RESUMO
Apoptotic cells contain nuclear autoantigens that may initiate a systemic autoimmune response. To explore the mechanism of antibody binding to apoptotic cells, 3H9, a murine autoantibody with dual specificity for phospholipids and DNA, was used. H chain mutants of 3H9 were constructed, expressed as single-chain Fv (scFv) in Escherichia coli, and assessed for binding to phosphatidylserine, an antigen expressed on apoptotic cells. Both 3H9 and its germline revertant bound to dioleoyl phosphatidylserine in ELISA, and binding was enhanced by beta 2 glycoprotein I (beta 2GPI), a plasma protein that selectively binds to apoptotic cells. Higher relative affinity for DOPS-beta 2GPI was achieved by the introduction of Arg residues into the 3H9 H chain variable region at positions previously shown to mediate DNA binding. Specificity of the two structurally most diverse scFv for apoptotic cells was shown by flow cytometry, and two populations of scFv-bound cells were identified by differences in propidium iodide staining. The results suggest that, in autoimmunity, B cells with Ig receptors for apoptotic cells and DNA are positively selected, and that the antibodies they produce have the potential to affect the clearance and processing of apoptotic cells.
Assuntos
Anticorpos Antinucleares/química , Anticorpos Antifosfolipídeos/química , Apoptose/imunologia , Glicoproteínas/imunologia , Fragmentos de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/química , Região Variável de Imunoglobulina/química , Fosfatidilserinas/imunologia , Sequência de Aminoácidos , Anticorpos Antinucleares/genética , Anticorpos Antinucleares/imunologia , Anticorpos Antifosfolipídeos/genética , Anticorpos Antifosfolipídeos/imunologia , DNA/imunologia , Humanos , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Células Jurkat , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , beta 2-Glicoproteína IRESUMO
Antibodies to DNA define an important autospecificity that arises in systemic lupus erythematosus (SLE). To elucidate the molecular features that may explain the pathogenesis of SLE, a heterologous system for expression of cloned V genes is often desirable. Here, a single-chain Fv coding domain was constructed by using the heavy- and light-chain V genes of a high-affinity site-directed mutant of the murine anti-dsDNA autoantibody, 3H9. This scFv was joined in frame to the c-jun leucine zipper for dimerization, and to two affinity tags, domain B of the staphylococcal protein A and a pentahistidine peptide, for purification. Dimerization of the scFv was determined by size-exclusion chromatography. The yields of the scFv following affinity purification on IgG agarose or Ni-NTA agarose were compared, and the activities of the resulting protein fractions were determined. A two-step purification of periplasmic extracts on Ni-NTA agarose and IgG agarose, followed by elution with 3.5 M MgCl(2), yielded scFv with the highest specific activity. The final purified material bound DNA by ELISA, electrophoretic mobility shift assay, and immunofluorescence of fixed Hep-2 cells. Antibodies purified in this fashion should have applications in structure/function studies in which it is essential to generate highly purified antigen-combining sites.
Assuntos
Marcadores de Afinidade/metabolismo , Anticorpos Antinucleares/isolamento & purificação , DNA/imunologia , Imunoglobulina G/química , Região Variável de Imunoglobulina/isolamento & purificação , Animais , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Autoanticorpos , Sequência de Bases , Cromatografia de Afinidade/métodos , Proteínas de Ligação a DNA , Dimerização , Durapatita , Escherichia coli/imunologia , Expressão Gênica , Humanos , Imunoglobulina G/genética , Zíper de Leucina , Camundongos , Níquel , Ácido Nitrilotriacético/análogos & derivados , Compostos Organometálicos , Periplasma , Engenharia de Proteínas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , SefaroseRESUMO
Antibodies to double-stranded DNA (dsDNA) are a defining feature of Systemic Lupus Erythematosus (SLE). The molecular characterization of anti-dsDNA autoantibodies reveals that they are actively selected for binding to antigen. Evidence for antigen selection includes the use of suitable rearrangement products, the switching of IgM isotype to IgG, and the acquisition of somatic mutations that raise the affinity for dsDNA. Through a process of specificity maturation, anti-dsDNA antibodies can arise from anti-single stranded DNA (ssDNA) antibodies that also occur in nonautoimmune individuals. To clarify circumstances leading to the initiation of systemic autoimmunity, we compare features of immune responses to nucleic acids that operate before and after disease develops. Evidence indicating that anti-dsDNA antibodies bind with DNA sequence preference is highlighted to propose that sequence-specific anti-dsDNA antibodies may be induced by an infectious agent and in turn may extend the response to endogenous nuclear antigens. Thus, sequence-specific anti-dsDNA B cells may provide an important stimulus to break the tolerance to self.
