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ABSTRACT: Anti-DNA antibodies contribute to the pathology of systemic lupus erythematosus. Their depositon in tissue lesions could result from localization of preformed immune complexes of antibodies with DNA or nucleosomes, or from cross-reaction of anti-DNA antibodies directly with tissue proteins. Structural analyses contribute to understanding their pathogenic potential. Primary structures of lupus immunoglobulin G double-stranded DNA-binding autoantibodies are determined by immunoglobulin genes with mutated variable region segments, indicative of selection by immunizing antigen. Arginine, lysine and asparagine residues in complementarity-determining region favor DNA binding. Heavy-chain variable regions make major contributions to DNA binding; affinity and specificity of binding are modulated or can be abrogated by the light-chain variable domain. Crytallographic structure is known for a few antibody-DNA complexes and several ligand-free Fab fragments. Computer modeling supplements this limited information. Structural information of lupus antibody interactions with both DNA and cross-reacting molecules will support use of ligands to inhibit tissue deposition of the antibodies and prevent lesion formation in lupus.
Cellular and Molecular Life Sciences CMLS 03/2003; 60(2):309-20. · 6.57 Impact Factor
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ABSTRACT: It has been proposed that autoreactivity of modest affinity contributes to positive selection of a preimmunization B cell repertoire, whereas high-affinity autoreactivity leads to negative selection. This hypothesis predicts that a B cell producing a physiologically selected unmutated ssDNA-binding Ab should be a precursor of cells that respond to diverse exogenous Ags. To test this prediction, we prepared transgenic mice bearing the rearranged V(H) domain of an IgM Ab from a nonautoimmune mouse immunized with a DNA-protein complex, poly(dC)-methylated BSA. The Ab, dC1, binds both poly(dC) and ssDNA. It is encoded by V(H) and V(L) gene segments with no mutations, suggesting that the producing cell may have been selected before and activated during immunization. The dC1V(H) transgene was targeted to the IgH locus. In heterozygous mice, on a nonautoimmune C57BL/6 background, the transgene allotype was expressed on B cell surfaces and in serum Ig, but about one-third of B cells expressed the endogenous allele instead. Total serum Ig concentrations were normal and included both transgene- and endogenous gene-coded IgM and IgG. The transgene V(H) D(H)J(H) was expressed in splenic IgM cDNA with few or no mutations, and in IgG cDNA with multiple mutations. The transgene allotype was also expressed in Abs formed on immunization with thyroglobulin, pneumococcal polysaccharide, and ssDNA-methylated BSA. Consistent with the hypothesis, cells with a rearranged autoreactive V(H) domain selected for reactivity with a form of ssDNA did serve as precursors for cells producing IgM and IgG Abs to diverse Ags.
The Journal of Immunology 11/2001; 167(7):3746-55. · 5.79 Impact Factor
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ABSTRACT: This protocol describes application of single cell reverse transcription polymerase chain reaction (RT-PCR) to the study of human immunoglobulin V region usage. The procedure begins with separation of peripheral blood mononuclear cells (PBMC) from human blood. The PBMC are stained with the B cell selective marker, anti-CD19. Stained B cells are sorted by flow cytometry and deposited, consecutively, one cell into each of an array of tubes. cDNA for one or more antibody variable regions (VH and/or VL) is synthesized with a primer (or primers) complementary to sequence(s) within the constant region (Cmu, Cgamma, Ckappa and/or Clambda). The cDNA is used as template for PCR amplification with gene or gene family specific primers. A second PCR is then performed with two nested primers to increase both the specificity and quantity of V region PCR products. The purified PCR products are sequenced directly and aligned to V region germline database and the Genbank database. Single cell RT-PCR is a fast and convenient way to analyze V region gene expression. It avoids the bias that may be introduced into V region cDNA library construction by the presence of highly variable levels of mRNA in different cells. The PCR products are obtained in quantities that can be cloned into bacterial expression vectors for production of recombinant V region protein domains.
