Journal of Protein Chemistry, Vol. 16, No. 2, 1997

Mechanism for Inhibition of Deoxyribonuclease Activity by Antisera Yuh-Fan Liu1 and Ta-Hsiu Liao^

Received November 1, 1996

The activity of bovine DNase, but not that of porcine DNase, is inhibited by antisera against bovine DNase, and vice versa. Inhibition of DNase is found in the immunoglobulin G-containing fractions, as shown by ion exchange chromatography. Inactive DNase, carboxymethylated specifically at the active site His134, competes with active DNase and reverses the antisera inhibition of DNase, suggesting that the epitode responsible for inhibition does not contain the active site His134. Alignment of the sequences of DNase of these two species shows that the greatest variation occurs between residues 153 and 163, within which are three consecutive peptide bonds, Lys-Trp-His-Leu, that are readily cleaved by trypsin, chymotrypsin, or thermolysin. The 8-hr digest of DNase by each of these three proteases has lost the ability to reverse antisera inhibition. The degree of antisera inhibition varies with the metal ion used as the activator for DNase-catalyzed reactions. When Mn2+, Co2+, or Mg2"1' plus Ca2+ are used as activators, inhibition is approximately 50%. When pBR322 plasmid is used as substrate, gel electrophoresis shows that the DNase-catalyzed DNA hydrolysis produces a significant amount of double-strand cuts with Mn2"1", Co2+, or Mg2* plus Ca2* as activators and antisera inhibit DNase action only on double-strand cuts. With only Mg2* as the activator no double-strand cuts are observed, either in the presence or absence of antisera, and the DNase activity is not significantly inhibited. We conclude that antisera inhibition is due to antibody binding of the DNase polypeptide chain within residues 153 and 163. These residues are not crucial for catalysis, but are required for DNA binding, which results in double-strand cuts. KEY WORDS: DNase; antisera inhibition; divalent metal ions; DNA scission.

(Junowicz and Spencer, 1973). However, there are large differences in their efficiencies of activation. The mode of action of DNase on duplex DNA also depends on which metal ion is used (Campbell and Jackson, 1980). The DNase-catalyzed hydrolysis of duplex DNA makes a significant number of double-strand cuts in the presence of Mn2+, Co2+, or Mg2"1" plus Ca2+, but only single-strand cuts when Mg2"1" is the activator. A previous study (Abe and Liao, 1983) showed that antisera against bovine DNase inhibits bovine, but not porcine DNase. Since the amino acid sequences of the two DNases are highly conserved (Paudel and Liao, 1986), particularly around the active site His134, the result of this study suggested that antisera act on sites

1. INTRODUCTION Divalent metal ions are required for activation of bovine pancreatic DNase (Kunitz, 1950) and these ions may have more than one function (Shack and Bynum, 1964; Melgar and Goldthwait, 1968; Wiberg, 1958). Several binding sites on the enzyme and/or on DNA have been suggested (Price, 1972; 1975; Douvas and Price, 1975; Jouve and Jouve, 1975; Price et al., 1969). The metal ions satisfying the requirement are Mg2+, Ca2+, Co2+, and Mn 2+ 1

Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei, Taiwan. 2 To whom correspondence should be addressed; e-mail: [email protected]. tw.

75 0277-8033/97/0200-0075S12.50/0 © 1997 Plenum Publishing Corporation

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other than the catalytic site. Herein we present attempts to correlate the antisera inhibition of DNase with DNase activation by metal ions and with the mode of DNase action on duplex DNA.

2. MATERIALS AND METHODS 2.1. Materials Calf thymus DNA and peroxidase-labeled goat antibody against rabbit IgG were obtained from Sigma. The pBR322 plasmid was from Promega. Antisera were prepared essentially as described previously (Abe and Liao, 1983). Porcine DNase was purified from crude extracts of porcine pancreas according to the procedure of Paudel and Liao (1986). Bovine pancreatic DNase was purified from the semipurified preparation of Worthington (Code DP) as described previously (Liao et al., 1991).

