Biogenesis and Proteolytic Processing of Lysosomal DNase II

Abstract

Deoxyribonuclease II (DNase II) is a key enzyme in the phagocytic digestion of DNA from apoptotic nuclei. To understand the molecular properties of DNase II, particularly the processing, we prepared a polyclonal antibody against carboxyl-terminal sequences of mouse DNase II. In the present study, partial purification of DNase II using Con A Sepharose enabled the detection of endogenous DNase II by Western blotting. It was interesting that two forms of endogenous DNase II were detected – a 30 kDa form and a 23 kDa form. Neither of those forms carried the expected molecular weight of 45 kDa. Subcellular fractionation showed that the 23 kDa and 30 kDa proteins were localized in lysosomes. The processing of DNase II in vivo was also greatly altered in the liver of mice lacking cathepsin L. DNase II that was extracellularly secreted from cells overexpressing DNase II was detected as a pro-form, which was activated under acidic conditions. These results indicate that DNase II is processed and activated in lysosomes, while cathepsin L is involved in the processing of the enzyme.

DNase II-dependent DNA digestion is required for DNA sensing by TLR9

“…4a). As reported previously 23 , the apparent molecular weight of DNase II was B28 kDa, which is smaller than the full-length DNase II. All the DNase II in BM-cDCs seems to be processed by cathepsin L 23 .…”

Lysosomal deoxyribonuclease II from the musselMytilus galloprovincialis: Characterization and seasonal activity

“…In contrast to the digestive glands, the gill lysate contained a single active DNase band of approximately 48 kDa, while the 37 kDa isoform observed in the digestive gland was not detected in the gills under the conditions of the DPZ assay, suggesting that post-translational proteolytic processing is not an essential step for the activation of DNase. This may reflect tissue-specific protein processing of the mussel DNase enzyme, as it was seen for the murine DNase II, where tissue-specific conditions influence DNase maturation and the 23 kDa form was found in the liver but not in the spleen (Ohkouchi et al 2013). Likewise, tissue-specific conditions in the gills could have contributed to the DNase's inability to reconstitute its enzyme activity following denaturation during zymogram analysis (Liao 1985).…”

“…In addition to the 48 and 37 kDa bands, a third (30 kDa) immunoreactive band was observed. This band of 30 kDa may be equivalent to the murine heavy-chain form (23 kDa) and α2 subunit of porcine splenic DNase II, products of lysosomal proteolytic processing (Ohkouchi et al 2013). The 30 kDa immunoreactive band could be derived from the proteolytic processing of the 37 kDa propeptide in lysosomes similarly to the processing of murine DNase, where the 23 kDa band is a proteolytic product of the 30 kDa form (Ohkouchi et al 2013).…”

“…The observed pattern of active enzyme bands is in accordance with mouse and porcine DNase II patterns, where multiple enzyme isoforms were detected. In mouse liver, DNase II is synthesized as a 45 kDa pro-chain form and processed to an active 30 kDa single-chain and a 23 kDa heavy-chain form during transport to lysosomes (Ohkouchi et al 2013). Similarly, porcine spleen DNase II is synthesized as a 46.5 kDa precursor polypeptide and post-translationally processed to a 35 kDa protein (Huang et al 2009).…”

“…Reports on the protein structure of DNase II remain equivocal. Molecular weights of human, porcine and murine DNase II differ among tissues, and identification of such lysosomal enzymes has suggested glycosylation as well as proteolytic processing during maturation in the endoplasmic reticulum, Golgi body and lysosomes (MacLea et al 2003;Huang et al 2009;Fischer et al 2011;Ohkouchi et al 2013).…”