Polyspecific binding of Escherichia coli beta-galactosidase by murine antibodies to DNA

To characterize further polyspecific interactions of antibodies to DNA, the binding of sera from autoimmune MRL-lpr/lpr mice to Escherichia coli beta-galactosidase (beta-gal) was analyzed. This protein was selected for study because of preliminary observations that sera from autoimmune mice bound unexpectedly to cloned fusion protein constructions containing beta-gal. Using ELISA assays, sera from MRL-lpr/lpr mice demonstrated high levels of antibodies to both DNA and beta-gal, in titers significantly greater than those of BALB/c controls. Affinity chromatography using beta-gal-Sepharose demonstrated that antibodies enriched for anti-beta-gal activity bound both DNA as well as beta-gal, indicating the presence of a population of cross-reactive anti-DNA antibodies. Furthermore, anti-DNA mAb of MRL-lpr/lpr strain origin also bound beta-gal by ELISA, although these levels were lower than those to DNA. Together, these results extend the range of polyspecific binding of murine anti-DNA antibodies to bacterial proteins. They further suggest caution in the interpretation of immunoassays using fusion protein constructions containing beta-gal, especially with sera from autoimmune mice.     

Identification and sequence of the VH gene elements encoding a human anti-DNA antibody

Antibodies to DNA similar to those found in patients with systemic lupus erythematosus (SLE) and autoimmune mice can be derived from the lymphocytes of normal individuals. It is not known whether these normal derived anti-DNA antibodies are made from the same VH gene elements as the anti-DNA antibodies made by SLE patients. To begin to answer this question, we examined mu chain cDNA clones from human hybrid clone C6B2 producing anti-DNA antibodies. The sequence of the 500 base pair restriction fragment containing the variable region (5' terminus) was determined and was sequenced. This antibody uses a VHII heavy chain subgroup gene, a J3 joining segment, a hitherto unknown D segment, and a previously reported leader sequence. Significant homology was found to a mouse anti-DNA antibody sequence in the use of VH subgroup in J3, and in the hypervariable regions with a shared Ser-Tyr construction in CDR1 and an identical five amino acid residue stretch in CDR2. Comparison with the limited sequence data of published SLE monoclonal anti-DNA antibodies, both human and mouse, suggests that this shared Ser-Tyr may be important in some but not all antibodies to DNA. Comparison of C6B2 antibody is made with other known antibody sequences with identification of those residues likely to be part of the antigen binding site.     

Evidence for structural plasticity of heavy chain complementarity-determining region 3 in antibody-ssDNA recognition

Anti-DNA antibodies play important roles in the pathogenesis of autoimmune diseases. They also represent a unique and relatively unexplored class of DNA-binding protein. Here, we present a study of conformational changes induced by DNA binding to an anti-ssDNA Fab known as DNA-1. Three crystal structures are reported: a complex of DNA-1 bound to dT3, and two structures of the ligand-free Fab. One of the ligand-free structures was determined from crystals exhibiting perfect hemihedral twinning, and the details of structure determination are provided. Unexpectedly, five residues (H97-H100A) in the apex of heavy chain complementarity-determining region 3 (HCDR3) are disordered in both ligand-free structures. Ligand binding also caused a 2-4A shift of the backbone of Tyr L92 and ordering of the L92 side-chain. In contrast, these residues are highly ordered in the Fab/dT3 complex, where Tyr H100 and Tyr H100A form intimate stacking interactions with DNA bases, and L92 forms the 5' end of the binding site. The structures suggest that HCDR3 is very flexible and adopts multiple conformations in the ligand-free state. These results are discussed in terms of induced fit and pre-existing equilibrium theories of ligand binding. Our results allow new interpretations of existing thermodynamic and mutagenesis data in terms of conformational entropy and the volume of conformational space accessible to HCDR3 in the ligand-free state. In the context of autoimmune disease, plasticity of the ligand-free antibody could provide a mechanism by which anti-DNA antibodies bind diverse host ligands, and thereby contribute to pathogenicity.     

