| Summary: | <p>Cattle present an extreme immunological model, wherein immunoglobulin sequence diversification is achieved not through recombinational diversity, as in humans and mice, but by great heterogeneity in the length of the complementarity determining region #3 of the heavy chain (CDR3H), and by somatic hypermutation independent of antigenic contact. Bovine CDR3H lengths span 5 - 72 residues, with those containing >48 labelled as ‘ultralong’ and assumed to interact with epitopes typically invisible to paratopes with shorter CDR loops. Cattle also demonstrate preferential combination of V(D)J rearrangements without antigenic stimulus, inferring a structural relationship between bovine immunoglobulins that is not yet understood. This project aims to advance understanding of these relationships from a structural and functional context.</p>
<p>The structures of several chain-exchanged ultralong antibodies were solved by X-ray crystallography, or predicted by AlphaFold, revealing an intimate contact between the CDR3L and the CDR3H. Varying the CDR3L sequence imparts an axial twist about the ultralong stalk domain, with minor accommodating atomic shifts at the distal, antigen-interacting knob domain, together with a subtle change in binding affinity by biolayer interferometry. Electron cryomicroscopy was used to assess whether chain-exchange resulted in any changes to the recognised epitope, but no significant differences were observed at the attained resolutions. Initial efforts with a non-ultralong antibody-antigen system are also described.</p>
<p>These observations imply bovine light chains principally contribute a structural role stabilising ultralong CDR3H, but their contribution in non-ultralong antibodies remains enigmatic. The limited impact of light chain selection on antibody function provides little insight to bovine B cell development. Bioengineers of the ultralong CDR3H may wish to consider the light chain sequence should their targeted epitope lie within deep groves of known geometry, however at present, light chain selection is only a minor consideration in the development of bovine ultralong CDR3H antibodies as diagnostic, therapeutic or research tools.</p>
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