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M Z Radic

Publications and source records attributed to M Z Radic.

28 records · Page 2Linked to original sources
PubMed1994

Genetic and structural evidence for antigen selection of anti-DNA antibodies.

The primary structure of anti-DNA antibodies is highly diverse, a result of different germline variable (V) gene use, different combinations of immunoglobulin gene segments, peculiar heavy chain complementarity determining region 3 (H-CDR3) segments, and somatic mutations. Nevertheless, tertiary structure predictions reveal common features that yield information about likely contact sites in the anti-DNA combining site. That these contacts are involved with DNA binding is supported by recurrent features of a newly compiled set of homology groups of 13 variable regions of heavy chains (VH) and 11 variable regions of light chains (VL), characteristic pattern of somatic mutations, and the results of site-directed mutagenesis. The role of antigen in the etiology of the autoimmune response is viewed in light of recent data on overlaps between anti-DNA and anti-nucleic acid binding protein specificities.

Amino Acid Sequence↗
PubMed1993

Residues that mediate DNA binding of autoimmune antibodies.

Somatic mutations to arginine (R) are a common feature of a subset of J558 H chain genes that code for the majority of high-affinity, anti-dsDNA antibodies in autoimmune MRL/lpr mice. To examine the consequences of such amino acid substitutions on DNA binding, we reverted three somatic mutations of a prototypic anti-dsDNA H chain gene, VH3H9, and assayed the effect of those reversions by expression in a V lambda 1 L chain-only plasmacytoma line. Reversion of R53 eliminated virtually all dsDNA binding and sharply reduced ssDNA affinity. While the complete germ-line revertant of VH3H9 retained a low level of DNA binding, the substitution of R96, a product of N base addition in the third complementarity determining region (CDR3), with glycine (G) was sufficient to abolish measureable DNA specificity. Antibodies with higher affinity for DNA were generated by introducing arginines into VH3H9 at any one of four positions where somatic mutations to arginine had been identified by sequencing other anti-dsDNA J558 H chain genes. All four arginine mutants showed affinity increments consistent with their direct involvement in DNA binding, although one such mutant, K64R, required the simultaneous reversion of an adjacent aspartic acid (D) to the germ-line glycine. Two variants with three nongerm-line arginines showed further improvements in DNA affinity suggesting that their contributions to DNA binding may be additive. Molecular modeling of antibody and mutant F(ab) structures and calculations of their electrostatic potentials were used as an aid in interpreting the results and in predicting the location and size of possible combining sites.

Amino Acid Sequence↗
PubMed1993

B lymphocytes may escape tolerance by revising their antigen receptors.

To explore mechanisms that prevent autoreactivity in nonautoimmune mice, endogenous immunoglobulin (Ig) light (L) chains that associate with a transgenic anti-DNA heavy chain were analyzed. The antibodies from splenic B cell hybridomas of such mice did not bind double-stranded DNA (dsDNA) and their L chain sequences showed a biased use of V kappa and J kappa gene segments. The 44 L chains in this survey were coded for by just 18 germline genes. Six of the genes, each belonging to a different V kappa group, were used more than once and accounted for three fourths of all sequences. Based on the distribution of V kappa genes, the L chain repertoire in this line of transgenic mice was estimated at 37 V kappa genes. The most frequently observed gene, a member of the V kappa 12/13 group, was identified in 16 hybrids. In addition, the majority of V kappa genes used J kappa 5. We interpret the skewed representation of V kappa and J kappa gene segments to result from negative selection. Based on the data, we suggest that V kappa rearrangements giving rise to anti-dsDNA reactivity are removed from the repertoire by a corrective mechanism capable of editing self-reactive Ig.

Animals↗
PubMed1992

Inhibition of abnormal T cell development and autoimmunity in gld mice by transgenic T cell receptor beta chain.

Mice homozygous for the gld (generalized lymphoproliferative disease) mutation developed systemic autoimmune disease and severe lymphadenopathy due to an age-related accumulation in the peripheral lymphoid organs of polyclonal T cells bearing a unique phenotype (CD4-CD8-TCR alpha beta+B220+). These T cells overexpress T cell receptor (TcR) alpha beta chain RNA, proto-oncogenes c-myb and fyn, and proliferate poorly in response to TcR-mediated stimulation. The origin of these T cells is poorly understood. To study the influence of a functionally rearranged TcR beta chain on the T cell developmental abnormality of the gld mutation and autoimmunity, we have backcrossed TcR V beta 8.1-transgenic mice to C3H-gld/gld to homozygosity (transgenic gld mice). In transgenic gld mice, lymphadenopathy was markedly inhibited and the accumulation of CD4-CD8- T cells did not occur, although the remaining T cells overexpressed c-myb and proliferated poorly in response to TcR occupancy. These features indicate that the pattern of proto-oncogene expression and abnormal function persist in phenotypically normal T cells in transgenic gld mice, and that these characteristics can be dissociated from the accumulation of CD4-CD8- T cells. The hypergammaglobulinemia and anti-double-stranded DNA (anti-dsDNA) antibody production was partially improved in transgenic gld mice, supporting the critical role of T cells in abnormal B cell activation described in autoimmunity-prone mice. To investigate further the mechanisms underlying the inhibition of CD4-CD8- T cell accumulation in transgenic gld mice, the fetal ontogeny of T cells in transgenic mice was compared with that of non-transgenic mice. In transgenic thymus, development of TcR alpha beta+ cells was accelerated as detected by earlier expression of CD4, CD8 and TcR in fetal thymus. In contrast, the number of TcR gamma delta+ cells was reduced. We suggest that altered T cell development in transgenic mice directly or indirectly inhibits the accumulation of abnormal T cells in gld mice.

