Intestinal DNases of 36 and 38.5kDa from the parasitic nematode Haemonchus contortus have non-classic DNase characteristics and produce DNA fragments with 3'-hydroxyls

Treatment with the anthelmintic fenbendazole induces fragmentation of genomic DNA in intestinal cells of Haemonchus contortus. This effect is characterized by DNA fragments with 3'-hydroxyls (OH). Investigation into DNases responsible identified intestinal DNase activities that produce DNA fragments with 3'-OH. However, this interpretation was complicated by a mixture of activities in the intestinal fractions evaluated. In addition, intestinal activities displayed non-classic characteristics. Here it is shown that heparin sulfate (HS) fractionation enriched for intestinal DNases that produce 3'-OH. The 2.0M NaCl fraction of HS contained DNase activity that produced 3'-OH with minimal contamination by activity that produced 3'-phosphates (P). 3'-OH were produced under acidic (pH 5.0) or neutral (pH 7.0) conditions by DNases in this fraction. These DNases were sensitive to EDTA under each condition. Furthermore, EDTA-sensitive DNase activity in this fraction digested H. contortus intestinal cell nuclear DNA in histological sections, producing 3'-OH under acidic and neutral conditions. DNases at 36 and 38.5kDa in this fraction each produced 3'-OH at pH 5.0 when gel eluted, and each activity was sensitive to EDTA. Hence, the 36 and 38.5kDa DNases in the 2.0M NaCl HS intestinal fraction have characteristics expected for candidate DNases that mediate DF in H. contortus intestinal cell nuclei induced by fenbendazole. DNase activity that produces 3'-OH under acidic condition with sensitivity to EDTA is unconventional for classic acidic or neutral DNases and is a unique finding for nematodes. Excretory/secretory products from the worm and whole worm lysates were also explored as sources to fractionate intestinal DNases identified. HS fractionation of those worm samples did not clearly resolve the intestinal DNases of interest, although DNases with distinct characteristics were identified in each source.     

Non-classic characteristics define prominent DNase activities from the intestine and other tissues of Haemonchus contortus

The anthelmintic fenbendazole (FBZ) induces nuclear DNA fragmentation (DF) in intestinal cells of Haemonchus contortus. The DNA fragments had 3'-OH, which suggests involvement of a neutral DNase. To identify candidate DNase(s) involved, DNase activity in H. contortus intestine and other worm fractions was characterized relative to classic DNases I (neutral) and II (acidic). Seven distinct DNase activities were identified and had Mrs of 34, 36, 37 or 38.5 kDa on zymographic analysis. The different activities were distinguished according to pH requirement, sensitivity to 10 mM EDTA and worm compartment. Activities of intestinal DNases at 34, 36 and 38.5 kDa were sensitive to EDTA at pH 5.0 and 7.0. Sensitivity to EDTA at pH 5.0 was unexpected compared to classic acidic DNase II activity, suggesting unusual properties of these DNases. In whole worms, however, the activities at 36 and 38.5 kDa were relatively insensitive to EDTA, indicating predominance of DNases that are distinct from the intestine. The activity at 37 kDa in excretory/secretory products had an acidic pH requirement and was insensitive to EDTA, resembling classic acidic DNase activity. Under conditions of pH 5.0 and 7.0, intestinal DNases produced 3'-ends that could be labeled by terminal deoxynucleotidyl transferase, indicating presence of 3'-OH. The labeling of 3'-ends at pH 5.0, again, was unexpected for acidic DNase activity. These results and several other activities suggest that multiple H. contortus DNases have characteristics distinct from the classic mammalian DNases I and II. Treatment of H. contortus with FBZ did not induce any detectable DNase activities distinct from normal intestine, although relative activities of intestinal DNases appear to have been altered by this treatment.     