Journal of Immunological Methods 11/2000; 244(1-2):217-25. · 2.20 Impact Factor
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The Israel Medical Association journal: IMAJ 10/2000; 2(9):703-7. · 1.02 Impact Factor
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Analytical Biochemistry 03/2000; 278(2):239-41. · 3.00 Impact Factor
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B D Stollar
Clinical Reviews in Allergy & Immunology 03/2000; 18(1):41-50. · 3.68 Impact Factor
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ABSTRACT: Immune responses change in aging humans, but it is not known whether there is an age-associated change in the expressed B cell repertoire. We compared Ig VH cDNA libraries from circulating B cells of five elderly and three young human adults. As in young persons, nearly two-thirds of the cDNA clones from older subjects had zero to three V(H) mutations, although there was more individual variation among the elderly. V(H)4 family expression increased in older subjects, both in unmutated and in mutated cDNA clones, whereas V(H)3 family expression predominated in young adults. To test for bias toward activated cells in the cDNA libraries, we studied two older persons by both cDNA library analysis and single-cell RT-PCR. In one subject, more than 85% of VH segments were unmutated by either analysis. In the second, mutated Ig segments were much more frequent in cDNA clones than in consecutive single cells; however, V(H) family usage and high representation of particular genes were similar in both analyses. While aging humans continue to produce naive B cells with unmutated Ig genes, a shift to greater use of the V(H)4 family members and expression of particular genes may reflect changes in selection of developing B cells before affinity maturation toward reactivity with foreign antigen.
Clinical Immunology 12/1999; 93(2):132-42. · 4.05 Impact Factor
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ABSTRACT: mAb Z22 is a highly selective IgG anti-Z-DNA Ab from an immunized C57BL/6 mouse. Previous studies showed that heavy chain CDR3 amino acids are critical for Z-DNA binding by the single chain variable fragment (scFv) comprising both V region heavy chain (VH) and V region light chain (VL) of mAb Z22 and that the VH domain alone binds Z-DNA with an affinity similar to that of whole variable fragment (Fv). To determine whether Z-DNA binding by VH alone and by Fv involves identical complementarity determining region residues, we tested effects of single or multiple amino acid substitutions in recombinant VH, scFv, and associated VH-VL heterodimers. Each recombinant product was a fusion protein with a B domain of Staphylococcal protein A (SPA). Z22VH-SPA alone was not highly selective; it bound strongly to other polynucleotides, particularly polypyrimidines, and ssDNA as well as to Z-DNA. In contrast, scFv-SPA or associated VH-VL dimers bound only to Z-DNA. VL-SPA domains bound weakly to Z-DNA; SPA alone did not bind. Introduction of multiple substitutions revealed that the third complementarity determining region of the heavy chain (CDR3H) was critical for both VH and scFv binding to Z-DNA. However, single substitutions that eliminated or markedly reduced Z-DNA binding by scFv instead caused a modest increase or no reduction in binding by VH alone. Association of VH-SPA with Z22VL-SPA restored both the effects of single substitutions and Z-DNA selectivity seen with Fv and intact Ab. Polypyrimidine and ssDNA binding by the isolated VH domain of immunization-induced anti-Z-DNA Ab resembles the activity of natural autoantibodies and suggests that VH-dependent binding to a ligand mimicked by polypyrimidines may play a role in B cell selection before immunization with Z-DNA.
The Journal of Immunology 05/1999; 162(8):4663-70. · 5.79 Impact Factor
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ABSTRACT: We have used single and multiple site-directed mutagenesis, and molecular modeling, to identify critical residues in the DNA binding site of MAb 2C10, an IgG anti-dsDNA autoantibody from an MRL/lpr lupus mouse. Simultaneous replacement of four Arg residues in the CDR3H abolished binding activity. With one exception, replacement of any one of these Arg residues reduced the activity to 20-50% of the unmutated scFv activity. Arg to Asp replacements had a slightly greater effect than Arg to Ala replacements. In the one exceptional case, replacement of Arg99 with Ala actually increased DNA binding five-fold and replacement by Asp had little effect. Mutation of Phe32 and Asn35 to A1a in CDRIH decreased DNA binding, whereas replacement of Arg31 with A1a had negligible effect. Ala substitution of any one of a cluster of Asp residues in CDR1L increased DNA binding three to six-fold, confirming previous findings that the L-chain of MAb 2C10 is not favorable for DNA binding. The L-chain does participate in shaping the selectivity of antigen binding, and mutation of CDR3L residue Asp92 or Asn93 caused a decrease in DNA binding activity. Directed mutagenesis, consistent with a molecular model, indicates that: several CDR amino acids contribute to DNA binding, without one residue dominating; both VH and VL CDR3 domains contribute to specificity of binding whereas the CDR1L hinders DNA binding. The results suggest a significant role for electrostatics in the interaction of DNA with MAb 2C10.
