Production and characterization of murine monoclonal anti-human DNase II antibodies,and their use for immunoaffinity purification of DNase II from human liver and urine

Four murine monoclonal anti-human deoxyribonuclease II (DNase II) antibodies were obtained from BALB/c mice immunized with human DNase II purified from human liver. Both single radial enzyme diffusion (SRED) and DNA-cast polyacrylamide gel electrophoresis (DNA-cast PAGE) were very useful for obtaining the DNase II-specific antibodies. All of the antibodies showed specific inhibition of human DNase II enzyme activity and specific immunostaining of the 32-kDa enzyme band, which is one of the three non-identical subunits of human DNase II molecule separated by sodium dodecyl sulfate (SDS)-PAGE followed by blotting on a transfer membrane. A formyl-cellulofine resin conjugated with each antibody specifically adsorbed and efficiently desorbed the active DNase II enzyme. Insertion of the immunoaffinity step in our purification procedure made the purification of human DNase II easier, faster and more effective than the conventional procedure.     

Biogenesis and Proteolytic Processing of Lysosomal DNase II

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

Drosophila acid DNase is a homolog of mammalian DNase II

Mammalian DNase II enzymes and the Caenorhabditis elegans homolog NUC-1 have recently been shown to be critically important during engulfment-mediated clearance of DNA. In this report, we describe the cloning and characterization of the gene encoding Drosophila DNase II. Database queries using the C. elegans NUC-1 protein sequence identified a highly homologous open reading frame in Drosophila (CG7780) that could encode a similar enzyme. Analysis of crude protein extracts revealed that wild-type Drosophila contain a potent acid endonuclease activity with cleavage preferences similar to DNase II/NUC1, while the same activity was markedly reduced in an acid DNase hypomorphic mutant line. Furthermore, the pattern of cleavage products generated from an end-labeled substrate by hypomorphic-line extracts was significantly altered in comparison to the pattern generated by wild-type extracts. Sequence analysis of CG7780 DNA and mRNA revealed that the hypomorphic line contains a missense mutation within the coding region of this gene. Additionally, Northern analysis demonstrated that CG7780 expression is normal in the mutant line, which in combination with the lowered/altered enzymatic activity and sequencing data suggested a defect in the CG7780 protein. To conclusively determine if CG7780 encoded the Drosophila equivalent of DNase II/NUC-1, transgenic lines expressing wild-type CG7780 in the mutant background were generated and subsequently shown to complement the mutant phenotype. Our results, therefore, provide compelling evidence that the predicted gene CG7780 encodes Drosophila DNase II (dDNase II), an enzyme related in sequence and activity to mammalian DNase II. Interestingly, overexpression of CG7780 both ubiquitously and in specific tissues failed to elicit any discernable phenotype.     

Human urine deoxyribonuclease II (DNase II) isoenzymes: a novel immunoaffinity purification, biochemical multiplicity, genetic heterogeneity and broad distribution among tissues and body fluids.

Deoxyribonuclease II (DNase II) was purified from the urine of a 48-year-old male (a single individual) using a column chromatography series, including concanavalin A-agarose and an immunoaffinity column utilizing anti-human spleen DNase II antibody, and was then characterized. Based on the catalytic properties of the purified enzyme, we have devised a technique of isoelectric focusing by thin-layer polyacrylamide gel electrophoresis (IEF-PAGE) combined with a specific zymogram method, for investigating the possible molecular heterogeneity of human DNase II. DNase II in urine as well as the purified form was found to exist in multiple forms with different pI values separable by IEF-PAGE within a pH range of 5-7. Since sialidase treatment of the urine sample induced simplification of the isoenzyme patterns with diminishment of anodal bands, it was clear that the multiplicity of the enzyme was in part due to differences in the sialic acid content. On screening of DNase II isoenzyme patterns in urine samples from more than 200 Japanese individuals, only the common isoenzyme pattern was observed and no electrophoretic variations were detected. However, genetic studies of urinary enzyme activity and comparative studies on the activity in urine, semen and leukocytes from the same individuals suggest that the enzyme activity level of DNase II may be under genetic control. The enzyme was widely distributed in human tissues and showed high activities in secretory body fluids such as breast milk, saliva, semen and urine, and leukocyte lysates.     

