Báo cáo khoa học: Murine serum nucleases – contrasting effects of plasmin and heparin on the activities of DNase1 and DNase1-like 3 (DNase1l3) doc

Murine serum nucleases – contrasting effects of plasmin and heparin on the activities of DNase1 and DNase1-like (DNase1l3) Markus Napirei, Sebastian Ludwig, Jamal Mezrhab, Thomas Klockl and Hans G Mannherz ă Abteilung fur Anatomie und Embryologie, Medizinische Fakultat, Ruhr-Universitat Bochum, Germany ă ă ă Keywords DNase1; DNase1l3; plasminogen system; serum; systemic lupus erythematosus Correspondence M Napirei, Abteilung fur Anatomie und ă Embryologie, Medizinische Fakultat, ă Ruhr-Universitat Bochum, Universitatsstraòe ă ¨ 150, D-44801 Bochum, Germany Fax: +49 2343214474 Tel: +49 2343223164 E-mail: markus.napirei@rub.de (Received November 2008, revised 27 November 2008, accepted 10 December 2008) doi:10.1111/j.1742-4658.2008.06849.x DNase1 is regarded as the major serum nuclease; however, a systematic investigation into the presence of additional serum nuclease activities is lacking We have demonstrated directly that serum contains DNase1-like (DNase1l3) in addition to DNase1 by an improved denaturing SDS-PAGE zymography method and anti-murine DNase1l3 immunoblotting Using DNA degradation assays, we compared the activities of recombinant murine DNase1 and DNase1l3 (rmDNase1, rmDNase1l3) with the serum of wild-type and DNase1 knockout mice Serum and rmDNase1 degrade chromatin effectively only in cooperation with serine proteases, such as plasmin or thrombin, which remove DNA-bound proteins This can be mimicked by the addition of heparin, which displaces histones from chromatin In contrast, serum and rmDNase1l3 degrade chromatin without proteolytic help and are directly inhibited by heparin and proteolysis by plasmin In previous studies, serum DNase1l3 escaped detection because of its sensitivity to proteolysis by plasmin after activation of the plasminogen system in the DNA degradation assays In contrast, DNase1 is resistant to plasmin, probably as a result of its di-N-glycosylation, which is lacking in DNase1l3 Our data demonstrate that secreted rmDNase1 and murine parotid DNase1 are mixtures of three different di-N-glycosylated molecules containing two high-mannose, two complex N-glycans or one high-mannose and one complex N-glycan moiety In summary, serum contains two nucleases, DNase1 and DNase1l3, which may substitute or cooperate with each other during DNA degradation, providing effective clearance after exposure or release from dying cells DNase1 (EC 3.1.21.1) is an endonuclease secreted into body fluids by a wide variety of exocrine and endocrine organs which line the gastrointestinal and urogenital tracts [1,2] By comparing serum from wild-type (WT) and DNase1 knockout (KO) mice, we have demonstrated previously that it is the major serum nuclease [3] A lack or decrease in serum DNase1 activity is associated with the development of systemic lupus erythematosus (SLE) like antinuclear autoantibodies (ANAs) directed against nucleosomes and their constituents, and immune complex-induced glomerulonephritis in humans and mice [4–6] Previously, we have reported that, in cooperation with different serine proteases, serum DNase1 degrades the chromatin of necrotic cells [3] Pure DNase1 hydrolyses ‘naked’ protein-free DNA with high efficiency, Abbreviations ANA, antinuclear autoantibodies; DNase1l3, DNase 1-like 3; DPZ, denaturing SDS-PAGE zymography; EndoH, endoglycosidase H; KO, knockout; NLS, nuclear localization signal; NPZ, native SDS-PAGE zymography; Pai-1, plasminogen activator inhibitor 1; pDNA, plasmid DNA; PNGaseF, peptide N-glycosidase F; rER, rough endoplasmic reticulum; rmDNase1 ⁄ rmDNase1l3, recombinant murine DNase1 ⁄ DNase1l3; rrDNase1l3, recombinant rat DNase1-like 3; SLE, systemic lupus erythematosus; SRED assay, single radial enzyme diffusion assay; TAE, Tris–acetate ⁄ EDTA; TBE, Tris–borate ⁄ EDTA FEBS Journal 276 (2009) 1059–1073 ª 2009 The Authors Journal compilation ª 2009 FEBS 1059 Murine serum nucleases M Napirei et al but efficient chromatin degradation depends on the proteolysis of DNA-bound proteins [3,7] Heparin promotes chromatin degradation by serum DNase1; however, the underlying mechanism for this activation is still unclear [3,7] Previously, we have observed that the serum of some DNase1 KO mice contains residual nucleolytic activity [7] In contrast with serum DNase1, this nucleolytic activity efficiently degrades chromatin by internucleosomal cleavage without proteolytic help, and is inhibited by heparin However, the conditions of occurrence and the identity of this additional serum nuclease have not been clarified to date, although preliminary data suggest that it displays biochemical characteristics of recombinant rat DNase1-like (rrDNase1l3; DNase c, DNase Y, LS-DNase, nhDNase) [7,8] DNase1l3 belongs to the DNase1 nuclease family, which consists of DNase1 and three further DNase1-like endonucleases (DNase1L1, DNase1L2 and DNase1l3) [8] Both DNase1 and DNase1l3 contain an N-terminal signal peptide for their translocation into the rough endoplasmic reticulum (rER) Indeed, they have been shown to be localized in the secretory compartment and secreted into the cell culture medium by transfected cells [7] In contrast with DNase1, DNase1l3 contains two nuclear localization signals (NLSs), which might explain its occurrence in the nucleus of certain cells [9] This finding seems to be important for the proposed role of DNase1l3 in chromatin cleavage during apoptosis, as described for several cell types in vitro [10–15] and in vivo [16,17] Traditionally, the presence of an NLS implies nuclear accumulation by active transport through the nuclear pores after binding of a specific importin to the NLS However, experiments employing murine and rat DNase1l3-green fluorescent protein constructs did not show any preferential nuclear localization of the fusion proteins after transfection of NIH3T3 cells [7] Instead, we observed secretion of these nucleases into the medium, which was abrogated after deletion of the N-terminal rER signal peptide It is therefore conceivable that the NLS of DNase1l3 might only be functional under special conditions, such as, for example, apoptosis, leading to the nuclear import of DNase1l3 after its release from the rER into the cytoplasm Macrophages of different organs have been shown to express DNase1l3 in vivo [18] Furthermore, DNase1l3 has been isolated from nuclei of rat thymocytes [19] and has been demonstrated to be involved in somatic hypermutation in stimulated B cells [20] These studies imply that DNase1l3 fulfils intra- and extracellular physiological functions in the immune system; however, the role of its presumed NLS in fulfilling the intracellular functions proposed is still unclear One of 1060 the extracellular functions might be the participation in the clearance of autoantigenic chromatin [21] In this work, we demonstrate that murine serum contains two chromatolytic activities with different properties Serum DNase1l3 degrades chromatin at internucleosomal sites on its own and is inhibited by proteolysis by plasmin In contrast, serum DNase1 degrades chromatin only in combination with proteases such as plasmin The plasmin resistance of DNase1 might be explained by its di-N-glycosylation, which is absent in DNase1l3 Heparin mimics the effect of proteases on DNase1-induced chromatolysis by displacing histones, whereas it inhibits DNase1l3 by binding We also describe an improvement of the denaturing SDS-PAGE zymography (DPZ) procedure originally described by Shiokawa et al [14], which allows the simultaneous detection of both nucleases in serum and tissue samples This test procedure might also be of clinical value, as reduced serum nuclease activity has been reported in patients