RESUMEN
When purified under rigorous conditions, some murine anti-double-stranded-DNA (anti-dsDNA) antibodies actually bind chromatin rather than dsDNA. This suggests that they may actually be antinucleosome antibodies that only appear to bind dsDNA when they are incompletely dissociated from nucleosomes. Experiments in murine models suggest that antibody-nucleosome complexes may play a crucial role in the pathogenesis of glomerulonephritis in systemic lupus erythematosus. Some human monoclonal anti-DNA antibodies are pathogenic when administered to mice with severe combined immunodeficiency (SCID). Our objective was to achieve stable expression of sequence-altered variants of one such antibody, B3, in Chinese hamster ovary (CHO) cells. Purified antibodies secreted by these cells were tested to investigate whether B3 is actually an antinucleosome antibody. The pathogenic effects of the antibodies were tested by implanting CHO cells secreting them into SCID mice. Purified B3 does not bind to dsDNA unless supernatant from cultured cells is added, but does bind to nucleosomes. The strength of binding to dsDNA and nucleosomes is dependent on the sequence of the light chain. Mice that received CHO cells secreting wild-type B3 developed more proteinuria and died earlier than control mice that received nonsecreting CHO cells or mice that received B3 with a single light chain mutation. However, none of the mice had histological changes or deposition of human immunoglobulin G in the kidneys. Sequence changes may alter the pathogenicity of B3, but further studies using different techniques are needed to investigate this possibility.
Asunto(s)
Anticuerpos Antinucleares/inmunología , Nucleosomas/inmunología , Proteinuria/etiología , Animales , Anticuerpos Antinucleares/biosíntesis , Anticuerpos Antinucleares/genética , Especificidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Células CHO/inmunología , Células CHO/trasplante , Línea Celular , Cricetinae , Cricetulus , Medios de Cultivo Condicionados/farmacología , ADN/inmunología , Modelos Animales de Enfermedad , Femenino , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/inmunología , Riñón/inmunología , Riñón/patología , Nefritis Lúpica/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Proteinuria/inmunología , Proteinuria/patología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , TransfecciónRESUMEN
Autoantibodies to a wide variety of antigens are associated with systemic lupus erythematosus (SLE). Antibodies to double-stranded DNA (anti-dsDNA) are thought to be particularly closely related to tissue damage and disease activity in SLE. Autoantibodies to histones, Sm and Ro are found in patients with SLE, but their role in pathogenesis is unclear. Using a transient expression system, we previously showed that particular sequence motifs in CDRs of light chains derived from the human Vlambda gene 2a2 are very important in determining their ability to form a DNA-binding site, when paired with the heavy chain of the human monoclonal anti-dsDNA antibody B3. These motifs are often sites of somatic mutation and/or contain arginine residues. In the experiments reported in this paper, the same expression system was used to show that these CDR motifs also affect binding to histones, Ro antigen and Sm antigen, but that binding to different antigens is affected in diverse ways by particular changes in the sequence of the CDRs. The heavy chain also plays a role in binding to these antigens. Pairing of the same range of 11 2a2 derived light chains with the heavy chain of a different anti-DNA antibody, 33.H11, gave reduced ability to bind DNA in comparison with the results obtained using the B3 heavy chain. Computer-generated models of the three-dimensional structures of these heavy/light chain combinations were used to define the positions occupied by the important sequence motifs at the binding sites of these antibodies, and to explain the different effects exerted by arginine residues at different positions in the light chains.
