Antibody-nucleic acid complexes. Conformational and base specificities associated with spontaneously occurring poly- and monoclonal anti-DNA antibodies from autoimmune mice

An enzyme-linked immunosorbent assay (ELISA) was developed to characterize spontaneously occurring, mono-and polyclonal anti-DNA antibodies. The assay consists of adsorbing single- (ss) and double-stranded (ds) DNA and various nucleoside-bovine serum albumin conjugates (e.g., A-, G-BSA, etc.) to microtiter wells and assesses the ability of various antibodies to bind to these immobilized antigens. The conformational and base specificity of two monoclonal antibodies (designated MRss-1 BWds-3) was examined in this manner. The exclusive binding of MRss-1 to ssDNA and guanosine-BSA (G-BSA) confirms our previous findings [Munns, T.W., Liszewski, M.K., Tellam, J.T., Ebling, F. M., & Hahn, B.H. (1982) Biochemistry 21,2929-2936] that this antibody recognizes single-stranded nucleic acids by virtue of their guanine content. The extensive binding of BWds-3 to dsDNA, its limited binding to ssDNA, and complete absence of binding to nucleoside-BSA antigens implied a double-stranded conformational specificity. Further, competitive studies with naturally occurring and synthetic alternating copolymers indicated that BWds-3 preferentially recognized the native dsDNA antigens. ELISA analysis of the spontaneously occurring, polyclonal anti-DNA antibodies from MRL/lpr and NZB/NZW-F1 mice revealed that the majority of anti-ssDNA antibodies bound to nucleoside-BSA conjugates. Anti-G antibodies were most prominent in both strains of mice, yet lesser and more variable quantities of anti-A, -C, -U, and -T antibodies were also detected. Preadsorption of serum with G-BSA/Sepharose resulted in the complete removal of anti-G antibodies and a 60% reduction in anti-ssDNA antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of saffron carotenoids as anticancer compounds with ctDNA, Oligo (dG.dC)15, and Oligo (dA.dT)15

Crocin and crocetin are two important natural saffron carotenoids, which, along with dimethylcrocetin (DMC) as a semi-synthetic product, are responsible for its color. Many biological properties of saffron have been reported, among which the anticancer property is the most important. Some anticancer drugs have direct interaction with DNA, and thus the present study attempted to investigate the interaction of three major saffron carotenoids-crocin, crocetin, and DMC--with calf thymus DNA (ctDNA) and oligonucleotides. The spectrophotometric data showed some changes in ctDNA absorption spectra due to the formation of complex with saffron extract and each of these three components. Also, all the three components caused the quenching of the fluorescence emission of ctDNA-ethidium bromide complex. The Scatchard analysis of these data indicated a noncompetitive manner for quenching, which is accompanied by the outside groove-binding pattern. The circular dichroism (CD) spectra also indicated the nonintercalative binding and induction of the conformational changes, and B to C transition in ctDNA structure and then unstacking of ctDNA bases at higher concentrations of the carotenoids. The CD spectra of G.C and A.T oligonucleotides after addition of these carotenoids indicated the transition from B- to C-DNA, which is very similar to the ctDNA spectral changes. The DeltaG(H(2)O), the best parameter for the estimation of macromolecule stability, was determined for ctDNA denaturation using dodecyl trimethylammonium bromide in the absence and presence of crocin, crocetin, or DMC. Our results showed a decrease in the Delta G(H(2)O), indicating the ctDNA destabilization due to its interaction with the mentioned ligands. In conclusion, the results show that saffron and its carotenoids interact with DNA and induce some conformational changes in it. Of these carotenoids, the order of potential of interaction with DNA is crocetin > DMC > crocin.     