Mammalian deoxyribonucleases I are classified into three types: pancreas, parotid, and pancreas-parotid (mixed), based on differences in their tissue concentrations

Deoxyribonuclease I (DNase I) activities were measured in 14 different tissues from humans and 5 other mammals (bovine, pig, rabbit, rat, and mouse) by using the single radial enzyme diffusion (SRED) method, which is a sensitive and nonradioactive assay for nucleases. The results indicated that these species are classifiable into three groups on the basis of their different tissue distributions of DNase I. In human and pig, the pancreas showed the highest activity of DNase I; in rat and mouse, the parotid glands showed the highest activity; and in bovine and rabbit, both pancreas and parotid glands showed high activity. Therefore we designated human and pig DNase I as pancreas type, rat and mouse DNase I as parotid type, and bovine and rabbit DNase I as pancreas-parotid (or mixed) type. DNase I of the pancreas type was more sensitive to low pH than the other types. DNase I of pancreas type is secreted into the intestinal tract under neutral pH conditions, whereas the other types are secreted from the parotid gland and have to pass through the very acidic conditions in the stomach. Differences in the tissue distribution and acid sensitivity of mammalian DNases I may provide important information about their digestive function from the evolutionary perspective.     

Isolation, characterisation and crystallization of deoxyribonuclease I from bovine and rat parotid gland and its interaction with rabbit skeletal muscle actin

A purification procedure is described yielding DNase I from bovine and rat parotid glands of high homogeneity. The apparent molecular masses of the DNases I isolated have been found by sodium dodecyl sulfate/polyacrylamide gel electrophoresis to be 34 and 32 kDa for bovine and rat parotid DNase I, respectively, and thus differ from the enzyme isolated from bovine pancreas (31 kDa). By a number of different criteria concerning their enzymic behaviour, the isolated enzymes could be clearly classified as DNases I, i.e. endonucleolytic activity preferentially on native double-stranded DNA yielding 5'-oligonucleotides, a pH optimum at about 8.0, the dependence of their enzymic activity on divalent metal ions, their inhibition by 2-nitro-5-thiocyanobenzoic acid and by skeletal muscle actin. Comparison of their primary structure by analysis of their amino acid composition and also two-dimensional fingerprints and isoelectric focusing indicate gross similarities between the enzymes isolated from bovine pancreas and parotid, but distinct species differences, i.e. between the enzymes isolated from bovine and rat parotid. All the DNases I are glycoproteins. From bovine parotid DNase I crystals suitable for X-ray structure analysis could be obtained. The DNases I from both parotid sources specifically interact with monomeric actin forming 1:1 stoichiometric complexes. Their binding constants to monomeric actin differ, being 2 X 10(8) M-1 and 5.5 X 10(6) M-1 for bovine and rat parotid DNase I, respectively. Only the enzyme isolated from bovine sources is able to depolymerize filamentous actin.     

Acid DNases and their interest among apoptotic endonucleases

Apoptosis is characterized by cell shrinkage, nuclear condensation and internucleosomal DNA cleavage. Besides the central role of caspases and other proteases, cell death triggers DNA degradation so that DNases have an active role in apoptotic cell death. The best-characterized apoptotic DNase is CAD, a neutral Mg-dependent endonuclease. Its activity is regulated by its inhibitor, ICAD, which is cleaved by caspases. Other neutral DNases have been shown to cleave nuclear DNA in apoptotic conditions: endonuclease G, GADD. In cells, the cytosolic pH is maintained to 7.2, mostly due to the activity of the Na(+)/H(+) exchanger. In many apoptotic conditions, a decrease of the intracellular pH has been shown. This decrease may activate different acid DNases, mostly when pH decreases below 6.5. Three acidic DNases II are so far known: DNase II alpha, DNase II beta and L-DNase II, a DNase II, derived from the serpin LEI (Leukocyte Elastase Inhibitor). Their activation during cell death is discussed in this review.     