Base specificity and idiotypy of anti-DNA autoantibodies reactive with synthetic nucleic acids

Synthetic nucleic acid reactivities and the distribution of idiotypes associated with poly(dA) and poly(dT) specificities were evaluated among both monoclonal and polyclonal anti-DNA antibodies from autoimmune New Zealand mice. Ten monoclonal anti-DNA antibodies (IgG2a or IgG2b), derived from NZB/NZW mice and reactive with natural DNA (duplex and/or heat-denatured), were found to collectively exhibit a diverse binding pattern with six deoxyribohomopolymers. Several monoclonal antibodies displayed reactivity with poly(dT) comparable to that with natural DNA. Serologic studies indicated that polyclonal anti-DNA autoantibodies from NZW/NZW mice and both parental strains also cross-reacted with various homopolymers and bound preferentially with those containing pyrimidines, particularly poly(dT), relative to purines. Detailed binding analyses with two poly(dT)-reactive monoclonal antibodies demonstrated that stable DNA/anti-DNA complexes were formed with synthetic oligomers containing six to 10 nucleotides; binding to such antigens was relatively insensitive to ionic strength and inversely dependent on temperature. Both antibodies exhibited preferential binding (greater than or equal to 10-fold) with poly(dT) relative to poly(dU), suggesting the importance of the C5-methyl group and/or helical conformation in pyrimidine base recognition. Idiotypes on poly(dA)-specific and poly(dT)-specific monoclonal antibodies were found to be reciprocally distinct, localized at or near active site residues, and expressed at low levels (less than 10 to 130 ng/ml) in anti-DNA sera from all three New Zealand strains. These findings suggest that: nucleotide base determinants are significantly involved in DNA/anti-DNA interactions; poly(dT) represents a major cross-reactive synthetic antigen; and idiotype expression among lupus autoantibodies which recognize such determinants may be diverse.     

Hybridoma anti-DNA autoantibodies from patients with rheumatoid arthritis and systemic lupus erythematosus demonstrate similar nucleic acid binding characteristics

Hybridoma anti-DNA antibodies have been generated from the fusion of the GM 4672 lymphoblastoid line with peripheral blood lymphocytes from four normal subjects, nine patients with rheumatoid arthritis (RA), and 13 patients with systemic lupus erythematosus (SLE). A total of 441 hybridoma clones were obtained, of which 37 secreted anti-DNA autoantibodies. The nucleic acid binding characteristics of the anti-DNA antibodies produced by two hybridomas from normal subjects, nine hybridomas from RA patients, and 18 hybridomas from SLE patients are reported. The hybridoma anti-DNA antibodies from all three groups showed similar antigen-binding characteristics for denatured DNA (dDNA), native DNA (nDNA), poly(I), poly(dT), and cardiolipin, by both direct binding and competitive binding analyses. One difference noted between normal-derived anti-DNA antibodies and autoimmune-derived antibodies was the inability of the former to react with z-DNA. However, this requires further substantiation with larger numbers of normal-derived clones. The broad overlap of reactivity to nucleic acid antigens among individual anti-DNA autoantibodies found in two clinically different autoimmune diseases, namely RA and SLE, suggests that the pathogenicity of anti-DNA autoantibodies may bear no relationship to their nucleic acid antigen-binding characteristics.     

Quantitative aspects of lupus anti-DNA autoantibody specificity

In this study we have attempted to define the cross-reactive potential of SLE anti-DNA antibodies (in 19 representative sera and plasmas) in both the solution phase and the solid phase. We used the Farr and RBC-CF solution phase assays to measure quantitatively the ability of a variety of negatively charged structurally unrelated molecules to inhibit antibody binding to both native DNA (nDNA) and denatured DNA (dDNA). The inhibitors used were of two types: 1) phospholipids (cardiolipin, phosphatidyl glycerol, and phosphatidic acid) and 2) repeating negatively charged molecules (poly-glutamic acid, heparin sulfate, and chondroitin sulfate). We found in both assays that the phospholipids could inhibit antibody binding to nDNA and dDNA, but a large excess (about 1500-fold) of these molecules was needed relative to DNA to achieve equivalent levels of inhibition. The repeating negatively charged molecules did not inhibit DNA binding at equivalent molar levels as the phospholipids; generally, at least a 10,000-fold excess was needed relative to the nucleic acids to achieve any appreciable inhibition. Results of a dDNA binding-inhibition solid-phase ELISA for cross-reactivity of the anti-DNA antibodies gave quite similar results. Finally, we found that eight of the SLE samples did have anti-cardiolipin antibodies, as demonstrated in a cardiolipin-based ELISA. These results suggest that previous reports describing an apparent cross-reactivity of anti-DNA antibodies may not represent physiologically relevant interactions between anti-DNA antibodies and non-nucleic acid antigens.     