Animals↗
PubMed1992

Hoechst 33258, distamycin A, and high mobility group protein I (HMG-I) compete for binding to mouse satellite DNA.

The experiments described were designed to test the hypothesis that the (A+T)-specific DNA binding ligands Hoechst 33258 and distamycin A affect the condensation of mouse centromeric heterochromatin by competing for binding to satellite DNA with one or more chromosomal proteins. The studies focused on the nonhistone chromosomal protein HMG-I since its binding properties predict it would be a target for competition. Gel mobility shift assays show that HMG-I forms specific complexes with satellite DNA and that the formation of these complexes is competed for by both Hoechst and distamycin. In addition, methidium propyl EDTA Fe(II) [MPE Fe(II)] footprints of ligand-satellite DNA complexes showed essentially the same protection pattern for both drugs and a similar, but not identical, HMG-I footprint. If these in vitro results reflect the in vivo situation then the incomplete condensation of centromeric heterochromatin observed when mouse cells are grown in the presence of either chemical ligand could be a consequence of competition for binding of HMG-I (and possibly other proteins) to satellite DNA.

Animals↗
PubMed1991

Expression of anti-DNA immunoglobulin transgenes in non-autoimmune mice.

Self-reactive B cells can be regulated by either deletion or inactivation. These manifestations of self-tolerance have been dramatically shown in transgenic mice in which the number of self-reactive cells has been artificially expanded. We have now extended these models to ask if B-cell tolerance as described for non-disease-associated antigens also operates for the targets of autoimmunity. The target we have chosen is DNA. Anti-DNA antibodies are diagnostic of certain autoimmune syndromes in humans and are a characteristic of the murine model of systemic autoimmunity, the MRl/lpr mouse. Antibodies to both single-stranded and double-stranded DNA have been implicated in disease. By generating anti-DNA transgenic mice, we have addressed the question of whether DNA-specific B cells are regulated in normal (non-autoimmune) mice. We indeed found that most transgenic B cells bind DNA, yet we failed to detect secreted anti-DNA. We suggest that as a consequence of their self-reactivity these B cells are developmentally arrested.

Animals↗
PubMed1991

Ig H and L chain contributions to autoimmune specificities.

An Ig H chain expression vector has been constructed by using the V region of 3H9, an antibody that binds ssDNA, dsDNA, and cardiolipin. The H chain construct was transfected into six hybridoma cell lines expressing Ig L chains. All resulting H and L chain combinations had at least some affinity for ssDNA, whereas five also bound dsDNA to a similar degree as 3H9. The loss of dsDNA binding was correlated with a single amino acid difference between two V kappa 8 L chains. A further characteristic of 3H9, its immunofluorescent staining pattern, was shared by four of the recombinant antibodies, whereas its specificity for cardiolipin was shared with five. The transfections reported here show that a V kappa 3 L chain confers specificity for an RNA-associated epitope and that a V kappa 21E L chain prevents cardiolipin binding. These experiments suggest that the 3H9 H chain contributes essential determinants required for binding to DNA as well as cardiolipin but that L chains can modulate or prevent this binding. L chains may also expand the specificity of a recombinant antibody.

Amino Acid Sequence↗
PubMed1990

Anti-DNA antibodies from autoimmune mice arise by clonal expansion and somatic mutation.

The proximate cause of autoantibodies characteristic of systemic autoimmune diseases has been controversial. One hypothesis is that autoantibodies are the result of polyclonal nonspecific B cell activation. Alternatively, autoantibodies could be the result of antigen-driven B cell activation, as observed in secondary immune responses. We have approached this question by studying monoclonal anti-DNA autoantibodies derived from unmanipulated spleen cells of the autoimmune MRL/lpr mouse strain. This analysis shows that anti-DNAs, like rheumatoid factors (19), are the result of specific antigen-driven stimulation. In addition, correlation of sequences with fine specificity shows that: (a) somatic mutations can cause specificity for dsDNA and that such mutations are selected for; (b) arginine residues play an important role in determining specificity; and (c) anti-idiotypes that recognize the majority of anti-DNA are probably not specific for any one family of V regions.

Amino Acid Sequence↗
PubMed1987

Curvature of mouse satellite DNA and condensation of heterochromatin.

Cloned, sequenced mouse satellite DNA exhibits properties characteristic of molecules that possess a stable curvature. Circularly permuted fragments containing the region predicted to bend were used to map the curvature relative to DNA sequence. The altered mobility of these fragments in polyacrylamide gels is reversed when gels are run in the presence of distamycin A, a drug that binds preferentially to AT-rich DNA. Treatment of living mouse cells with this drug dramatically reduces the condensation of centromeric heterochromatin, the exclusive location of satellite sequences. In situ hybridization of satellite probes to extended chromosomes at the electron microscope level shows that satellite does not comprise a single block but is distributed throughout the centromere region. Based on these experiments, we hypothesize that the structure of mouse satellite DNA is an important feature of centromeric heterochromatin condensation.

Animals↗