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 3 (DNase1l3)
Markus Napirei, Sebastian Ludwig, Jamal Mezrhab, Thomas Klo
¨
ckl and Hans G. Mannherz
Abteilung fu
¨
r Anatomie und Embryologie, Medizinische Fakulta
¨
t, Ruhr-Universita
¨
t Bochum, Germany
DNase1 (EC 3.1.21.1) is an endonuclease secreted into
body fluids by a wide variety of exocrine and endo-
crine organs which line the gastrointestinal and uro-
genital 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]. Previ-
ously, we have reported that, in cooperation with
different serine proteases, serum DNase1 degrades the
chromatin of necrotic cells [3]. Pure DNase1 hydro-
lyses ‘naked’ protein-free DNA with high efficiency,
Keywords
DNase1; DNase1l3; plasminogen system;

serum; systemic lupus erythematosus
Correspondence
M. Napirei, Abteilung fu
¨
r Anatomie und
Embryologie, Medizinische Fakulta
¨
t,
Ruhr-Universita
¨
t Bochum, Universita
¨
tsstraße
150, D-44801 Bochum, Germany
Fax: +49 2343214474
Tel: +49 2343223164
E-mail:
(Received 8 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 3
(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 mur-
ine DNase1 and DNase1l3 (rmDNase1, rmDNase1l3) with the serum of
wild-type and DNase1 knockout mice. Serum and rmDNase1 degrade chro-
matin 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 sensitiv-
ity 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 com-
plex 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.
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
but efficient chromatin degradation depends on the
proteolysis of DNA-bound proteins [3,7]. Heparin pro-
motes chromatin degradation by serum DNase1; how-
ever, 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 internucleos-
omal cleavage without proteolytic help, and is inhib-
ited 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 3 (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 endonucleas-
es (DNase1L1, DNase1L2 and DNase1l3) [8]. Both
DNase1 and DNase1l3 contain an N-terminal signal
peptide for their translocation into the rough endoplas-
mic 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 find-
ing 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 locali-
zation of the fusion proteins after transfection of NIH-
3T3 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 thymo-
cytes [19] and has been demonstrated to be involved in
somatic hypermutation in stimulated B cells [20]. These
studies imply that DNase1l3 fulfils intra- and extracel-
lular physiological functions in the immune system;
however, the role of its presumed NLS in fulfilling the
intracellular functions proposed is still unclear. One of
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 prote-
ases 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 ⁄ 6
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 previ-
ous studies, we found that chromatin breakdown by
the serum of WT mice was not inhibited by the addi-
tion of aprotinin [3]. Instead, we found that aprotinin
accelerated and equalized the overall nucleolytic activi-
ties 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 addi-
tion of aprotinin, thereby masking the inhibitory effect
of aprotinin on DNase1 ⁄ plasmin-induced chromatoly-
sis as described previously [3]. These results contradict
our previous studies, which demonstrated that chroma-
tin degradation by the sera of WT mice was com-
pletely 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
Murine serum nucleases M. Napirei et al.
1060 FEBS Journal 276 (2009) 1059–1073 ª 2009 The Authors Journal compilation ª 2009 FEBS
DNase1 KO mice (Fig. 1A). As it is assumed that, in
addition to DNase1, the residual serum nuclease activ-
ity 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
by activation of DNase1 ⁄ plasmin-dependent chromatin
breakdown. Employing aprotinin and heparin in par-
allel, we found that, in the sera of DNase1 KO mice,
the inhibitory effect of heparin blocked the accelerat-
ing 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 chro-
matin degradation (as described previously [3]) over-
rides its inhibitory effect on DNase1l3-like activity and
the inhibitory effect of aprotinin on DNase1 ⁄ plasmin-
dependent 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 hepa-