Spontaneous shift of anti-DNA antibody idiotypes in systemic lupus erythematosus

Spontaneous shift in Id expression of polyclonal anti-DNA antibodies in a patient, BS, with SLE was investigated. BS had active lupus nephritis in 1982 and developed central nervous system lupus in 1986 without evidence of active nephritis. Two rabbit polyclonal anti-Id (BS-82 and BS-86 R-anti-Id) were raised against affinity-purified anti-DNA antibodies prepared from 1982 serum (BS-82) and 1986 serum (BS-86), respectively. In addition, murine monoclonal anti-Id was prepared against BS-82 Id. Direct binding assays showed that all three anti-Id had preferential binding to the immunizing anti-DNA antibodies (the homologous Id) and poor binding to anti-DNA antibodies prepared from the different dated sample of BS. This was confirmed by inhibition assays of binding of anti-Id to the homologous Id by various Id. Moreover, inhibition assays of binding of various Id to DNA by the R-anti-Id showed that the R-anti-Id was the most effective inhibitor for the homologous Id. Testing for Id expression in serial (1982 to 1986) serum samples of BS with the R-anti-Id as probes showed that BS-82 Id declined and was undetectable after October, 1984, whereas BS-86 Id was first detectable in July, 1985, and increased by June, 1986. These results clearly demonstrate spontaneous shifts in Id expression of human anti-DNA antibodies. The phenomenon of Id shift should be considered in any future strategy for the diagnosis and therapy of human autoimmune disease by anti-Id.     

Responsiveness of autoimmune and normal mice to nucleic acid antigens

To test the possibility that anti-DNA antibody formation in autoimmune disease is related to an exceptional responsiveness to nucleic acid immunogens, we examined the ability of normal and autoimmune strains of mice to produce antibodies after immunization with DNA and synthetic helical DNA analogues. Autoimmune mice (MRL/++) did not respond significantly to denatured DNA-methylated BSA in adjuvant (in comparison with adjuvant alone). They did, however, respond to helical structures that differed from B-DNA, including poly-(dG) . poly(dC), poly(dT-dG) . poly(dC-dA), and left-handed Z-DNA. Normal mice (C57BL/6) responded to denatured DNA and, after immunization with the other polymers, produced antibodies of equal or higher titer than those of MRL/++ animals. Neither strain responded to native DNA. The induced anti-nucleic acid antibodies were highly selective and reacted only with the immunogen. In contrast, both lupus autoantibodies and antisera from normal animals that received adjuvant alone cross-reacted with multiple nucleic acid antigens. This result, and the finding that MRL/++ mice were poor responders to nucleic acid immunogens, suggest that different clones and pathways are involved in nucleic acid-induced and spontaneous anti-DNA antibody formation.     

Specific and shared idiotypes found on hybridoma anti-DNA autoantibodies derived from rheumatoid arthritis and systemic lupus erythematosus patients

The idiotype determinants found on hybridoma anti-DNA autoantibodies produced from the fusion of peripheral blood lymphocytes from 13 systemic lupus erythematosus (SLE) and five rheumatoid arthritis (RA) patients with the GM 4672 human lymphoblastoid line were analyzed. A total of 47 SLE and 21 RA hybridomas were studied, of which 26 SLE and 10 RA produced anti-DNA autoantibodies. Rabbit antisera, raised to six of the SLE hybridoma anti-DNA IgM antibodies, were rendered idiotype specific by multiple absorptions on human IgM and IgG immunoabsorbent columns. In direct binding radioimmunoassays, all six anti-idiotype antisera reacted specifically with the anti-DNA antibody used as immunogen. In competition studies, five anti-idiotype antisera were able to inhibit the binding of their homologous idiotype to DNA-coated tubes. In addition, DNA and polynucleotides inhibited the binding of the five idiotypes to anti-idiotype-coated tubes, suggesting that these anti-idiotypes react with idiotype determinants located within the antigen-combining sites of the anti-DNA antibody molecules. Shared idiotypes were detected among the 68 hybridoma antibodies by direct binding studies on anti-idiotype-coated tubes. Our results revealed that 58% (21/36) of the anti-DNA antibodies and 16% (5/32) of the non-DNA-binding antibodies reacted with at least one anti-idiotype serum. Five anti-idiotype antisera reacted only with hybridoma anti-DNA antibodies from SLE patients. The other anti-idiotype antiserum reacted with both SLE- and RA-derived hybridoma anti-DNA and non-DNA-binding antibodies. These studies indicate that some anti-idiotype antisera may detect specific idiotypes found only on SLE-derived anti-DNA auto-antibodies, whereas other antisera detect shared idiotypes found on both RA and SLE DNA-binding and non-DNA-binding antibodies.     

