Epitopes described in "Equilibrium binding studies of recombinant anti-single-stranded DNA Fab. Role of heavy chain complementarity-determining regions."

Article Authors:A A Komissarov; M J Calcutt; M T Marchbank; E N Peletskaya; S L Deutsher
Article Title:Equilibrium binding studies of recombinant anti-single-stranded DNA Fab. Role of heavy chain complementarity-determining regions.
Reference Detail
Reference ID:1012487
Abstract:We previously isolated nucleic acid-binding antibody fragments (Fab) from bacteriophage display libraries representing the immunoglobulin repertoire of automimune mice to expedite the analysis of antibody-DNA recognition. In the present study, the binding properties of one such anti-DNA Fab, high affinity single-stranded (ss) DNA-binding Fab (DNA-1), were defined using equilibrium gel filtration and fluorescence titration. Results demonstrated that DNA-1 had a marked preference for oligo(dT) (100 nM dissociation constant) and required oligo(dT) >5 nucleotides in length. A detailed analysis of the involvement of the individual heavy chain (H) complementarity-determining regions (CDR) ensued using previously constructed HCDR transplantation mutants between DNA-1 and low affinity ssDNA-binding Fab (D5), a Fab that binds poorly to DNA (Calcutt, M. J. Komissarov, A. A., Marchbank, M. T., and Deutscher, S. L. (1996) Gene (Amst.) 168, 9-14). Circular dichroism studies indicated that the wild type and mutant Fab studied were of similar overall secondary structure and may contain similar combining site shapes. The conversion of D5 to a high affinity oligo(dT)-binding Fab occurred only in the presence of DNA-1 HCDR3. Results with site-specific mutants in HCDR1 further suggested a role of residue 33 in interaction with nucleic acid. The results of these studies are compared with previously published data on DNA-antibody recognition.
Affiliations:Department of Biochemistry, University of Missouri School of Medicine, Columbia 65212, USA.
Reference Type:Literature
PubMed ID:8647821
Journal:J Biol Chem
Journal Volume:271
Article Pages:12241-6
Journal ISSN:0021-9258
Article Chemical List:Immunoglobulin Fab Fragments;Immunoglobulin Variable Region;Ligands;DNA
Article MeSH List:Amino Acid Sequence; Animals; Binding Sites; DNA(immunology; isolation & purification ); Immunoglobulin Fab Fragments(metabolism ); Immunoglobulin Variable Region(immunology ); Ligands; Mice; Molecular Sequence Data; Protein Binding; Spectrometry, Fluorescence
Curation Last Updated:2016-04-11 20:57:41