Deoxyribonuclease activities in Myxococcus coralloides D

Myxococcus coralloides D produced cell-bound deoxyribonucleases (DNases) during the exponential phase of growth in liquid medium. DNase activity was much higher than that detected in other myxobacterial strains and was fractionated into three different peaks by filtration through Sephadex G-200. The DNases were named G, M and P. The optimum temperatures were 37°C, 33°C and 25°C respectively, although high activities were recorded over the temperature range 20–45°C. The pH range of high activity was between 6·0 and 9·0, with an optimum for each DNase at 8·0. DNases M and P were strongly inhibited by low concentrations of NaCl, but activity of DNase G was less affected by NaCl. The three activities required divalent metal ions as cofactors (especially Mg²⁺ and Mn²⁺); however, other metal ions (Fe²⁺, Ni²⁺, Zn²⁺) were inhibitors. The molecular weights were estimated by gel filtration chromatography and SDS-PAGE as 44 kDa (DNase G), 49 kDa (DNase M) and 39 kDa (DNase P).     

Deoxyribonuclease activities in Myxococcus coralloides D

Myxococcus coralloides D produced cell-bound deoxyribonucleases (DNases) during the exponential phase of growth in liquid medium. DNase activity was much higher than that detected in other myxobacterial strains and was fractionated into three different peaks by filtration through Sephadex G-200. The DNases were named G, M and P. The optimum temperatures were 37 degrees C, 33 degrees C and 25 degrees C respectively, although high activities were recorded over the temperature range 20-45 degrees C. The pH range of high activity was between 6.0 and 9.0, with an optimum for each DNase at 8.0. DNases M and P were strongly inhibited by low concentrations of NaCl, but activity of DNase G was less affected by NaCl. The three activities required divalent metal ions as cofactors (especially Mg2+ and Mn2+); however, other metal ions (Fe2+, Ni2+, Zn2+) were inhibitors. The molecular weights were estimated by gel filtration chromatography and SDS-PAGE as 44 kDa (DNase G), 49 kDa (DNase M) and 39 kDa (DNase P).     

Purification and characterization of the Ca2+ plus Mg2+-dependent endodeoxyribonuclease from calf thymus chromatin

Ca2+ plus Mg2+-dependent endodeoxyribonuclease was extracted from calf thymus chromatin and purified to a state free from contamination by other DNases. This DNase required both Ca2+ and Mg2+, or Mn2+ alone for its activity and the optimum pH for activity was at 6.5-7.5. No specificity for the 5'-base was observed. The molecular weight of the DNase was estimated to be about 25,000-30,000 by glycerol gradient centrifugation. Actin and antibody for pancreatic DNase (DNase I) did not inhibit the enzyme, whereas both strongly inhibited DNase I, suggesting that these two DNases are different enzymes.     

Susceptibility of mammalian deoxyribonucleases I (DNases I) to proteolysis by proteases and its relationships to tissue distribution: biochemical and molecular analysis of equine DNase I

Equine (Equus caballus) deoxyribonuclease I (DNase I) was purified from the parotid gland, and its 1295-bp cDNA was cloned. The mature equine DNase I protein consisted of 260 amino acid residues. The enzymatic properties and structural aspects of the equine enzyme were closely similar to those of other mammalian DNases I. Mammalian DNases I are classified into three types--pancreatic, parotid and pancreatic-parotid-based on their tissue distribution; as equine DNase I showed the highest activity in the parotid gland, it was confirmed to be of the parotid-type. Comparison of the susceptibility of mammalian DNases I to proteolysis by proteases demonstrated a marked correlation between tissue distribution and sensitivity/resistance to proteolysis; pancreatic-type DNase I shared properties of resistance to proteolysis by trypsin and chymotrypsin, whereas parotid-type DNase I did not. In contrast, pancreatic-parotid-type DNase I exhibited resistance to proteolysis by pepsin, whereas the other enzyme types did not. However, site-directed mutagenesis analysis revealed that only a single amino acid substitution could not account for acquisition of proteolysis resistance in the mammalian DNase I family during the course of molecular evolution. These properties are compatible with adaptation of mammalian DNases I for maintaining their activity in vivo.     

