The first crystal structure of CD8 from a cartilaginous fish

IntroductionCartilaginous fishes are the most evolutionary-distant vertebrates from mammals and possess an immunoglobulin (Ig)- and T cell-mediated adaptive immunity. CD8 is the hallmark receptor of cytotoxic T cells and is required for the formation of T cell receptor-major histocompatibility complex (TCR-MHC) class I complexes. MethodsRACE PCR was used to obtain gene sequences. Direct dilution was applied for the refolding of denatured recombinant CD8 protein. Hanging-drop vapor diffusion method was performed for protein crystallization. ResultsIn this study, CD8 alpha and CD8 beta orthologues (termed ScCD8 alpha and ScCD8 beta) were identified in small-spotted catshark (Scyliorhinus canicula). Both ScCD8 alpha and ScCD8 beta possess an extracellular immunoglobulin superfamily (IgSF) V domain as in previously identified CD8 proteins. The genes encoding CD8 alpha and CD8 beta are tandemly linked in the genomes of all jawed vertebrates studied, suggesting that they were duplicated from a common ancestral gene before the divergence of cartilaginous fishes and other vertebrates. We determined the crystal structure of the ScCD8 alpha ectodomain homodimer at a resolution of 1.35 angstrom and show that it exhibits the typical topological structure of CD8 alpha from endotherms. As in mammals, the homodimer formation of ScCD8 alpha alpha relies upon interactions within a hydrophobic core although this differs in position and amino acid composition. Importantly, ScCD8 alpha alpha shares the canonical cavity required for interaction with peptide-loaded MHC I in mammals. Furthermore, it was found that ScCD8 alpha can co-immunoprecipitate with ScCD8 beta, indicating that it can form both homodimeric and heterodimeric complexes. ConclusionOur results expand the current knowledge of vertebrate CD8 dimerization and the interaction between CD8 alpha with p/MHC I from an evolutionary perspective.