rin and inhibited by aprotinin as a result of plasmin
inhibition, and DNase1l3-like activity, which is inhib-
ited by heparin and activated by aprotinin. As aproti-
nin is a serine protease inhibitor, it is conceivable that
the DNase1l3-like nuclease might be sensitive to prote-
olysis or indirectly inhibited by proteolysis of DNA-
bound 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 demon-
strated 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 1 (Pai-1)
to the sera of DNase1 KO mice completed chromatoly-
sis 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 superna-
tants containing the secreted recombinant nucleases.
A
B
C
Fig. 1. Murine serum contains two chromatolytic activities with dif-
ferent properties. Digestion of nuclear chromatin by serum from
WT and DNase1 KO mice. (A) Isolated MCF-7 nuclei were incu-
bated with 2.5% (v ⁄ v) serum concentrations for 8 h at 37 °C. Apro-
tinin 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 2 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.
M. Napirei et al. Murine serum nucleases
FEBS Journal 276 (2009) 1059–1073 ª 2009 The Authors Journal compilation ª 2009 FEBS 1061
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 rmDN-
ase1, but inhibited that by rmDNase1l3 at low concen-
trations. 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 break-
down by rmDNase1, leading to a random DNA cleav-
age 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, internu-
cleosomal 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 cleav-
age by rmDNase1, possibly by inducing an alteration
in the chromatin structure itself, and inhibits chroma-
tin and pDNA cleavage by rmDNase1l3. This inhibi-
tion might be caused by direct binding of heparin to
DNase1l3 and ⁄ or an alteration of the chromatin struc-
ture (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 inhibit-
ing 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 break-
down 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 proba-
bly 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
A
C
B
D
Fig. 2. 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 2 lL of cell culture
supernatants, respectively. Incubation for 30 min at 37 °Cin10m
M
Tris ⁄ HCl pH 7.0, 2 mM MnCl
2
and 2 mM CaCl
2
. (B–D) Chromatin

digestion of isolated MCF-7 nuclei by rmDNase1 and rmDNase1l3:
5 lL of cell culture supernatants were employed for 2 h at 37 °C.
(B) In contrast with pDNA, nuclear chromatin digestion by rmDN-
ase1 was enhanced by heparin, leading to a random DNA cleavage
pattern. In accordance with pDNA, chromatin digestion by rmDN-
ase1l3 was inhibited by heparin. Aprotinin had no effect on chroma-
tin digestion by the two recombinant nucleases. (C) Chromatin
digestion by rmDNase1 and rmDNase1l3 in the presence of plas-
min or thrombin. Plasmin and thrombin induced internucleosomal
chromatin degradation by rmDNase1, whereas rmDNase1 per-
formed it alone. Plasmin, but not thrombin, inhibited chromatin deg-
radation by rmDNase1l3. Conditions as in (B). (D) Pre-incubation of
rmDNase1l3, but not rmDNase1, with plasmin for 30 min 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.
Murine serum nucleases M. Napirei et al.
1062 FEBS Journal 276 (2009) 1059–1073 ª 2009 The Authors Journal compilation ª 2009 FEBS
attack by rmDNase1, most probably by proteolysis of
histone H1 as shown previously [3]. In contrast, inter-
nucleosomal 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 break-

down by rmDNase1l3. These results also indicate that
plasmin, but not thrombin, proteolytically attacks and
inactivates rmDNase1l3 but not rmDNase1. To dem-
onstrate this conclusion more directly, we pre-incu-
bated both recombinant nucleases with plasmin for
30 min 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 chroma-
tin cleavage, demonstrating that rmDNase1 is not
degraded by plasmin (Fig. 2D). In contrast, pre-incu-
bation of rmDNase1l3 with plasmin inhibited subse-
quent chromatolysis, demonstrating that rmDNase1l3
is degraded by plasmin during the pre-incubation per-
iod (Fig. 2D).
Activation of plasminogen depletes the
DNase1l3-like activity of murine serum
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 activ-
ity is sensitive to proteolysis by plasmin. This is sup-
ported 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 sub-
jected serum frozen at )20 °C to thawing to room
temperature and subsequently stored it at 4 °C, it
lost its DNase1l3-like activity within 2 weeks (Fig. 3).
Thus, serum from DNase1 KO mice completely lost
its ability to induce chromatolysis, whereas serum
from WT mice still contained DNase1 ⁄ plasmin-
dependent chromatolytic activity, which was inhibited
by aprotinin and Pai-1 as described previously [3].
From these data, we conclude that the storage con-
ditions 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 hep-
arin-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.
Fig. 3. Activation of plasminogen depletes the DNase1l3-like activ-
ity of murine serum. Chromatin digestion by serum from WT and
DNase1 KO mice [2.5% (v ⁄ v) serum concentration, 8 h of incuba-
tion at 37 °C]. Top panel: serum was stored at )20 °C, thawed to
room temperature and analysed directly. Bottom panel: identical
sera analysed after 2 weeks of storage at 4 °C. The addition of
aprotinin and Pai-1 demonstrated the presence of a protease-sensi-
tive 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 activ-
ity, which is inhibited by aprotinin and Pai1, remained in the serum
from the WT mouse.
M. Napirei et al. Murine serum nucleases
FEBS Journal 276 (2009) 1059–1073 ª 2009 The Authors Journal compilation ª 2009 FEBS 1063
To address this question in more detail, we incu-
bated 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 hepa-
rin is induced by a transition of protein-complexed
(chromatin) to protein-free DNA. Whether this transi-
tion 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).
Establishing DPZ for the detection of rmDNase1
and rmDNase1l3
In previous experiments, we were unable to detect nuc-
leases 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
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 proce-
dure 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-mercapto-
ethanol during electrophoresis and all further incuba-
tion 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 MnCl
2
⁄ CaCl
2
instead of MgCl
2
⁄ CaCl
2
,
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 Mg
2+
or Mn
2+