Structure of (dG)n.(dC)n under superhelical stress and acid pH

We have recently shown that a (GA)n.(TC)n tract undergoes a sharp structural transition under superhelical stress (V.I. Lyamichev, S.M. Mirkin and M.D. Frank-Kamenetskii, J. Biomol. Struct. Dyn. 2,327 (1985]. Unlike the well studied transitions to the cruciform and to the Z form, this novel transition was strongly pH-dependent. We have found the (dG)n.(dC)n insert to undergo a pH-dependent structural transition similar to that of the (GA)n.(TC)n tract. These new data meet our earlier expectations and disagree with the data of D.E. Pulleyblank, D.B. Haniford and A.R. Morgan, Cell 42, 271 (1985). We conclude that a novel DNA structure (the H-form) is typical of homopurine-homopyrimidine mirror repeats (the H palindromes) under superhelical stress and/or acid pH. In the H-form the homopyrimidine strand forms a hairpin while half of the homopurine strand interacts with the hairpin forming a triplex, the other half of the homopurine strand being unstructured (V.I. Lyamichev, S.M. Mirkin and M.D. Frank-Kamenetskii, J. Biomol. Struct. Dyn. 2, 3, 667 (1986].     

Magnesium-dependent supercoiling-induced transition in (dG)n.(dC)n stretches and formation of a new G-structure by (dG)n strand.

Plasmids containing (dG)27.(dC)27 inserts (pPG27), (dG)37.(dC)37 inserts (pPG37), and (dG)24C(dG)21.(dC)24G(dC)21 inserts (pPG46C) were constructed for the study of structural transitions within (dG)n.(dC)n stretches. Two-dimensional gel electrophoresis has shown that a Mg2+-dependent supercoiling-induced structural transition takes place at pH 8 in plasmid pPG46C. The transition occurs at -0=0.06 and involves a supercoiling release corresponding to 5 superhelical turns. After denaturation of the restriction fragments containing (dG)n.(dC)n inserts, the strands do not renature completely and (dG)n-containing strand migrates in PAGE much faster than the (dC)n-containing one. Chemical modification experiments with the (dG)n-strand have revealed the periodic nature of the protection of guanines against dimethyl sulfate methylation. The (dG)n strand in the presence of Mg2+ forms complexes with the complementary (dC)n strand, which differ from the native duplex in mobility. We believe these effects to be due to the formation of an intrastrand structure within the (dG)n strand stabilized by G.G interactions (we called it G-structure), which in the presence of Mg2+ forms an interstrand complex. with the (dC)n strand.     

Cloning of B cells from autoimmune MRL-lpr/lpr and MRL.xid mice

The relationship between colony formation (cloning) of B cells and their activation in murine autoimmunity was investigated in MRL-lpr/lpr and MRL.xid mice. Cells from MRL-lpr/lpr mice showed similar requirements for in vitro growth as normal CBA/J and BALB/c cells, with maximal colony formation in the presence of the supporting factors lipopolysaccharide and sheep red blood cells. The frequency of colony-forming cells from MRL-lpr/lpr spleens or hapten-specific B-cell preparations was slightly greater than the two normal control strains, with this difference significant only for a comparison of BALB/c and MRL-lpr/lpr spleens. In contrast, MRL-lpr/lpr mice bearing the xid gene for B-cell immunodeficiency (MRL.xid) had markedly reduced B-cell colony formation. These mice nevertheless expressed anti-DNA antibodies, although at levels reduced from that of MRL-lpr/lpr controls. These results indicate that enhanced in vitro colony formation need not accompany B-cell hyperactivity in murine autoimmune disease and that autoantibody production can occur in mice with impairment in this growth property.     

Endonuclease G: a (dG)n X (dC)n-specific DNase from higher eukaryotes.

An endonuclease activity (termed endonuclease G) that selectively cleaves DNA at (dG)n X (dC)n tracts has been partially purified from immature chicken erythrocyte nuclei. Sites where n greater than or equal to 9 are cleaved in a manner that resembles types II and III restriction nucleases. The nicking rate of the G-strand is 4- to 10-fold higher than that of the C-strand depending on the length of the (dG)n X (dC)n tract and/or nucleotide composition of the flanking sequences. Endonuclease G hydrolyzes (dG)24 X (dC)24 of supercoiled DNA in a bimodal way every 9-11 nucleotides, the maxima in one strand corresponding to minima in the opposite, suggesting that it binds preferentially to one side of the double helix. The nuclease produces 5' phosphomonoester ends and its activity is dependent on Mg2+ or Mn2+. The wide distribution and high relative activity of endonuclease G in a variety of tissues and species argues for a general role of the enzyme. The striking correlation between genetic instability and poly(dG) X poly(dC) tracts in DNA suggests that these sequences and endonuclease G are involved in recombination processes.     