2.4. Enzyme-Linked Inununosorbent Assay (ELISA) The procedure for ELISA was essentially according to Engvall (1980). In wells of the microtiter plates, bovine or porcine DNase was first coated for 2 hr at 37°C in the coating buffer (3 mM NaN3, 0.035 M NaHCO3, 0.014 M Na2CO3, pH 9.6) and then blocked with 0.5% skim milk powder in phosphate-buffered saline, pH 7.2, overnight at 4°C. The coated antigen was incubated at 37°C with serial diluted antisera for 1 hr and then washed four times with 0.05% Tween-20 in phosphate-buffered saline, pH 7.2. Antibodies bound to antigens were determined by 1 hr incubation with the peroxidaselabeled goat antibody against rabbit IgG. After another wash step, the wells were covered with the substrate solution, which was 2,2'-azinobis(3ethlbenothiazoline-6-sulfonic acid) (0.4 mg/ml) in 0.1 M phosphate/citrate buffer, pH 4.0, containing 0.03% H2O2. The absorbance at 405 nm after 5 min was measured on the ELISA plate reader (Bio-Tek EL312).

2.2. Preparation of CM-His134-DNase 3. RESULTS Bovine CM-His134-DNase was prepared as described by Price et al. (1969). Purified bovine pancreatic DNase (1 mg/ml) was equilibrated at 25°C in 0-05 M Tris-HCl, pH7-2, 4mM CuCl2, for at least 15 min. lodoacetate was then added and the solution was kept in the dark. At suitable intervals aliquots were removed and assayed. DNase activity was determined by the hyperchromicity assay of Kunitz (1950) as modified by Liao (1974). The DNase activity of CM-His134-DNase was less than 1 % of that of unmodified DNase.

2.3. Assay of Single and Double Cuts of DNA In a typical assay, 9-2 mg/ml of pBR322 plasmid DNA in 0-1 M HEPES, 0-1 M NaCl, pH 7-2, and 3-6 mM divalent metal ions was incubated at 25°C with 7-lmU/ml DNase. At various times, 14-ju.l aliquots were removed and added to 3 /A! of 50 mM EDTA, pH 7-2, 25% glycerol, and 0-01% bromophenol blue on ice. All samples were resolved into their component species by electrophoresis on 1% agarose slab gels using a TAE running buffer containing 2-5 mg/ml of ethidium bromide (Manitis et al., 1982). Total electrophoresis time was 60 min.

3.1. Cross-Reaction Between Antisera Against Bovine and Porcine DNase In the immuno-cross-reaction, as analyzed by ELISA (Fig. la), both the anti-bovine and the anti-porcine DNase sera have a higher titer for its own antigen than for its counterpart, suggesting that the two DNase molecules of different species share some common antigenic determinants. However, other epitope sites, not common to both DNase molecules, may also exist. As for DNase activity, the anti-bovine DNase antisera inhibit 72% of the bovine DNase, but not the porcine DNase activity (Fig. Ib, right panel), whereas antisera against porcine DNase inhibit 61% of the porcine DNase, but not the bovine DNase (Fig. Ib, left panel). 3.2. Nature of the Inhibition Factor When the 0-50% ammonium sulfate fraction of the anti-bovine DNase antisera is applied to a DEAE-cellulose column, the elution profile (Fig. 2) shows that the inhibition of DNase activity occurs in fractions 40-56, which contain antibodies against DNase as shown by ELISA (absorbance at 405 nm). The proteins in these fractions show

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Fig. 1. (a) ELISA of bovine and porcine DNases with the anti-bovine DNase and anti-porcine DNase antisera. Dilutions of the antisera were made with phosphate-buffered saline, pH 7.2. Antigens were coated 2pmol/well and peroxidase-labeled goat antibody against rabbit IgG was diluted 1:500. Readings at 405 nm were made 5 min after H2O2 was added, (b) Inhibition of DNase activity by antisera. To 1 ml of the assay solution (Liao, 1974) was added 10/j.1 of antibovine, antiporcine, or preimmune (as control) serum and 10/zl of bovine (11.4 U/ml) or porcine (17.4 U/ml) DNase.

mainly immunoglobulin G on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (data not shown). Thus, it is the antibody, not other serum factors, in the anti-DNase antisera that inhibits the DNase activity. 3.3. Reversal of Antisera-Inhibited DNase Activity The inhibition of DNase activity by antisera is reversed with increasing amounts of the DNaseinactive CM-His134-DNase (Fig. 3a), suggesting that the chemically modified CM-His134-DNase retains a peptide segment that can compete with the intact DNase for antibody binding. Proteolytic (trypsin, chymotrypsin, or thermolysin) digests of DNase can also reverse the inhibition. However, the reversal ability decreases with increasing proteolysis time (Fig. 3b). All 8-hr digests are unable to reverse the inhibition. When the 1-hr tryptic digest (DNaseinactive), having 70% reversal ability, is passed through a gel filtration column (Sephadex G-25), only fractions in the void volume have reversal

