CRITICAL BINDING-SITE AMINO-ACIDS OF ANTI-Z-DNA SINGLE-CHAIN FV MOLECULES - ROLE OF HEAVY AND LIGHT-CHAIN CDR3 AND RELATIONSHIP TO AUTOANTIBODY ACTIVITY

Bacterial expression of single chain variable fragment (scFv) domains was used to assess Ag-binding contributions of specific regions and re sidues in a mouse mAb to Z-DNA (AbZ22). A variant scFv (Z3-3) that did not bind Z-DNA had the Z22 light chain but differed from the Z22 heav y chain at four complimentarity determining region 3 (CDR3), one FR4 a nd five V-H segment residues. Gene segment swapping and site-directed mutagenesis indicated that the major contribution of the Z22 heavy cha in is its CDR3. A scFv with the CDR3H-FR4H of Alo Z22 and the V-H segm ent of Z3-3 had the same selective high affinity Z-DNA binding as Z22. Some Z-DNA binding was retained even when the CDR3H-FR4H of Ab Z22 wa s combined with a V-H segment that shared only 44% sequence identity w ith Z22. Directed mutations indicated further that residues N99 and S9 8 in heavy chain CDR3 and F96 in light chain CDR3 were particularly im portant for Ag binding. Certain substitutions in CDR3H converted the h ighly selective Z22 Fv into a polyreactive Fv with autoantibody-like b inding to B-DNA and denatured DNA. In a graphic molecular model, heavy chain N99 protrudes from the CDR3 loop at the base of the Ag-binding groove, and the light chain F96 is barely exposed on the base oi this groove; the light chain F96 may be important in heavy chain-light chai n association. Autoantibody and immunization-induced Ab to nucleic aci d can be built on a very similar framework and differ by a small numbe r of amino acid CDR3H residues.