(Received for publication, September 13, 1995; and in revised form, January 17, 1996)
From the
Nucleic acid specificity was tested for two monoclonal
anti-double-stranded DNA autoantibodies, 2C10 and H241, derived from
two lupus-prone MRL/Mp-lpr/lpr mice. Antibody 2C10 bound
double-stranded oligonucleotides with a preference for dA-dT over dG-dC
base pairs and did not bind single-stranded oligonucleotides.
Distamycin A, an antibiotic that binds to the minor groove, inhibited
2C10 binding of double-stranded DNA, suggesting that this antibody
interacts with dA-dT base pairs in the minor groove. Antibody H241
binding was previously shown to have a dG-dC preference and to involve
both major and minor grooves. In attempted footprinting assays, both
2C10 and H241 markedly enhanced rather than protected against cleavage
of DNA by hydroxyl radical-generating systems. With 2C10, this
enhancement effect was observed only when hydroxyl radical generation
was associated with oxidation of Fe(II). In contrast, H241 enhancement
occurred in the presence of HO
and ascorbate or
UV light irradiation and did not depend on added metal ion. The
enhancement sites were related to the antibody binding specificities.
The oligonucleotide 5`-AAAATATATATTT-3` was a much more effective
inhibitor of the 2C10 enhancement than of the H241 effect, whereas the
oligonucleotide 5`-GGGGCGCGCGCCC-3` was a much more effective inhibitor
of the H241 enhancement. In addition, the enhanced cleavage occurred
preferentially at dA-dT-rich regions with 2C10 and at dG-dC-rich
regions with H241. These findings raise the possibility that anti-DNA
autoantibodies could enhance DNA damage in inflammatory lesions in
which hydroxyl radicals are generated.
Autoantibodies to dsDNA ()are characteristic of the
autoimmune disease systemic lupus erythematosus. Increased production
of anti-dsDNA antibodies reflects or predicts periods of active
clinical disease(1) , and anti-DNA antibodies can contribute to
pathogenesis of lesions. There are several possible bases for their
contribution to tissue damage. Anti-DNA antibodies are concentrated in
immune complexes in glomerular lesions of lupus nephritis, where they
may initiate inflammatory reactions(2, 3) . It was
originally proposed that glomerular lesions resulted from deposition of
DNA
anti-DNA complexes that formed in the circulation (2, 3) , but many anti-DNA antibodies cross-react with
other negatively charged polymers or membrane proteins (4, 5, 6) and may bind directly to structures
present in the glomerular basement membrane(4, 7) . As
well as initiating inflammation, anti-DNA antibodies may cause cell
damage more directly. Some anti-DNA antibodies bind to cell surfaces
and initiate complement-dependent cytotoxicity(8) , whereas
others penetrate the membrane of living cells and may reach the nucleus (8, 9, 10) . It has also been reported that
some anti-DNA autoantibodies present in sera of systemic lupus
erythematosus patients can catalyze the hydrolytic cleavage of
DNA(11, 12) .
Because some, but not all, anti-DNA antibodies are pathogenic, there has been much interest in whether the pathogenicity is related to binding specificity, either for DNA epitopes or for cross-reactive structures. Thus, many anti-DNA autoantibodies have been analyzed extensively at the level of primary structure and modeling of their binding sites (13, 14) or, when possible, by x-ray crystallography(15, 16) . In addition, DNA-binding sites of monoclonal anti-DNA antibodies and epitopes have been mapped by competitive immunoassay with polynucleotides or oligonucleotides(17, 18) . High resolution footprinting, measuring protection against chemical cleavage of DNA, has been useful in identifying the epitopes for autoantibodies to single-stranded DNA (19) and experimentally induced antibodies to Z-DNA (20) .
In a previous study, we had examined the
specificity of a pathogenic monoclonal IgG anti-dsDNA antibody, H241,
derived from an MRL/Mp-lpr/lpr lupus mouse. In competitive
immunoassays with a series of synthetic double-stranded
oligonucleotides, H241 bound to a (dG-dC) or (dG-dC)
core in the center of a base-paired
octadecanucleotide(21) . This selectivity was also reflected in
the very marked preference of this antibody for poly(dG-dC) over
poly(dA-dT). A second IgG antibody, 2C10, isolated from a different
MRL/Mp-lpr/lpr mouse, had a very different selectivity,
markedly favoring poly(dA-dT) over poly(dG-dC)(22) .