ability (data not shown), suggesting that partially digested large peptides in the digest contain the segment responsible for reversal of antisera inhibition. 3.4. Effects of Metal Ions on the Antisera Inhibition As shown in Table I, the degree of DNase inhibition varies with the metal ions used. For the reaction with Mn 2+ , Co2+, or Mg2+ plus Ca2+ as activators, the amount of inhibition is over 50%. With Mg2+ alone the inhibition is, within experimental error, insignificant. 3.5. Inhibition of Double-Strand Cuts by Antisera Using the supercoiled plasmid DNA as substrate, Campbell and Jackson (1980) described in analytical method differentiating single-strand nicks from double-strand cuts. Based on this method, the pBR322 plasmid DNA is used as substrate for DNase in the presence and absence of antisera with

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Fig. 2. Anion-exchange chromatography of anti-bovine DNase antibody. The 0-50% ammonium sulfate fraction of antisera was applied to a DEAE-cellulose column ( 2 X 8 cm) equilibrated with 10 mM Tris-HCl, pH 8.0. The column was washed with 30 ml of the equilibrating buffer. The chromatogram shown was obtained after a linear gradient elution with 0-0.3 M NaCl in 10 mM Tris-HCl, pH 8.0. Fraction size, 0.5ml. The ELISA results are expressed as absorbance at 405 nm. The pooled fraction 40-56 is termed "the DEAE-purifled antibody." Onetenth dilution of the antibody gave a maximum reading in ELISA with 30 ng of coated bovine DNase per well.

various metal ions as activators (Fig. 4). The results show that inhibition of double-strand cuts occurs in the presence of Mn2+, Co2+, or Mg2+ plus Ca2+, while with Mg2+ alone there are no double-strand cuts with or without antisera. 4. DISCUSSION The two antisera, one against bovine DNase and the other against porcine DNase, are cross-reactive by the titer assay, but are not cross-reactive by the DNase activity assay (Fig. 1). Because alignment of the amino acid sequences of bovine and porcine DNases shows that the His134-active-site containing peptides are highly conserved (Paudel and Liao, 1986) and because the inactive CM-His134-DNase reverses inhibition (Fig. 3a), the epitope for antisera binding must not include His134. In the sequence alignment between the bovine and porcine species there are 56 amino acid changes, most of which are conservative. The three regions that are considered nonconservative are residues 121-127, 153-163, and 188-191. The 153-163 region has the highest degree of variation, five amino acid changes. Within the 153-163 region there is a Lys-Trp-His-Leu sequence whose peptide

Fig. 3. (a) Reversal of antibody-inhibited DNase activity by CM-His134-DNase. The concentration of CM-His134-DNase was 210/j.g/ml, equivalent to 210 U/ml active DNase. The assay buffer was 100 mM HEPES, pH 7.2, 100 mM NaCl, 5 mM Mn2+. (b) Reversal of antibody-inhibited DNase activity by protease digests of DNase. Protease concentrations, Img/ml; 10/x.l added. Digestion conditions: 37°C, 100mM Tris-HCl, pH 8.0. Digested DNase concentrations, 71 /xg/ml. For both (a) and (b) the DEAE-purified antibody was used. Table I. Effects of Metal Ions on Antisera Inhibition of DNA Hydrolysis Catalyzed by DNase" Rate6 X 103

4mMMg 2 + 10mMMg 2+ + 5 mM Ca2+ 4 mM Mn2+ 4mMCo 2 + 4mMCa 2 +

DNase

DNase + antibody

Inhibition by antibody (%)

73

65

11

112 210 94 0

50 111 43 0

55 47 54

" To 1 ml of the DNase assay solution (0.1 M Tris-HCl, pH 7.0, 50 /j.g of calf thymus DNA) containing various metal ions was added 0.21 unit of DNase or 0.21 unit of DNase plus 10 ^1 of the DEAE-purified antibody. * The change of absorbance at 260 nm per min.

