Studies On The Crystal Structure And Evolution Of Two IgSF Molecules In Amphioxus And Biomphalaria Glabrata

Immune system is the most important structure of the body’s self defensive, and to exploring the origins and evolution of the immune system has always been a hot topic in immunology research. Members of the immunoglobulin superfamily involved in almost all the immune response, however when IgSF structures appeard in the immune system and how gradually evolved into the core structure is still unknown.In order to discover the origin of IgSF immune-related molecules and novel immune response types with molecular diversity in lower invertebrates and analyze evolutionary characteristics of the immune system and antigen receptor molecules, we selected original chordates cephalochordata amphioxus, which is the species with the special evolutionary status, and molluscs Biomphalaria glabrata to study. We intend to illustrate the structural characteristic and evolutionary pattern of these ancient IgSF immune molecules in the immune by structural biology study.We found a novel IgSF molecule termed Amphi-IgSF-V, and resolved the crystal structure with1.95A resolution. Amino acid homologies between Amphi-IgSF-V and V regions from the opossum TCRδ chain and human Ig light chain are more than30%. The3D structure of Amphi-IgSF-V is consistent to the typical structure of the IgSF V type molecule, and share very similar conformation (RMSD<1.2) to structures of IgSF V domains of mouse Ig heavy chain, human TCR a chain, bony fish NITR and so on. Amphi-IgSF-V is the membrane surface molecule with only a single IgSF V domain of its extracellular region which was fist found in amphioxus. There are four tyrosine in the intracellular region and the last two contained an ITAM-like motif. The J-chain like region (PGDGSDNKLvV) is more similar to the typical one compared to the VCBP3which is the only resolved IgSF V molecular in amphioxus before. The side chain of Arg33, Phe38and Pro88of Amphi-IgSF-V in dimer and the two molecules formed the "three-lays peaking" structure which is the typical structure of the antigen receptor molecules.These findings suggest that the Amphi-IgSF-V molecule may be a primitive ancestor of immune receptors in amphioxus, and provides a new approach for the study of the immune system as well as emphasis on the immune-related IgSF V-type molecule evolution.Fibrinogen related proteins (FREPs) are a class of defensive molecules which was confirmed in gene, transcription and protein aggregation level has diversity characteristic in Biomphalaria glabrata. In this thesis, we have chosen the FREP3VV (include FREP3V1and FREP3V2) which is the variable region of FREP3, FREP4and the IgSF domain of FREP2to study. We expressed and crystallized FREP3V1, FREP3V2, and reached effective diffraction data in2.8A and2.1A respectively, then we mutated effective methionines in FREP3V1and FREP3V2, and also reached effective diffraction data in1.7A and2.0A respectively; FREP2V was expressed and crystallized, then received a set of effective diffraction data in2.6A; We rescued recombinant baculovirus stable expressed FREP3VV and FREP4protein and constructed a method to get soluble FREP3VV protein, this can be helpful to resolved FREP3VV structural and carry functional studies. For these results, it becomes possible to resolve the structure of FREP molecular, and provides a method foundation for the study structural characteristics and evolution patterns of the IgSF Ⅴ in invertebrate immune system along the evolutionary process.From above, our studies on the crystal structure and the evolution of two IgSF molecules in Cephalochordate and Mollusca, showed the results that would promote the development in the field of IgSF Ⅴ type immune-related molecule structural research.