Equilibrium binding studies of recombinant anti-single-stranded DNA Fab. Role of heavy chain complementarity-determining regions.
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.
Komissarov
A A
AA
Department of Biochemistry, University of Missouri School of Medicine, Columbia 65212, USA.
Calcutt
M J
MJ
Marchbank
M T
MT
Peletskaya
E N
EN
Deutsher
S L
SL
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004247:DNA / Q000276:immunology* / Q000302:isolation & purification; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000276:immunology*; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D008024:Ligands; D004247:DNA