Messenger ribonucleoprotein complexes isolated with oligo(dT)-cellulose chromatography from kidney polysomes

As an initial step towards understanding the role of mRNP complexes in translational regulation during compensatory renal hypertrophy, characteristics of polysome-associated mRNP isolated by affinity chromatography were studied. Renal mRNP contained 15–30% of the counts after a 1 hr pulse with ³H-orotic acid; it sedimented mainly between 10S and 100S and had a buoyant density of 1.42–1.44 g/cm³. RNA derived from the mRNP sedimented between 5S and 40S on sucrose density gradients, with the greatest radioactivity in the region of 15S. After labeling with ³H-adenine for 1 hr, up to 17% of the radioactivity present in the mRNP-associated RNA was resistant to digestion by pancreatic and T1 ribonucleases. The mRNP protein moiety contained six polypeptides with molecular weights 69,000, 75,000, 80,000, 100,000, 109,000, and 118,000 daltons, which were undetected in the material not binding to oligo(dT)-cellulose.     

Recurrent utilization of genetic elements in V regions of antinucleic acid antibodies from autoimmune mice.

Two IgG anti-DNA and two IgG anti-RNA autoantibodies derived from lupus prone NZB/NZW F1 mice have been analyzed for their Ag fine specificities and for their H and L chain V-region sequences. A remarkable similarity of VH gene sequences with previously sequenced antinucleic acid autoantibodies (Eilat, D., D. M. Webster and A. R. Rees. J. Immunol. 141:1745, 1988) was noted. This finding indicates that a small number of unique VH genes is involved in this autoimmune response and that the sequences of these genes are correlated with the different specificities for the autoantigen. The VK sequences appeared, by computer search, to be selected nonrandomly, but their use was not restricted to autoantibodies. An additional striking feature was evident in the construction of the D region elements, giving rise to CDR3 peptides that can interact with DNA and RNA. These constructs probably include D-D fusion products, which are relatively rare in Ig rearrangements.     

Development of the B cell anti-DNA repertoire in (NZB x NZW)F1 mice. Relationship with the natural autoimmune repertoire.

The relationship between pathologic anti-DNA and natural autoantibodies (Auto Ab) remains unclear. In particular, it has not yet been elucidated whether pathologic anti-DNA antibodies originate from and are regulated by the pool of natural Auto Ab. To address this question, a large number of Ig-secreting hybridomas were derived from the unstimulated splenocytes of B/W mice, newborn to 12 mo of age, and their binding activities against a panel of self-Ag (DNA, actin, tubulin, myosin, and myoglobin), isotype, idiotypic determinants, and VH gene utilization were analyzed. A progressive increase in the number of Ig-secreting clones was observed and associated with a constant proportion (approximately 6%) of autoreactive B cell clones. However, dramatic changes in the pool of autoreactive B cell hybridomas were observed as the disease evolved, including the selective maintenance of IgM anti-DNA polyspecific antibodies, reduction in percentage of polyspecific IgM mAb with no DNA-binding activity, and the production of IgG anti-DNA antibodies of the IgG2 class. The kinetics, immunochemical properties, and idiotypic analysis of polyspecific IgM mAb with DNA-binding activity strongly suggest that they belong to natural Auto Ab and constitute the precursors of pathologic IgG anti-DNA antibodies. In addition, and IgM polyspecific antibody was demonstrated to bind IgG anti-DNA mAb through F(ab')2 interactions suggesting a regulatory role of natural antibodies and their participation in the control of pathologic Auto Ab production.     

Lymphocytes binding polyriboadenylic acid and synthesizing antibodies to nucleic acids in autoimmune and normal mice.

