Identification Of Immunostimulatory Sequence-specific DNA-binding Proteins

Immunostimulatory sequence (155), unmethylated CpG dinucleotides in particular base contexts, are relatively common in bacterial DNA but rare in vertebrate DNA, and can be detected as an infectious danger signal by the vertebrate innate immune system. In the present study, we tried to identify ISS-specific receptors by searching for DNA-binding proteins specifically recognizing ISS to provide basis for further studies on the molecular mechanisms of 155. For this purpose, the Yeast-One-Hybrid system using 155 as bait was established and adopted in screening of a human bone marrow eDNA library. Among four dual-positive clones obtained so far, two encode light chain of immunoglobulin (Ig) homologous to an anti-DNA and anti-HBsAg (HBsAb) respectively, suggesting a direct interaction between some of Igs and 155. To further confirm the ISS-Ig interaction, DNA-Igs interaction was further studied by enzyme-linked immunosorbant assay (ELISA) and electrophoretic mobility shift assay (EMSA). Since it has been proved that both DNA uptake and acidification inside the endosome are required for triggering of 155-dependent activation signaling, suggesting that 155 engage its putative receptor at acidic pH, our ELISA and EMSA were carried out at different pH (pH7.4-5.8). The ELISA revealed that different kinds of Igs such as HBsAb and an Anti-V5 monoclonal antibody, shown only negligible reactivity with plasmid DNA at pH7.4 , but bound to DNA with remarkable higher affinity at acidic PH; more importantly, the plasmids inserted with more copies of 155 shown higher reactivity with these Igs than their blank vectors. The EMSA found the ISS-containing oligodeoxynucleotides (CpG-ODN) bound to varied Igs, including HBsAb, anti-human-IgG1, anti-human-LILA-DR and anti-CMV mAbs, also in a pH-dependent way, while inversion of CpG into GpC decrease the reactivity of ODN with these Igs. These results support existence of pH-dependent ISS-Igs interaction. In consideration of the crucial role of membrane Ig (mlg) in B cell activation, these results suggest that mlg may be a candidate ISS receptor in ISS-induced B cell activation, so the roles of mlg in ISS-induced B cell activation need further study.