with SLE and in lupus-prone mice [21] Results Murine serum contains two chromatolytic activities with different properties Freshly prepared serum was collected from C57BL ⁄ WT and DNase1 KO mice and employed in nuclear chromatin digestion assays We found that all sera derived from DNase1 KO mice contained residual nuclease activity (Fig 1A) In contrast with our previous studies, we found that chromatin breakdown by the serum of WT mice was not inhibited by the addition of aprotinin [3] Instead, we found that aprotinin accelerated and equalized the overall nucleolytic activities of sera from both mouse strains, leading to an accumulation of mononucleosomal DNA fragments (Fig 1A) These data imply that the residual serum nuclease activity found in DNase1 KO mice also occurred in WT mice, and was activated by the addition of aprotinin, thereby masking the inhibitory effect of aprotinin on DNase1 ⁄ plasmin-induced chromatolysis as described previously [3] These results contradict our previous studies, which demonstrated that chromatin degradation by the sera of WT mice was completely inhibited by aprotinin [3], and imply that, in the earlier study, the second nuclease activity of murine serum was not always detectable In accordance with our previous studies, we found that heparin accelerated chromatin degradation by the sera of WT mice [3], whereas it inhibited that catalysed by the residual serum nuclease activity found in FEBS Journal 276 (2009) 1059–1073 ª 2009 The Authors Journal compilation ª 2009 FEBS M Napirei et al A B C Fig Murine serum contains two chromatolytic activities with different properties Digestion of nuclear chromatin by serum from WT and DNase1 KO mice (A) Isolated MCF-7 nuclei were incubated with 2.5% (v ⁄ v) serum concentrations for h at 37 °C Aprotinin equalized the internucleosomal chromatin degradation by sera from both mouse genotypes, whereas heparin inhibited that by serum from DNase1 KO mice, but enhanced that by WT serum (B) Pure serum with 2–8 h of incubation at 37 °C under otherwise identical conditions Chromatin degradation in the serum of a WT mouse proceeded to completion with ongoing incubation time, whereas it stopped in serum from a DNase1 KO mouse (C) Pure serum with h of incubation at 37 °C Chromatin degradation in the serum of a DNase1 KO mouse was accelerated by the addition of aprotinin and the specific inhibitor for the activation of the plasminogen system Pai-1 DNase1 KO mice (Fig 1A) As it is assumed that, in addition to DNase1, the residual serum nuclease activity detectable in DNase1 KO mice also occurs in WT mice, the acceleration of chromatin degradation by WT serum in the presence of heparin must be caused Murine serum nucleases by activation of DNase1 ⁄ plasmin-dependent chromatin breakdown Employing aprotinin and heparin in parallel, we found that, in the sera of DNase1 KO mice, the inhibitory effect of heparin blocked the accelerating effect of aprotinin on the residual nuclease activity (Fig 1A) As heparin inhibits rrDNase1l3, as shown previously [3], we concluded that the residual serum nuclease activity was caused by the presence of a DNase1l3-like nuclease In WT serum, the accelerating effect of heparin on DNase1 ⁄ plasmin-dependent chromatin degradation (as described previously [3]) overrides its inhibitory effect on DNase1l3-like activity and the inhibitory effect of aprotinin on DNase1 ⁄ plasmindependent chromatolysis (see Fig 3) In summary, these and our previous experiments indicate that murine serum contains two chromatolytic activities with opposite activation properties: DNase1 ⁄ plasmin activity, which is activated by heparin and inhibited by aprotinin as a result of plasmin inhibition, and DNase1l3-like activity, which is inhibited by heparin and activated by aprotinin As aprotinin is a serine protease inhibitor, it is conceivable that the DNase1l3-like nuclease might be sensitive to proteolysis or indirectly inhibited by proteolysis of DNAbound structural proteins To test whether the chromatolytic activities were also active in undiluted serum, we added cell nuclei directly into pure serum The data obtained demonstrated complete chromatin degradation by WT serum in an internucleosomal manner, which was less efficient and did not proceed to completion in serum from DNase1 KO mice (Fig 1B) However, the addition of aprotinin or plasminogen activator inhibitor (Pai-1) to the sera of DNase1 KO mice completed chromatolysis to mononucleosomes and even to oligonucleotides (Fig 1C) These experiments demonstrate that the DNase1l3-like nuclease of murine serum is sensitive to proteolysis by plasmin or inhibited by proteolysis of DNA-bound structural proteins DNA digestion by murine serum nucleases in comparison with recombinant murine DNase1 (rmDNase1) and rmDNase1l3 To clarify the effect of heparin and aprotinin on the mode of chromatolysis by serum from WT and DNase1 KO mice in more detail, we investigated their influence on rmDNase1 and rmDNase1l3 in plasmid DNA (pDNA) and chromatin digestion assays For this purpose, we transiently transfected NIH-3T3 cells with expression vectors for the murine DNase1 and DNase1l3 cDNA, and collected cell culture supernatants containing the secreted recombinant nucleases FEBS Journal 276 (2009) 1059–1073 ª 2009 The Authors Journal compilation ª 2009 FEBS 1061 Murine serum nucleases M Napirei et al First, we evaluated the effect of heparin in DNA digestion assays As shown in Fig 2A, heparin had no stimulating effect on pDNA degradation by rmDNase1, but inhibited that by rmDNase1l3 at low concentrations These results imply that heparin had no effect on protein-free DNA and did not stimulate the activity of rmDNase1 directly, but inhibited rmDNase1l3 Employing both recombinant nucleases in chromatin digestion assays, we found that, in contrast with pDNA digestion, chromatin breakdown by pure rmDNase1 was weak in comparison with that by pure rmDNase1l3, which efficiently degraded chromatin in an internucleosomal manner (Fig 2B) In contrast with pDNA digestion, heparin activated chromatin breakdown by rmDNase1, leading to a random DNA cleavage pattern (DNA smear in the agarose gel; Fig 2B), as described previously for serum from WT mice [3] In accordance with the pDNA digestion assay, internucleosomal chromatin breakdown by rmDNase1l3 was inhibited by heparin (Fig 2B) In summary, these data demonstrate that heparin has opposing effects on these nucleases: it enhances chromatin but not pDNA cleavage by rmDNase1, possibly by inducing an alteration in the chromatin structure itself, and inhibits chromatin and pDNA cleavage by rmDNase1l3 This inhibition might be caused by direct binding of heparin to DNase1l3 and ⁄ or an alteration of the chromatin structure (see below) Employing aprotinin in pDNA (data not shown) and chromatin digestion (Fig 2B) assays using both recombinant nucleases, we did not observe any effect on their nucleolytic activities This result suggests that the apparently stimulating effect of aprotinin on serum DNase1l3-like activity (see Fig 1A,C) and its inhibiting effect on serum DNase1 (as described previously [3] and Fig 3) are facilitated by the inhibition of serum proteases Therefore, we repeated the chromatin digestion assays employing both recombinant nucleases in the presence of either thrombin or plasmin (Fig 2C) As described previously [3,7], we found that thrombin as well as plasmin induced chromatin breakdown by pure rmDNase1, leading to internucleosomal chromatin cleavage comparable with that induced by pure rmDNase1l3 alone (Fig 2C) Plasmin was found to be much more efficient than thrombin (Fig 2C) The simultaneous addition of aprotinin inhibited the promoting effect of plasmin on chromatin breakdown by rmDNase1, whereas the action of thrombin was only slightly inhibited (Fig 