Primary structures and chain dominance of anti-DNA antibodies

Using several anti-DNA autoantibodies, we analyzed the relative involvement of heavy and light chains in their interactions with DNA. We previously obtained eight hybridomas producing monoclonal anti-DNA autoantibodies by fusing spleen cells from an MRL-lpr/lpr mouse with myeloma cells. The chain dominance was analyzed by UV cross-linking experiments, in which the antibodies were covalently cross-linked with radioisotope-labeled oligonucleotides by short-wavelength UV-light, and the cross-linked H and L chains were analyzed by SDS-PAGE and densitometric scanning. Among these, three were found to be heavy chain dominant antibodies in which heavy chains are dominantly involved in DNA binding. The other five were co-dominant antibodies in which both heavy and light chains are involved in DNA binding. To determine the factor(s) that can explain the chain dominance in DNA binding, we determined the amino acid sequences of the variable regions of both heavy (VH) and light (VL) chains of all eight monoclonal antibodies. By analyzing the data, we were able to draw the following conclusions: (1) The arginine residues are found in the CDR3 regions of both VH and VL of the co-dominant antibodies; whereas, the same residues are found only in the CDR3s of VH, but not in VL, of the heavy chain dominant antibodies. (2) The net charges of the V regions affect the chain dominance. From the results of this study it is suggested that the presence of arginine residue in CDR3 is a critical factor in determining chain-dominance, as well as DNA binding of anti-DNA antibodies in general.     

Specificity analysis of human anti-DNA antibodies

Human hybrids producing anti-DNA antibodies were generated by the fusion of pokeweed mitogen-stimulated splenic lymphocytes from a child with sickle cell anemia to GM4672. Of 19 hybrids, three (15%) produced anti-DNA antibody as detected by an enzyme linked immunosorbent assay. One subclone from each of these three hybrids was then characterized. All produced IgM antibody in large amounts ranging from 22 to 266 micrograms/ml per million cells per 24 hr. All three antibodies bound both double- and single-stranded DNA. Competitive inhibition assays revealed the greatest inhibition of DNA binding with the ribohomopolymers polyinosinic and polyguanylic acid. A complex pattern of cross-reactivity with various other polynucleotides and with some phospholipids was observed. Subtle differences were found among the three antibodies in light chain class and some of the binding specificities. By using a modified Farr assay, all three monoclonals were found to be of low to intermediate affinity. These results confirm that anti-DNA antibodies apparently equivalent to those seen in patients with SLE can be derived from "normal" nonautoimmune individuals.     

Molecular dynamics of immune complex of photoadduct-containing DNA with Fab-Anti-DNA antibody fragment

Antibodies to DNA play an important role in the pathogenesis of autoimmune diseases. The elucidation of structural mechanisms of both the antigen recognition and the interaction of anti-DNA antibodies with DNA will help to understand the role of DNA-containing immune complexes in various pathologies and can provide a basis for new treatment modalities. Moreover, the DNA-antibody complex is an analog of specific intracellular DNA-protein interactions. In this work, we used in silico molecular dynamic simulations of bimolecular complexes of the dsDNA segment containing the Fab fragment of an anti-DNA antibody to obtain the detailed thermodynamic and structural characteristics of dynamic intermolecular interactions. Using computationally modified crystal structure of the Fab-DNA complex (PDB ID: 3VW3), we studied the equilibrium molecular dynamics of the 64M-5 antibody Fab fragment associated with the dsDNA fragment containing the thymine dimer, the product of DNA photodamage. Amino acid residues that constitute paratopes and the complementary nucleotide epitopes for the Fab-DNA construct were identified. Stacking and electrostatic interactions were found to play the main role in mediating the most specific antibodydsDNA contacts, while hydrogen bonds were less significant. These findings may shed light on the formation and properties of pathogenic anti-DNA antibodies in autoimmune diseases, such as systemic lupus erythematosus associated with skin photosensitivity and DNA photodamage.     

