Abstract
Class-I Restricted T Cell-Associated Molecule (CRTAM) is a protein that is expressed after T cell activation. The interaction of CRTAM with its ligand, nectin-like 2 (Necl2), is required for the efficient production of IL-17, IL-22, and IFNγ by murine CD4 T cells, and it plays a role in optimal CD8 T and NK cell cytotoxicity. CRTAM promotes the pro-inflammatory cytokine profile; therefore, it may take part in the immunopathology of autoimmune diseases such as diabetes type 1 or colitis. Thus, antibodies that block the interaction between CRTAM and Necl2 would be useful for controlling the production of these inflammatory cytokines. In this work, using bioinformatics predictions, we identified three short disordered epitopes (sDE1-3) that are located in the Ig-like domains of murine CRTAM and are conserved in mammalian species. We performed a structural analysis by molecular dynamics simulations of sDE1 (QHPALKSSKY, Ig-like V), sDE2 (QRNGEKSVVK, Ig-like C1), and sDE3 (CSTERSKKPPPQI, Ig-like C1). sDE1, which is located within a loop of the contact interface of the heterotypic interaction with Nectl2, undergoes an order–disorder transition. On the contrary, even though sDE2 and sDE3 are flexible and also located within loops, they do not undergo order–disorder transitions. We evaluated the immunogenicity of sDE1 and sDE3 through the expression of these epitopes in chimeric L1 virus-like particles. We confirmed that sDE1 induces polyclonal antibodies that recognize the native folding of CRTAM expressed in activated murine CD4 T cells. In contrast, sDE3 induces polyclonal antibodies that recognize the recombinant protein hCRTAM-Fc, but not the native CRTAM. Thus, in this study, an exposed disordered epitope in the Ig-like V domain of CRTAM was identified as a potential site for therapeutic antibodies.
Highlights
Disordered proteins (IDPs) are molecules that undergo conformational transition; they do not adopt a unique folded structure in their native state because they are highly dynamic and rich in polar residues [1,2,3,4]
Some proteins have well-conformed domains and disordered segments in their structures that are similar to disordered proteins; these sequences are known as intrinsically disordered regions (IDRs) [13]
IDRs in immunogenic linear B-cell epitopes have been identified for the neuraminidase and hemagglutinin proteins of the avian influenza virus H5N1 and H9N2, and they were suggested as candidates to produce a vaccine for human and poultry use [18]
Summary
Disordered proteins (IDPs) are molecules that undergo conformational transition; they do not adopt a unique folded structure in their native state because they are highly dynamic and rich in polar residues [1,2,3,4] This characteristic allows IDPs to act as hub or scaffold molecules in protein–protein interaction networks with different ligands (binding promiscuity) and participate in the modulation of many cellular processes, such as cell cycle regulation [5], signaling pathways, transcription, and translation [6,7,8]. Some proteins have well-conformed domains and disordered segments in their structures that are similar to disordered proteins; these sequences are known as intrinsically disordered regions (IDRs) [13] Typical characteristics of these IDRs include solvent accessibility and transitional order–disorder states in the absence of ligands. Since IDRs can be binding sites for protein–protein interactions and can be B-cell epitopes, we focused on identifying exposed epitopes with short IDRs in the extracellular region of CRTAM to generate antibodies for future therapeutic applications in treating inflammatory diseases
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