Splenic lymphocytes from both normal and autoimmune mice bind significant quantities of polyriboadenylic acid (poly rA) when incubated with radiolabeled poly rA for 40 min at 37 degrees C. This poly rA binding is specifically inhibited by an excess of nonradioactive poly rA and by anti-mouse immunoglobulin. Poly rA binding is decreased by exposing spleen cells to Pronase and is restored by subsequent culture for 18 to 72 hr. Poly rA-binding activity is associated more with bone marrow-derived than with thymus-derived lymphocytes. These results suggest the presence of autoantigen-binding lymphocytes in normal as well as autoimmune mice. Furthermore, spleen cells from normal and autoimmune mice cultured for 72 hr synthesize and secrete antibodies to poly rA and DNA. These antibodies can be recovered from the culture supernatants by a solid immunoadsorbent technique and antibody immunoprecipitation. The synthesis of antibodies to nucleic acids by normal spleen cells suggests that autoreactive lymphocytes may be released from normal immunoregulatory control during in vitro culture conditions.     

Comparative study of the interaction of meso-tetrakis (N-para-trimethyl-anilium) porphyrin (TMAP) in its free base and Fe derivative form with oligo(dA.dT)15 and oligo(dG.dC)15

Interaction between a cationic porphyrin and its ferric derivative with oligo(dA.dT)₁₅ and oligo(dG.dC)₁₅ was studied by UV–vis spectroscopy, resonance light scattering (RLS), and circular dichroism (CD) at different ionic strengths; molecular docking and molecular dynamics simulation were also used for completion. Followings are the observed changes in the spectral properties of meso-tetrakis (N-para-trimethyl-anilium) porphyrin (TMAP), as a free-base porphyrin with no axial ligand, and its Fe derivative (FeTMAP) upon interaction with oligo(dA.dT)₁₅ and oligo(dG.dC)₁₅: (1) the substantial red shift and hypochromicity at the Soret maximum in the UV–vis spectra; (2) the increased RLS intensity by increasing the ionic strength; and (3) an intense bisignate excitonic CD signal. All of them are the reasons for TMAP and FeTMAP binding to oligo(dA.dT)₁₅ and oligo(dG.dC)₁₅ with the outside binding mode, accompanied by the self-stacking of the ligands along the oligonucleotide helix. The CD results demonstrated a drastic change from excitonic in monomeric behavior at higher ionic strengths, which indicates the groove binding of the ligands with oligonucleotides. Molecular docking also confirmed the groove binding mode of the ligands and estimated the binding constants and energies of the interactions. Their interaction trend was further confirmed by molecular dynamics technique and structure parameters obtained from simulation. It showed that TMAP reduced the number of intermolecular hydrogen bonds and increased the solvent accessible surface area in the oligonucleotide. The self-aggregation of ligands at lower concentrations was also confirmed.     

Differential stabilization by netropsin of inducible B-like conformations in deoxyribo-, ribo- and 2'-deoxy-2'-fluororibo-adenosine containing duplexes of (dA)n . (dT)n and (dA)n . (dU)na.

Six polynucleotide duplexes containing polydeoxyadenylic acid, polyadenylic acid or poly-2'-deoxy-2'-fluoro-adenylic acid in one strand, and polydeoxyuridylic acid or polydeoxythymidylic acid in the other strand have been studied by circular dichroism, ionic strength dependence of melting temperatures and binding of the DNA specific antibiotic netropsin. Circular dichroism spectra of (dA)n . (dT)n and (dA)n . (dU)n indicated the presence of the B-form of DNA, while those of (dAfl)n . (dT)n and (rA)n . (dT)n (and the corresponding (dU)n hybrids) indicated the presence of the A-form. (dAfl)n . (dT)n and (dAfl)n . (dU)n bound netropsin only slightly less than the (dA)n containing duplexes, while replacement by (rA)n decreased netropsin binding to a large degree. Since netropsin requires B-DNA for binding, it is concluded that the A to B transition is facilitated in the case of fluorine substitution in the sugar moiety, while the 2'-OH group greatly limits this conformational change.     