DNase,RNase, and phosphatase activities of antibodies formed upon immunization by DNA,DNase I,and DNase II

Relative DNase, RNase (efficiency of hydrolysis of ribo- and deoxyribooligonucleotides (ON)), and phosphatase (removal of the ON 5′ terminal phosphate) catalytic activities of antibodies (AB) obtained after rabbit immunization by DNA, DNase I, and DNase II were compared. It is shown that electrophoretically homogeneous preparations of polyclonal AB from non-immunized rabbits did not exhibit such activities. Immunization of rabbits by DNA, DNase I, and DNase II results in generation of IgG abzymes that exhibit high activity in the ON hydrolysis reaction and even higher activity in cleavage of 5′ terminal phosphate of ON. In this case K _m values for supercoiled plasmid DNA and ON found in reactions of their AB-dependent nuclease hydrolysis and phosphatase cleavage of 5′ terminal phosphate differ by 2–4 orders of magnitude. This shows that nuclease and phosphatase activities belong to different abzyme fractions within polyclonal AB. Thus, in this work data indicative of the possibility of a formation of antibodies exhibiting phosphatase activity after immunization of animals with DNA, DNase I, and DNase II, were obtained for the first time. Possible reasons for production of AB with phosphatase activity after immunization of rabbits with these immunogens are discussed.     

A third DNA-dependent ATPase from Bacillus cereus free of ATP-dependent DNase activity

The purification of ATP-dependent DNase from Bacillus cereus led to the isolation and characterization of a third DNA-dependent ATPase. The enzyme called ATPase III has been purified free of nuclease activity. None of the expected ATPases proved to be identical with ATP-dependent DNase-DNA-dependent ATPase. Separation of ATPase I, II and III and a DNase specific for single-stranded DNA from the same source excludes the possibility of ATP-dependent DNase being the action of a single enzyme molecule.     

Implications of CAD and DNase II in ischemic neuronal necrosis specific for the primate hippocampus

The exact molecular mechanism of ischemic neuronal death still remains unclear from rodents to primates. A number of studies using lower species animals have suggested implication of apoptosis cascade, while using monkeys the authors recently claimed necrosis cascade by calpain-induced leakage of lysosomal cathepsins (calpain-cathepsin hypothesis). This paper is to study implications of apoptotic versus necrotic cascades for the development of hippocampal CA1 neuronal death in the primate brain undergoing complete global ischemia. Here, we focused on two terminal cell death effectors; caspase-activated DNase (CAD) and lysosomal enzyme DNase II, in the monkey CA1 sector undergoing 18 min ischemia. The expressions of their mRNA and proteins, and the subcellular localizations as well as ultrastructure and specific DNA gel electrophoresis were examined. Expression of CAD was much less in the normal brain, compared with the lymph node or heart tissues. On day 1 after ischemia, however, CAD mRNA and protein were significantly increased in the CA1 sector, and then CAD protein immunohistochemically showed a translocation from the perikarya into the nucleus. Activated DNase II protein was significantly increased on days 2 and 3 after ischemia, and also showed a similar translocation indicating lysosomal leakage. Although the post-ischemic CA1 neurons showed positive terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining on days 3-5, they showed eosinophilic coagulation necrosis on light microscopy, and frank membrane disruption and mild chromatin condensation on electron microscopy. Furthermore, DNA smear pattern typical for necrosis was observed instead of DNA laddering. These data altogether suggest that the post-ischemic CA1 neuronal death of the monkey occurs not by apoptosis but by necrosis with participations of lysosomal enzymes DNase II and cathepsins as well as CAD. The interactions between apoptotic (caspase-3 and CAD) and necrotic (calpain, cathepsin and DNase II) cascades should be studied further.     

Molecular, biochemical and immunological analyses of canine pancreatic DNase I

The DNase I from canine pancreas was purified 260-fold to electrophoretic homogeneity with a 35% yield using three-step column chromatography. The activity of the purified enzyme was completely inhibited by 20 mM EDTA, an antibody specific to the purified enzyme and G-actin. A 1,373-bp cDNA encoding canine DNase I was constructed from the total canine pancreatic RNA using a rapid amplification of cDNA ends method, followed by sequencing. The mature canine DNase I protein was found to consist of 262 amino acids. A survey of DNase I in 13 different canine tissues revealed the highest levels of both DNase I enzyme activity and gene expression in the pancreas; therefore, the canine DNase I is of the pancreatic type. Phylogenetic and sequence identity analyses, studies of immunological properties and the tissue-distribution patterns of DNase I indicated that the canine enzyme is more closely related to the human DNase I than to other mammalian DNases I. Therefore, canine DNase I is found to be one of the best substitutes in studies of human DNase I.     