ions. Thus, the pH optimum of rmDNase1
Fig. 5. Influence of Mn
2+
and Mg
2+
ions on the activity of rmDN-
ase1 and rmDNase1l3. pDNA digestion employing cell culture
supernatants containing rmDNase1 (0.1 lL supernatant, 10 min of
incubation at 37 °C) or rmDNase1l3 (1 lL supernatant, 30 min of
incubation at 37 °C). Influence of the pH value and ion composition:
Assays were performed in 10 m
M buffers with different pH values
(acetate ⁄ NaOH, Mes ⁄ NaOH or Tris ⁄ HCl) in the presence of either
2m
M MgCl
2
⁄ 2mM CaCl
2
(top panel) or 2 mM MnCl
2
⁄ 2mM CaCl
2
(bottom panel).
Fig. 4. Heparin displaces core histones from chromatin and alters
nuclear structure. Western blot analysis of assay supernatants con-
taining 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).
Murine serum nucleases M. Napirei et al.

1064 FEBS Journal 276 (2009) 1059–1073 ª 2009 The Authors Journal compilation ª 2009 FEBS
was in the range pH 6.5–7.5 in the presence of Mg
2+
,
and shifted by one pH unit in the presence of Mn
2+
(pH 7.5–8.5). Similarly, the pH optimum of rmDN-
ase1l3 in the presence of Mg
2+
was shifted from
pH 4.5–5.5 to pH 5.5–6.5 by Mn
2+
. Although the
activity of rmDNase1 in the presence of Mn
2+
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-termi-
nal 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 hybrid-
ization 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
B
C
D
Fig. 6. 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 condi-
tions (see Materials and methods). (A) Anal-
ysis of spleen and kidney tissue extracts
from WT and DNase1 KO mice prepared in
either TET or RIPA buffer. In spleen and kid-
ney 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 repre-
sents DNase1l3, as it was also detectable in
the liver of both mice, co-migrated with
rmDNase1l3, displayed a higher activity in
the presence of Mn
2+
instead of Mg
2+
, and
was inhibited by heparin. (C, D) Murine
serum possesses two nucleases, DNase1l3
and DNase1, which co-migrate with rmDN-
ase1 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.
M. Napirei et al. Murine serum nucleases
FEBS Journal 276 (2009) 1059–1073 ª 2009 The Authors Journal compilation ª 2009 FEBS 1065

extracts was also present in the liver (Fig. 6B). Fur-
thermore, this nuclease co-migrated with rmDNase1l3,
displayed an enhanced activity in the presence of
Mn
2+
in comparison with Mg
2+
ions, and was inhib-
ited 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 hepa-
rin (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, employ-
ing 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 com-
parison 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 chromatog-
raphy, 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-ter-
minal 25 amino acid residues of mDNase1l3, which
are unique for this nuclease among the members of the
DNase1 family. Purification by affinity chromatogra-
phy 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
the corresponding NIH-3T3 cell extract and that
mDNase1l3 purified from WT serum with high speci-
ficity 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 rmDN-
ase1 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
A
B
Fig. 7. Immunodetection of DNase1l3 after purification from serum
by heparin-Sepharose. (A) DPZ under reducing conditions. Murine
DNase1l3 was purified from 1 mL of serum collected from DNase1
KO mice by heparin-Sepharose affinity chromatography. Serum
samples (2 lL) taken pre- and post-chromatography reveal the effi-
cient binding of DNase1l3 to heparin. Binding remained stable dur-
ing 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 1
M NaCl. (B) DNase1l3 of 0.5 mL of serum col-
lected 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 fibro-
blasts transiently transfected with mDNase1 or mDNase1l3 as a
control.

