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 domains associated stoichiometrically to reconstitute the Ag binding site of Z22, as judged by specific reactivity with Z-DNA and anti-Z22 ld Abs. The apparent Kd of the Z22 VH-VL association was 5.47 x 10(-8) M, measured by surface plasmon resonance. A replacement at VL position 96, which reduced Ag binding affinity of a single chain Fv (clone LZ1-2) by two orders of magnitude, did not reduce the affinity of interaction between the VH and VL domains (apparent Kd = 1.93 x 10(-8) M for VH association with LZ1-2). Fab prepared from native Z22 bound specifically to a 30-bp Z-DNA oligonucleotide with an apparent Kd = 1.56 x 10(-8) M. The VH domain alone bound Z-DNA specifically with an affinity similar to that of the Fab or Fv's of Z22 (Kd = 1.68 x 10(-8) M), whereas Z22 VL domain alone did not interact with nucleic acids. Z22 VH binding to Ag was inhibited by association with the mutant LZ1-2 VL. These results indicate that the Z22 H chain makes important contributions to specific binding of Z-DNA. Although the L chain does not add greatly to the binding energy, an appropriate L chain is required to permit Ag binding in the Fv domain. These in vitro results resemble the in vivo modulation of H chain autoreactivity that occurs with L chain substitution in receptor editing.
Polymenis
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Department of Biochemistry, Tufts University School of Medicine, Boston, MA 02111.
Stollar
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D000595:Amino Acid Sequence; D000818:Animals; D000974:Antibodies, Antinuclear / Q000235:genetics / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000378:metabolism; D000915:Antibody Affinity; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D004247:DNA / Q000276:immunology* / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D000974:Antibodies, Antinuclear; D000911:Antibodies, Monoclonal; D017931:DNA Primers; D018076:DNA, Complementary; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D004247:DNA