Human urine DNase I: immunological identity with human pancreatic DNase I, and enzymic and proteochemical properties of the enzyme

DNase I in human urine was purified to an electrophoretically homogeneous state by column chromatographies on DEAE-lignocellulose, hydroxyapatite, DEAE-cellulose, Sephadex G-75 and elastin-celite. The purified enzyme was immunologically identical with human pancreatic DNase I, but not with bovine pancreatic DNase I. The molecular weight and isoelectric point of the enzyme were estimated to be 4.1 X 10(4) and 3.6, respectively. The amino acid analysis revealed that 1 mol of the enzyme contained 8 mol of half-cystine. The N-terminal amino acid was identified as leucine by the dansyl chloride method. The enzyme was active in the presence of Mg2+, Co2+, or Mn2+, The optimum pH was around 6.5. The enzyme was stable in the pH range from 5.0 to 9.0 and at temperatures lower than 45 degrees C. The rate of hydrolysis of native DNA by the enzyme was twice as fast as that observed with heat-denatured DNA. This enzyme exhaustively degraded about 20% of the phosphodiester bonds in native DNA. The enzyme also degraded poly(dA) and poly(dT), but hardly degraded poly(dG) and poly(dC).     

Characterization of two DNase gamma-specific monoclonal antibodies and the in situ detection of DNase gamma in the nuclei of apoptotic rat thymocytes

Two novel monoclonal antibodies (mAbs), hg302 and hg303, raised against a synthetic peptide corresponding to the basic domain of human DNase gamma, are characterized in detail. In Western blot analysis, hg303 recognizes both wild type and C-terminal Myc-His-tagged human DNase gamma, but does not cross-react with human DNase I family members, DNase I, DNase X, or DNAS1L2. On the other hand, dot blot analysis reveals the fine specificity of hg302; it recognizes human, mouse, and rat DNase gamma, but not other DNase I family DNases under non-denaturing conditions. Furthermore, hg302 efficiently immunoprecipitates wild type, but not C-terminal Myc-His-tagged, human DNase gamma from cell lysates. In immunohistochemical analysis, hg302 strongly recognizes DNase gamma in the nuclei of X-ray irradiation-induced apoptotic, but not normal rat thymocytes. The specific detection of DNase gamma in apoptotic nuclei is confirmed by indirect-immunofluorescence analysis using TNF-alpha-induced apoptotic HeLa S3 cells transfected with DNase gamma. These results, together with the observations that DNase gamma is present in normal thymocytes and its activity is unchanged during the apoptosis, suggest that some molecular change(s), which triggers the activation of DNase gamma, occurs in response to apoptotic stimuli in the basic domain, and hg302 specifically recognizes the activated DNase gamma in immunohistochemical analysis.     

Carp hepatopancreatic DNase I: biochemical,molecular, and immunological properties

A survey of DNase I in nine different carp tissues showed that the hepatopancreas has the highest levels of both DNase I enzyme activity and gene expression. Carp hepatopancreatic DNase I was purified 17,000-fold, with a yield of 29%, to electrophoretic homogeneity using three-step column chromatography. The purified enzyme activity was inhibited completely by 20 mM EDTA and a specific anti-carp DNase I antibody and slightly by G-actin. Histochemical analysis using this antibody revealed the strongest immunoreactivity in the cytoplasm of pancreatic tissue, but not in that of hepatic tissue in the carp hepatopancreas. A 995-bp cDNA encoding carp DNase I was constructed from total RNA from carp hepatopancreas. The mature carp DNase I protein comprises 260 amino acids, the same number as the human enzyme, however, the carp enzyme has an insertion of Ser59 and a deletion of Ala225 in comparison with the human enzyme. These alterations have no influence on the enzyme activity and stability. Three amino acid residues, Tyr65, Val67, and Ala114, of human DNase I are involved in actin binding, whereas those of carp DNase I are shifted to Tyr66, Val68, and Phe115, respectively, by the insertion of Ser59: the decrease in affinity to actin is due to one amino acid substitution, Ala114Phe. The results of our phylogenetic and immunological analyses indicate that carp DNase I is not closely related to the mammalian, avian or amphibian enzymes, and forms a relatively tight piscine cluster with the tilapia enzyme.     