The Applicability of Agar-Diffusion Test in Measuring Deoxyribonucleases in Pig Intestinal Content

The suitability of an agar-diffusion test (ADT) using toluidine blue deoxyribonucleic acid agar (TDA) for measuring DNase activity in pig intestinal contents was investigated. The ADT was compared with a spectrophotometrical method. Distinct metachromatic zones around wells in the DNA-containing agar, into which the intestinal content was applied, indicated DNase activity. The DNase activity was determined semiquantitatively by making serial twofold dilutions of the intestinal content. The spectrophotometrical method was optimal at pH 7.2. The ADT proved to be most sensitive at pH 5.6. The ability of the 2 methods employed to measure low concentrations of DNases was equal. However, the ADT was considered more suitable than the spectrophotometrical method because ADT measured reduced amounts of enzyme. DNase activity was demonstrated throughout the small intestine and in the large intestine. By the zymogram technique, at least 3 different DNases could be demonstrated in the lower parts of the small intestine, 1 of which could be of extrapancreatic origin.     

An extracellular endodeoxyribonuclease from Streptomyces aureofaciens

Several extracellular DNases were detected after cultivation of Streptomyces aureofaciens B96 under submerged conditions. These DNases are nutritionally regulated and high content of amino acid nitrogen in cultivation medium repress their production. By varying cultivation conditions, there remained only two extracellular nuclease activities. The major one, extracellular endodeoxyribonuclease SaD I, was purified to homogeneity by ammonium sulfate precipitation, adsorption on Spheron, chromatography on Superose-12P followed by FPLC on MonoQ and final purification on HiTrapQ. The molecular weight of the purified SaD I determined by SDS-PAGE was 31 kDa. The DNase hydrolyses endonucleolytically both double-stranded and single-stranded circular and linear DNA. It does not cleave RNA and does not exhibit phosphodiesterase nor phosphomonoesterase activity. It requires a divalent cation (Zn2+, Co2+, Mn2+, Mg2+) and its activity optimum is at neutral pH (pH 7.2). The optimal temperature for DNA cleavage was 40 degrees C. Activity was strongly inhibited in the presence of phosphate, Hg2+, chelating agents or iodoacetate, but it was stimulated by addition of dimethyl sulphoxide.     

An enzyme from the earthworm Eisenia fetida is not only a protease but also a deoxyribonuclease

The earthworm enzyme Eisenia fetida Protease-III-1 (EfP-III-1) is known as a trypsin-like protease which is localized in the alimentary canal of the earthworm. Here, we show that EfP-III-1 also acts as a novel deoxyribonuclease. Unlike most DNases, this earthworm enzyme recognizes 5′-phosphate dsDNA (5′P DNA) and degrades it without sequence specificity, but does not recognize 5′OH DNA. As is the case for most DNases, Mg²⁺ was observed to markedly enhance the DNase activity of EfP-III-1. Whether the earthworm enzyme functioned as a DNase or as a protease depended on the pH values of the enzyme solution. The protein acted as a protease under alkaline conditions whereas it exhibited DNase activity under acid conditions. At pH 7.0, the enzyme could work as either a DNase or a protease. Given the complex living environment of the earthworm, this dual function of EfP-III-1 may play an important role in the alimentary digestion of the earthworm.     

The immunological and structural comparisons of deoxyribonucleases I. Glycosylation differences between bovine pancreatic and parotid deoxyribonucleases

A rabbit antiserum against bovine pancreatic DNase A is used to study the immunological reaction of DNases I. As shown by double immunodiffusion, bovine pancreatic DNases A, B, C, and D are immunologically identical, so are DNases from bovine pancreas and parotid and from ovine pancreas. These DNases also behave similarly in immunotitration of DNase activity and all are tightly bound to the immunoaffinity medium, requiring an acidic buffer with 10% ammonium sulfate to dissociate. On the other hand, porcine pancreatic and malted barley DNases that do not form precipitin lines remain active in solution with the antibody; however, in spite of the lack of inhibition these DNases are retarded (but not tightly bound) in immunoaffinity chromatography, suggesting interaction with the antibody. In thin layer isoelectric focusing, the parotid DNase, purified with the immunoaffinity technique, shows only two major active components whose isoelectric points correspond to those of DNases A and C of bovine pancreas. As estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of parotid DNase is 34,000, approximately 3,000 more than that of the pancreatic enzyme. However, both parotid and pancreatic DNases have the same NH2-terminal leucine, an identical COOH-terminal amino acid sequence, nearly identical amino acid compositions, and almost the same peptide maps. The molecular weight difference is due to differences in the carbohydrate side chains. Results of peptide analyses indicate that parotid DNase contains two glycopeptides; pancreatic DNase has only one. In addition, both parotid glycopeptides contain glucosamine and galactosamine while the pancreatic glycopeptide has only glucosamine.     

