Nucleosome‐specific antibody from an autoimmune mrl/mp‐lpr/lpr mouse

Abstract

Objective. To characterize the binding properties and variable-region sequences of LG4-1, a monoclonal antibody from an autoimmune MRL/Mp-lpr/lpr mouse that reacts specifically with nucleosome core particles and represents a new antinuclear antibody specificity.Methods. The reactivity of the antibody against various nuclear substrates was determined using an enzymatic immunoassay, and the variable-region genes were sequenced from messenger RNA, using the dideoxy chain termination method.Results.LG4-1 was found to react with nucleosome core particles but not with individual histones and DNA, or with various histone-histone and histoneDNA complexes. It was demonstrated that this antibody is encoded by a combination of variable-region genes and gene segments that have undergone few somatic mutations.Conclusion. The nucleosome core particle ex- Nucleosomes, the fundamental repeating units of the chromatin fiber, are formed of complexes of histones and DNA (1,2). The nucleosome core particle is composed of a central tetramer of 2 molecules each of histones H3 and H4 flanked by 2 dimers of histones H2A and H2B and surrounded by 2 superhelical turns of approximately 146 basepairs of DNA (2). The full nucleosome contains a molecule of histone H1 located outside of each core particle at the point where DNA enters and exits the nucleosome (1). The core histones (H2A, H2B, H3, and H4) have a heterogeneous distribution of amino acids, with basic residues concentrated in the amino-terminal third of the molecule while the rest of the molecule resembles a globular protein (H2A and H3 also have short, basic carboxyterminal domains). The histones in the core particle interact strongly through their globular domains, and only their unstructured basic regions are accessible to trypsin digestion (3). Thus, protease-digested nucleosomes composed of core histone-limit-peptides (corresponding to the globular domains) and DNA maintain an overall structure similar to that of native nucleosomes (3).Nucleosomes and their subcomponents can also be readily reconstituted in vitro, yielding complexes with structural properties similar to those of their native counterparts (4). For instance, H2A and H2B or H3 and H4 can spontaneously reassociate under physiologic conditions to form H2A-H2B dimers and H3,-H4, tetramers, respectively (5-7). The complete histone octamer will reassociate if the repulsive electrostatic interactions between cationic

An epichromatin epitope: Persistence in the cell cycle and conservation in evolution

“…ELISA data 11,13,14,17,18 demonstrate that the chromatin epitope recognized by PL2-6 is a conformational one, involving a ternary complex of H2A, H2B and DNA. Even though the epitope is present in the binary complex of H2A and H2B, reactivity is augmented by complexing with (mixed sequence) DNA on a surface, as in ELISA plates, or in solution.…”

“…This implies that the epichromatin epitope is present in the conserved globular regions of histones H2A and H2B, which primarily reside within the nucleosome cores. Even though the ELISA reactivity of PL2-6 with uncomplexed H2A or H2B is very weak, 17 it is able to yield ( Fig. 5 and Sup.…”

“…Even though the epitope is present in the binary complex of H2A and H2B, reactivity is augmented by complexing with (mixed sequence) DNA on a surface, as in ELISA plates, or in solution. Another observation (MM unpublished), using a protocol employed with a different antibody, 38 is that trypsin removal of the histone basic tails in nucleosomes does not eliminate the ELISA reactivity of the epitope. This implies that the epichromatin epitope is present in the conserved globular regions of histones H2A and H2B, which primarily reside within the nucleosome cores.…”

“…Most of our current knowledge about the binding specificity of the epichromatin antibody (PL2-6) is derived from ELISA studies. 11,13,14,17,18 We know, based upon ELISA quantitation, that PL2-6 binds strongly to mononucleosomes and to a ternary complex of histones H2A + H2B + DNA, weakly to H2A + H2B and very weakly to H3 + H4 + DNA, individual histones or DNA alone. In the present immunoblotting results with both native Hela and recombinant H2A and H2B.…”

Significance of anti-nuclear and anti-extracellular matrix autoantibodies for albuminuria in murine lupus nephritis; a longitudinal study on plasma and glomerular eluates in MRL/l mice

“…Lately, it was found that the prevalence of antinucleosome and anti-H2A/H2B-DNA subnucleosome reactivity is higher than that of anti-dsDNA and anti-histone reactivity [11,12]. In addition, we [13] and others [14,15] have identified MoAbs derived from lupus mice which were reactive only with the intact nucleosome and not with its components, DNA or histones. A correlation between both plasma anti-H2A/H2B-DNA subnucleosome and anti-nucleosome reactivity with nephritis has been described [11], and recently we showed that anti-nucleosome antibodies complexed to nucleosomal antigens are able to bind to the GBM after renal perfusion in rats [13].…”