Competitive assays of antibody 2C10 with synthetic oligonucleotides
indicated that it could bind well to alternating dA-dT, to stretches of
(dA)
(dT)
, or to
(dA-dG)
(dC-dT) sequences, (
)so it was more difficult
to assign a single kind of epitope to it. Recently, we tried to apply a
DNA footprinting assay to observe the DNA sites recognized by these two
monoclonal anti-dsDNA antibodies more directly. Unexpectedly, we found
that these two anti-dsDNA antibodies enhanced rather than protected
against the cleavage of DNA by hydroxyl radical-generating systems. The
two antibodies differed in the conditions under which they were
effective and in the sites of DNA cleavage they supported.
Figure 4:
Enhancement of hydroxyl radical cleavage
of DNA by anti-dsDNA antibodies. The 274-bp dsDNA probe was incubated
with anti-dsDNA antibody 2C10 (A) or H241 (B) or with
a control myeloma protein, MPC11, for 30 min in PBS. Then a 100
µM concentration of the divalent metal ion indicated at
the top of each lane was added. Four min later, an excess of
NaEDTA and thiourea was added to quench the reaction. In C, the dsDNA probe was incubated with H241 for 30 min, and the
complexes were then incubated further with no additions (lane
1) or with 1 mM sodium ascorbate plus 9 mM H
O
(lane 2); ascorbate alone (lane 3); H
O
alone (lane 4);
or ascorbate plus H
O
plus 10 µM EDTA-chelated Fe(II) (lane 5), Mg(II) (lane 6),
Zn(II) (lane 7), or Ca(II) (lane 8). Four min later,
the reaction was quenched by EDTA and thiourea. In D, the
dsDNA probe was incubated with MPC11 or 2C10 for 30 min and then for 4
min with no additions (lane 1) or with ascorbate and
H
O
(lanes 2 and 4) or
ascorbate plus H
O
plus EDTA-chelated Fe(II) (lanes 3 and 5).
Figure 5:
Dependence of the enhanced DNA cleavage on
the hydroxyl radical concentration. The DNA probe was cleaved by
Udenfriend's system (28) with anti-dsDNA antibody 2C10 (A) or H241 (B) or with a control myeloma protein,
MPC11. The standard reaction mixture (1 ) contained 10
µM Fe(II), 20 µM Na
EDTA, 9 mM H
O
, and 1 mM sodium ascorbate (A, lanes 2 and 6; and B, lane
4). These concentrations were 0.25
(B, lane
2), 0.5
(B, lane 3), 2
(A, lane 3 and 7), and 4
(A, lanes 4 and 8) the standard. Lanes 1 and 5 in A and lane 1 in B are
uncleaved controls. Shown in C is the densitometric estimation
of the amounts of the starting DNA incubated with the hydroxyl
radical-generating mixture and H241 (
), 2C10 (
), or MPC11
(
).
Figure 6:
Time course of the enhanced DNA cleavage
in the presence of anti-dsDNA antibodies. A, the DNA probe was
incubated with anti-dsDNA antibody 2C10 or H241 or with a control
myeloma protein, MPC11, and then cleaved by the reaction with the
standard concentration of Udenfriend's system reagents (28) for continuous hydroxyl radical generation along with
oxidation of Fe(EDTA). The reaction time was varied
from 0 to 480 s as indicated. B, shown is the densitometric
estimation of the amounts of the starting DNA in reactions with H241
(
), 2C10 (
), and MPC11
(
).
Figure 7:
Inhibition of the DNA cleavage enhancement
effect of anti-dsDNA antibodies by oligonucleotides. Oligonucleotide K5
(5`-AAAATATATATTT-3`) or K6 (5`-GGGGCGCGCGCCC-3`) was added to the DNA
cleavage reaction using Udenfriend's system (28) in the
presence of anti-dsDNA antibodies 2C10 and H241 or a control myeloma
protein, MPC11. Based on the densitometric estimation of the amounts of
the starting materials, percent inhibition was calculated as follows:
inhibition = (1 - ((cleavage with anti-DNA and inhibitor)
- (cleavage with MPC11))/((cleavage with anti-DNA) -
(cleavage with MPC11))) 100.
