Crystal Structure And Evolutionary Mechanism Studies Of Amphioxus CAL And Zebrafish PTX Molecules

The adaptive immune system (AIS), which is characterized by the diversified antigen receptors (BCRs and TCRs), lymphocytes and various intricate rearrangement mechanisms, arose in cartilaginous fish approximately500million years ago. Till now, all the components of this sophisticated defence system restrictedly exist in jawed vertebrates, leaving the origin of suddenly and rapidly developed highly complex TCR-BCR based the AIS untouched. However, the same immunoglobulin superfamily V sets which are used by BCRs and TCRs for antigen recognition, the similar rearrangement mechanisms and the close linkage of BCR and TCR genes in genome of jawed vertebrates, such as a shark, lead to the universal agreement that the diversity antigen receptors of jawed vertebrates share a common ancestral IgSF domain containing gene in lower chordate. Here, we focused on a common ancestor of all vertebrates-amphioxus, whose genome has been sequenced in2008and it shows that amphioxus is the most ancient living fossil-species before the2rounds of whole genome duplication. Search of common ancestral molecule-like (CAL) in amphioxus, and structural determination and functional analysis of it will reflect the characteristics of the common ancestral molecule of IgSF. Also it will give insight in to understanding of evolutionary origin of adaptive immune system.Short-chain pentraxins (PTXs), though the key component of innate immune system, have similar functions with antibodies. It can both trigger the classical complement pathway through Clq and activate opsonisation and phagocytosis by interacting with Fc receptors (FcRs) as well. As having all these components, zebrafish is the optimum species in low vertebrates for structural studying and adaptive immunity-interaction relationship analysis of PTX. So, here the structures of amphioxus CAL and zebrafish PTX, which are the two key molecules during immune evolution, has been determined. By conbining methods of bioinformatics, molecular immunology and structural biology, the functions of them have been further analyzed.Firstly, in the genome and transcriptome of amphioxus, a common ancestral molecule-like (CAL) has been found. According to the genomic analysis, CAL is in the immune-function related region. To our surprise, RSS-like sequences can be found in CAL introns2,3and4. Furthermore CART motifs are also found in CAL transmembrane region. Then, CAL was expressed, purified and crystallized. The crystal structures show that CAL is composed by two independent immunoglobulin domains, which connect together to form an IgV and IgC tandem structure. Till now, this is the most ancient determined IgV-C molecule. CAL possesses typical structural features of mammal adaptive receptors, including the conserved framework motifs, hydrophobic core, J chain like segment, diversified CDR-analogous loops and even the3-layer packing dimerization pattern. Furthermore, CALs are confirmed to be specifically expressed in the primitive immune organs-digestive tracts (gills, guts and hepatic cecum). Particularly, they are in the lumens of these organs, where are continuously exposed to various micro-organisms. The sub-cellular localization of CAL in intestinal epithelial cell showed that they were primarily localized in the cell protrusions, secretory granules and cilia, which were related with immune functions. Secondly, the crystal structures of Dare-PTX and Dare-PTX-Ca have been determined here for the first time. Dare-PTX is a trimer, which is a new form of determined PTX structures. In the structures, Dare-PTX has a deep and narrow ligand-binding pocket (LBP) in the recognition face. In the effector face, a new Clq binding mode and partial FcR-binding sites have been identified.In conclusion, the research about CAL here show us an immune function related IgV-C molecule found in amphioxus, which carries on somatic mutations and has typic structural characteristics and basal genomic elements of adaptive immune receptors. These will help our understanding of the evolution and the origin of jawed vertebrates AIS. The structures of Dare-PTX show us a primitive PTX in bony fish evolved450million years ago and its co-evolution relationship with AIS.