NOD2 precipitates. HEK-293 cells were stably transduced with retroviral constructs for CBP-SBP-NOD2 or the empty CBP-SBP construct serving as a vector control. HEK cells were then transiently transfected with a plasmid expressing myc-tagged OAS2. NOD2 and OAS2 status is indicated above each lane. A) The expression of NOD2 and OAS2 in lysates of transfected cells was determined by immunoblotting with anti-NOD2 (polyclonal from Cayman) and anti-myc (9E10, Sigma-Aldrich). As a loading control, the membrane was immunoblotted with a β-actin antibody. Lysates from untransduced and untransfected HEK cells were included as a reference. B) Precipitates were prepared by addition of streptavidin-Sepharose beads, and bound proteins were then identified by immunoblotting with NOD2- and myc-specific antibodies (shown adjacent to each panel).

Figure 3

NOD2 co-immunoprecipitates with endogenous OAS2. A) Lysates from HEK293 cells transfected with myc-tagged human OAS2 were immunoprecipitated with 56/3 (left two lanes) or with control monoclonal antibody (anti-IκBα) (right two lanes) and assayed for OAS2 activity in the absence or presence of poly I:C (50 µg/ml) (indicated at bottom of each lane) and [α-³²P]ATP. Reaction products were separated by electrophoresis and detected by autoradiography. The location of 2’-5’-oligoadenylates containing 2, 3, 4,..etc adenylate units are indicated by arrows. B) THP1 cells were stably transduced with empty retroviral vector (V) or vector containing wild type NOD2 (N) (indicated above each lane). Lysates immunoblotted with anti-NOD2 or anti-β-actin confirmed appropriate expression of transduced NOD2 (lanes 1 and 2). Lysates were then immunoprecipitated with anti -OAS2 (56/3) (lanes 3 and 5) or control antibody anti -IκBα (lanes 4 and 6), electrophoresed and immunoblotted with anti-NOD2 or anti-β-actin.

Figure 4

Overexpression of NOD2 enhances RNase-L function in response to poly(I:C). THP-1 cells stably transduced with empty vector control (VC) or NOD2 were then treated for 3 hours with poly(I:C) (25 µg/ml) in Mirus TKO transfection reagent. Total RNA was isolated and northern blot analysis was performed with an oligonucleotide probe specific for 18S rRNA.