Murine serum nucleases M. Napirei et al.
1066 FEBS Journal 276 (2009) 1059–1073 ª 2009 The Authors Journal compilation ª 2009 FEBS
N-terminal signal peptide. In contrast, mDNase1
migrated at  37 kDa, although the calculated molecu-
lar 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 pos-
sesses two potential N-glycosylation sites (Asn-X-
Ser ⁄ 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, lead-
ing 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 molecu-
lar mass of non-treated rmDNase1 ( 37 kDa)]. A very
minor proportion possessed two high-mannose N-gly-
cans (both cleavable by EndoH, leading to migration of
EndoH-treated rmDNase1 at  29 kDa, which is con-
sistent 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-gly-
cosylation 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 gly-
cosylated 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 stud-
ies on the characterization of the nucleolytic activities
of DNase1 and DNase1l3. By comparing the proper-
ties of rmDNase1 and rmDNase1l3 in the hydrolysis
of pDNA and chromatin with those of serum col-
lected 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 mur-
ine and human sera contain both DNase1 and

A
B
Fig. 8. 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 pos-
sessed two high-mannose N-glycans, leading to complete de-N-gly-
cosylation 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 calcu-
lated molecular mass of 33.1 kDa, which is consistent with the
estimated molecular mass of rmDNase1l3 ( 34 kDa) from the
zymograms.
M. Napirei et al. Murine serum nucleases
FEBS Journal 276 (2009) 1059–1073 ª 2009 The Authors Journal compilation ª 2009 FEBS 1067
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 rmDN-

ase1l3 harbour different properties with regard to
DNA substrates. Thus, rmDNase1l3 cleaves protein-
free 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 Mn
2+
instead of Mg
2+
, in addition to Ca
2+
-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
Ca
2+
⁄ Mg
2+
-dependent ssDNA nuclease with high
activity at low ionic strength. Furthermore, it has been
reported that Mn
2+
, in contrast with Mg
2+
, has dif-
ferent 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
Mn
2+
from GC pairs leads to conformational changes
of the double helix, which are interpreted as tilting of
the bases of locally Mn
2+
-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 Mn
2+
. The activating effect of Mn
2+
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 inhib-
ited 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 chromatoly-
sis, provided that the activation of the plasminogen
system occurs or other proteases are present. This

dependence may explain previous observations indicat-
ing that normal physiological concentrations of
DNase1 in human serum are insufficient to degrade
DNA [26].
Histone degradation by proteases is not necessary
for chromatolysis by rmDNase1l3. Recombinant mur-
ine 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-activa-
tor of DNase c, leading to the degradation of pDNA
and chromatin at physiological ionic strength. They
hypothesized that DNase c might compete with his-
tone H1 for DNA binding and, after histone H1 dis-
placement, 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 cleav-
age by DNase1l3 [23]. Indeed, an altered DNA confor-
mation seems to be crucial for efficient DNA
hydrolysis by rmDNase1l3, as demonstrated by our
observation of enhanced cleavage of pDNA and chro-
matin in the presence of Mn

2+
. From these findings,
we propose that the activating mode of histone H1
and Mn
2+
on DNA hydrolysis by rmDNase1l3 may
be caused by their similar influence on DNA confor-
mation 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 hepa-
rin-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
Murine serum nucleases M. Napirei et al.
1068 FEBS Journal 276 (2009) 1059–1073 ª 2009 The Authors Journal compilation ª 2009 FEBS
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 dis-
placement 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 gen-
erating 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 intracellu-
lar endonuclease [29]. However, the present data demon-
strate 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 sys-
tem 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 previ-
ous studies, serum DNase1l3 was underestimated or not
discovered. Thus, our data demonstrate that both serum

