Characterization of Gene p306 Expressed in Monocyte Derived Dendritic Cells

Dendritic cells (DCs) play a significant role in the initiation and maintenance of primary immune responses. The identification of new genes that are involved in DC biology is crucial for a better understanding of the unique DC functions. We approached this issue by generating a substractive cDNA library based on suppression hybridization between monocyte derived DCs (mDCs) cDNA and the reference monocyte cDNA. Among various differentially in mDC expressed genes, we identified the gene p306 with unknown functions. In the following, we characterized the gene p306 and the translated protein splice variants. We confirmed the p306 expression in mDCs on mRNA level by RACE and RT-PCR with subsequent sequencing. Besides the 2 known splice variants, we found a third novel form. Two splice variants were expressed in activated myeloid and plasmacytoid DCs sorted from blood. To characterize the p306 protein forms, we analyzed the hypothetical proteins by various prediction programs. We found that the N-terminus of the protein isoforms is homologue to a DNA-binding protein. Structural features are an oligonucleotide-binding fold with an integrated zinc ribbon. To detect p306 protein expression, we generated a polyclonal antibody and used it in Western blotting experiments. We were able to detect 3 protein bands with sizes of 36 kDa, 84 kDa and about 200 kDa, which differed from the expected sizes of 20 kDa and 111 kDa. We found that the unexpected sizes are in part due to N-glycosylation using the inhibitor tunicamycin. When analyzing cell lysates and nuclear extracts, we found that the 36 kDa protein form is expressed exclusively in the nucleus, while other forms could be detected in nucleus and cytosol. Interestingly, activation of mDCs with Toll like receptor ligands Pam³Cys, Poly I:C, LPS and R848 selectively upregulated the expression of the 84 kDa form in the nucleus. DC treatment with IL-10 or Imatinib, compounds that inhibit DC differentiation and function, abolished the expression of all 3 protein forms. In order to analyze the possible effect of proteases on the post-transcriptional regulation of the protein expression and generation of the identified splice variants, mDCs were treated with several protease inhibitors. DC treatment with cathepsin/subtilisin inhibitor led to the downregulation of all 3 protein variants, indicating that p306 might be involved in the regulation of apoptosis induction. The pan-caspase inhibitor zVAD caused the upregulation of all protein forms. In conclusion, we identified a novel gene p306 that is differentially expressed in mDC, which encodes for at least 3 different splice variants and protein forms with diverse cellular localization. Although the functions of the p306 proteins still have to be determined, the gene p306 is probably involved in binding nucleic acids and in regulating apoptosis induction.