2C), which is most probably explained by the fact that aprotinin inhibits plasmin with higher specificity than thrombin These results strongly suggest that these proteases render internucleosomal regions accessible for nucleolytic 1062 A B C D Fig DNA digestion facilitated by rmDNase1 and rmDNase1l3 (A) Effect of increasing amounts of heparin on pDNA digestion by rmDNase1 and rmDNase1l3, employing 0.1 and lL of cell culture supernatants, respectively Incubation for 30 at 37 °C in 10 mM Tris ⁄ HCl pH 7.0, mM MnCl2 and mM CaCl2 (B–D) Chromatin digestion of isolated MCF-7 nuclei by rmDNase1 and rmDNase1l3: lL of cell culture supernatants were employed for h at 37 °C (B) In contrast with pDNA, nuclear chromatin digestion by rmDNase1 was enhanced by heparin, leading to a random DNA cleavage pattern In accordance with pDNA, chromatin digestion by rmDNase1l3 was inhibited by heparin Aprotinin had no effect on chromatin digestion by the two recombinant nucleases (C) Chromatin digestion by rmDNase1 and rmDNase1l3 in the presence of plasmin or thrombin Plasmin and thrombin induced internucleosomal chromatin degradation by rmDNase1, whereas rmDNase1 performed it alone Plasmin, but not thrombin, inhibited chromatin degradation by rmDNase1l3 Conditions as in (B) (D) Pre-incubation of rmDNase1l3, but not rmDNase1, with plasmin for 30 at 37 °C prior to the addition of MCF-7 cell nuclei inhibited chromatin cleavage The addition of aprotinin after pre-incubation did not restore chromatin cleavage, demonstrating that the inhibition is caused by proteolysis of rmDNase1l3 by plasmin during the pre-incubation period FEBS Journal 276 (2009) 1059–1073 ª 2009 The Authors Journal compilation ª 2009 FEBS M Napirei et al Murine serum nucleases is degraded by plasmin during the pre-incubation period (Fig 2D) Activation of plasminogen depletes the DNase1l3-like activity of murine serum Fig Activation of plasminogen depletes the DNase1l3-like activity of murine serum Chromatin digestion by serum from WT and DNase1 KO mice [2.5% (v ⁄ v) serum concentration, h of incubation at 37 °C] Top panel: serum was stored at )20 °C, thawed to room temperature and analysed directly Bottom panel: identical sera analysed after weeks of storage at °C The addition of aprotinin and Pai-1 demonstrated the presence of a protease-sensitive DNase1l3-like nuclease activity in the serum from the DNase1 KO mouse, which disappeared after thawing and prolonged storage of the serum The DNase1 ⁄ plasmin-dependent chromatolytic activity, which is inhibited by aprotinin and Pai1, remained in the serum from the WT mouse attack by rmDNase1, most probably by proteolysis of histone H1 as shown previously [3] In contrast, internucleosomal chromatin cleavage by rmDNase1l3 was inhibited by plasmin, but not by thrombin (Fig 2C) Furthermore, the simultaneous addition of aprotinin restored internucleosomal chromatin cleavage by rmDNase1l3 in the presence of plasmin (Fig 2C) These data reveal that the proteolysis of histones is apparently not necessary and that their intact nature does not inhibit internucleosomal chromatin breakdown by rmDNase1l3 These results also indicate that plasmin, but not thrombin, proteolytically attacks and inactivates rmDNase1l3 but not rmDNase1 To demonstrate this conclusion more directly, we pre-incubated both recombinant nucleases with plasmin for 30 at 37 °C, and subsequently added cell nuclei alone or in combination with aprotinin We found that pre-incubation of rmDNase1 with plasmin had no effect on its ability to cause internucleosomal chromatin cleavage, demonstrating that rmDNase1 is not degraded by plasmin (Fig 2D) In contrast, pre-incubation of rmDNase1l3 with plasmin inhibited subsequent chromatolysis, demonstrating that rmDNase1l3 Our finding that the serine protease inhibitor aprotinin and the inhibitor for the activation of the plasminogen system Pai-1 maintained the chromatolytic activity of diluted and undiluted serum of DNase1 KO mice (Fig 1A,C) implies that DNase1l3-like nuclease activity is sensitive to proteolysis by plasmin This is supported by the observation that rmDNase1l3 is inactivated by the addition of plasmin (Fig 2C,D) Therefore, the inability of serum from DNase1 KO mice to cleave chromatin after prolonged incubation (Fig 1B) indicates that DNase1l3 is inactivated by plasminogen activation during the nuclear chromatin degradation assay These data explain why, in previous experiments, the sera of WT and DNase1 KO mice were depleted in DNase1l3-like nuclease [3] Indeed, when we subjected serum frozen at )20 °C to thawing to room temperature and subsequently stored it at °C, it lost its DNase1l3-like activity within weeks (Fig 3) Thus, serum from DNase1 KO mice completely lost its ability to induce chromatolysis, whereas serum from WT mice still contained DNase1 ⁄ plasmindependent chromatolytic activity, which was inhibited by aprotinin and Pai-1 as described previously [3] From these data, we conclude that the storage conditions are crucial for the maintenance of the serum DNase1l3-like nuclease, whereas DNase1 is much more stable Heparin displaces core histones from chromatin and alters nuclear structure In a previous study, we showed that the activation of the plasminogen system leads to proteolysis of histone H1 of necrotic cells when incubated in the presence of murine serum [3] Proteolysis of histone H1 renders internucleosomal regions accessible to nucleolytic attack by serum DNase1, leading to internucleosomal chromatin breakdown In addition, we found that heparin-promoted chromatin degradation by WT serum was accompanied by a switch in the cleavage pattern from internucleosomal to random Our experiments using pDNA showed that heparin had no direct effect on rmDNase1 The random cleavage pattern of nuclear chromatin suggests that, in addition to H1, the nucleosomal core histones (histones H2A ⁄ H2B ⁄ H3 and H4) are displaced from chromatin FEBS Journal 276 (2009) 1059–1073 ª 2009 The Authors Journal compilation ª 2009 FEBS 1063 Murine serum nucleases M Napirei et al Fig Heparin displaces core histones from chromatin and alters nuclear structure Western blot analysis of assay supernatants containing MCF-7 cell nuclei and increasing amounts of heparin using an anti-histone H3 serum that cross-reacted with further core histones (murine histone H3, 15.4 kDa; histone H2A ⁄ H2B,  14 kDa; histone H4,  11.4 kDa) To address this question in more detail, we incubated cell nuclei in the presence of increasing amounts of heparin, and subsequently analysed the supernatants by immunoblotting for the presence of core histones, which might have diffused out of the nuclei As expected, increasing amounts of heparin led to an enhanced dissociation of nucleosomal core histones from chromatin (Fig 4) These results support the assumption that the enhanced chromatolysis by rmDNase1 and serum DNase1 in the presence of heparin is induced by a transition of protein-complexed (chromatin) to protein-free DNA Whether this transition is also the cause of the inhibition of chromatolysis by rmDNase1l3 or serum DNase1l3-like nuclease remains speculative As the hydrolysis of protein-free pDNA by rmDNase1l3 is also inhibited by heparin, it is conceivable that heparin, at least, inhibits DNase1l3 directly, for example by binding to the nuclease (see below) assay does not allow the identification of the residual nuclease activity by, for example, the estimation of the molecular mass of the nuclease Therefore, we attempted to establish a DPZ procedure for the identification of both serum nucleases employing cell culture supernatants of cells transiently expressing mDNase1 and mDNase1l3 We employed the DPZ procedure of Shiokawa et al [14], and found that the detection of both nucleases in cell culture supernatants became possible, whereas