nucleases are involved in chromatolysis under physio-
logical ion concentrations and composition in vitro.
Serum nucleases seem to fulfil intra- as well as extra-
vascular 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 auto-
antigen formation; (c) clearance of circulating immune
complexes composed of ANA and their DNA-contain-
ing 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 ‘suppres-
sion of antinuclear autoimmunity’. Indeed, it has been
shown that many patients with SLE [5,30] and SLE-
prone 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 spleno-
cytes and bone marrow-derived macrophages [21].
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 hepatocellu-
lar necrosis by an overdose of acetaminophen, nucleos-

omal chromatin fragments accumulate in the blood of
DNase1 KO mice, whereas, in WT mice, these frag-
ments 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 extracel-
lular DNase1 penetrates and accumulates within the
nuclei of necrotic cells [3]. Necrotic cells induce inflam-
mation, 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 conceiv-
able that DNase1l3 is secreted by macrophages
recruited into inflamed tissues. Thus, DNase1l3 may
function as the primary chromatolytic activity generat-
ing nucleosomal fragments, which are subsequently
further degraded by DNase1. Activation of the plas-
minogen 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 mDN-
ase1l3 by plasmin; however, it is still unclear whether
this is also true for the in vivo situation. The exact con-
centrations 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 degr-
adation. 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 nuc-
leases 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
M. Napirei et al. Murine serum nucleases
FEBS Journal 276 (2009) 1059–1073 ª 2009 The Authors Journal compilation ª 2009 FEBS 1069
(DNase1l3) of a C57BL ⁄ 6 mouse using the RNeasy Mini
Kit (Qiagen, Hilden, Germany). The cDNAs were gener-
ated by reverse transcription of 2 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 pCAP
s
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 per-
formed according to the instructions of the German Collec-
tion of Microorganisms (Braunschweig, Germany) in 90%
(v ⁄ v) DMEM high-glucose (4.5 gÆL
)1
) medium containing
10% (v ⁄ v) heat-inactivated fetal bovine serum Gold, 2 mm
l-glutamine, 1 mm sodium pyruvate and 1% (v ⁄ v) strepto-
mycin ⁄ 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 trans-
fection using the MATra-A reagent (IBA BioTAGnology,
Go
¨
ttingen, Germany) according to the manufacturer’s

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 ⁄ 6 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 tho-
racic bleeding after opening the thorax and setting a cut into
the heart. The blood was transferred into a microcentrifuge
tube and allowed to coagulate for 1–2 h at 4 °C. Subse-
quently, the blood was centrifuged for 10 min 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, 5 mm EDTA and 1% (v ⁄ v) protease
inhibitor cocktail (Sigma-Aldrich)], incubated for 30 min on
ice, subsequently mixed with 100 lLof2· 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
5 min 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 homoge-
nizing 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 sub-
sequently frozen and thawed twice, incubated on ice for
30 min, and the cell debris was sedimented by centrifugation
at 21 000 g for 10 min at 4 °C. The protein content of the
supernatants was determined by the standard Bradford pro-
cedure [33], and the samples were adjusted to a concentration
of 8 mgÆmL
)1
using lysis buffer. For DPZ, 100 l L of the tis-
sue samples were mixed with 100 lLof2· SDS gel-loading
buffer, heated for 5 min to 95 °C, cooled to room tempera-
ture, 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 lgÆmL
)1
calf thy-
mus DNA (D1501, Sigma-Aldrich). Serum samples were
prepared using SDS gel-loading buffer without 2-mercapto-
ethanol as described above, and loaded onto the zymo-
grams. 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 electro-
phoresis, 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, 3 mm
CaCl
2
,3mm MgCl
2
and 10 mm sodium azide. Subse-
quently, 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 lgÆmL
)1
ethidium
Murine serum nucleases M. Napirei et al.
1070 FEBS Journal 276 (2009) 1059–1073 ª 2009 The Authors Journal compilation ª 2009 FEBS
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. Zymo-
grams 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 5 m m
2-mercaptoethanol at 50 °C for altogether 1 h. Nuclease
refolding was performed by incubating the gels in 150 mL
of 10 mm Tris ⁄ HCl pH 7.8 containing 1 mm 2-mercapto-
ethanol 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, 1 mm 2-mercaptoethanol and either 3 mm
MnCl
2
⁄ 3mm CaCl
2
(optimal for DNase1l3 activity) or
3mm MgCl
2
⁄ 3mm CaCl
2
(optimal for DNase1 activity)
for 24–48 h at 37 °C. To specifically inhibit DNase1l3 activ-
ity, 50 UÆmL
)1
of heparin was added to the reaction buffer.
Heparin-Sepharose affinity chromatography
Murine serum DNase1l3 was purified using heparin-Sepha-
rose (Amersham Biosciences Europe GmbH, Freiburg,
Germany) in either a batch or column procedure. Serum
was diluted with 5 vol of binding buffer [20 m m Tris ⁄ HCl
pH 7.5, 0.15 m NaCl, 5% (v ⁄ v) glycerol, 0.1 mm EDTA
and 1 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 1 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
1mm 2-mercaptoethanol] containing different concentra-
tions of NaCl (0.3–2 m). Standard elution was performed
with the concentration of 1 m NaCl. Subsequently, the sam-
ples 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).
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-
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, 1 lg of parotid
protein was treated in an assay of 50 lL with either 100 U
PNGaseF or 100 U EndoH for 15 min or 2 h at 37 °C.
Five microlitres of the samples were mixed with 100 lLof
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 superna-
tants containing the recombinant nucleases, 0.1 lL (rm-

