Biogenesis and proteolytic processing of lysosomal DNase II.

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

Anti-DNase II polyclonal antibody detects recombinant and endogenous DNase II proteins.

<p>A, Mock transfected (lane 1, 3) or transiently transfected (lane 2, 4) 293FT cells with a plasmid for expression of the mouse DNase II protein with a FLAG-His tag at the carboxyl-terminus. The cell lysates were analyzed by Western blotting using anti-FLAG M2 antibody (lanes 1, 2) or anti-DNase II antibody (lanes 3, 4). Closed arrowhead; DNase II-FLAG-His protein detected by both antibodies, open arrowheads; DNase II-FLAG-His protein detected only by anti-DNase II antibody. Asterisk indicates non-specific detection of protein bands. B, Analyses of Con A-eluted fractions. Spleen lysates from <i>DNase II^+/+ IFN-IR^−/−</i> mice and <i>DNase II^−/− IFN-IR^−/−</i> mice were partially purified with Con A Sepharose. Fractions eluted with 0.1 (lanes 1, 6), 0.2 (lanes 2, 7), 0.3 (lanes 3, 8), 0.4 (lanes 4, 9), and 0.5 M (lanes 5, 10) α-methyl-D-mannoside were concentrated using a trichloroacetic acid precipitation, and the samples were analyzed by Western blotting using either the anti-DNase II antibody (upper panel), or anti-cathepsin D antibody (lower panel). Cathepsin D was used to confirm that a lysosomal glycosylated protein was extracted in the lysates and purified by Con A Sepharose. C, DNase activity of the eluted fractions. The eluted fractions from Con A Sepharose were directly assayed for DNase activity as described in the experimental procedures. N: negative control experiment without lysates.</p

Immunohistochemical detection of DNase II.

<p>The spleen (A, C) and bone marrow of the femur (B, D) from <i>DNaseII^+/+IFN-IR^−/−</i> mice (A, B) and <i>DNaseII^−/− IFN-IR^−/−</i> mice (C, D) were stained with anti-DNase II antibody. In the spleen, immunoreactivity for DNase II appeared as granules in the red pulp (A, arrowheads). Similar granular staining was detected in the bone marrow (B, arrowhead). No such granular staining was found in the spleen or femur of <i>DNaseII^−/− IFN-IR^−/−</i> mice (C, D). Cryosections of the spleen (E–L) and bone marrow of the femur (M–T) from wt mice were stained with anti-DNase II (green) and anti-CD68 (red) (E–H and M–P) or with anti-DNase II (green), anti-cathepsin L (red), and anti-lamp1 (blue) (I–L and Q–T). Panels H and P are high magnification images of the insets of panels G and O, respectively. Nuclei were stained by DAPI (gray). Asterisks in K, L, S, T indicate lumen of heterophagolysosomes. Double-immunogold labeling of DNase II (5 nm) and lamp-1 (10 nm) (U) or CD68 (10 nm) (V, W) in mouse spleen. Positive signals indicating DNase II (black arrows) were localized in lamp1- (red arrows) or CD68- (red arrows) positive lysosomes (light green overlay) (U, V). DNase II was detected in abundant amounts in relatively larger CD68-positive heterophagolysosomes (light pink overlay) (W). N: nucleus. Bars: A–D = 20 µm; E–G and M–O = 10 µm; H–L and P–T = 2 µm; U–W = 500 nm.</p

Secretion of DNase II and its DNase activity under acidic conditions.

<p>A, B, C, Extracellular DNase II purification from culture media of COS-1 cells or COS-1 cells stably expressing DNase II-FLAG-His (DNase II/COS-1) was performed as described in the experimental procedures. Con A fractions or anti-FLAG IP fractions were subjected to SDS-PAGE followed by Western blotting using anti-DNase II antibody (A), or anti-FLAG antibody (B). No DNase II forms were detected in the media that were obtained from non-transfected wild-type (WT) COS-1 cells, while intense bands immunoreactive to anti-DNase II or anti-FLAG antibody appeared at a molecular mass of 45 kDa from DNase II/COS-1 cells (A, B). DNase II assay was performed using each of the fractions (C). COS-1 cells or DNase II/COS-1 cells were cultured in DMEM (lane 1), with mannose-6-phosphate (M6P, lane 2), protease inhibitor cocktail (P.I., lane 3), or M6P and P.I. (lane 4). In transfected COS-1 cells, plasmid DNA was completely degraded at pH 4.7 in each lane applied (C). On the other hand, in WT COS-1 cells, plasmid DNA was weakly degraded in the Con A fractions at pH 4.7, but not in anti-FLAG IP fractions. D, DNase activity was measured after pre-incubation (see in experimental procedures). Plasmid DNA was significantly degraded when Con A fractions (left panel) or FLAG IP fraction (right panel) from transfected COS-1 cells were once pre-incubated at pH 4.7 for 2 hours and further incubated for 3 hours at pH 6.5 or pH6.0, respectively, whereas it was not degraded when the same fractions were pre-incubated only at pH 6.5. N: negative control experiment without a Con A fraction.</p

Subcellular fractionation of DNase II overexpressed in COS-1 cells.

<p>Subcellular fractionation of COS-1 cells stably overexpressing DNase II-FLAG-His was performed as described in the experimental procedures. Two µg of the samples from PNS, a lysosomal fraction and a microsomal fraction, were subjected to SDS-PAGE followed by Western blotting. Cathepsin D and Bip were used as markers of the lysosome and microsome (endoplasmic reticulum), respectively. Closed arrowhead; 45 kDa of proDNase II-FLAG-His protein, open arrowheads; 30 kDa and 23 kDa of processed forms of DNase II-FLAG-His proteins. Asterisk indicates non-specific detection of protein bands.</p

Processing of 30-kDa to 23-kDa DNase II proteins in lysosomes.

<p>A, Effect of proteinase inhibitors on processing of 30-kDa to 23-kDa DNase II proteins. COS-1 cells stably expressing DNase II-FLAG-His were cultured in the presence of a proteinase inhibitor cocktail, pepstatin A or E-64d. Closed arrowhead; proDNase II protein, open arrowheads; processed forms of DNase II proteins. B, C, Processing of 30 kDa DNase II in lysosomes was altered in cathepsin L-deficient mice. The liver and spleen from each mouse were applied to Con A Sepharose and analyzed by Western blotting as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059148#s2" target="_blank">materials and methods</a>. Closed arrowhead; 30 kDa of processed form of DNase II protein, open arrowheads; 23 kDa of processed form of DNase II protein. C, Ratios of the amount (density) of the 23-kDa protein to that of the 30-kDa protein in the liver and spleen obtained from wild-type (wt), CB−/− and CL−/− mice, and from a wt mouse at postnatal day 23 (p23) and a CD−/− mouse at p23. Results (n = 3) are recorded as the mean ± S.D.; *P<0.05 versus wild type in Student’s <i>t</i>-test.</p