Single-base oligodeoxyguanosine-binding proteins on nonspecific cytotoxic cells: identification of a new class of pattern-recognition receptors.

Abstract

The present study was designed to identify a possible new class of pathogen-recognition proteins that bind single-base oligodeoxynucleotide (ODN) ligands. Binding by the teleost natural killer cell equivalent [referred to as nonspecific cytotoxic cells (NCC)] was compared with mammalian cells (mouse RAW264.7 cells and human THP-1 cells). The ODN analysed were composed of 20-mers of guanosine (dG20), adenosine (dA20), thymidine (dT20) or cytosine (dC20). Binding studies first determined the 50% saturation levels for NCC (1.25 microg/ml), RAW264.7 (0.2 microg/ml) and THP-1 (0.8 microg/ml). Binding by dG20 to all the three cell types was saturable. Ligand blots of NCC membrane lysates with biotinylated dG20 revealed two different major molecular weight species (16-18 and 29 kDa) of binding proteins. The 29-kDa protein was identified with the help of Western blot analysis using a polyclonal antibody specific to an NCC antimicrobial protein (ncamp-1). The membrane expression of the 29-kDa ncamp-1 was determined by the binding of surface-biotinylated NCC membrane proteins with digoxigenin dG20 followed by immunoprecipitation using anti-digoxigenin agarose beads. The 29 and 14-18 kDa NCC membrane proteins were cross-reactive using Western blot examination with a polyclonal anti-histone 1 antibody. Function studies revealed that dG20 activated a twofold upregulation of membrane binding by homologous dG20-biotin. dG20 also stimulated NCC-increased membrane expression of NCC receptor protein 1. Additional experiments were performed to determine the DNase sensitivity of the different ODN. dG20 appeared to be more resistant to DNase treatment, compared to dC20, dA20 and dT20. The single-base ODN-binding proteins may represent a new class of pattern-recognition receptors that are involved in innate anti-bacterial resistance mediated by NCC.