DNase1) or 1 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 2 mm CaCl
2
and
2mm MgCl
2
) and incubated at 37 °C for 10 min (rmDNase1)
or 30 min (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 5 min and subjected to 1%
(w ⁄ v) Tris–acetate ⁄ EDTA (TAE)-agarose gel electrophoresis.
Nuclear chromatin digestion assay
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 10
5
cell nuclei diluted in 200 lL of reaction buffer (10 mm
Tris ⁄ HCl, 50 mm NaCl, 2 mm MgCl
2
,2mm CaCl
2
, 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, Ger-
many), 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.
Anti-mDNase1l3 serum production
For the generation of a polyclonal rabbit anti-mDNase1l3
serum, the last 25 amino acids of murine DNase1l3 were
M. Napirei et al. Murine serum nucleases
FEBS Journal 276 (2009) 1059–1073 ª 2009 The Authors Journal compilation ª 2009 FEBS 1071
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 Num-
ber 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 GST-
mDNase1l3 fusion protein was dialysed against NaCl ⁄ P
i
,
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 col-
lected and prepared after 3 months, and employed in
immunoblotting.
Immunoblotting
For the detection of histone displacement from chromatin
by heparin, 5 · 10
5
MCF-7 cell nuclei were incubated in
100 lLof10mm Hepes pH 7.0, 50 mm NaCl, 40 mm
b-glycerophosphate, 1% (v ⁄ v) protease inhibitor cocktail,
2mm CaCl
2
and 2 mm MgCl
2
for 2 h at 37 °C in the pres-
ence of increasing amounts of heparin. Subsequently, the
cell nuclei were sedimented by centrifugation for 15 min 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 sub-
jected to 15% (w ⁄ v) SDS-PAGE, followed by protein trans-
fer 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 pow-
der dissolved in Tris-buffered saline supplemented with
0.05% (v ⁄ v) Tween-20. Immunodetection of histone H3
using the alkaline phosphatase protocol, with 5-bromo-4-
chloro-3-indolyl phosphate and nitroblue tetrazolium as
detection substrates, was performed according to Sambrook
et al. [35]. As primary antibody, a polyclonal goat anti-his-
tone H3 serum (sc-8654; Santa Cruz Biotechnology Inc.,
Santa Cruz, CA, USA) was employed at a dilution of
1 : 250 overnight at 4 °C. As a secondary antibody, a
chicken anti-goat IgG conjugated with alkaline phosphatase
(sc-2965; Santa Cruz Biotechnology) was employed at a
dilution of 1 : 2000 for 1 h at room temperature.
For anti-mDNase1l3 immunoblotting, samples were
subjected to 10% (v ⁄ v) SDS-PAGE, followed by protein
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 electro-
transfer. 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 1 : 2000 over-
night at 4 °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 1 : 2000 for 1 h at room temperature.
Acknowledgements
The authors thank Rana Houmany, Eva Maria Kon-

ieczny and Swantje Wulf for excellent technical assis-
tance 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).
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