the method usually performed in our laboratory only allowed the efficient detection of DNase1 The main differences between the two methods, which led to the detection of DNase1l3, are as follows: (a) strict maintenance of the reducing conditions by the presence of 2-mercaptoethanol during electrophoresis and all further incubation steps (washing out SDS from gels, nuclease refolding and reaction within gels); (b) removal of SDS by heat and not by dissolved milk powder; (c) nuclease refolding and reaction in the absence of milk powder (for details, see Materials and methods) Experiments to optimize the DPZ procedure demonstrated that, in the presence of MnCl2 ⁄ CaCl2 instead of MgCl2 ⁄ CaCl2, detection of rmDNase1l3 was preferentially enhanced (see later) This finding was analysed in more detail using pDNA digestion assays (Fig 5) Indeed, we found that the pH optimum and nucleolytic activity of both nucleases varied in the presence of either Mg2+ or Mn2+ ions Thus, the pH optimum of rmDNase1 Establishing DPZ for the detection of rmDNase1 and rmDNase1l3 In previous experiments, we were unable to detect nucleases other than DNase1 in murine serum by native SDS-PAGE zymography (NPZ) and DPZ or the single radial enzyme diffusion (SRED) assay [7] Failure of detection of mDNase1l3, in contrast with mDNase1, by NPZ (performed at pH 8.6) is most probably explained by its strong basic pI of 8.7, in contrast with the acidic pI of 4.9 of mDNase1 For the SRED assay, we found that the failure of detection of DNase1l3-like nuclease activity in murine serum was most probably caused by its sensitivity to proteolysis Thus, freshly prepared sera of DNase1 KO mice loaded onto SRED gels displayed residual nuclease activity, which was inhibited by heparin (data not shown) However, this 1064 Fig Influence of Mn2+ and Mg2+ ions on the activity of rmDNase1 and rmDNase1l3 pDNA digestion employing cell culture supernatants containing rmDNase1 (0.1 lL supernatant, 10 of incubation at 37 °C) or rmDNase1l3 (1 lL supernatant, 30 of incubation at 37 °C) Influence of the pH value and ion composition: Assays were performed in 10 mM buffers with different pH values (acetate ⁄ NaOH, Mes ⁄ NaOH or Tris ⁄ HCl) in the presence of either mM MgCl2 ⁄ mM CaCl2 (top panel) or mM MnCl2 ⁄ mM CaCl2 (bottom panel) FEBS Journal 276 (2009) 1059–1073 ª 2009 The Authors Journal compilation ª 2009 FEBS M Napirei et al Murine serum nucleases was in the range pH 6.5–7.5 in the presence of Mg2+, and shifted by one pH unit in the presence of Mn2+ (pH 7.5–8.5) Similarly, the pH optimum of rmDNase1l3 in the presence of Mg2+ was shifted from pH 4.5–5.5 to pH 5.5–6.5 by Mn2+ Although the activity of rmDNase1 in the presence of Mn2+ was increased only slightly, rmDNase1l3 displayed strongly enhanced nucleolysis Furthermore, we found by pDNA digestion assays that increasing concentrations of Tris (approximately half activity in the presence of 80 mm Tris) and NaCl (approximately half activity in the presence of 50 mm NaCl) had a greater inhibitory influence on rmDNase1l3 than on rmDNase1 (no inhibitory influence of Tris and approximately half activity in the presence of 150 mm NaCl) (data not shown) Detection of DNase1 and DNase1l3 in murine serum and tissues by DPZ To clarify that the DNase1l3-like nuclease in murine serum is indeed DNase1l3, we investigated, by the improved DPZ procedure (reducing conditions), serum samples and tissue extracts of kidney (high DNase1 content [2]) and spleen (high DNase1l3 content [8]) from WT and DNase1 KO mice (Fig 6A) We used TET and RIPA as extraction buffers (see Materials and methods), and found that nuclease detection was more efficient using RIPA buffer (Fig 6A) Detection of DNase1 in kidney samples was verified by its absence in samples of DNase1 KO mice Furthermore, we detected a nuclease signal in spleen and kidney samples of both mice of approximately 34 kDa, which corresponds to the estimated molecular mass of 33.1 kDa for mature mDNase1l3 (without the N-terminal signal peptide of 25 amino acids in length) Indeed, the expression of DNase c (DNase1l3) in human spleen and kidney has been verified previously by RNA dot blot analysis [8], and by RNA in situ hybridization for LS-DNase (DNase1l3) in Rhesus monkey macrophages of the spleen marginal zones, red pulp and the mesangium of the kidney [18] Previously, expression of LS-DNase has also been shown for hepatic Kupffer cells [8] By analysing spleen and liver tissue extracts from WT and DNase1 KO mice, we found that the 34 kDa nuclease detectable in spleen A Fig Detection of DNase1 and DNase1l3 in murine serum and tissues by DPZ (A–C) Modified DPZ under reducing conditions (D) Conventional DPZ under non-reducing conditions (see Materials and methods) (A) Analysis of spleen and kidney tissue extracts from WT and DNase1 KO mice prepared in either TET or RIPA buffer In spleen and kidney of both mice, a  34 kDa nuclease was detected DNase1 was only detectable in the kidney extract of the WT mouse and displayed a molecular mass of  37 kDa (B) The 34 kDa nuclease most probably represents DNase1l3, as it was also detectable in the liver of both mice, co-migrated with rmDNase1l3, displayed a higher activity in the presence of Mn2+ instead of Mg2+, and was inhibited by heparin (C, D) Murine serum possesses two nucleases, DNase1l3 and DNase1, which co-migrate with rmDNase1 and rmDNase1l3, respectively Human serum also contains DNase1l3; however, hDNase1 is only detectable by DPZ under non-reducing conditions Again, mDNase1l3 and hDNase1l3, by contrast with mDNase1, are inhibited by heparin C B FEBS Journal 276 (2009) 1059–1073 ª 2009 The Authors Journal compilation ª 2009 FEBS D 1065 Murine serum nucleases M Napirei et al extracts was also present in the liver (Fig 6B) Furthermore, this nuclease co-migrated with rmDNase1l3, displayed an enhanced activity in the presence of Mn2+ in comparison with Mg2+ ions, and was inhibited by the addition of heparin (Fig 6B) From these data, we conclude that the detected nuclease must be DNase1l3 Employing serum from WT and DNase1 KO mice, we demonstrated that murine serum indeed contains both DNase1, as deduced from its absence in the serum of DNase1 KO mice, and DNase1l3 (Fig 6C) Again, serum DNase1l3 co-migrated with rmDNase1l3 and, in contrast with DNase1, was inhibited by heparin (Fig 6C) In addition, we found that human serum also contains DNase1l3 (Fig 6C,D), which was also inhibited by heparin (Fig 6C) However, in contrast with mDNase1, detection of hDNase1 by DPZ was only possible under non-reducing conditions, employing the method usually performed in our laboratory (Fig 6C,D) Interestingly, rmDNase1 and DNase1 present in murine kidney extracts and serum displayed a higher molecular mass of  37 kDa in DPZ, in comparison with the calculated molecular mass of 29.8 kDa for mature mDNase1 (Fig 6A–D) Immunodetection of DNase1l3 after its purification from serum by heparin-Sepharose In order to provide further proof that the additional serum nuclease detected by DPZ is indeed DNase1l3, and to evaluate whether its inhibition by heparin is caused by direct binding, we attempted to purify the DNase1l3-like nuclease from the serum of DNase1 KO mice employing heparin-Sepharose affinity chromatography, and to detect it by immunoblotting using a polyclonal anti-mDNase1l3 serum This antibody was produced by immunizing rabbits with a fusion protein consisting of glutathione S-transferase and the C-terminal 25 amino acid residues of mDNase1l3, which are unique for this nuclease among the members of the DNase1 family Purification by affinity chromatography revealed that the DNase1l3-like serum nuclease indeed bound to heparin with high specificity, as revealed by its elution from heparin-Sepharose only at high ionic strength (Fig 