The centromeric region of chromosome 7 from MRL mice (Lmb3) is an epistatic modifier of Fas for autoimmune disease expression

Lupus is a prototypic systemic autoimmune disease that has a significant genetic component in its etiology. Several genome-wide screens have identified multiple loci that contribute to disease susceptibility in lupus-prone mice, including the Fas-deficient MRL/Fas(lpr) strain, with each locus contributing in a threshold liability manner. The centromeric region of chromosome 7 was identified as a lupus susceptibility locus in MRL/Fas(lpr) mice as Lmb3. This locus was backcrossed onto the resistant C57BL/6 (B6) background, in the presence or absence of Fas, resulting in the generation of B6.MRLc7 congenic animals. Detailed analysis of these animals showed that Lmb3 enhances and accelerates several characteristics of lupus, including autoantibody production, kidney disease, and T cell activation, as well as accumulation of CD4(-)CD8(-) double-negative T cells, the latter a feature of Fas-deficient mice. These effects appeared to be dependent on the interaction between Lmb3 and Fas deficiency, as Lmb3 on the B6/+(Fas-lpr) background did not augment any of the lupus traits measured. These findings confirm the role of Lmb3 in lupus susceptibility, as a modifier of Fas(lpr) phenotype, and illustrate the importance of epistatic interaction between genetic loci in the etiology of lupus. Furthermore, they suggest that the genetic lesion(s) in MRLc7 is probably different from those in NZMc7 (Sle3/5), despite a significant overlap of these two intervals.     

A poly(dT)-stimulated ATPase activity associated with simian virus 40 large T antigen.

Highly purified SV40 large T antigen exhibits an ATPase activity which can be stimulated approximately 7-fold by the DNA homopolymer poly(dT). The poly(dT)-stimulated enzyme can hydrolyze various ribonucleotide and deoxyribonucleotide triphosphates, with ATP and dATP serving as the best substrates. Purified large T antigen hydrolyzes ATP to ADP and Pi, with a maximum specific activity of 13.5 mumol of inorganic phosphate released per h per mg of protein. Of the various natural and synthetic polynucleotides tested, poly(dT) was by far the best activator. Long chain poly(dT) molecules are much more effective activators than are short chain length oligo(dT) molecules. The highly purified large T antigen contains no detectable protein kinase activity.     

Human and murine anti-DNA antibodies induce the production of anti-idiotypic antibodies with autoantigen-binding properties (epibodies) through immune-network interactions

To examine the potential role of immune-network interactions in the production of lupus autoantibodies, normal NZW rabbit antibody responses were analyzed after immunization with one of the following Ig preparations: human lupus serum anti-dsDNA antibodies, human lupus serum anti-ssDNA antibodies, a mixture of human lupus serum anti-dsDNA and anti-ssDNA antibodies, the MRL-lpr/lpr anti-dsDNA mAb H241, and the MRL-lpr/lpr anti-ssDNA mAb H130. Four of five rabbits produced Ig typical of lupus autoantibodies: individual rabbit Ig cross-reacted with multiple autoantigens including nucleic acids, cardiolipin, SmRNP, glomerular extract, laminin, and exogenous Ag. Rabbit anti-Id against human anti-dsDNA antibodies were highly specific for dsDNA. Notably, in each serum the autoantibody activity was confined to the anti-Id Ig fraction. A similar spontaneously occurring Id-anti-Id interaction was also found between anti-ssDNA and anti-dsDNA antibodies isolated from an individual lupus patient. These results indicate that lupus autoantibodies which share Ag binding properties with pathogenic Ig, including both cross-reactive and anti-dsDNA antibodies, can induce the production of Ig with similar autoantigen binding properties through immune-network interactions. This phenomenon, if unregulated, could lead to the amplification of pathogenic autoantibody production in individuals with systemic lupus.     

Interclonal and intraclonal diversity among anti-DNA antibodies from an (NZB x NZW)F1 mouse

The immunologic basis for the generation of autoantibodies that are characteristic of systemic autoimmunity in mice and humans remains obscure. Experiments directed toward the analysis of serum antibody and the cell populations that combine to generate antibody in autoimmune mice have led to the proposition that autoantibody production, including anti-DNA, results from the nonselective, polyclonal activation of B cells. The present results from the molecular analyses of anti-DNA autoantibodies from an individual (NZB x NZW)F1 autoimmune mouse, however, are inconsistent with a clonally nonselective model for autoantibody production and are most consistent with a clonally selective, Ag-driven model for anti-DNA autoantibody production. These results demonstrate that Ig V region structures contributed by germ-line V region genes; recombinational diversity, including unusual DH gene usage and DH-DH recombination; and somatic mutation during B cell clonal expansion are all important for generating antibody and presumably B cell Ig receptor specificity for nucleic acids including native, duplex DNA.