Figure 8:
DNA cleavage pattern enhanced by anti-DNA
antibodies. A, the DNA probe was cleaved by hydroxyl radical
generated by reduction of HO
in
Udenfriend's system (28) in the presence of anti-DNA
antibodies 2C10 and H241 or a control myeloma protein, MPC11. G, Maxam-Gilbert's G-reaction; G-control,
Maxam-Gilbert's G-reaction without alkaline cleavage. B,
densitometric analysis corresponds to the position of the
oligonucleotide insert described under ``Materials and
Methods.'' The ordinate represents the relative
sensitivity to the cleavage calculated as follows: relative cleavage
sensitivity = (density of cleaved ladder in the presence of
anti-DNA)/(density of cleaved ladder in the presence of MPC11). Hatched columns, H241; closed columns,
2C10.
Figure 9:
Pattern of DNA cleavage by hydroxyl
radical produced along with oxidation of Cu(I) in the presence of 2C10.
The ordinate represents the relative sensitivity to the
cleavage calculated as described for Fig. 8. DNA was incubated
for 4 min in a mixture of 1 µM copper sulfate, 0.1 mM sodium ascorbate, and 0.9 mM HO
.
Figure 1:
Competitive ELISA for 2C10. A,
antibody 2C10 was preincubated with various double- or single-stranded
oligonucleotides, and the mixture was added to microtiter plate wells
coated with native calf DNA. , 5`-CGCGCATATATATATGCGCG-3` and
3`-GCGCGTATATATATACGCGC-5`;
, 5`-CGCGCATAGATCTATGCGCG-3` and
3`-GCGCGTATCTAGATACGCGC-5`;
, 5`-CGCGCAGAGAGAGAGGCGCG-3`;
,
5`-CGCGCCTCTCTCTCTGCGCG-3`;
, 5`-CGCGCAGAGAGAGAGGCGCG-3` and
3`-GCGCGTCTCTCTCTCCGCGC-5`. B, 2C10 was preincubated with
polynucleotides, and the mixture was added to microtiter plate wells
coated with native DNA. +, poly(dA-dT);
, poly(dG-dC);
, poly(dG-dmC). Competitive ELISA was performed as
described(21) .
For further experiments, we
used a 274-bp dsDNA probe purified from a modified plasmid as described
under ``Materials and Methods.'' First we confirmed the
binding of the antibodies to this DNA probe with an assay for
retardation of migration in gel electrophoresis. Antibody 2C10 shifted
the mobility of the P-labeled DNA almost completely at a
DNA/antibody molar ratio of 1:40 (Fig. 2A). Radioactive
oligonucleotide hardly entered the gel when the ratio was 1:80,
presumably because of the formation of larger immune complexes. H241
caused partial retardation at a ratio of 1:20 or 1:40 and complete
retardation at 1:80 (Fig. 2B). (A ratio of 1:40 with
H241 caused nearly complete retardation in some experiments (Fig. 3, lane 7).) Based on these observations, we
carried out the following experiments at a DNA/antibody ratio of 1:80
unless otherwise indicated.
Figure 2: Gel retardation assay for the binding of anti-dsDNA antibodies to the 274-bp dsDNA probe. DNA was incubated with anti-dsDNA antibody 2C10 (A) or H241 (B) or with a control myeloma protein, MPC11, and loaded on a 10% polyacrylamide gel with a 4% stacking gel. Molar ratios of DNA to antibody are indicated at the top of each lane.
Figure 3: Effect of distamycin A on the binding of 2C10 and H241 to dsDNA. Antibodies 2C10 (lanes 2-5) and H241 (lanes 7-10) were incubated with the 274-bp dsDNA probe alone at DNA/antibody ratios of 1:40 (lanes 2 and 7) and 1:80 (lanes 4 and 9) or with a DNA/distamycin A mixture at DNA/antibody ratios of 1:40 (lanes 3 and 8) and 1:80 (lanes 5 and 10). The mixtures were then loaded on a 10% polyacrylamide gel with a 4% stacking gel. Lanes 1 and 6 are the DNA without antibodies.