7A) This result indicates that inhibition of this nuclease by heparin is caused by a direct interaction Next, we purified the DNase1l3-like nuclease from 0.5 mL of WT serum and, after further concentration, equal parts of the sample were used in immunoblotting and DPZ Cell extracts of NIH-3T3 fibroblasts transiently transfected with mDNase1 or mDNase1l3 were employed as control We found that the anti-mDNase1l3 serum recognized mDNase1l3 in 1066 A B Fig Immunodetection of DNase1l3 after purification from serum by heparin-Sepharose (A) DPZ under reducing conditions Murine DNase1l3 was purified from mL of serum collected from DNase1 KO mice by heparin-Sepharose affinity chromatography Serum samples (2 lL) taken pre- and post-chromatography reveal the efficient binding of DNase1l3 to heparin Binding remained stable during two washing steps with 0.2 M NaCl (fractions I and II) Elution (fractions III–VII, 10-fold enrichment in comparison with the original serum) with increasing amounts of NaCl revealed a strong affinity of DNase1l3 to heparin, which could only be effectively dissolved by the addition of M NaCl (B) DNase1l3 of 0.5 mL of serum collected from WT mice was purified by heparin-Sepharose affinity chromatography, and the two halves were employed in DPZ under reducing conditions (top panel) and in immunoblotting (bottom panel) against mDNase1l3, using cell extracts of NIH-3T3 fibroblasts transiently transfected with mDNase1 or mDNase1l3 as a control the corresponding NIH-3T3 cell extract and that mDNase1l3 purified from WT serum with high specificity in comparison with mDNase1, which was only detected by DPZ (Fig 7B) Murine DNase1 is di-N-glycosylated, whereas murine DNase1l3 is not N-glycosylated DPZ demonstrated a higher molecular mass of rmDNase1 and DNase1 present in murine serum and kidney in comparison with rmDNase1l3 and DNase1l3 detected in murine serum, spleen, kidney and liver samples (Fig 6) Murine DNase1l3 migrated at an expected molecular mass of  34 kDa in DPZ, which is consistent with the calculated molecular mass of 33.1 kDa for the mature mDNase1l3, i.e without its FEBS Journal 276 (2009) 1059–1073 ª 2009 The Authors Journal compilation ª 2009 FEBS M Napirei et al N-terminal signal peptide In contrast, mDNase1 migrated at  37 kDa, although the calculated molecular mass for the mature enzyme without its N-terminal signal peptide is 29.8 kDa As it has been described that bovine DNase1 displays tissue-specific mono- or di-N-glycosylation of the high mannose or complex type [22], we analysed rmDNase1 and rmDNase1l3 for the presence of N-glycosylation Murine DNase1 possesses two potential N-glycosylation sites (Asn-XSer ⁄ Thr) at Asn18 and Asn106, whereas murine DNase1l3 possesses one potential site at Asn283 (the numbering refers to the amino acid sequence of the mature protein without the N-terminal signal peptide) [7] However, Asn283 is not conserved between DNase1l3 of mouse, rat and humans [7] We treated both nucleases with endoglycosidase H (EndoH), which cleaves high-mannose and, in part, hybrid N-glycans, or with peptide N-glycosidase F (PNGaseF), which cleaves all forms of N-glycans, and subsequently performed DPZ (Fig 8) We found that rmDNase1l3 is apparently not N-glycosylated, whereas rmDNase1 is di-N-glycosylated (Fig 8A) Obviously, secreted rmDNase1 is a mixture of molecules differing in the composition of the two N-glycosylation sites Approximately half of the molecules possessed one high-mannose and one complex N-glycan [only the high-mannose N-glycan was cleavable by EndoH, leading to migration of EndoH-treated rmDNase1 between di- ( 37 kDa) and de- ( 29 kDa) N-glycosylated rmDNase1 at  35 kDa) The other half possessed two complex N-glycans [not cleavable by EndoH, leading to migration of EndoH-treated rmDNase1 at the molecular mass of non-treated rmDNase1 ( 37 kDa)] A very minor proportion possessed two high-mannose N-glycans (both cleavable by EndoH, leading to migration of EndoH-treated rmDNase1 at  29 kDa, which is consistent with the calculated molecular mass for mature mDNase1) (Fig 8A) As expected, PNGaseF cleaved both N-glycans, leading to completely de-N-glycosylated rmDNase1 (Fig 8A) In order to verify that di-N-glycosylation of mDNase1 also occurs in vivo, we repeated the experiments with murine parotid gland DNase1 and obtained identical results (Fig 8B) These data suggest that, after transfection, rmDNase1 is glycosylated in a random manner by NIH-3T3 cells, and in vivo by the exocrine cells of the parotid gland, and furthermore demonstrate that the putative glycosylation site of DNase1l3 is not recognized Discussion In the present work, we continued our previous studies on the characterization of the nucleolytic activities Murine serum nucleases A B Fig Murine DNase1 is di-N-glycosylated, whereas mDNase1l3 is not DPZ of rmDNase1 and rmDNase1l3 (A) and murine parotid gland DNase1 (B) treated with EndoH or PNGaseF Recombinant murine DNase1l3 is not N-glycosylated [bottom panel of (A)] Recombinant murine DNase1 [top panel of (A)] and parotid gland DNase1 (B) represent a mixture of di-N-glycosylated molecules Approximately one-half of the molecules possessed two complex N-glycans (resistant to deglycosylation by EndoH; molecular mass  37 kDa); the other half possessed one high-mannose and one complex N-glycan (leading to mono-N-glycosylation after EndoH treatment, molecular mass  35 kDa); a very minor proportion possessed two high-mannose N-glycans, leading to complete de-N-glycosylation by EndoH comparable with that by PNGaseF treatment (resulting in a molecular mass of 29.8 kDa as calculated from the sequence of mature mDNase1) Mature mDNase1l3 has a calculated molecular mass of 33.1 kDa, which is consistent with the estimated molecular mass of rmDNase1l3 ( 34 kDa) from the zymograms of DNase1 and DNase1l3 By comparing the properties of rmDNase1 and rmDNase1l3 in the hydrolysis of pDNA and chromatin with those of serum collected from WT and DNase1 KO mice, we were able to clarify the identity of the nucleolytic activities of murine serum Our new experiments prove that murine and human sera contain both DNase1 and FEBS Journal 276 (2009) 1059–1073 ª 2009 The Authors Journal compilation ª 2009 FEBS 1067 Murine serum nucleases M Napirei et al DNase1l3 Our previous studies concentrated on the characterization of the properties of serum DNase1, although we suspected the presence of an additional nuclease with biochemical properties of rrDNase1l3 in murine serum [7] Properties of DNase1 and DNase1l3 in the hydrolysis of DNA substrates Our data demonstrate that rmDNase1 and rmDNase1l3 harbour different properties with regard to DNA substrates Thus, rmDNase1l3 cleaves proteinfree pDNA with a lower efficiency in comparison with rmDNase1, but degrades chromatin more rapidly as a result of preferential cleavage at internucleosomal sites Our experiments show that the presence of Mn2+ instead of Mg2+, in addition to Ca2+-ions enhances, in particular, DNase1l3 activity over a broad pH range Previously, Mizuta et al [23] have reported that recombinant human DNase c (DNase1l3) is a Ca2+ ⁄ Mg2+-dependent ssDNA nuclease with high activity at low ionic strength Furthermore, it has been reported that Mn2+, in contrast with Mg2+, has different effects on DNA conformation: (a) it leads to toroidal condensates of supercoiled pDNA, resulting in more extensive digestion by S1 nuclease [24]; and (b) it affects the CD spectra, especially of GC-rich native DNA, by binding to the GC pairs in addition to the phosphate groups [25] Proton displacement by Mn2+ from GC pairs leads to conformational changes of the double helix, which are interpreted as tilting of the bases of locally Mn2+-chelated regions [25] These data may explain why DNase1l3, in particular, which has been described