Expression and characterization of a DNase I-Fc fusion enzyme

Recombinant human deoxyribonuclease I (DNase I) is an important clinical agent that is inhaled into the airways where it degrades DNA to lower molecular weight fragments, thus reducing the viscoelasticity of sputum and improving the lung function of cystic fibrosis patients. To investigate DNases with potentially improved properties, we constructed a molecular fusion of human DNase I with the hinge and Fc region of human IgG1 heavy chain, creating a DNase I-Fc fusion protein. Infection of Sf9 insect cells with recombinant baculovirus resulted in the expression and secretion of the DNase I-Fc fusion protein. The fusion protein was purified from the culture medium using protein A affinity chromatography followed by desalting by gel filtration and was characterized by amino-terminal sequence, amino acid composition, and a variety of enzyme-linked immunosorbent assays (ELISA) and activity assays. The purified fusion contains DNase I, as determined by a DNase I ELISA and an actin-binding ELISA, and an intact antibody Fc region, which was quantified by an Fc ELISA, in a 2:1 stoichiometric ratio, respectively. The dimeric DNase I-Fc fusion was functionally active in enzymatic DNA digestion assays, albeit about 10-fold less than monomeric DNase I. Cleavage of the DNase I-Fc fusion by papain resulted in a specific activity comparable to the monomeric enzyme. Salt was inhibitory for wild type monomeric DNase I but actually enhanced the activity of the dimeric DNase I-Fc fusion. The DNase I-Fc fusion protein was also less Ca2+-dependent than DNase I itself. These results are consistent with a higher affinity of the dimeric fusion protein to DNA than monomeric DNase I. The engineered DNase I-Fc fusion protein described herein has properties that may have clinical benefits.     

Purification and properties of Epstein-Barr virus DNase expressed in Escherichia coli.

A cDNA corresponding to the BGLF5 open reading frame of the Epstein-Barr virus (EBV) genome and coding for an early DNase was inserted into the procaryotic expression vector pKK223-3. One bacterial clone producing the expected 52-kilodalton DNase was used as a source of EBV DNase. The 52-kilodalton Dnase was purified in the active form to near homogeneity by ammonium sulfate precipitation and successive chromatographies on phosphocellulose, DNA-cellulose, and gel filtration columns. The purified enzyme exhibited both exonuclease and endonuclease activities, an absolute requirement for divalent cations, an alkaline pH preference, and a typical residual activity in presence of 300 mM KCl. Moreover, the enzyme was specifically inhibited by human sera with high antibody titers to EBV early antigens. These properties are similar to those observed for EBV-induced DNase from lymphoblastoid cell extracts. In addition, the enzyme was recognized by both immunoglobulin G and A serum fractions from patients with nasopharyngeal carcinoma (NPC). From these results and previous studies which demonstrated the value of antibody titers to this viral DNase as an NPC marker, it appears that EBV-encoded DNase produced in a heterologous expression system could be used in the development of a specific and early NPC diagnosis test.     

DNase II is a member of the phospholipase D superfamily

MOTIVATION: DNase II is an endodeoxyribonuclease involved in apoptosis and essential for the mammalian development. Despite the understanding of biochemical properties of this enzyme, its structure and relationships to other protein families remain unknown. RESULTS: Using protein fold-recognition we found that DNase II exhibits a catalytic domain common to the phospholipase D superfamily. Our model explains the available experimental data and provides the first structural platform for sequence-function analyses of this important nuclease.     

Chromatin structure of the chicken beta-globin gene region. Sensitivity to DNase I, micrococcal nuclease, and DNase II

We have examined in some detail the chromatin structure of a 6.2 kilobase pair (kbp) chromosomal region containing the chicken beta-globin gene. The chromatin structure was probed with three nucleases, DNase I, micrococcal nuclease, and DNase II, and the rate of digestion of specific subfragments of the region was compared with the rate of bulk DNA digestion. We have characterized the rate of digestion of each fragment in terms of a sensitivity factor which measures the sensitivity of a fragment to a particular nuclease relative to bulk DNA. The sensitivity factors were determined by a least squares curve fitting method based on target analysis. In nuclei isolated from 14-day-old chicken embryo red blood cells, the entire 6.2-kbp region shows approximately a 10- to 20-fold increase in sensitivity to DNase I, a 3-fold increased sensitivity to micrococcal nuclease, and a 6-fold increased sensitivity to DNase II. In addition to the adult beta-globin gene, this region contains 5' and 3' flanking sequences, the 5' half of the inactive, embryonic globin gene, epsilon, and some repeated sequences. There is no obvious correlation between these genetic elements and the overall chromatin structure as measured by the nuclease sensitivity. This same region shows little or no special sensitivity in nuclei isolated from 14-day-old chicken embryo brain. Furthermore, fragments of the inactive ovalbumin gene show little or no sensitivity in either red blood cells or brain. These results support the conclusion that the entire 6.2-kbp region is largely packaged as active chromatin in 14-day-old chicken embryo red blood cells.     

Developmental regulation of topoisomerase II sites and DNase I-hypersensitive sites in the chicken beta-globin locus.