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Fig. 4. DNase hydrolysis of a supercoiled plasmid. Bottom: (a, b) 1.2 mM Mn2+, (c, d) 1.2 mM Co2+. Top: (a,b) 10 mM Mg2^, (c,d) 10 mM Mg2+ plus 5mM Ca2+. For both top and bottom: (a,c) lOmU/ml DNase, (b, d) lOmU/ml DNase plus the DEAE-purified antibody (Ab-DNase). The plasmid DNA was pBR322. I, II, and L represent the supercoiled, relaxed, and linear forms of the plasmid, respectively; M indicates the DNA size makers. Single-strand cuts lead to the relaxed form, whereas double-strand cuts lead to the linear form. Reaction and assay conditions were as described in Section 2.

bonds are readily cleaved by trypsin, chymotrypsin, or thermolysin. The 8-hr digests by each of these three proteases are unable to reverse antisera inhibition (Fig. 3b). These facts suggest that the inhibitory effect is due to antibody binding of DNase in the 153-163 region. Since this region is

not essential for enzymatic catalysis, the inhibition is probably due to a decrease in catalytic efficiency. Wiberg (1958) has proposed, based on the efficiency of activation by metal ions, that DNase has two sites for metal ions. At site I the efficiency is Mn2+ = Ca2+ > Mg2+, the same order as the ionic

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Fig. 5. (a) A two-site model of DNase to explain antisera inhibition of DNase activity, (b) Conformation of the backbone of DNase in its complex with actin. Only a portion of the actin backbone trace is shown in white. The residues around the catalytic-site cleft (arrow) are marked in red and the two Ca2+-binding sites in purple. The Val154-Met164 region responsible for antibody binding is shown in yellow.

Inhibition of DNase by Antisera radii, Mn2+ > Ca2+ > Mg2+. At site II the efficiency is Mn 2+ >Mg 2+ »Ca 2+ , an order similar to their tendency to form coordinate complexes. More than one site for metal ions has also been suggested by physical data (Price, 1972, 1975; Douvas and Price, 1975; Jouve and Jouve, 1975; Price et al., 1969). We propose for the metal-ion binding a model (Fig. 5a) in which site II, the catalytic site, accommodates metal ions such as Mn2+, Co2+, or Mg2+, while site I, the modulating site, binds Mn2+, Co2+, or Ca2+. The effect of antisera, either on hydrolysis rates or on the mode of action of double-strand cuts, is in accord with the binding efficiency for site I. On the three-dimensional structure (Fig. 5b), the catalyticsite cleft formed by the side chains of His252, His134, Glu39, Gly78, and Asp212 is away from site I and from the actin-binding site (Kabsch et al., 1990). However, the 153-163 region is close to site I, which is at or near one of the two Ca2+-binding sites involving the side chains of Glu112, Asp107, Phe109, and Asp" (Fig. 5b). Thus, the binding of Ca2+ or other metal ions at site I may be prevented by antibody binding to region 153-163. As a result, the DNase molecule loses its ability to modulate and to perform double-strand cuts. EcoRI restriction endonuclease has a homodimeric structure that enables the enzyme to make double-strand cuts on duplex DNA (McClarin et al., 1986). Fungal DNase also has a homodimeric structure (Chen et al., 1993) which makes a significant number of double-strand cuts, regardless of the metal ions used. Probably this dimeric structure accounts for the fact that fungal DNase hydrolyzes DNA almost seven times faster than does bovine DNase. In the case of bovine DNase, because CM-His134-DNase does not inhibit doublestrand cuts when it is mixed with active DNase (date not shown), the heterodimer of CM-His134DNase/DNase must not occur. In an experiment in which the DNase amount was reduced 100-fold to minimize possible dimerization, the number of double-strand cuts remained unchanged (data not shown). Thus, double-strand cutting is not caused by dimerization. Since DNA hydrolysis is due to monomeric action, the cutting of the second strand may occur in a processive way. That is, the enzyme may remain bound to DNA during many reaction cycles. This view is supported by the finding (Campbell and Jackson, 1980) that at the end of each double-strand cut there is a single-strand region of 11 nucleotide that is about equal to one turn of duplex DNA. Perhaps metal-ion binding at

81 site I converts DNase into a conformation with a much higher affinity with DNA, thus enabling the enzyme to cut the duplex DNA at almost the same position along the duplex ladder. Endogeneous Ca2+, Mg2+-DNase may be involved in nuclear DNA degradation during apoptosis (Peitsch et al., 1993; Polzar et al., 1993). This DNase was identified as DNase type I, the same type as the DNases used in this investigation. Thus, it is possible that site I in DNase, which regulates the double cuts through Ca2+ binding, may be one of the factors in apoptosis.

ACKNOWLEDGMENTS We thank Dr. Roger E. Koeppe of Oklahoma State University for critical reading of the manuscript. This work was supported in part by a grant from the National Science Council of ROC (#NSC83-0412-B002-208).

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