Saturday, September 7, 2024
9/7/2024
Abstract Antibodies play a pivotal role in the adaptive immune system due to their ability to specifically bind to a diverse array of antigens. There are two structurally diverse forms of antibodies that have arisen throughout evolution: 1) immunoglobulin (Ig)-based antibodies, which are found in gnathostomes (i.e., jawed vertebrates); and 2) variable lymphocyte receptor (VLR)-based antibodies which are found in agnathans (i.e., jawless vertebrates). Gnathostomes generate Ig-based antibodies by two diversification methods. Primary diversification of Ig-based antibodies occurs via V(D)J recombination where the variable antigen-binding exon is assembled from various gene segment options to generate a diversity of binding specificities in developing B lineage cells. Secondary diversification of Ig-based antibodies occurs through somatic hypermutation (SHM) which is mediated by activation induced cytidine deaminase (AID) in activated germinal center B cells (mammals) or B cell-rich lymphoid structures early in life (chickens). Agnathans have an alternative form of leucine rich repeat (LRR)-based antibodies called variable lymphocyte receptors (VLRs). While there are many convergent properties of Ig and VLR antibodies, there has been no evidence of SHM in VLRs despite the existence of a homolog to AID in lamprey, which opens doors to evaluate the impact of Ig- and LRR-based structures in tolerating diversification through an SHM-mediated protein-variation mechanism. Preliminary computational data suggest that the diverse structures of Ig- and LRR-based antibodies may vary in their capacity to maintain structural integrity during hypermutation, a process crucial for the expression of antibody diversity. These results are consistent with a hypothesis that structural stability constraints may play a role in real-time protein mutability potential. This proposal will test that hypothesis that Ig-based antibodies are more mutationally tolerant than LRR-based antibodies, with two specific aims: (i) determine the mutational tolerance of Ig-based and LRR-based antigen receptors in vitro, and (ii) define diversity and maturation of Ig and VLR substrates in vivo. Aim 1 will evaluate the ability for large mutation libraries of sets of three Igs and three VLRs to continue to be expressed and bind antigen. Aim 2 will compare VLR diversification and affinity maturation to a single chain immunoglobulin-based antibody (i.e., VHH) to immunogens in vivo. The expected outcome of this proposal will be to (i) deliver biophysical insights into the aptitude of evolutionarily distinct but functionally convergent adaptive immune receptors for mutation-driven diversification, and (ii) begin to inform optimal substrate design receptive to mutation-library-driven biotechnological evolution of biomedically relevant substrates.
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