DOMAIN INTERACTIONS AND ANTIGEN-BINDING OF RECOMBINANT ANTI-Z-DNA ANTIBODY VARIABLE DOMAINS - THE ROLE OF HEAVY AND LIGHT-CHAINS MEASURED BY SURFACE-PLASMON RESONANCE

The heavy (H) and light(L) chain V domains of anti-Z-DNA mouse mAb Z22 were expressed separately in bacteria. When mixed in vitro, the V dom ains associated stoichiometrically to reconstitute the Ag binding site of Z22, as judged by specific reactivity with Z-DNA and anti-Z22 Id A bs. The apparent K-d of the Z22 V-H-V-L association was 5.47 x 10(-8) M, measured by surface plasmon resonance. A replacement at V-L positio n 96, which reduced Ag binding affinity of a single chain Fv (clone LZ 1-2) by two orders of magnitude, did not reduce the affinity of intera ction between the V-H and V-L domains (apparent K-d = 1.93 X 10(-8) M for V-H association with LZ1-2). Fab prepared from native Z22 bound sp ecifically to a 30-bp Z-DNA oligonucleotide with an apparent K-d = 1.5 6 X 10(-8) M. The V-H domain alone bound Z-DNA specifically with an af finity similar to that of the Fab or Fv's of Z22 (K-d = 1.68 X 10(-8) M), whereas Z22 V-L domain alone did not interact with nucleic acids. Z22 V-H binding to Ag was inhibited by association with the mutant LZ1 -2 V-L. These results indicate that the Z22 H chain makes important co ntributions to specific binding of Z-DNA. Although the L chain does no t add greatly to the binding energy, an appropriate L chain is require d to permit Ag binding in the Fv domain. These in vitro results resemb le the in vivo modulation of H chain autoreactivity that occurs with L chain substitution in receptor editing.