Molecular Immunology 01/1999; 35(18):1207-17. · 2.90 Impact Factor
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ABSTRACT: Previous studies showed that VH domains of several anti-DNA Abs can bind DNA in the absence of VL. In the current work, we tested the VH autoreactive potential more generally, examining VH domains that did not come from known autoantibodies. Using a bacterial expression system, we produced 11 fusion proteins, each containing a VH domain and a B domain of staphylococcal protein A. The VH domains were coded in cDNA libraries from circulating B cells of healthy young adult humans. Thus, binding properties of the Ig molecules from which they came were unknown. The B cells had not been stimulated in vitro. Seven cDNA clones combined the frequently expressed VH3-23 gene segment with varied DH and JH segments. The other clones contained unmutated VH3-7, VH3-9, VH3-53, and VH4-39 segments. We compared these bacterial expression products with single-chain Fv, VH and VL domains of IgM mAb 18/2, a VH3-23-encoded, DNA-binding autoantibody. Submicromolar concentrations of 5 of the 11 VH domains bound to ssDNA. Those and one more also bound to immobilized poly(dT), and two bound to circular plasmid dsDNA. Soluble poly(dT) was the most potent inhibitor in competitive ELISA. Seven of the VH domains also bound to immobilized nuclear ribonucleoprotein, four to histone and none to thyroglobulin. Two interacted with the matrix of a Sephacryl S-100 column. The polyreactive autoantigen-binding properties of these VH domains raise the question of whether these properties may play a role in the formation of the VH repertoire of circulating B cells.
The Journal of Immunology 09/1998; 161(3):1274-83. · 5.79 Impact Factor
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Annals of the New York Academy of Sciences 05/1997; 815:30-9. · 3.15 Impact Factor
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B D Stollar
Lupus 02/1997; 6(3):346-8. · 2.34 Impact Factor
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B D Stollar
Lupus 02/1997; 6(3):327-8. · 2.34 Impact Factor
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B D Stollar
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ABSTRACT: Bacterial production of recombinant Fab or Fv domains of antibodies is an important tool for analyzing structural correlates of antigen binding or idiotype expression. Bacterial products may be Fab or Fv molecules that assemble from separate chains in the periplasm or a single-chain Fv protein. This article describes properties and applications of a plasmid vector used for production of single-chain Fv (scFv). The expression cassette, initially designed for production of the Fv domain of anti-Z-DNA mAb Z22, has a bacterial secretion signal that permits secretion of soluble scFv into growth medium. A single B domain of staphylococcal protein A facilitates affinity purification and generic assay of products independent of antigen-binding activity. Gene segment swapping and directed mutagenesis identified structural features important for Z-DNA binding and revealed the structural similarity of autoantibody and immunization-induced antibody. The H and L regions of mAb Z22 are readily replaced by those of any cloned Ig, a library of V regions, or other proteins. Modified forms of the vector code for production of separate H or L chain V regions, which can associate with each other to form functional Fv complexes. Although there is large variation in the yield with different V regions, most clones provide enough soluble product for antigen-binding assays. Current developments are aimed at increasing the yield consistency to allow production of enough material for three-dimensional structural analysis.
Methods 02/1997; 11(1):12-9. · 4.01 Impact Factor
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ABSTRACT: Antibodies H241 and 2C10 are lupus mouse IgG autoantibodies that bind native DNA. In previous experiments, oligonucleotide antigens affinity-labeled both H and L chains of H241 but only the H chain of antibody 2C10. Primary structures of the V regions of the 2C10 H and L chains and the H241 L chain, determined from cDNA, help to explain the previous affinity-labeling experiments. The 2C10 L chain CDRs had several Asp residues and a net negative charge of five, whereas the 2C10 H chain CDRs had four Arg residues and a net positive charge of five. The L chain CDRs of H241 had a net positive charge of one. [The H241 H chain cDNA sequence was published previously by Gangemi et al. (1993) J. Immun. 151, 4660-4671]. Plasmid vectors were used for bacterial expression of H and L chains of 2C10 alone and in combinations in single chain Fv (scFv) molecules. The H chain alone bound native DNA as well as or better than the H-plus-L chain scFv. The H chain alone also bound Z-DNA. Combination of the 2C10 H chain with the L chain of an anti-Z-DNA antibody maintained the selectivity for Z-DNA, whereas its combination with the 2C10 L chain (in the 2C10 Fab) yielded selective B-DNA binding. The results with 2C10 match other examples in which the H chain is sufficient for DNA binding but selectivity is modulated by the L chain. The H chain binding to autoantigen may reflect selective events in early stages of B cell development.