Characterization of human DNase I family endonucleases and activation of DNase gamma during apoptosis

We describe here the characterization of the so far identified human DNase I family DNases, DNase I, DNase X, DNase gamma, and DNAS1L2. The DNase I family genes are found to be expressed with different tissue specificities and suggested to play unique physiological roles. All the recombinant DNases are shown to be Ca(2+)/Mg(2+)-dependent endonucleases and catalyze DNA hydrolysis to produce 3'-OH/5'-P ends. High activities for DNase I, DNase X, and DNase gamma are observed under neutral conditions, whereas DNAS1L2 shows its maximum activity at acidic pH. These enzymes have also some other peculiarities: different sensitivities to G-actin, aurintricarboxylic acid, and metal ions are observed. Using a transient expression system in HeLa S3 cells, the possible involvement of the DNases in apoptosis was examined. The ectopic expression of each DNase has no toxic effect on the host cells; however, extensive DNA fragmentation is observed only in DNase gamma-transfected cells after the induction of apoptosis. Furthermore, DNase gamma is revealed to be located at the perinuclear region in living cells, and to translocate into the nucleus during apoptosis. Our results demonstrate that DNase I, DNase X, DNase gamma, and DNAS1L2 have similar but unique endonuclease activities, and that among DNase I family DNases, DNase gamma is capable of producing apoptotic DNA fragmentation in mammalian cells.     

Purification and analysis of DNases of Tritrichomonas foetus: evidence that these enzymes are glycoproteins

Tritrichomonas foetus is the causative agent of trichomoniasis. In cattle, infection results in economic losses to the beef and dairy industries due to abortion and infertility. Soluble DNases of T. foetus that play a role in pathogenesis and are potential therapeutic targets, were extracted and purified utilising lectin affinity chromatography. The DNases were bound to and eluted from Concanavalin A (Con A)-sepharose indicating that they are glycoproteins with -linked mannose or glucose residues. The nature of the glycans carried on the eluted proteins in the fraction containing DNase activity was assessed using an enzyme-linked lectin assay. The lectin binding studies predict the presence of both N- and O-type glycans. Manganese was a potent (33%) activator of the DNase(s) whereas zinc inhibited enzyme activity by approximately 66%. The DNase(s) had a pH optimum of 4 and a molecular weight of 160 kDa. The DNase(s) were able to completely degrade DNA from animal, plant, fungal, yeast and bacterial sources, but did not significantly degrade RNA.     

Deoxyribonucleases from rat brain

Two distinctly different DNases were isolated from rat brain and could be separated easily by ammonium sulphate fractionation. One of the DNases acts optimally at pH 5.0 hydrolysing preferentially native DNA and requiring an optimal Mg²⁺ concentration of about 0.03 m . The other DNase has its optimal pH between 7.4 and 8.9, acts preferentially on heat-denatured DNA and requires a lower Mg²⁺ concentration, the optimum being 1 × 10^?4m . Cerebellum from adult rat brain has a lower acid DNase activity and higher alkaline DNase activity, and therefore has a higher ratio of alkaline DNase to acid DNase than the other areas of brain studied. This unique activity ratio in cerebellum of adult rat brain was not observed in infant rat brain.     

Possible Relationship Between Yeast Deoxyribonucleases and Deoxyribonucleic Acid Yield

The deoxyribonucleic acid (DNA) yield and deoxyribonuclease (DNase) activity of several yeasts were correlated. Debaryomyces castellii and Debaryomyces franciscae were found to contain active DNases which carry out DNA hydrolysis, whereas the amounts of DNA as determined by extraction with Sarkosyl buffer (pH 7.8) were found to be small. On the other hand, Candida parapsilosis, Saccharomyces carmosousae, and Lodderomyces elongisporus produced no detectable DNases active at pH 7.8, and their DNA yield was correspondingly high. L. elongisporus was found to possess DNase only at pH 4.0.     

