Structure-function studies on a polyreactive (natural) autoantibody. Polyreactivity is dependent on somatically generated sequences in the third complementarity-determining region of the antibody heavy chain.
SMI is a previously characterized IgM kappa polyreactive (natural) autoantibody. The variable regions of the heavy and light chains of SMI are respectively encoded by a nonmutated VH1 gene, designated 51p1, and a conserved nonmutated V kappa gene, designated Humkv325. These V genes seem to be over-represented in the autoimmune and fetal B cell repertoires, 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 underestimates the contribution of the somatically generated heavy chain third complementarity-determining region (HCDR3) to autoantibody specificity. 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 acid residue. This allowed us to examine the relative contribution of selected 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 substitution within the HCDR3 could dramatically alter the specificity of this autoantibody. Some substitutions abrogated the reactivity with all the tested Ags, whereas others changed the affinity or spectrum of reactivity for certain self-Ags. These results demonstrate that the autoantibody-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.
Martin
T
T
Laboratory of Immunopathology, Central Hospital, University Hospitals of Strasbourg, France.
Crouzier
R
R
Weber
J C
JC
Kipps
T J
TJ
Pasquali
J L
JL
D000818:Animals; D001323:Autoantibodies / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain; D005803:Genes, Immunoglobulin; D006801:Humans; D007113:Immunity, Innate; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007075:Immunoglobulin M / Q000235:genetics / Q000378:metabolism; D007145:Immunoglobulin kappa-Chains / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000235:genetics / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship
D001323:Autoantibodies; D017931:DNA Primers; D018076:DNA, Complementary; D007143:Immunoglobulin Heavy Chains; D007075:Immunoglobulin M; D007145:Immunoglobulin kappa-Chains