Preincubation of the 274-bp DNA probe with distamycin A, an antibiotic that binds in the minor groove with preference for dA-dT-rich sequences (27) , caused marked inhibition of antibody 2C10 binding, but did not affect H241 binding (Fig. 3). Together with ELISA data described above, this result suggests that 2C10 recognizes double helical structure of DNA and makes contacts preferentially with regions of dA-dT base pairs in or over the minor groove.
A mixture of Fe(II)
chelated with NaEDTA, H
O
, and
ascorbate, known as Udenfriend's system(28) , is a
convenient reagent for continuous production of hydroxyl radical. Thus,
we tested the effect of anti-DNA antibodies in the mixture of
H
O
and ascorbate with or without divalent metal
ions and Na
EDTA (Fig. 4, C and D).
Antibody H241 markedly enhanced the cleavage of DNA by
H
O
and ascorbate, and this effect was not
dependent on the presence of any metal ions (Fig. 4C).
In fact, the presence of Fe(II) partially inhibited the enhancement by
H241 (Fig. 4C). In contrast, enhanced DNA cleavage by
2C10 was observed in the presence of Fe(II), ascorbate, and
H
O
, but not with ascorbate and
H
O
alone (Fig. 4D) or
ascorbate, H
O
, and other metals (data not
shown). The control antibody MPC11 did not enhance cleavage under any
of these conditions (Fig. 4D).
The next experiment measured the time dependence of this effect of anti-DNA antibodies in the standard reaction mixture (Fig. 6). The reaction was rapid in the presence of H241; >50% of the starting DNA was degraded within 15 s. The reaction with 2C10 was slower under this assay condition, but there was still significant enhancement of cleavage over a 2-8-min period.
As a further test of this interpretation, the extents of cleavage at specific sites in the DNA sequence were compared in the analytical sequencing gels (Fig. 8). The susceptibility for hydroxyl radical cleavage in the presence of 2C10 and H241 showed a reciprocal pattern. The enhancement with antibody 2C10, relative to the reaction in the absence of antibody, was greatest in dA-dT-rich sequences, whereas the enhancement with antibody H241 was greatest in dG-dC-rich sequences, again indicating that increased susceptibility for cleavage is related to the specific binding sites of each antibody. In this experiment, >95% of the DNA was uncut following the hydroxyl radical reaction with a control antibody, MPC11. This condition is consistent with single-hit kinetics(29) , supporting the conclusion that the result truly reflects substrate specificity.
These experiments indicate that two anti-DNA autoantibodies
from lupus mice enhance the degradation of DNA by hydroxyl
radical-generating systems. Steps were taken to test whether the effect
was truly due to the antibody rather than a contaminant that might have
been present in an immune complex. The antibodies were highly purified,
as described under ``Materials and Methods.'' Their purity
was confirmed by SDS-polyacrylamide gel electrophoresis, which detected
no contamination by other DNA-binding proteins. Further verification
was obtained by counting the number of IgG-nucleosome complexes or free
nucleosomal particles among IgG particles in a fixed area in an
electron micrograph of a preparation that included mica flakes for
quick-freezing and deep-etching. In the case of monoclonal anti-DNA
antibodies obtained after a wash with a high salt concentration,
neither IgG-nucleosome complexes nor free nucleosomes were found among
80 or more IgG particles, whereas 42 out of 80 IgG particles were
associated with nucleosomes in the case of monoclonal antibodies
obtained by the conventional affinity chromatography without a high
salt wash. ()The effectiveness of this washing is consistent
with the previous demonstration of salt sensitivity of DNA binding by
antibody 2C10 (31) and other anti-DNA
autoantibodies(32) . Removal of the DNA also removes proteins
that may be associated with it in DNA-antibody complexes.
Specificity of the effects provided more direct evidence for the role of the antibody-binding sites in this phenomenon. The relative effectiveness of oligonucleotides as inhibitors of the enhancement corresponded with their effectiveness as inhibitors of DNA binding. Both the cleavage enhancement and DNA binding by antibody 2C10 were inhibited preferentially by the AT oligonucleotide, and both activities of H241 were inhibited preferentially by the GC oligonucleotide. Furthermore, enhancement occurred preferentially at dA-dT segments of the test DNA with antibody 2C10, but at dG-dC segments with H241. The sequence specificity was not absolute, and neither were the binding differences(21) . The effect was not due to uncontrolled conditions of the hydroxyl-generating system; unlike the antibodies, the Cro protein protected its specific target under the same conditions, just as expected in a footprinting assay.
