STRUCTURE-FUNCTION STUDIES ON A POLYREACTIVE (NATURAL) AUTOANTIBODY -POLYREACTIVITY IS DEPENDENT ON SOMATICALLY GENERATED SEQUENCES IN THE3RD COMPLEMENTARITY-DETERMINING REGION OF THE ANTIBODY HEAVY-CHAIN

SMI is a previously characterized IgM kappa polyreactive (natural) aut oantibody. The variable regions of the heavy and light chains of SMI a re respectively encoded by a nonmutated V(H)1 gene, designated 51p1, a nd a conserved nonmutated V kappa gene, designated Humkv325. These V g enes seem to be over-represented in the autoimmune and fetal B cell re pertoires, and to be frequently expressed in malignant B cells during certain lymphoid proliferations such as chronic lymphocytic leukemia. Polyreactive natural autoantibodies are thought to rely mainly on the use of such V genes in germ-line configuration. However, this model un derestimates the contribution of the somatically generated heavy chain third complementarity-determining region (HCDR3) to autoantibody spec ificity. We used oligonucleotide site-directed mutagenesis to permute the sequence of the SMI-HCDR3 to generate a family of mutant proteins, each of which differed from the original SMI-IgM kappa by one amino a cid residue. This allowed us to examine the relative contribution of s elected amino acid residues in this region to the binding affinity of SMI against a panel of self-Ags. We found that a single amino acid sub stitution within the HCDR3 could dramatically alter the specificity of this autoantibody. Some substitutions abrogated the reactivity with a ll the tested Ags, whereas others changed the affinity or spectrum of reactivity for certain self-Ags. These results demonstrate that the au toantibody-binding activity of these conserved autoantibody-associated germ-line V genes is dependent upon heavy chain junctional sequences that are generated somatically during Ig gene rearrangement.