to have a higher affinity and ⁄ or cleavage activity towards ssDNA, is activated in the presence of Mn2+ The activating effect of Mn2+ was used by us to optimize the detection of DNase1l3 in pDNA and chromatin digestion assays, as well as in DPZ In accordance with Mizuta et al [23], we found that high ionic strength (NaCl or Tris) more strongly inhibited the activity of rmDNase1l3 than of rmDNase1 Nevertheless, our experiments demonstrate that, in undiluted serum, DNase1l3 is sufficiently active to facilitate chromatolysis at physiological ionic strength and composition Similarly, the murine serum DNase1 concentration is also sufficient to induce chromatolysis, provided that the activation of the plasminogen system occurs or other proteases are present This dependence may explain previous observations indicating that normal physiological concentrations of DNase1 in human serum are insufficient to degrade DNA [26] 1068 Histone degradation by proteases is not necessary for chromatolysis by rmDNase1l3 Recombinant murine DNase1l3 and DNase1l3 from other species are able to induce internucleosomal chromatin degradation on their own [7,23] Indeed, Mizuta et al [23] have demonstrated that histone H1 functions as a co-activator of DNase c, leading to the degradation of pDNA and chromatin at physiological ionic strength They hypothesized that DNase c might compete with histone H1 for DNA binding and, after histone H1 displacement, will gain access to and hydrolyse chromatin DNA This competition seems to be conceivable, as rmDNase1l3 (pI 8.7) has an estimated charge of +6.7 at pH 7.0; thus, it is a basic protein, like the histones, at physiological pH values In contrast, rmDNase1 (pI 4.9) is an acidic protein with a charge of )9.4 at pH 7.0, which might explain the opposite behaviour of the two nucleases despite their structural similarities [7] Alternatively, it has been proposed that histone H1 binding to internucleosomal regions might generate ssDNA portions, which are preferred targets for cleavage by DNase1l3 [23] Indeed, an altered DNA conformation seems to be crucial for efficient DNA hydrolysis by rmDNase1l3, as demonstrated by our observation of enhanced cleavage of pDNA and chromatin in the presence of Mn2+ From these findings, we propose that the activating mode of histone H1 and Mn2+ on DNA hydrolysis by rmDNase1l3 may be caused by their similar influence on DNA conformation and not by displacement of histone H1 Our data demonstrate that heparin inhibits the cleavage of chromatin and protein-free pDNA by rmDNase1l3, whereas it activates chromatolysis and does not influence pDNA digestion by rmDNase1 As heparin is a negatively charged sulfated polysaccharide, a direct interaction with polyanionic DNA can be excluded Therefore, we propose that heparin binds directly to and inhibits DNase1l3 This assumption is consistent with the fact that DNase1l3 binds to heparin-Sepharose at physiological pH values [27] Indeed, we were able to verify this interaction by purifying the DNase1l3-like nuclease activity of murine serum through heparin-Sepharose affinity chromatography Subsequent immunoblotting, employing an antibody generated against a peptide comprising the last 25 C-terminal amino acids of mDNase1l3, demonstrated that the second nucleolytic activity of murine serum is identical to that of DNase1l3 In contrast, DNase1 is negatively charged at physiological pH values and is not inhibited by heparin Previously, we suspected that the activating effect of heparin on chromatolysis by serum DNase1 might be caused by hyperactivation of the plasminogen system [3] However, our present data FEBS Journal 276 (2009) 1059–1073 ª 2009 The Authors Journal compilation ª 2009 FEBS M Napirei et al show that, in the absence of the plasminogen system, heparin has the same activating effect on rmDNase1 Therefore, its effect is most probably caused by displacement of the positively charged histones from chromatin, thereby generating protein-free DNA, which is more efficiently cleaved by DNase1, leading to a switch from an internucleosomal to a random chromatin cleavage pattern Indeed, in agreement with Hildebrand et al [28], we verified the displacement of all histones by heparin Thus, heparin mimics the effect of proteases on DNase1-induced chromatolysis by generating stretches of protein-free chromosomal DNA Serum DNase1 and DNase1l3 might suppress antinuclear autoimmunity DNase1l3 was originally isolated from rat thymus, implied to be essential for chromatin degradation during apoptosis, and therefore regarded as a purely intracellular endonuclease [29] However, the present data demonstrate that DNase1l3 is also secreted in vivo into murine and human serum in addition to DNase1 We found that, as a result of the activation of the plasminogen system in the in vitro chromatin digestion assays, murine serum DNase1l3 is rapidly degraded by plasmin, whereas di-N-glycosylation probably protects DNase1 from proteolysis These findings explain why, in previous studies, serum DNase1l3 was underestimated or not discovered Thus, our data demonstrate that both serum nucleases are involved in chromatolysis under physiological ion concentrations and composition in vitro Serum nucleases seem to fulfil intra- as well as extravascular functions in vivo: (a) clearance of chromatin released into the circulation by dying cells to prevent occlusion of capillaries by DNA clots; (b) clearance of nuclear debris within inflamed tissues to prevent autoantigen formation; (c) clearance of circulating immune complexes composed of ANA and their DNA-containing antigens to prevent their renal deposition; and (d) clearance of deposited nuclear antigens and immune complexes from basement membranes to suppress inflammation caused by hypersensitivity reactions [4] All of these functions can be summarized as ‘suppression of antinuclear autoimmunity’ Indeed, it has been shown that many patients with SLE [5,30] and SLEprone mice [4–6] display a lack or decrease in serum DNase1 The participation of murine DNase1l3 in the suppression of antinuclear autoimmunity has also been postulated Thus, it has been shown that SLE-prone MRL-lpr and NZB ⁄ W F1 mice possess a homozygous missense mutation within the DNase1l3 gene, resulting in a reduced activity of DNase1l3 secreted by splenocytes and bone marrow-derived macrophages [21] Murine serum nucleases The specific roles of the two serum nucleases in vivo have been investigated poorly to date For serum DNase1, it has been shown that it is involved in the degradation of chromatin released from necrotic cells (hepatocytes) Thus, after the induction of hepatocellular necrosis by an overdose of acetaminophen, nucleosomal chromatin fragments accumulate in the blood of DNase1 KO mice, whereas, in WT mice, these fragments disappear as a result of further degradation to oligonucleotides [31] These data imply that DNase1 present in the serum and ⁄ or liberated from necrotic hepatocytes degrades chromatin to oligonucleotides Indeed, in vitro data have demonstrated that extracellular DNase1 penetrates and accumulates within the nuclei of necrotic cells [3] Necrotic cells induce inflammation, accompanied by an increased permeability of blood vessels Thus, it is conceivable that serum DNase1 diffuses into necrotic tissues The same can be hypothesized for DNase1l3 Furthermore, it is conceivable that DNase1l3 is secreted by macrophages recruited into inflamed tissues Thus, DNase1l3 may function as the primary chromatolytic activity generating nucleosomal fragments, which are subsequently further degraded by DNase1 Activation of the plasminogen system at sites of necrosis and inflammation is a well-known phenomenon [32] However, plasmin activity is tightly controlled by extracellular protease inhibitors to prevent extensive