Isolation and characterization of multiple forms of ovine pancreatic deoxyribonuclease. Chromatograhpic behavior of the enzyme on concanavalin A-agarose and carboxymethylcellulose columns.

A new procedure has been devised for the purification of ovine DNase, including (NH/4)2SO4 fractionation, two steps of CM-cellulose chromatography, concanavalin A-agarose chromatography, and gel filtration on Sephadex G--100. The enzyme, like bovine DNase, exhibits multiplicity due to changes in the primary structure and the sugar structure of the carbohydrate moiety. Unlike bovine DNase, ovine DNase does not have sialic acid in any of its multiple forms. Concanavalin A-agarose is useful in the purification of not only ovine but also bovine DNase. For ovine DNase, it is a necessary and key step of purification; for bovine DNase, it can be used to purify commercial preparations of DNase free from proteases in a single step as judged by its stability in Ca2+-free media at pH 8.0. The purified enzyme has a specific activity equal to that of a highly purified DNase and presumably contains predominantly DNases A and C. Two of the four forms of ovine DNase have been purified to apparent homogeneity and subjected to chemical analysis. The present results show that bovine and ovine DNases have indistinguishable molecular weights and identical end groups, suggesting that they may have the same number of amino acid residues. The amino acid composition indicates that two enzymes may have six residues of amino acids subject to substitution which can be explained by single base changes in their genetic code words. Amino acid analyses also indicate that the most likely difference between two forms of ovine DNase is the substitution of Leu for Arg.     

A single amino acid substitution can shift the optimum pH of DNase I for enzyme activity: biochemical and molecular analysis of the piscine DNase I family

We purified four piscine deoxyribonucleases I (DNases I) from Anguilla japonica, Pagrus major, Cryprus carpio and Oreochromis mossambica. The purified enzymes had an optimum pH for activity of approximately 8.0, significantly higher than those of mammalian enzymes. cDNAs encoding the first three of these piscine DNases I were cloned, and the sequence of the Takifugu rubripes enzyme was obtained from a database search. Nucleotide sequence analyses revealed relatively greater structural variations among the piscine DNase I family than among the other vertebrate DNase I families. From comparison of their catalytic properties, the vertebrate DNases I could be classified into two groups: a low-pH group, such as the mammalian enzymes, with a pH optimum of 6.5-7.0, and a high-pH group, such as the reptile, amphibian and piscine enzymes, with a pH optimum of approximately 8.0. The His residue at position 44 of the former group is replaced by Asp in the latter. Replacement of Asp44 of piscine and amphibian DNases I by His decreased their optimum pH to a value similar to that of the low-pH group. Therefore, Asp44His might be involved in an evolutionarily critical change in the optimum pH for the activity of vertebrate DNases I.     

Characteristics of DNase activities in excretory/secretory products of infective larvae of Haemonchus contortus

While multiple DNase activities occur in the excretory/secretory products (ESPs) of the adult Haemonchus contortus, the DNase activities in ESPs of the infective larvae (L3) have not been studied. Thus, the DNase activities in ESPs of H. contortus L3 were investigated and compared to those of adults for developmental stage-specific analysis. The DNase activities had relative molecular masses (M rs) of 34 and 36 kDa upon zymographic analysis at pH 5.0 and 7.0 when the larvae were incubated for over 48 h. The 34 and 36 kDa DNases of L3 ESPs were also detected in adult ESPs with similar characteristics. However, the 37 and 38.5 kDa DNases of the adult ESPs were not detected in the L3 ESPs. Since the 37 and 38.5 kDa DNase activities were mainly detected in adult ESPs, these activities appear to be specific to the adult stage whereas the other ESP DNase activities appear to be expressed during multiple stages of the parasite's life cycle. While the difference in DNase activities of L3 and adults remains obscure, the role of DNase in larval development should be further clarified and the identification of stage-specific developmental markers will lead to the discovery of specific factors that stimulate larval development.