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B D Stollar

Publications and source records attributed to B D Stollar.

At least 37 records · Page 2Linked to original sources
PubMed1996

Enhancement of oxidative cleavage of DNA by the binding sites of two anti-double-stranded DNA antibodies.

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 en- hanced 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 H2O2 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.

Animals↗
PubMed1996

Heavy chain dominance in the binding of DNA by a lupus mouse monoclonal autoantibody.

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.

Amino Acid Sequence↗
PubMed1995

Kinetic analysis of the interactions of recombinant human VpreB and Ig V domains.

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.

Animals↗
PubMed1995

Domain interactions and antigen binding of recombinant anti-Z-DNA antibody variable domains. The role of heavy and light chains measured by surface plasmon resonance.

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.

Amino Acid Sequence↗
PubMed1994

Critical binding site amino acids of anti-Z-DNA single chain Fv molecules. Role of heavy and light chain CDR3 and relationship to autoantibody activity.

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.

Amino Acid Sequence↗
PubMed1994

Molecular analysis of anti-DNA antibodies.

There are two major reasons for interest in antibodies to DNA: autoantibodies to DNA are characteristic of the autoimmune disease systemic lupus erythematosus and contribute to its pathology; and both autoantibodies and experimentally induced antibodies to nucleic acids serve as useful biochemical reagents. Physical biochemistry and molecular biology are applied increasingly to questions of the structure and origin of antibodies to DNA and how antibodies recognize specific structures in DNA. Cloning of cDNA for Ig V regions of anti-DNA antibodies reveals that some cells making IgG disease-related autoantibodies are derived from precursor cells that produce natural autoantibodies unrelated to disease. In addition, some immunization-induced antibodies to DNA use gene segments similar to those found in autoantibodies. Cloning and expression of recombinant V regions allow detailed analysis of antibody structures required for DNA binding. Domain swapping and mutagenesis with vectors for bacterial expression of single chain Fv molecules revealed the importance of CDR3 sites of both H and L chain V regions for specific antigen binding by antibody to Z-DNA. In certain autoantibodies, the H chain plays a dominant role in determining DNA binding. Molecular analysis opens doors to studies of normal immune tolerance and its loss in autoimmune disease as well as to development of antibodies with modified specificity.

Animals↗
PubMed1994

Heavy-chain directed B-cell maturation: continuous clonal selection beginning at the pre-B cell stage.

A number of laboratories have demonstrated a biased representation of certain V-region segments in the primary B-cell repertoire. This may reflect clonal selection at the pre-B cell stage of differentiation. Here, Robert Schwartz and David Stollar suggest that pre-B cells undergo positive selection directed by the presence of surface heavy chain with low affinity to autoantigen. This mechanism would account for the anti-self property of the pre-immune B-cell repertoire.

Animals↗
PubMed1994

Dipeptidyl peptidase IV (DP IV) activity in serum and on lymphocytes of MRL/Mp-lpr/lpr mice correlates with disease onset.

DP IV (CD26), a serine protease expressed on activated T cells, participates in immune responses in vivo as well as in vitro. We measured cell surface and serum DP IV in mice of the autoimmune MRL/Mp-lpr/lpr (MRL/l) strain, which is characterized by massive T cell proliferation and production of anti-nuclear autoantibodies. The mass of inguinal lymph nodes correlated with serum DP IV activity. Furthermore, serum DP IV activity increased markedly in parallel with the acceleration of lymph node swelling and anti-nDNA antibody production. Serum DP IV activity in 16-week-old MRL/l mice reached levels up to three higher than those in age-matched MRL/Mp- +/+ mice or BALB/c mice. Immunohistochemical staining and flow cytometric analysis identified DV IV on surfaces of lymphocytes from the enlarged lymph nodes of MRL/l mice. Subcutaneous injection of the mechanism-based inhibitor, Pro-boroPro, reduced protease activity in serum and cell suspensions prepared from spleen and lymph nodes, confirming the identity of the enzyme as DP IV. These results indicate that the massively accumulating lymphocytes of MRL/l mice have a property characteristic of activated T cells, although they express little surface CD4 or CD8 and do not produce IL-2. DP IV may participate in the role these cells play in the pathogenesis of MRL/l autoimmune disease.

Aging↗
PubMed1993

A majority of Ig H chain cDNA of normal human adult blood lymphocytes resembles cDNA for fetal Ig and natural autoantibodies.

Certain Ig VH gene segments, with few or no mutations, recur frequently in natural autoantibodies, fetal antibodies, and products of B cell tumors. The goal of this study was to determine whether similar Ig gene segment usage occurs in normal human adult PBL. Extending previous analyses, 105 randomly picked H chain V region clones of representative cDNA libraries from PBL were sequenced. Clones were from: IgM and IgG libraries from one RNA sample of a normal adult; a second IgM library from the same subject 11 mo later; and one IgM library from a second subject. Although some clones had clear evidence of mutation, 48 of 77 IgM clones (62%) shared 99% or more identity with known germline VH segments, and most of these had no mutations in the CDR3 portion of the JH segment. Certain VH gene segments, expressed in autoantibodies and fetal antibodies, occurred at high frequency in these libraries. Fourteen of the clones with 99% identity to known VH segments had CDR3 segments identical to portions of known germline DH gene sequences; two such clones had no N nucleotides at the VHDH or DHJH junctions. IgG-encoding sequences had more mutations than IgM-encoding sequences. JH and DH usage was not random. The circulating B cell population may represent a distinct compartment, with a large proportion of cells similar to those of the fetal and natural autoantibody repertoire. Polyreactive Ig products of these circulating cells may serve a screening function, binding and delivering diverse Ag to secondary lymphoid tissues where more highly selective antibodies are formed to foreign or self-Ag.