tissue damage Our in vitro results demonstrate the degradation of mDNase1l3 by plasmin; however, it is still unclear whether this is also true for the in vivo situation The exact concentrations of released and activated enzymes within inflamed tissues, the order of their appearance and the duration of their activity have not been clarified to date Further in vivo experiments are necessary to resolve these questions In summary, our data reveal, for the first time, that serum contains two nucleases – DNase1 and DNase1l3 – which display different substrate specificities Both nucleases may complement or substitute each other under certain conditions during chromatin degradation It is hoped that future studies on DNase1, DNase1l3 and double KO mice will provide further insight into the exact function and role of the two nucleases in the prevention of antinuclear autoimmunity Materials and methods Cloning of murine DNase1 and DNase1l3 expression vectors For cloning of the murine DNase1 and DNase1l3 cDNA, total RNA was isolated from kidney (DNase1) or spleen FEBS Journal 276 (2009) 1059–1073 ª 2009 The Authors Journal compilation ª 2009 FEBS 1069 Murine serum nucleases M Napirei et al (DNase1l3) of a C57BL ⁄ mouse using the RNeasy Mini Kit (Qiagen, Hilden, Germany) The cDNAs were generated by reverse transcription of lg of RNA employing the Omniscript RT Kit (Qiagen) and oligo(dT) primers (12–18 nucleotides; Sigma-Aldrich, Taufkrichen, Germany) RT-PCR of DNase1 cDNA (Genbank Accession Number NM010061, nucleotides 250–1185) was performed using the N-terminal primer 5¢-GACTGCTGCAGAATTCTCAG ATTGGCT-3¢ and the C-terminal primer 5¢-GTGGAT GCGGCCGCACCAGAAGCA-3¢ containing EcoRI and NotI restriction sites generated by site-directed mutagenesis RT-PCR of DNase1l3 cDNA (Genbank Accession Number AF047355, nucleotides 153–1114) was performed using the N-terminal primer 5¢-GAAGTCCCAGGAATTCAAAGA TGT-3¢ and the C-terminal primer 5¢-GCGTGAT ACCCGGGAGCGATTG-3¢ containing EcoRI and SmaI restriction sites generated by site-directed mutagenesis Both cDNAs were first subcloned blunt-end into the MluNI site of the vector pCAPs using the PCR Cloning Kit (Roche Diagnostics GmbH, Mannheim, Germany), subsequently isolated by EcoRI ⁄ NotI cleavage and, finally, cloned into the EcoRI ⁄ NotI sites of the vector pDsRed-N1 (Clontech, Heidelberg, Germany), from which the cDNA of the red fluorescent protein was eliminated by EcoRI ⁄ NotI cleavage Cell transfection Cultivation of NIH-3T3 fibroblasts (ACC59) was performed according to the instructions of the German Collection of Microorganisms (Braunschweig, Germany) in 90% (v ⁄ v) DMEM high-glucose (4.5 gỈL)1) medium containing 10% (v ⁄ v) heat-inactivated fetal bovine serum Gold, mm l-glutamine, mm sodium pyruvate and 1% (v ⁄ v) streptomycin ⁄ neomycin (all reagents from PAA Laboratories GmbH, Pasching, Austria) Transient transfection of the cells with expression vectors for murine DNase1 and DNase1l3 cDNA was performed by magnet-assisted transfection using the MATra-A reagent (IBA BioTAGnology, Gottingen, Germany) according to the manufacturers ă instructions Transient gene expression was allowed for 48 h, and the cell culture medium containing the secreted nucleases was divided into aliquots and stored at )20 °C until use Preparation of serum and tissue samples WT and DNase1 KO mice of the inbred strain C57BL ⁄ were bred in our animal facility The mice were allowed free access to standard laboratory chow and water, and kept in a light ⁄ dark cycle for 12 h All animal procedures carried out in this work conformed with German Animal Protection Law Blood was collected from ether-anaesthetized animals by thoracic bleeding after opening the thorax and setting a cut into the heart The blood was transferred into a microcentrifuge 1070 tube and allowed to coagulate for 1–2 h at °C Subsequently, the blood was centrifuged for 10 at 600 g and the serum was transferred into a fresh tube and stored at )20 °C until use For DPZ, 10 lL of serum were mixed with 90 lL of RIPA buffer [10 mm Tris ⁄ HCl pH 7.2, 150 mm NaCl, 0.1% (w ⁄ v) SDS, 1% (v ⁄ v) Triton X-100, 1% (w ⁄ v) sodium deoxycholate, mm EDTA and 1% (v ⁄ v) protease inhibitor cocktail (Sigma-Aldrich)], incubated for 30 on ice, subsequently mixed with 100 lL of 2· SDS gel-loading buffer [100 mm Tris ⁄ HCl pH 6.8, 4% (w ⁄ v) SDS, 0.2% (w ⁄ v) bromophenol blue, 20% (v ⁄ v) glycerol and 350 mm 2-mercaptoethanol] and 20 lL of these samples were loaded onto the zymograms after heating the samples to 95 °C for and cooling to room temperature The organs of the dead mice were removed, snap-frozen in liquid nitrogen and stored at )80 °C Tissue extracts were prepared by homogenizing the organs in lysis buffer for 30 s using a rotor-stator (Ultra-Turrax T8 homogenizer; IKA Labortechnik, Staufen, Germany) at maximal power (level 6) Either TET buffer [10 mm Tris ⁄ HCl pH 8.0, 20 mm EDTA, 0.5% (v ⁄ v) Triton X-100 and 1% (v ⁄ v) protease inhibitor cocktail] or RIPA buffer (see above) was used as lysis buffer Samples were subsequently frozen and thawed twice, incubated on ice for 30 min, and the cell debris was sedimented by centrifugation at 21 000 g for 10 at °C The protein content of the supernatants was determined by the standard Bradford procedure [33], and the samples were adjusted to a concentration of mgỈmL)1 using lysis buffer For DPZ, 100 lL of the tissue samples were mixed with 100 lL of 2· SDS gel-loading buffer, heated for to 95 °C, cooled to room temperature, and 20 lL (80 lg protein) were loaded onto the zymograms DPZ Non-reducing conditions Standard SDS-PAGE gels, according to Laemmli [34], were prepared with 4% (v ⁄ v) collecting gels without DNA and 10% (v ⁄ v) resolving gels containing 200 lgỈmL)1 calf thymus DNA (D1501, Sigma-Aldrich) Serum samples were prepared using SDS gel-loading buffer without 2-mercaptoethanol as described above, and loaded onto the zymograms As a molecular mass marker, the PageRulerÔ Prestained Protein Ladder (MBI Fermentas, St Leon-Rot, Germany) was used Electrophoresis was carried out at 80 V using Tris ⁄ glycine electrophoresis buffer [25 mm Tris, 192 mm glycine, 0.1% (w ⁄ v) SDS, pH 8.7] After electrophoresis, SDS was removed and the proteins were refolded by washing the gels overnight with 5% (w ⁄ v) milk powder dissolved in 150 mL of 10 mm Tris ⁄ HCl pH 7.8, mm CaCl2, mm MgCl2 and 10 mm sodium azide Subsequently, nuclease reaction was performed by incubating the gels in the same buffer without milk powder for 24–48 h at 37 °C Nuclease activities were detected as dark unstained areas after staining the gels with 0.5 lgỈmL)1 ethidium FEBS Journal 276 (2009) 1059–1073 ª 2009 The Authors Journal compilation ª 2009 FEBS M Napirei et al bromide and photographing the gels on a UV-light transilluminator Reducing conditions DPZ under reducing conditions, according to the method described by Shiokawa et al [14], was modified as described in the Results section and performed as follows Zymograms were prepared as described above for non-reducing DPZ Samples were prepared in SDS gel-loading buffer containing 2-mercaptoethanol as described above, and loaded onto the zymograms Electrophoresis was carried out at 80 V using Tris ⁄ glycine electrophoresis buffer and, after electrophoresis, SDS was removed by washing the gels twice with 150 mL of 10 mm Tris ⁄ HCl pH 7.8 and mm 2-mercaptoethanol at 50 °C for altogether h Nuclease refolding was performed by incubating the gels in 150 mL of 10 mm Tris ⁄ HCl pH 7.8 containing mm 2-mercaptoethanol for 24 h at 37 °C Nuclease reaction was performed by incubating the gels in 150 mL of 10 mm Tris ⁄ HCl pH 7.8, mm 2-mercaptoethanol and either mm MnCl2 ⁄ mm CaCl2 (optimal for DNase1l3 activity) or mm MgCl2 ⁄ mm CaCl2 (optimal for DNase1 activity) for 24–48 h at 37 °C To