A human anti-insulin IgG autoantibody apparently arises through clonal selection from an insulin-specific "germ-line" natural antibody template. Analysis by V gene segment reassortment and site-directed mutagenesis.
We analyzed the structural correlates underlying the insulin-dependent selection of the specific anti-insulin IgG1 kappa mAb13-producing cell clone, derived from a patient with insulin-dependent diabetes mellitus treated with recombinant human insulin. First, we cloned the germ-line genes that putatively gave rise to the expressed VH and V kappa segments and used them to generate the full (unmutated) "germ-line revertant" of the "wild-type" (somatically mutated) mAb13, using recombinant PCR methods and an in vitro human C gamma 1 and C kappa expression system. The full "germ-line revertant" bound insulin specifically and in a dose-saturable fashion, but with a relative avidity (AVrel) more than three-fold lower than that of its wild-type counterpart (Avrel, 1.69 x 10(-8) vs 4.91 x 10(-9) g/microliters). Second, we established, by reassorting wild-type and germ-line revertant forms of the mAb13 VH and V kappa segments, that the increased Avrel for insulin of mAb13 when compared with its full "germ-line revertant" counterpart was entirely dependent on the mutations in the VH not those in the V kappa chain. Third, we determined, by site-directed mutagenesis experiments, that of the three mutations in the mAb13 VH segment (Ser-->Gly, Ser-->Thr, and Ser-->Arg at positions 31, 56, and 58, respectively), only Arg58 was crucial in increasing the mAb13 Avrel (from 1.44 x 10(-8) to 5.14 x 10(-9) g/microliters) and affinity (Kd, from 189 to 59 nM) for insulin. The affinity enhancement mediated by the VH segment Arg58 residue reflected about a threefold decrease in dissociation rate constant (Koff, from 4.92 x 10(-3) to 1.54 x 10(-3) s-1) but not an increase in association rate constant (Kon, from 2.60 x 10(4) to 2.61 x 10(4) M-1 s-1), and it contrasted with the complete loss of insulin binding resulting from the substitution of the VH segment Asn52 by Lys. The present findings suggest that human insulin, a self Ag, has the potential to recruit a natural autoantibody-producing cell precursor expressing a specific surface receptor for Ag in unmutated configuration, and drive it through affinity maturation. They also show that binding of insulin by such a receptor can be enhanced or completely abrogated by a single amino acid change.
Ichiyoshi
Y
Y
Department of Pathology, Cornell University Medical College, New York, NY 10021.
Zhou
M
M
Casali
P
P
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000916:Antibody Diversity; D000937:Antigen-Antibody Reactions; D001323:Autoantibodies / Q000276:immunology*; D001327:Autoimmune Diseases / Q000188:drug therapy / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D003922:Diabetes Mellitus, Type 1 / Q000188:drug therapy / Q000235:genetics / Q000276:immunology; D015329:Gene Rearrangement, T-Lymphocyte; D006801:Humans; D006825:Hybridomas; D007074:Immunoglobulin G / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D007145:Immunoglobulin kappa-Chains / Q000276:immunology*; D007328:Insulin / Q000276:immunology* / Q000627:therapeutic use; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000627:therapeutic use; D016415:Sequence Alignment; D017385:Sequence Homology; D013698:Templates, Genetic
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D007328:Insulin; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins