The I binding specificity of human VH 4-34 (VH 4-21) encoded antibodies is determined by both VH framework region 1 and complementarity determining region 3.
Essentially all cold agglutinins (CA) with red blood cell I/i specificity isolated from patients with CA disease stemming from lymphoproliferative disorders utilize the VH 4-34 (VH 4-21) gene segment. This near universality of the restricted use of a single gene segment is substantially greater than that demonstrated for other autoantibodies. The monoclonal antibody 9G4 exclusively binds VH 4-34 encoded antibodies and serves as a marker for the VH 4-34 gene segment. Previous studies form our laboratory localized the 9G4 reactive area to framework region 1 (FR1). In the present study, the relative roles of VH FR1, heavy (H) chain complementarity determining region 3 (CDRH 3) and the light (L) chain in I antigen binding were investigated. Mutants containing FR1 sequences from the other VH families, CDRH 3 exchanges, and combinatorial antibodies involving L chain interchanges were produced in the baculovirus system and tested in an I binding assay. The data indicate that FR1 of the VH 4-34 gene segment and the CDRH 3 are essential for the interaction between CA and the I antigen, with the CDRH 3 being fundamental in determining the fine specificity of antigen binding (I versus i). Mutants with substantially altered CDRH 1 and CDRH 2 regions bind I as long as the FR1 is VH 4-34 encoded and the CDRH 3 has a permissive sequence. Light chain swaps indicate that even though antigen binding is predominantly mediated by the H chain, the association with antigen can be abrogated by an incompatible L chain. The necessity for VH 4-34 FR1 explains the almost exclusive use of the VH 4-34 gene segment in cold agglutinins. We hypothesize that, as a general phenomenon, the H chain FR1 of many antibodies may be important in providing the contact required for the close association of antibody with antigen, while the CDRH 3 dictates the fine specificity and strenght of binding.
Li
Y
Y
Department of Microbiology, The University of Texas, Southwestern Medical Center, Dallas, 75235, USA.
Spellerberg
M B
MB
Stevenson
F K
FK
Capra
J D
JD
Potter
K N
KN
D000373:Agglutinins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000744:Anemia, Hemolytic, Autoimmune / Q000276:immunology; D000918:Antibody Specificity; D001323:Autoantibodies / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001666:Binding Sites, Antibody / Q000235:genetics; D002240:Carbohydrate Sequence; D003450:Cryoglobulins; D005803:Genes, Immunoglobulin / Q000235:genetics*; D006028:Glycosphingolipids / Q000378:metabolism*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation
D000373:Agglutinins; D001323:Autoantibodies; D003450:Cryoglobulins; D006028:Glycosphingolipids; C078965:I-antigen; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; C029443:cold agglutinins