Adult↗
PubMed1993

High-frequency representation of a single VH gene in the expressed human B cell repertoire.

Idiotype (Id) 16/6 marks a variable (V) region structure that occurs frequently in the human immunoglobulin repertoire. The basis of the Id has been traced to a germline heavy chain gene segment, VH18/2 (VH26). To pursue the molecular basis for the frequency of Id 16/6, we have analyzed polymerase chain reaction-generated C mu, C gamma, and VH3 family V gene libraries derived from the circulating and tonsillar B cells of four normal individuals and from the B cells of two patients with active systemic lupus erythematosus (SLE). The frequency of VH18/2 in these libraries was compared with three control VH genes, VH56P1, VH21/28, and VHA57. Plaque lifts from C mu and C gamma VH cDNA libraries were screened with gene-specific oligonucleotide probes. The frequency of VH18/2 ranged from 4 to 10% of JH+ plaques (two of five times that of control VH genes). In four VH3 family-specific libraries derived from rearranged DNA, VH18/2 represented 19-33% of VH3+ plaques. Hybridizing VH18/2 plaques were 98-100% homologous to the germline VH gene; mutations when present were often in framework 3. Extensive variation was seen in the complementarity determining region 3 sequences of these rearranged V genes. The high frequency of VH18/2 expression in the B cell repertoire was confirmed by sequencing randomly picked JH+ plaques. In two patients with active SLE the frequency of use of VH18/2 was not greater than that observed in normal subjects. These results show that VH18/2 is overrepresented in the B cell repertoire of normal subjects and suggest that the immune repertoire may be dominated by relatively few V genes.

Amino Acid Sequence↗
PubMed1993

Role of mouse VH10 and VL gene segments in the specific binding of antibody to Z-DNA, analyzed with recombinant single chain Fv molecules.

A plasmid vector was constructed for the expression of a single chain Fv domain of mouse mAb to Z-DNA (antibody Z22), which is encoded by VH10 and V kappa 10 gene family members along with Dsp2, JH4, and J kappa 4 segments. The vector coded for a PhoA secretion signal, VH segment, flexible peptide linker, VL segment, (His)5, and a protein A domain. Unique restriction sites allowed exchange of the segments as cassettes. Bacteria transformed with the vector secreted soluble recombinant Fv with specific Z-DNA-binding activity. When the L chain of Z22 was replaced with a library of splenic VL cDNA from a mouse immunized with Z-DNA, only a light chain closely resembling that of the original Z22 (differing at six amino acid positions) yielded Fv with Z-DNA-binding activity. The Fv with this L chain replacement had a lowered affinity, but remained selective for Z-DNA. Replacement of the Z22 H chain with a mixture of 11 VH10-encoded H chains yielded two Z-DNA binding clones, but they bound B-DNA and denatured DNA as well as Z-DNA. The replacement clones indicate the importance of the H chain CDR3 and particular VH-VL combinations in formation of specific antibodies to Z-DNA.

Amino Acid Sequence↗
PubMed1993

The targets and genes for antibodies to Z-DNA.

Antibodies to Z-DNA serve as models for recognition of nucleic acid structure by proteins that use peptide loops rather than helix-loop-helix, zinc-finger or other recurrent motifs for DNA binding. Anti-Z-DNA antibodies have been elicited in rabbits, mice and goats by injection of brominated poly(dG-dC), a stable form of Z-DNA. Detailed studies of two mouse monoclonal antibodies to Z-DNA, Z22 and Z44, have been reported. Epitopes on Z-DNA have been mapped by both serological reactions and n.m.r. spectroscopy. Antibodies Z22 and Z44 recognize different sites on the Z-DNA. They are both encoded by members of the VH10 gene family, which also encodes known autoantibodies to DNA. Vectors for bacterial expression of single-chain Fv molecules have been used to determine the importance of specific H- and L-chain combinations and particular CDR3 sequences for Z-DNA binding. Changes in the H-chain CDR3 cause a decrease in the highly selective Z-DNA binding and an increase in autoantibody-like binding of B-DNA and single-stranded DNA.

Amino Acid Sequence↗
PubMed1992

Immunogenicity of Z-DNA depends on the size of polynucleotide presented in complexes with methylated BSA.

The importance of polynucleotide size for immunogenicity was tested with size-fractionated Z-DNA. High molecular weight Z-DNA, larger than 1000 bp, was fragmented by digestion with micrococcal nuclease. Fractions corresponding to less than 60, 60-120, 100-200, 200-400 and 400-900 bp were isolated by gel filtration on Sepharose 4B. These fractions and the greater than 1000 bp Z-DNA were mixed with methylated BSA and the complexes were injected into C57BL/6 mice with RIBI adjuvant. Only one of four mice responded to the less than 60 bp immunogen. All the fractions larger than 60 bp induced specific anti-Z-DNA antibodies, mostly of IgG isotype, in all animals injected. Fractions larger than 200 bp induced antisera of higher titer than did 60-120 or 100-200 bp fractions. All positive sera reacted with Z-DNA but not with B-DNA and only very weakly with denatured DNA. In competitive assays, similar concentrations of fragments larger than 60 bp inhibited binding to immobilized Z-DNA. A higher concentration of less than 60 bp fragments was required for competitive binding. Even for a highly immunogenic nucleic acid that differs from the B-DNA conformation, a polynucleotide larger than 100 bp is much more immunogenic than smaller fragments.

Animals↗