Structural features of Dnase1L3 responsible for serum antigen clearance

Abstract

Autoimmunity develops when extracellular DNA released from dying cells is not cleared from serum. While serum DNA is primarily digested by Dnase1 and Dnase1L3, Dnase1 cannot rescue autoimmunity arising from Dnase1L3 deficiencies. Dnase1L3 uniquely degrades antigenic forms of cell-free DNA, including DNA complexed with lipids and proteins. The distinct activity of Dnase1L3 relies on its unique C-terminal Domain (CTD), but the mechanism is unknown. We used multiple biophysical techniques and functional assays to study the interplay between the core catalytic domain and the CTD. While the core domain resembles Dnase1, there are key structural differences between the two enzymes. First, Dnase1L3 is not inhibited by actin due to multiple differences in the actin recognition site. Second, the CTD augments the ability of the core to bind DNA, thereby facilitating the degradation of complexed DNA. Together, these structural insights will inform the development of Dnase1L3-based therapies for autoimmunity.

Deficiency of macrophage-derived Dnase1L3 causes lupus-like phenotypes in mice

“…We next measured serum Dnase1 activity, which is the ability of nucleases to degrade naked DNA. Dnase1 activity was measured by plasmid degradation, which can be accomplished by both Dnase1 and Dnase1L3 present in the serum [2,15,16]. We observed no difference in the Dnase1 activity between WT and cKO mouse sera (Fig 1B).…”

“…Commercial HEp-2 cells and ANA staining reagents were from MBL Bion (Des Plaines, IL). GFP-Lamp1 cloned in pCS2+ was used for Dnase1 assays, as previously described [15]. Mouse Dnase1L3 lacking the signal sequence was cloned into p202 using BamHI and XhoI restriction sites.…”

“…Mouse Dnase1L3 lacking the signal sequence was cloned into p202 using BamHI and XhoI restriction sites. Recombinant human Dnase1L3 in p202 was previously described [15]. Recombinant proteins were purified as previously described [15].…”

“…We observed no difference in the Dnase1 activity between WT and cKO mouse sera (Fig 1B). We were unable to measure Dnase1L3-specific activity with either our immune complex degradation assay or barrier to transfection assay [15] because confounders in the serum interfered with these assays. Overall, these data indicate that total serum Dnase1 activity was not altered by macrophage-specific loss of Dnase1L3.…”

“…While Dnase1 has been implicated in lupus-like phenotypes in mice [13], recent work showed that B6 mice lacking Dnase1 do not develop lupus-like phenotypes, nor does loss of Dnase1 exacerbate lupus-like phenotypes in Dnase1L3 -/mice [14]. One key difference between Dnase1 and Dnase1L3 is a disordered Cterminus that enables digestion of DNA complexed with proteins and lipids [2,15,16]. While both nucleases can degrade plasmid DNA, Dnase1L3 has specific activity against complexed DNA that is essential for preventing SLE.…”