Preferential recognition of specific DNA motifs by anti-double-stranded DNA autoantibodies.
Although antibodies (Ab) specific for double-stranded (ds) DNA are thought to be involved in the etiopathogenesis of systemic lupus erythematosus (SLE), the fine structure of their DNA targets remains elusive. We have adapted a polymerase chain reaction (PCR)-assisted immunoprecipitation method to define the binding sites in DNA sequences recognized by high affinity anti-dsDNA Ab of SLE patients. SLE sera were used to bind templates from a pool of double-stranded oligonucleotides (ON). A central part of 20 base-pair random sequence was flanked by restriction endonuclease recognition sites and sequences complementary to predefined PCR primers. Immunoselected ON were precipitated, isolated from the immune complexes and then subjected to a further immunoprecipitation step after amplification by PCR. After five cycles of immunoprecipitation and PCR, the resulting ON were cloned. Sequence analysis revealed that sera from SLE patients and two human monoclonal anti-dsDNA Ab obtained from SLE patients preferentially select sequences expected to form non-B-DNA structures. Inhibition studies of the Farr assay confirmed the increased affinity of the selected epitopes for anti-DNA Ab as compared to random B-DNA.
Herrmann
M
M
Institute of Clinical Immunology and Rheumatology, Department of Medicine III, Erlangen, Germany.
Winkler
T H
TH
Fehr
H
H
Kalden
J R
JR
D000974:Antibodies, Antinuclear / Q000276:immunology*; D000918:Antibody Specificity; D001324:Autoantigens / Q000737:chemistry / Q000276:immunology; D001482:Base Composition; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000276:immunology* / Q000648:ultrastructure; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D008180:Lupus Erythematosus, Systemic / Q000276:immunology*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction / Q000379:methods
D000974:Antibodies, Antinuclear; D001324:Autoantigens; D017931:DNA Primers; D004247:DNA