Lack of promiscuity in autoantigen-specific H and L chain combinations as revealed by human H and L chain "roulette".
Individual H or L chains from a human autoantibody were used to search for other L or H chains that could form antigen-binding fragments, Fab, with the same specificity. The parent Fab (SP1.2) exhibits high affinity binding for thyroid peroxidase (TPO), a 107-kDa protein that is the major autoantigen in human autoimmune thyroiditis. This autoantibody "roulette," performed by using Ig H and L chain gene libraries expressed in bacteria, increased the frequency of TPO-binding clones in the new libraries. However, the frequency was still much lower than would be the case if promiscuous combinations with a variety of H or L chains were compatible with specific Ag binding. Nucleotide sequence analysis of the H and L chains of the new TPO-binding clones revealed even more restriction. Thus, with the SP1.2 H chain, all 11 new Fab utilized L chains from the same V kappa 1 family germline gene as SP1.2 itself. Similarly, five of six H chains "captured" by the SP1.2 L chain were very closely related to the SP1.2 H chain. However, one totally different H chain was isolated: SP4.6 has a VH region that differs substantially from that of SP1.2. SP4.6 also has a distinct D region, uses a different JH, and, unlike SP1.2, which is an IgG1, belongs to subclass IgG4. The affinities for TPO of SP4.6 (with the different H chain) and SP1.20 (which had the least mutated L chain germline gene) were similar to that of SP1.2 (approximately 10(-10) M). As expected, the SP1.2 and SP1.20 Fab, which have the same H chain and closely related L chains, bound to the same domain on TPO. However, a similar domain on TPO was recognized by both SP4.6 and SP1.2, despite the fact that their V, D, and J regions are quite different. This observation raises the possibility that the L chain is critical in defining epitope specificity, even in the presence of completely different D regions and nonidentical VH regions.
Portolano
S
S
Thyroid Molecular Biology Unit, Veterans' Administration Medical Center, San Francisco, CA 94121.
Chazenbalk
G D
GD
Hutchison
J S
JS
McLachlan
S M
SM
Rapoport
B
B
D000595:Amino Acid Sequence; D000915:Antibody Affinity; D001323:Autoantibodies / Q000235:genetics / Q000276:immunology*; D001324:Autoantigens / Q000276:immunology*; D001483:Base Sequence; D015723:Gene Library; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology*; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000276:immunology; D007453:Iodide Peroxidase / Q000276:immunology*; D008969:Molecular Sequence Data
D001323:Autoantibodies; D001324:Autoantigens; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D007453:Iodide Peroxidase