Asunto(s)
Anticuerpos Monoclonales/genética , Arginina/genética , Autoantígenos , ADN/inmunología , Histonas/inmunología , ARN Citoplasmático Pequeño , Ribonucleoproteínas Nucleares Pequeñas/inmunología , Ribonucleoproteínas/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Arginina/metabolismo , Sitios de Unión de Anticuerpos/genética , Sitios de Unión de Anticuerpos/inmunología , Células COS , Ensayo de Inmunoadsorción Enzimática , Técnicas de Transferencia de Gen , Humanos , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteínas Nucleares snRNPRESUMEN
INTRODUCTION: Previous studies have suggested the importance of somatic mutations and arginine, asparagine and lysine residues in the complementarity determining regions (CDRs) of antiphospholipid antibodies (aPL) implicated in the pathogenesis of the antiphospholipid antibody syndrome. The relative contributions of the heavy and light chains of aPL in binding to cardiolipin (CL) were assessed by pairing the heavy and light chains of two IgG, beta(2)GPI dependent aPL (IS4 and CL24) with different partner chains from other IgG, beta(2)GPI independent aPL (UK4) and anti-DNA antibodies (B3 and 33H11). METHODS: Four heavy (V(H)) and five light (V(L)) chain variable sequences from three aPL and two anti-DNA antibodies were cloned into expression vectors containing appropriate gamma(1), lambda or kappa constant region cDNA. Paired combinations of heavy and light chain expression plasmids were transfected into COS-7 cells allowing transient expression of whole IgG molecules, which were harvested and tested for the ability to bind CL and DNA by enzyme-linked immunosorbant assay (ELISA). RESULTS: Whole IgG was produced from 19 heavy/light chain combinations. IS4V(H) was dominant in conferring the ability to bind CL with four of the five V(L) tested. The identity of the V(L) region paired with IS4V(H) was important in determining the strength of binding to CL. IS4V(H) contains multiple arginine residues in CDR3, which may have accumulated due to antigen driven selection. It is likely that these arginine residues may interact with CL. The combination B3V(H)/B3V(L) also bound CL, but none of the other 14 combinations showed any binding in this assay. CONCLUSION: Whole IgG molecules capable of binding CL were produced by in vitro expression in COS-7 cells. Arginine residues play important roles in binding to CL and double-stranded DNA. However, different patterns of mutation to arginine are associated with binding to each of these antigens.
Asunto(s)
Anticuerpos Antifosfolípidos/química , Cardiolipinas/metabolismo , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/química , Secuencia de Aminoácidos , Animales , Anticuerpos Antifosfolípidos/metabolismo , Células COS , Regiones Determinantes de Complementariedad , ADN/metabolismo , Ensayo de Inmunoadsorción Enzimática , Glicoproteínas/metabolismo , Humanos , Inmunoglobulina G/análisis , Cadenas Pesadas de Inmunoglobulina/metabolismo , Cadenas Ligeras de Inmunoglobulina/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , beta 2 Glicoproteína IRESUMEN
OBJECTIVE: Previous studies have suggested the importance of somatic mutations and certain residues in the complementarity determining regions (CDRs) of antiphospholipid antibodies (aPL) implicated in the pathogenesis of antiphospholipid antibody syndrome (APS). The authors tested this hypothesis by carrying out a systematic analysis of all published aPL sequences. METHODS: Each aPL variable region sequence was aligned to the closest germline counterpart in the VBASE Sequence Directory by using DNAPLOT software, allowing analysis of nucleotide homology and distribution of somatic mutations. The probability that this distribution arose as a result of antigen-driven accumulation of replacement mutations in the CDRs was tested statistically. RESULTS: There was no preferential gene or family use in the 36 aPL sequences identified. Immunoglobulin (Ig) M aPL had few somatic mutations compared with IgG. Of the IgG aPL, 9 of 14 showed evidence of antigen-driven accumulation of replacement mutations in the CDRs. Multinomial analysis allowed a clearer statistical identification of sequences that had been subject to antigen drive. The more specific IgM aPL and some IgG aPL displayed an accumulation of arginine, asparagine, and lysine residues in CDRs. CONCLUSIONS: High-specificity binding in IgG aPL, but not in more specific IgM aPL, is conferred by antigen-driven somatic mutation. This may in part be caused by an accumulation of arginine, asparagine, and lysine residues in the CDRs, which are germlines encoded in the more specific IgM aPL, but often arise because of somatic mutation in IgG aPL. RELEVANCE: An understanding of the role of arginine, asparagine, and lysine residues in the binding of pathogenic aPL to phospholipids, and to beta(2)-glycoprotein I, may eventually help in the development of drugs to interfere with those interactions, and thereby improve the treatment of antiphospholipid antibody syndrome.