A conserved anti-DNA antibody idiotype associated with nephritis in murine and human systemic lupus erythematosus

In order to identify unique structural features of pathogenic autoantibodies to DNA in SLE, a murine anti-anti-DNA (anti-Id) mAb (mAb 1C7) was produced in response to immunization of lupus mice with a syngeneic anti-DNA mAb (mAb 3E10). Immunization of lupus mice with mAb 3E10 inhibited production of native anti-DNA antibodies, suppressed development of lupus kidney disease (nephritis), and induced production of anti-anti-DNA (anti-Id) antibodies. mAb 1C7 bound F(ab')2 fragments of mAb 3E10, and it bound other murine anti-DNA mAb, but not murine mAb or polyclonal serum antibodies unreactive with DNA. Moreover, binding of mAb 1C7 anti-Id to mAb 3E10 was inhibited by DNA, suggesting anti-Id binding within or near the binding site for DNA. Furthermore, mAb 1C7 bound serum IgG immunoglobulins from 9/12 patients with lupus nephritis and serum anti-DNA antibodies compared to only 3/12 SLE patients with comparable serum levels of anti-DNA antibodies, but without nephritis (p = 0.04), and only 1/53 SLE patients without serum anti-DNA antibodies, 0/49 patients with rheumatoid arthritis, and 1/47 healthy subjects (p less than 0.001). These results provide evidence that mAb 1C7 identifies a conserved Id associated with anti-DNA antibodies in murine and human SLE and may be useful as a structural probe to characterize pathogenic anti-DNA antibodies in SLE.     

Slipped (CTG).(CAG) repeats of the myotonic dystrophy locus: surface probing with anti-DNA antibodies

At least 15 human diseases have been associated with the length-dependent expansion of gene-specific (CTG).(CAG) repeats, including myotonic dystrophy (DM1) and spinocerebellar ataxia type 1 (SCA1). Repeat expansion is likely to involve unusual DNA structures. We have structurally characterized such DNA, with (CTG)(n).(CAG)(n) repeats of varying length (n=17-79), by high-resolution gel electrophoresis, and have probed their surfaces with anti-DNA antibodies of known specificities. We prepared homoduplex S-DNAs, which are (CTG)x.(CAG)y where x=y, and heteroduplex SI-DNAs, which are hybrids where x>y or x相似文献   

IgG anti-DNA autoantibodies within an individual autoimmune mouse are the products of clonal selection

Although mice from almost all inbred strains produce IgM anti-DNA antibody in response to B cell mitogens, only (NZB x NZW)F1 mice and mice from other strains that are genetically predisposed to autoimmunity spontaneously produce anti-DNA antibody of the IgG isotype. Because (NZB x NZW)F1 mice display marked B cell hyperactivity, anti-DNA antibody production in these mice has been thought to result from spontaneous, polyclonal B cell activation. Although this may be true for IgM anti-DNA antibodies, our results demonstrate that IgG anti-DNA antibodies are not polyclonal. Rather, IgG anti-DNA autoantibodies within an individual autoimmune mouse are oligoclonal and somatically mutated. These results demonstrate that IgG anti-DNA autoantibodies are the products of clonally selective B cell stimulation and exhibit the same characteristics as secondary immune antibodies to conventional immunogens: they are IgG, they are clonally restricted, and they are somatically mutated.     

Monoclonal autoantibodies to subnucleosomes from a MRL/Mp(-)+/+ mouse. Oligoclonality of the antibody response and recognition of a determinant composed of histones H2A, H2B, and DNA.