We have mapped DNase I-hypersensitive sites and topoisomerase II (topo II) sites in the chicken beta-globin locus, which contains four globin genes (5'-rho-beta H-beta A-epsilon-3'). In the 65 kilobases (kb) mapped, 12 strong hypersensitive sites were found clustered within the 25-kb region from 10 kb upstream of rho to just downstream of epsilon. The strong sites were grouped into several classes based on their tissue distribution, developmental pattern, and location. (i) One site was present in all cells examined, both erythroid and nonerythroid. (ii) Three sites, located upstream of the rho-globin gene, were present at every stage of erythroid development, but were absent from nonerythroid cells. (iii) Four sites at the 5' ends of each of the four globin genes were hypersensitive only in the subset of erythroid cells that were transcribing or had recently transcribed the associated gene. (iv) Another three sites, whose pattern of hypersensitivity also correlated with expression of the associated gene, were found 3' of rho, beta H, and epsilon. (v) A site 3' of beta A and 5' of epsilon was erythroid cell specific and present at all developmental stages, presumably reflecting the activity of this enhancer throughout erythroid development. We also mapped the topo II sites in this locus, as determined by teniposide-induced DNA cleavage. All strong teniposide-induced cleavages occurred at DNase I-hypersensitive sites, while lesser amounts of cleavage were observed in transcribed regions of DNA. Most but not all of the DNase I-hypersensitive sites were topo II sites. These data are consistent with the hypothesis that, in vivo, topo II preferentially acts on nucleosome-free regions of DNA but suggest that additional topo II regulatory mechanisms must exist.     

DEVELOPMENTAL STUDIES OF THE DIPTERAN SALIVARY GLAND : II. DNase Activity in Chironomus thummi

The deoxyribonuclease (DNase) activity of the dipteran (Chironomus thummi) salivary gland, measured both enzymatically and immunochemically, increases about 7-fold with the onset of metamorphosis. The increase in DNase activity occurs at a time when the activities of other enzymes and the total protein content are decreasing. The increased DNase activity is followed by glandular destruction. It is suggested that the alterations of this activity may be regulated by the activities of specific chromosomal sites, and that the enzyme may, at least in part, account for the glandular destruction observed at the time of increased enzyme activity.     

Purification of topoisomerase II from amsacrine-resistant P388 leukemia cells. Evidence for two forms of the enzyme

Topoisomerase II was purified from an amsacrine-resistant mutant of P388 leukemia. A procedure has been developed which allows the rapid purification of nearly homogeneous enzyme in quantities sufficient for enzyme studies or production of specific antisera. The purified topoisomerase II migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as two bands with apparent molecular masses of 180 (p180) and 170 kDa (p170); both proteins unknotted P4 DNA in an ATP-dependent manner and displayed amsacrine-stimulated covalent attachment to DNA. Staphylococcus V8 protease cleavage patterns of p170 and p180 showed distinct differences. Specific polyclonal antibodies to either p170 or p180 recognized very selectively the form of the enzyme used to generate the antibodies. Immunoblotting with these specific antibodies showed that both p180 and p170 were present in cells lysed immediately in boiling sodium dodecyl sulfate. Comparison of the purified topoisomerase II from amsacrine-resistant P388 with that from amsacrine-sensitive P388 demonstrated that each cell type contained both p180 and p170; however, the relative amounts of the two proteins were consistently different in the two cell types. The data strongly suggest that p170 is not a proteolytic fragment of p180. Thus, P388 cells appear to contain two distinct forms of topoisomerase II.     

DNase II deficiency impairs innate immune function in Drosophila

DNase II enzymes are highly conserved proteins that are required for the degradation of DNA within phagolysosomes. Engulfment of apoptotic cells and/or bacteria by phagocytic cells requires the function of DNase II to completely destroy ingested DNA. Mutation of the dnase II gene results in an increase of undegraded apoptotic DNA within phagocytic cells in mice and nematodes. Additionally, reduction of DNase II enzymatic activity in Drosophila melanogaster has been shown to lead to increased accumulation of DNA in the ovaries. Due to the importance of DNA clearance during infection, we hypothesized that a severe reduction of DNase II activity would result in diminished immune function and viability. To test this hypothesis, we knocked down DNase II activity in flies using RNAi. As expected, expression of a dnase II-RNAi construct in flies resulted in a dramatic reduction of DNase II activity and a significant decrease in total hemocyte numbers. Furthermore, infection of dnase II-RNAi flies with Gram negative or positive bacteria resulted in a severe reduction in fly viability. These results confirm that DNase II and the ability to clear macromolecular DNA is essential for maintaining proper immune function in Drosophila.