Extensive studies have demonstrated that hydroxyl radical generated by the Fenton reaction modifies bases and cleaves DNA(33, 34, 35, 36) . The ability of bound protein to protect against this cleavage is the basis of high resolution footprinting assays in vitro(19, 26) , and protection against oxidative species is afforded within cells by histones in condensed chromatin(37) . On the other hand, DNA-nuclear matrix protein association presents hypersensitive sites for oxidative damage, possibly related to the binding of copper ions(37, 38) . Metal-binding proteins such as ferritin (39) also mediate increased DNA cleavage, as can lactoferrin with copper bound to its surface(40) , an inorganic polymer that binds both iron and DNA (41) or small DNA-binding molecules such as adriamycin(42) . These proteins, polymers, and small molecules may focus the Fenton reaction on simultaneously bound DNA. In addition, binding of iron to DNA may determine cleavage sites(34) .
It is not known how antibodies 2C10 and H241
enhanced DNA cleavage, but there were differences in the mechanisms
operating with the two antibodies. Because the enhancement with
antibody 2C10 occurred either with simple addition of Fe(II) to the
DNA-antibody complexes or with Fe(II) and the
HO
/ascorbate mixture, but not with the
copper/peroxide/ascorbate reagent or peroxide/ascorbate alone, it is
possible that antibody 2C10 selectively bound both DNA and Fe(II),
concentrating hydroxyl radical generation near the target sites. In
analogy, deliberate incorporation of a metal-binding site into an
antibody Fv domain has been proposed as a means of generating a
designed metalloenzyme(43) .
Antibody H241 acted
differently, as it was effective with HO
and
ascorbate without added metal ion. The presence of iron actually
reduced the antibody-mediated enhancement, whereas other ions had no
effect. Antibody H241 (and perhaps 2C10 as well) may have facilitated
cleavage by distorting the DNA structure, making target sites more
accessible. X-ray crystallographic analysis of an autoantibody to
single-stranded DNA, for example, revealed antibody stabilization of an
unpredicted conformation of oligo(dT) in the immune
complex(16) . Comparable structural data are not available for
antibodies 2C10 and H241 and their complexes with DNA. The effect is
not a property of all anti-DNA antibodies because hydroxyl radical
generation has been used effectively for protection-based footprinting
of anti-single-stranded DNA autoantibody epitopes(19) .
Because an excess of antibody was present in the solution and the precise number of cleavage events was not known, it is not clear whether the rate enhancement involved turnover, as in enzyme catalysis, or whether it was stoichiometric. The antibodies alone did not hydrolyze DNA in the time course of these reactions, up to 30 min. Hydrolysis of DNA by purified IgG and Fab fragments from systemic lupus erythematosus patients' sera has been detected by sensitive assays (11, 12) . In addition, Paul and co-workers have reported hydrolysis of vasointestinal peptide by IgG autoantibodies and their Fab fragments (44, 45) or isolated light chains (46) and by the purified (47) or recombinant (48) light chain of a monoclonal antibody from a mouse immunized with this peptide. Additional study will determine the extent to which autoantibodies provide new approaches to isolation of catalytic antibodies, adding to the large number that have been induced with haptens based on transition state analogues (49) or obtained by selection from combinatorial libraries(50) .
Antibody that increases the sensitivity of DNA to cleavage by hydroxyl radical or other reactive oxygen species could have an effect in vivo in sites of inflammation, where such species are generated. For example, higher than normal oxygen radical production has been detected in both bronchial alveolar cells and polymorphonuclear cells from patients with systemic lupus erythematosus(51) . Increased numbers of chromosomal breaks and increased sensitivity to UV light, with involvement of reactive species, have also been detected and suggested as a basis for the well known sensitivity of these patients to UV irradiation(52, 53) . Protection by superoxide dismutase provided evidence that reactive oxygen species were involved in the damage by UV radiation(52, 54) . The presence of anti-DNA antibodies such as 2C10 or H241 could enhance the sensitivity of DNA to such damage. In turn, hydroxyl radical-mediated damage to DNA may increase the binding by anti-DNA antibodies (55, 56) and increase the immunogenicity of DNA (56) .