MRL/Mp(-)+/+ mice produce antinuclear antibodies and develop a spontaneous autoimmune syndrome with lupus-like nephritis. We obtained a panel of seven histone-reactive IgG mAb from a single MRL/Mp(-)+/+ mouse. These antibodies do not react significantly with DNA or individual histones, but bind strongly to the histone H2A-H2B dimer and even more strongly to the H2A-H2B-DNA complex. These antibodies also bind to whole nuclei when tested by immunofluorescence, indicating that they recognize an epitope accessible in chromatin. The V region sequences of these antibodies have been determined. The H chain third complementarity-determining regions of these antibodies are similar to those found in anti-DNA antibodies even though the antibodies in our panel do not react with DNA in the absence of histones, suggesting that DNA is part of the subnucleosome epitope. Several of these antibodies are clonally related, supporting the hypothesis that the activation of these clones is Ag-driven. Analysis of the sequences of these antibodies indicates that they derive from autoreactive B cells that were clonally expanded and whose V region genes have undergone numerous somatic mutations.     

SLE血清中抗-DNA抗体分离和性质的研究

本文用国产高分子树脂(T)接枝小牛胸腺DNA,通过亲合层析从系统性红斑狼疮SLE患者血清中纯化出抗-ds DNA抗体和抗-ss DNA抗体。酶联免疫吸附分析(ELISA)的研究表明:SLE抗-DNA抗体和DNA结合的差异性很大,是高度非均一性的。抗-ss DNA抗体不仅组成成分比抗-ds DNA抗体复杂,ss DNA/抗-ssDNA亲合能力也明显高于ds DNA/抗-ds DNA。纯化的抗-DNA抗体以IgG类抗体占主导,同时也有其它类型抗体存在(例如IgM等)。抗-ds DNA抗体有较抗-ss DNA抗体高的IgG含量(两者的IgG/IgM分别是7.0和4.0),说明IgG抗-DNA抗体更倾向于同dsDNA结合。     

Clearance kinetics and immunochemistry in rabbits of soluble antibody/DNA immune complexes. Effects of antibody class and DNA conformation

We examined the clearance kinetics in rabbits of soluble antibody/DNA immune complexes (IC) containing either IgG or IgM anti-DNA antibodies. Differences in the complement-mediated binding of these IC to rabbit blood cells (platelets) were also studied. Complexation of either double-stranded (ds) or single-stranded (ss) DNA with IgG anti-DNA tends to preclude in vivo DNA recognition mechanisms; the DNA is cleared as part of an IC at a rate slower than that of free DNA. Binding of ds- or ssDNA by IgM anti-DNA antibodies leads to formation of IC which are cleared more like free DNA, and this effect is most evident for ssDNA. However, although both IgG- and IgM-containing IC bound rapidly to blood cells in vivo, significant differences in their immunochemistry were apparent. For example, the DNA in IgM-containing IC was more susceptible to both in vivo and in vitro degradation. In addition, the binding of IgM-containing IC to rabbit platelets and human red blood cells was considerably more labile. Based on this systematic investigation of the soluble antibody/DNA IC that can potentially form in the circulation of a patient with systemic lupus erythematosus, it should be possible to formulate predictions regarding the relative pathogenic potential of these IC.     

Detection of masked anti-DNA antibodies in lupus sera by a monoclonal anti-idiotype

We report here the use of a monoclonal anti-idiotype 3I to human anti-DNA antibodies to detect in serum idiotype-positive antigen-binding antibodies lacking DNA-binding activity as measured by conventional antigen binding assays. We studied paired serum samples from 13 patients with systemic lupus obtained at two times in the course of their disease: in each patient, one serum sample has anti-DNA activity and the second serum sample has no anti-dsDNA activity detectable by Millipore filter, ELISA, or Crithidia assay. Reactivity with 3I as detected with a radioimmunoassay (RIA) was present in all 13 sera with anti-dsDNA activity. Six patients showed a decrease in 3I reactivity to normal levels in the second serum sample, in which anti-dsDNA antibodies were not detectable by conventional antigen-binding assays. The other seven patients' second serum sample continued to show elevated 3I reactivity by RIA even though no anti-dsDNA activity was apparent. When the 3I-reactive antibodies from these latter patients' sera were eluted from a 3I affinity column, they revealed DNA-binding activity. Furthermore, dsDNA binding by these sera was apparent when they were displayed on Western blots of isoelectric focusing gels run in 8 M urea and incubated with radiolabeled dsDNA. These results indicate that the 3I anti-idiotype can detect anti-DNA antibodies in some sera of SLE patients that lack anti-DNA activity by ordinary assays. These antibodies may be inhibited in binding dsDNA by excess antigen or autologous anti-idiotype, and their DNA binding activity can be unmasked by procedures promoting immune complex dissociation.