The Structural And Functional Research Of AaHIV And Acucetin, And The Structural Biology Research Of ScFv-A21

As people living standard rise, diseases and the health have become a growing concern. So the research of proteins related to diseases has become more and more important. Metalloproteinases play a key role in normal physiological activity, such as: ADAM 17 is not only a tumour necrosis factor-a convertase(TACE), but also participate in the activating of Epidermal Growth Factor Receptor (EGFR) ligands; ADAM10 is necessary in signalling via the Notch pathways; ADAM1 and ADAM2 play roles in spermatogenesis and sperm function, potentially by effecting maturation of sperm and their adhesion and migration in the uterus. When their functions are dysregulated, they are linked to pathological states including cancer, cardiovascular disease, asthma, Alzheimer's disease. So it is important to exploring the structure-function relationship of their homologs and theirs. This thesis presents the research of the structure and function of AaHIV, a homolog from the venom of Agkistrodon acutus.AaHIV is a P-III type snake venom metalloproteinase, which is constituted of metalloproteinase, disintegrin-like and cysteine-rich domains. In physiological conditions, it could degrade itself to release a product named acucetin which contains intact disintegrin-like domain and cysteine-rich domain. AaHIV and acucetin could completely inhibit the aggregation of platelets induced by horm collagen, partially inhibit the aggregation of platelets induced by ADP, but could not affect the aggregation of platelets induced by restrocetin. Some cellular experiments showed that they could bind the platelets and the CHO (Chinese hamster ovary) cell expressing the integrin aIIbβ3, but only AaHIV could bind the CHO cell expressing integrin a2β1 weakly. This chapter also introduces the process of solving the crystal structures of AaHIV and acucetin. Based on the crystal structure of AaHIV, the analysis of the process of AaHIV auto-degradation and solution state suggests a new model of the protein of the ADAM/reprolysin subfamily targeting their substrates. In the model these proteins function as a dimmer, and the disintegrin-like domain from one molecule binds the target by recognizing the specific binding site and the metalloproteinase of another molecule hydrolyzes the target by the Zn2+ active site. At the same time this chapter discusses the modes of AaHIV and acucetin interacting with their targets on the platelet, and proposes several possible sites of AaHIV binding integrin aIIbβ3 based on the structure of AaHIV and acucetin. The structural basis of interaction of AaHIV with vWF has been discussed too.The thesis also presents another protein named ScFv-A21 which is a single chain antibody of epidermis growth factor receptor ( EGFR ) named P185/Her2 and related to some tumors. Her2 functions as a dimer with other members (Herl, Her3, Her4) of the family of epidermis growth factor receptor. And it is found highly expressing in the tumor cells of the patients suffering from diverse tumors, so its several antibodies, such as Herceptin, have been applied to treat some tumor. The research group of Professor Liu Jing prepared a monoclonal antibody named A21 which could affect the growth of some tumor cells. And its single chain antibody ( ScFv-A21 ) also was prepared to detect the structure basic of the interaction of A21 and Her2. After screening of ScFv-A21 crystallization conditions, we obtained its crystal and solved its structure. Based on the crystal structures of ScFv-A21 and Her2, the Docking experiment was carried out. From the result of the Docking the residues participating in the antigen-antibody interaction have been found and the related residues of Her2 provide a new target site for exploring new antibodies of anti-tumor function. On the other side the discovering of the residues of ScFv-A21 interacting with Her2 affords some structure basic for the reconstruct of A21.