specifically inhibit DNase1l3 activity, 50 mL)1 of heparin was added to the reaction buffer Murine serum nucleases berg, Germany) Subsequently, the samples were mixed with an equal volume of 2· SDS gel-loading buffer, heated to 95 °C, cooled to room temperature, and 20 lL of the samples were subjected to DPZ as described above For the deglycosylation of murine parotid DNase1, lg of parotid protein was treated in an assay of 50 lL with either 100 U PNGaseF or 100 U EndoH for 15 or h at 37 °C Five microlitres of the samples were mixed with 100 lL of 1· SDS gel-loading buffer, heated to 95 °C, cooled to room temperature and 20 lL of the samples were subjected to DPZ under reducing conditions as described above pDNA digestion assay To examine the nucleolytic activities of cell culture supernatants containing the recombinant nucleases, 0.1 lL (rmDNase1) or lL (rmDNase1l3) of the supernatants was added to 20 lL of substrate solution (100 ng pDNA dissolved in 10 mm Tris ⁄ HCl pH 7.0, containing mm CaCl2 and mm MgCl2) and incubated at 37 °C for 10 (rmDNase1) or 30 (rmDNase1l3) Buffer and ion compositions, as well as the addition of additives, were varied as indicated in the Results section and the figure legends Thereafter, the samples were heated to 65 °C for and subjected to 1% (w ⁄ v) Tris–acetate ⁄ EDTA (TAE)-agarose gel electrophoresis Heparin-Sepharose affinity chromatography Nuclear chromatin digestion assay Murine serum DNase1l3 was purified using heparin-Sepharose (Amersham Biosciences Europe GmbH, Freiburg, Germany) in either a batch or column procedure Serum was diluted with vol of binding buffer [20 mm Tris ⁄ HCl pH 7.5, 0.15 m NaCl, 5% (v ⁄ v) glycerol, 0.1 mm EDTA and mm 2-mercaptoethanol] and added to the Sepharose (0.1–1 mL, depending on the batch or column procedure) Subsequently, the Sepharose was washed with 10 vol of washing buffer (20 mm Tris ⁄ HCl pH 7.5, 0.2 m NaCl, 0.1 mm EDTA and mm 2-mercaptoethanol) and the bound proteins were eluted with elution buffer [20 mm Tris ⁄ HCl pH 7.5, 5% (v ⁄ v) glycerol, 0.1 mm EDTA and mm 2-mercaptoethanol] containing different concentrations of NaCl (0.3–2 m) Standard elution was performed with the concentration of m NaCl Subsequently, the samples were desalted and concentrated using Ultracel YM-10 MicroconÒ Centrifugal Filter Devices (Millipore GmbH, Eschborn, Germany) All buffers were supplemented with protease inhibitor cocktail (Sigma-Aldrich) Isolation of MCF-7 cell nuclei was performed as described previously [2] The cell nuclei were treated with either cell culture supernatants containing the recombinant nucleases or serum derived from WT or DNase1 KO mice [3] Five microlitres of the cell culture supernatants were added to 105 cell nuclei diluted in 200 lL of reaction buffer (10 mm Tris ⁄ HCl, 50 mm NaCl, mm MgCl2, mm CaCl2, pH 7.0) and incubated at 37 °C for 1–2 h Serum was either employed at a concentration of 2.5% (v ⁄ v) in an assay described for the cell culture supernatants (various times of incubation at 37 °C as indicated in the figure legends) or cell nuclei were directly added to undiluted serum and incubated at 37 °C for 2–8 h Some reaction samples also contained murine Pai-1 (Calbiochem Novabiochem, Schwalbach, Germany), heparin (LiqueminÒ; Hoffmann La Roche, Grenzach Wyhlen, Germany), thrombin, plasmin or aprotinin (all supplied by Sigma-Aldrich) at the concentrations indicated in the figure legends Subsequent to the incubation step at 37 °C, nuclear DNA was isolated using a QIAamp DNA Blood Mini Kit (Qiagen), and the DNA was analysed by 1.5% (w ⁄ v) Tris– borate ⁄ EDTA (TBE)-agarose gel electrophoresis Deglycosylation Aliquots of cell culture supernatants (30 lL) containing rmDNase1 or rmDNase1l3 were treated with different amounts of either EndoH or PNGaseF according to the manufacturer’s instructions (New England Biolabs, Heidel- Anti-mDNase1l3 serum production For the generation of a polyclonal rabbit anti-mDNase1l3 serum, the last 25 amino acids of murine DNase1l3 were FEBS Journal 276 (2009) 1059–1073 ª 2009 The Authors Journal compilation ª 2009 FEBS 1071 Murine serum nucleases M Napirei et al cloned in fusion to glutathione S-transferase Using the vector pDs-mDNase1l3 (see above) as a template and employing the N-terminal (5¢-CAGTTGAGTTTAAGCTA CAGT-3¢) and C-terminal (5¢-GGCTCGAGGATACCTA GGAGC-3¢) primers containing EcoRI and XhoI restriction sites, respectively, generated by site-directed mutagenesis, the mDNase1l3 cDNA sequence (Genbank Accession Number AF047355, nucleotides 1115–1128) was amplified by PCR and cloned into the EcoRI ⁄ XhoI sites of the vector pGEX-4T2 of the GST Gene Fusion System (Amersham Biosciences Europe GmbH) The fusion protein was expressed using Escherichia coli BL21 bacteria and, after harvesting and lysing of the bacteria, it was purified employing Glutathione Sepharose 4B (Sigma-Aldrich, Deisenhofen, Germany) in a batch procedure according to the manufacturer’s instructions The purified GSTmDNase1l3 fusion protein was dialysed against NaCl ⁄ Pi, and a rabbit was immunized by subcutaneous injection of, first, 200 lg and then twice at 1-month intervals 100 lg protein dissolved in TiterMax GoldÒ (HiSS Diagnostics GmbH, Freiburg, Germany) Blood and serum were collected and prepared after months, and employed in immunoblotting transfer onto a poly(vinylidene difluoride) membrane using CAPS buffer [10 mm 3-(cyclohexamine)propane-3-sulfonic acid, 10% (v ⁄ v) methanol, pH 11.0] for semidry electrotransfer Blotting membranes were blocked as described above and immunodetection was performed using the ECL detection system (Amersham Biosciences Europe GmbH) As primary antibody, the polyclonal rabbit anti-mDNase1l3 serum (see above) was used at a dilution of : 2000 overnight at °C As a secondary antibody, an anti-rabbit IgG conjugated with horseradish peroxidase (Cell Signaling Technologies Inc., Danvers, MA, USA) was employed at a dilution of : 2000 for h at room temperature Acknowledgements The authors thank Rana Houmany, Eva Maria Konieczny and Swantje Wulf for excellent technical assistance and Dr Dirk Eulitz for providing the murine DNase1 cDNA This work was supported by a grant from the FoRUM programme of the Ruhr-University Bochum (F505-2006) References Immunoblotting For the detection of histone displacement from chromatin by heparin, · 105 MCF-7 cell nuclei were incubated in 100 lL of 10 mm Hepes pH 7.0, 50 mm NaCl, 40 mm b-glycerophosphate, 1% (v ⁄ v) protease inhibitor cocktail, mm CaCl2 and mm MgCl2 for h at 37 °C in the presence of increasing amounts of heparin Subsequently, the cell nuclei were sedimented by centrifugation for 15 at 21 000 g The supernatants were mixed with 0.25 vol of 5· SDS gel-loading buffer, and 20 lL of the samples were subjected to 15% (w ⁄ v) SDS-PAGE, followed by protein transfer onto a poly(vinylidene difluoride) membrane using the PerfectBlueÔ Semi-Dry-Electroblotter (Peqlab, Erlangen, Germany) and 48 mm Tris, 39 mm glycine, 0.037% (w ⁄ v) SDS and 20% (v ⁄ v) methanol as transfer buffer Blotting membranes were blocked with 3% (w ⁄ v) low-fat milk powder dissolved in 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Authors Journal compilation ª 2009 FEBS 1073 ... rmDNase1l3 ( 34 kDa) from the zymograms of DNase1 and DNase1l3 By comparing the properties of rmDNase1 and rmDNase1l3 in the hydrolysis of pDNA and chromatin with those of serum collected from WT and. .. (rrDNase1l3; DNase c, DNase Y, LS-DNase, nhDNase) [7,8] DNase1l3 belongs to the DNase1 nuclease family, which consists of DNase1 and three further DNase1- like endonucleases (DNase1L1, DNase1L2 and. .. nucleases in the prevention of antinuclear autoimmunity Materials and methods Cloning of murine DNase1 and DNase1l3 expression vectors For cloning of the murine DNase1 and DNase1l3 cDNA, total RNA was