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
M. Polymenis et Bd. Stollar, 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, The Journal of immunology, 152(11), 1994, pp. 5318-5329
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.