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

In contrast with serum DNase1, this nucleolytic activity efficiently degrades chromatin by internucleos-omal cleavage without proteolytic help, and is inhib-ited by heparin.. These data i

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: markus.napirei@rub.de

(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.

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

DNase1KO 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

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 proteprote-olysis 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.

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 C in 10 m M

Tris ⁄ HCl pH 7.0, 2 m M MnCl 2 and 2 m M 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) Chromachroma-tin digestion by rmDNase1 and rmDNase1l3 in the presence of plas-min or thrombin Plasplas-min 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.

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 37C, 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, demonstrachroma-ting 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 4C, 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.

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 inhibitransi-tion 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 MnCl2⁄ CaCl2instead 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

Fig 5 Influence of Mn 2+ and Mg 2+ ions on the activity of rmDN-ase1 and rmDNrmDN-ase1l3 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

2 m M MgCl2⁄ 2 m M CaCl2(top panel) or 2 m M MnCl2⁄ 2 m M CaCl2 (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).

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

rmDN-ase1l3 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-termi-nal sigN-termi-nal 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 rmDNrmDN-ase1l3, 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.

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

Mn2+in comparison with Mg2+ 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 DNrmDN-ase1 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 washdur-ing 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.

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 activitstud-ies

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.

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 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

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

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

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 hishis-tone 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 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 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