Antibodies that are specific for a single amino acid interchange in a protein epitope use structurally distinct variable regions.

Abstract

SummaryWe have analyzed how the immune system generates antibodies that are specific for analogues of an epitope on the influenza virus hemagglutinin (HA) that differ solely by the presence of Asp or Gly at amino acid 225 . Most antibodies induced in response to HA(Asp225) use one of a few closely related variable (V) region structures that are encoded by characteristic VH/Vk gene segment combinations. Remarkably, none of these VH/Vk combinations was induced in response to HA(Gly225) . Instead of modifying the HA(Asp225)-specific V regions byjunctional variation or somatic mutation to recognize the altered epitope, new VH/Vk combinations were used. The expression of unique VH/Vk combinations appears to confer exquisite specificity to the selection of HAspecific B cells from the pre-immune repertoire.n both B and T lymphocytes, specific recognition is mediated through clonally distributed heterodimeric receptors. For each type of receptor, two polypeptide chains are generated by somatic rearrangement of separate V, J, and in the case of VH and VA D gene segments (reviewed in reference 1). Crystallographic analyses of antibody molecules have demonstrated that the regions of greatest sequence variability, called complementarity-determining regions (CDR), form loops (2, 3); for each polypeptide, CDR1 and CDR2 are encoded by the V gene segment, whereas the junctionally encoded CDR3 is somatically generated during V (D) J gene assembly (1,4,5). The spatial arrangement of the amino acid residues in these loops establishes the specificity of antibody molecules for protein molecules.Previous studies have clearly demonstrated that somatic alterations of V region sequences byjunctional variation and somatic mutation can play an important role in determining the specificity of antibody molecules (6-8) . For example, somatic alterations of antibodies that recognize the influenza virus A/PR/8/34 hemagglutinin (PR8 HA)' can influence their interaction with the HA, as measured by their ability to crossreact with mutant viruses that possess different amino acid substitutions in the antibody combining site (9-14) . Somatic alterations of V region sequences can also change the specificity of antibody molecules such that they recognize different antigens. An example of this is the somatic mutation of a phosphocholine-specific antibody to acquire specificity for DNA (15). Similarly, somatic mutation of V region se-I Abbreviations used in this paper. HA, hemagglutinin ; HAU, hemagglutinating units . quences can alter antibody specificity for analogues of the hapten arsonate (16) . However, the extent to which somatic alterations of V region sequences can give rise to antibodies that are specific for different protein antigens has been unclear. This question is of particular interest in view of the extensive interactions that take place in the combining site between antibodies and protein epitopes. Analyses of complexes formed between antibodies and protein antigens have demonstrated interactions between 15 and 22 amino acids on both ...

Specificity-Based Negative Selection of Autoreactive B Cells during Memory Formation

“…We previously showed that the majority of the Ab response to the T3 HA in BALB/c mice cross-reacts with the PR8 HA, while ϳ15% is directed to epitopes that include aa 225, uses structurally distinct variable regions, and does not cross-react with the PR8 HA (38). To examine the effects of the neo self-PR8 HA on B cell repertoire formation, we immunized HACII, HA104, and BALB/c mice with T3 virus and determined the frequency of B cells that can react with T3 and PR8 viruses.…”

Functional Consequences of the Developmental Arrest and Follicular Exclusion of Anti-Double-Stranded DNA B Cells

“…The H-and L-chain V regions of the hybridoma Abs were sequenced from mRNA according to the protocol described (23). Briefly, cytoplasmic RNA was isolated and constant region-specific primers were used to direct synthesis of cDNA copies of the H-(C1) and L-(C1 and C) chain V regions.…”

“…Briefly, cytoplasmic RNA was isolated and constant region-specific primers were used to direct synthesis of cDNA copies of the H-(C1) and L-(C1 and C) chain V regions. The cDNA was then amplified using the constant region primers in conjunction with VH5Ј1 for H-chains and 1L or L5 primers for Lchains (23). Amplification products were sequenced by automated analysis (The Wistar Institute Nucleic Acid Facility).…”

Analysis of idiotope structure of ovarian cancer antibodies: recognition of the same epitope by two monoclonal antibodies differing mainly in their heavy chain variable sequences

“…For example, antibodies against a(I -•6) dextrans use difTerent VH and VL genes to form combining sites specific for the inlernal linear sequence [27], Diverse V,i and V] genes can be used to produce antibodies reactive with a defined protein epitope [28]. Also, two structurally different antibodies have been characterized that are specific for an epitope of the infiuenza virus haemagglutinin protein different for only one amino acid [29], Although the epitope in ihis case was very similar, the antibodies were shown to lie structurally very different.…”