Molecular Immunology 03/1996; 33(2):197-210. · 2.90 Impact Factor
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B D Stollar
Annals of the New York Academy of Sciences 10/1995; 764:265-74. · 3.15 Impact Factor
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ABSTRACT: The surrogate light chain, composed of VpreB and lambda 5/14.1 proteins, is selectively expressed on B cell precursors, and is important for B cell development. The surrogate light chain associates with cell surface mu-chains on preB cells, but little is known about the ligand specificity and affinity of VpreB binding. To analyze its interactions with Igs, we made recombinant human VpreB protein and measured its affinity for H and L chain V domains using surface plasmon resonance. The recombinant VpreB protein existed as a homodimer in solution. VpreB chains associated with each other with an apparent Kd = 5 x 10(-7) M, and bound to a human VH domain, a mouse VH domain, and a human VL domain with a similar affinity. VpreB protein also bound to human Fab fragments of IgG with an apparent Kd = 6 x 10(-7) M, but showed a very low affinity for human Fc fragments of IgG. VpreB-Fab complex formation was reproduced by the formation of a trimolecular VpreB-VH-VL complex. Thus, VpreB proteins can associate with each other and also form complexes with Ig at sites different from those involved in VH-VL interaction. By flow cytometry, biotinylated VpreB protein bound to surface Ig-positive B cells but not T cells. Receptors that contain VpreB could be cross-linked by either Ig or by self-association.
The Journal of Immunology 09/1995; 155(3):1218-28. · 5.79 Impact Factor
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ABSTRACT: The heavy (H) and light (L) chain V domains of anti-Z-DNA mouse mAb Z22 were expressed separately in bacteria. When mixed in vitro, the V domains associated stoichiometrically to reconstitute the Ag binding site of Z22, as judged by specific reactivity with Z-DNA and anti-Z22 ld Abs. The apparent Kd of the Z22 VH-VL association was 5.47 x 10(-8) M, measured by surface plasmon resonance. A replacement at VL position 96, which reduced Ag binding affinity of a single chain Fv (clone LZ1-2) by two orders of magnitude, did not reduce the affinity of interaction between the VH and VL domains (apparent Kd = 1.93 x 10(-8) M for VH association with LZ1-2). Fab prepared from native Z22 bound specifically to a 30-bp Z-DNA oligonucleotide with an apparent Kd = 1.56 x 10(-8) M. The VH domain alone bound Z-DNA specifically with an affinity similar to that of the Fab or Fv's of Z22 (Kd = 1.68 x 10(-8) M), whereas Z22 VL domain alone did not interact with nucleic acids. Z22 VH binding to Ag was inhibited by association with the mutant LZ1-2 VL. These results indicate that the Z22 H chain makes important contributions to specific binding of Z-DNA. Although the L chain does not add greatly to the binding energy, an appropriate L chain is required to permit Ag binding in the Fv domain. These in vitro results resemble the in vivo modulation of H chain autoreactivity that occurs with L chain substitution in receptor editing.
The Journal of Immunology 04/1995; 154(5):2198-208. · 5.79 Impact Factor
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Methods in molecular biology (Clifton, N.J.) 02/1995; 51:265-79.
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ABSTRACT: Bacterial expression of single chain variable fragment (scFv) domains was used to assess Ag-binding contributions of specific regions and residues in a mouse mAb to Z-DNA (AbZ22). A variant scFv (Z3-3) that did not bind Z-DNA had the Z22 light chain but differed from the Z22 heavy chain at four complimentarity determining region 3 (CDR3), one FR4 and five VH segment residues. Gene segment swapping and site-directed mutagenesis indicated that the major contribution of the Z22 heavy chain is its CDR3. A scFv with the CDR3H-FR4H of Ab Z22 and the VH segment of Z3-3 had the same selective high affinity Z-DNA binding as Z22. Some Z-DNA binding was retained even when the CDR3H-FR4H of Ab Z22 was combined with a VH segment that shared only 44% sequence identity with Z22. Directed mutations indicated further that residues N99 and S98 in heavy chain CDR3 and F96 in light chain CDR3 were particularly important for Ag binding. Certain substitutions in CDR3H converted the highly selective Z22 Fv into a polyreactive Fv with autoantibody-like binding to B-DNA and denatured DNA. In a graphic molecular model, heavy chain N99 protrudes from the CDR3 loop at the base of the Ag-binding groove, and the light chain F96 is barely exposed on the base of this groove; the light chain F96 may be important in heavy chain-light chain association. Autoantibody and immunization-induced Ab to nucleic acid can be built on a very similar framework and differ by a small number of amino acid CDR3H residues.
The Journal of Immunology 07/1994; 152(11):5318-29. · 5.79 Impact Factor
