The structural basis for DNA binding by an anti-DNA autoantibody

Jang, Young-Ju; Sanford, David G.; Chung, Hee Yong; Baek, Seung‐Hoon; Stollar, B. David

doi:10.1016/s0161-5890(98)00095-9

Cited by 38 publications

(51 citation statements)

References 33 publications

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“…In these experiments, we studied further the fine specificity of the 2C10, especially on the role of negatively-charged Asp cluster in CDR1L or Asp and Asn residues in CDR3L using mutant scFvs. The mutants of the CDR1L or CDR3L which contain normal VH of the 2C10 were previously produced by replacement of the amino acid residues in the CDRs with Ala by site-directed mutagenesis (Jang et al, 1998). Fig.…”

Section: Resultsmentioning

confidence: 99%

“…of triplicate samples and experiments were repeated three times. (Jang et al, 1998). The DNA is ten base pairs of alternating (dA-dC)·(dG-dT) with a B-DNA conformation.…”

Section: Discussionmentioning

confidence: 99%

“…Mutated scFv constructs were transformed, expressed, and purified as described in Jang et al (1998). Briefly, E. coli BL2l(DE) pLysE cells transformed with the scFvs were grown in the presence of IPTG for induction.…”

Section: Bacterial Expression Purification and Characterization Of mentioning

confidence: 99%

“…To show the residues of CDR1L and CDR3L more closely in the structure of scFv, computer modeling was performed based on homology to known antibody structures using the composer module of the program Sybyl (Tripos Inc.) as described in Jang et al (1998).…”

Section: Computer Modelingmentioning

confidence: 99%

“…The results indicated that VL of 2C10 might be involved in determining the fine specificities for antigen binding. In the successive studies, we showed that CDR1L residues of the 2C10 scFv interact unfavorably with DNA, reducing the affinity of the antibody for all DNAs, whereas CDR3L residues form favorable contacts that increase the affinity (Jang et al, 1998).…”

Section: Introductionmentioning

confidence: 99%

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Modulation of fine specificity of anti-DNA antibody by CDR3L residues

Jang¹,

Sanford²

2001

Molecular Immunology

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Section: Resultsmentioning

confidence: 99%

“…of triplicate samples and experiments were repeated three times. (Jang et al, 1998). The DNA is ten base pairs of alternating (dA-dC)·(dG-dT) with a B-DNA conformation.…”

Section: Discussionmentioning

confidence: 99%

Section: Bacterial Expression Purification and Characterization Of mentioning

confidence: 99%

Section: Computer Modelingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Modulation of fine specificity of anti-DNA antibody by CDR3L residues

Jang¹,

Sanford²

2001

Molecular Immunology

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Diverse roles for the third complementarity determining region of the heavy chain (H3) in the binding of immunoglobulin Fv fragments to DNA, nucleosomes and cardiolipin

2000

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Autoantibodies to DNA and chromatin employ junctional diversity and somatic mutations to generate or enhance antigen recognition. To define the role of diversity generating mechanisms in the etiology of autoantibodies to nuclear antigens, the heavy (H) chain of a murine autoantibody, 3H9, was used in its somatically mutated or germ‐line form in conjunction with its own or with heterologous CDR3 (H3) domains. The resulting H chains were expressed together with the 3H9 light (L) chain as single‐chain Fv (scFv) in Escherichia coli and assayed for binding to DNA, nucleosomes, or cardiolipin by enzyme‐linked immunosorbent assay. All recombinant scFv exhibited nearly identical binding to cardiolipin. In contrast, the binding to nuclear antigens was drastically reduced by the reversion of mutations in 3H9 or the exchange of H3, such that only 3H9 itself bound strongly to single‐stranded DNA, double‐stranded DNA and nucleosomes. The results illustrate diverse interactions between a single combining site and different autoantigens. The analysis of these interactions suggests that the 3H9 VH domain, as encoded by the germ line, directs binding to cardiolipin, whereas structural determinants of H3, in concert with the remainder of the combining site, guide the maturation of antibody binding toward nuclear autoantigens.

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Analysis of autoimmune bone marrow by antibody‐phage display: Somatic mutations and third complementarity‐determining region arginines in anti‐DNA γ and κ V genes

Seal

Hoet

Raats

et al. 2000

Arthritis & Rheumatism

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Objective. To examine anti-double-stranded DNA (anti-dsDNA) IgG autoantibodies from the bone marrow of individuals with systemic lupus erythematosus (SLE).Methods. A library of single-chain variable fragments (scFv) was constructed from SLE bone marrow complementary DNA of ␥, , and isotype by cloning into the pHENIX phagemid vector. The library was screened with dsDNA in solution, and 2 anti-DNA phage, DNA1 and DNA4, were isolated and their Ig V genes sequenced. Soluble scFv corresponding to DNA1 and DNA4, and their heavy (H)-and light (L)-chain recombinants, were prepared, purified, and analyzed for binding to DNA by enzyme-linked immunosorbent assay.Results. DNA1 and DNA4 used different Ig H-chain (3-30 and 5-51, respectively) and L-chain (DPK15 and DPK22, respectively) V genes. The ratios of replacement mutations to silent mutations in DNA1 and DNA4 suggest that their V genes were selected for improved antigen binding in vivo. The recombinant between DNA4VH and DNA1VL showed the highest relative affinity for both single-stranded DNA and dsDNA. These 2 Ig subunits contained third complementarity-determining region arginines and had acquired the majority of replacement mutations.Conclusion. Anti-dsDNA IgG autoantibodies from the bone marrow of SLE patients exploit diverse V genes and cationic V-D-J and V-J junctions for DNA binding, and accumulate replacement mutations that enhance binding.Anti-double-stranded DNA (anti-dsDNA) antibodies represent an important specificity among the diverse autoantibodies that are found in systemic lupus erythematosus (SLE), yet their molecular characterization remains incomplete (for review, see ref. 1). AntidsDNA IgG autoantibodies use a variety of recurrent heavy (H)-and light (L)-chain V-region (V H and V L ) genes, and anti-dsDNA-combining sites often acquire somatic replacement mutations during the process of clonal expansion (2,3). Frequently, these replacement mutations generate arginine, asparagine, or lysine side chains whose functional groups are suitable for contacts with DNA. These replacement mutations, and the arginine residues found in the third complementaritydetermining region (CDR3) of many anti-DNA H and L chains, are consistent with positive selection of antidsDNA autoantibodies for improved binding to DNA. Selection for DNA binding was demonstrated in murine anti-dsDNA autoantibodies by reversion of somatic replacement mutations and by in vitro mutagenesis of arginines at the V-D-J junction (4,5).The detailed characterization of anti-dsDNA V genes has been further advanced by the expression of antibody fragments on the surface of bacteriophage (3,6). This heterologous expression system affords the opportunity to screen a large, highly diverse crosssection of the V-gene repertoire.

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The structural basis for DNA binding by an anti-DNA autoantibody

Cited by 38 publications

References 33 publications

Modulation of fine specificity of anti-DNA antibody by CDR3L residues

Modulation of fine specificity of anti-DNA antibody by CDR3L residues

Diverse roles for the third complementarity determining region of the heavy chain (H3) in the binding of immunoglobulin Fv fragments to DNA, nucleosomes and cardiolipin

Analysis of autoimmune bone marrow by antibody‐phage display: Somatic mutations and third complementarity‐determining region arginines in anti‐DNA γ and κ V genes

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