Isolation and functional characterisation of cyokines from the spiny dogfish Squalus acanthias.

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Elasmobranchs (sharks, skates and rays) are the earliest group in phylogeny to have all of the molecules of adaptive immunity also observed in mammals (MHC, TCR, Immunoglobulin). For this reason this group has been extensively studied to try to understand how the adaptive immune system evolved. Work to date has found 3 immunoglobulin isotypes in sharks; the so-called 'conventional' heavy-light chain isotypes IgM (which occurs in a pentameric and monomeric form) and IgW (the shark IgD orthologue). The third, non-conventional, shark specific isotype is called IgNAR (novel antigen receptor) and is of special interest as it is a heavy chain homodimer that naturally lacks light chain. EM studies have shown that each heavy chain has one variable (V) domain tethered to 5 (C) constant domains via a flexible, hinge-like region however dimerization of the V regions is not required for high-affinity antigen binding. Crystal structures of selected IgNAR V regions in complex with their antigen showed that the presence of germline-encoded non-canonical cysteines and a truncation through CDR2/framework region (Fr)2 give the V's a very small size (12 kDa) and extremely compact domain structure. Binding to antigen is mediated mainly by the highly diverse CDR3, which is generated through the rearrangement of 3 D regions. The long (9-24 aa) CDR3 loop protrudes from the top of the V domain to interact with antigen, and in some instances, has been shown to insert itself into clefts and active sites on the antigen surface to provide high affinity binding. In some instances this has the effect of blocking the enzymatic function of the target molecule. Due to their high affinity binding, targeting of active sites, very small size and general robustness in harsh environments there is significant interest within Wyeth towards the development of these molecules as human therapeutics. The shark basic biology group was established within Wyeth Research Aberdeen to support this aim by studying B-cell development and isotype choice in the spiny dogfish (Squalus acanthias), specifically looking at the best way to induce the production of antigen-specific IgNAR V regions, either in vivo or in vitro. Whilst molecular approaches allow us to begin to address this aim there is a real and pressing need to establish methods for the long-term culture of shark B-cells in vitro. For this we need to isolate and study the cytokines which may be involved in lymphocyte growth and development in sharks. Thus, for this work we propose to locate (via gene synteny) and sequence a number of cytokine genes from spiny dogfish which, from their role in other species, should drive B-cell growth and differentiation. We will then focus our attention on a couple of these molecules to produce as recombinant proteins and study their effects on shark B-cells in vitro.