Molecular characterization of cold agglutinins

An unusual bias involving the exclusive usage of the V4-34 gene segment by pathogenic antibodies with I/i antigen specificity has been documented in the literature. In addition, all unmutated and several mutated V4-34 encoded antibodies have been shown to be reactive with the anti-idiotypic monoclonal antibody 9G4. The 9G4 Id, therefore, is a marker for V4-34 gene segment expression. Based on these two correlations, it became vital to localize and characterize the nature of the 9G4 Id and to determine the relationship between the Id and I binding. Mutational analysis indicated that the 9G4 Id is located in framework region 1 (FR1) of V4-34 encoded antibodies. Two distinct sections of FR1, encompassing amino acid residues 6-12 and 23-25, form the 9G4 Id. Mutational analysis demonstrated that both FR1 and CDRH3 were required for I binding. When either one was disrupted, the mutant antibody could not bind I. This indicates that I binds through a framework region, and not exclusively through CDRH3. This renders the I interaction with the V4-34 encoded portion of immunoglobulins unconventional, with characteristics similar to superantigen binding to immunoglobulin through FR. When the FR1 DNA sequence of V4-34 was exchanged for FR1 sequences from other VH families I binding was lost, providing a structural explanation for this restricted VH usage. An understanding of the localization and structure of the 9G4 Id and the requirements of V4-34 encoded antibodies for I binding provide insights into the structure of pathogenic antibodies and their requirements for binding antigen. This information should be useful in analyzing new interactions such as the lytic activity of some V4-34 encoded antibodies for B cells.