Expression, Purification, And Small Angle X-ray Scattering (SAXS) Analysis of Vascular Endothelial Growth Factors, Their Receptor And Complex

Autor: Yi-Jang Chen, 陳奕璋
Rok vydání: 2012
Druh dokumentu: 學位論文 ; thesis
Popis: 100
Vascular endothelial growth factors (VEGFs) regulate blood and lymphatic vessel development and homeostasis. The binding of VEGFs with their cognate receptors (VEGF receptors, VEGFRs) will induce the ligand-mediated receptor dimerization which will activate intracellular tyrosine kinase of receptors undergoing auto-phosphorylation and propagate signal-transduction cascade. In mammalians, there are five members of VEGFs (VEGF-A, B, C, D and PlGF) and three members of VEGFRs (VEGFR-1, Flt1; VEGFR-2, KDR/Flk1; and VEGFR-3, Flt4). VEGFRs belong to type V receptor tyrosine kinases which are characterized with seven immunoglobulin-like domains in their extraceullar domains. VEGF-C and VEGF-D were secreted by endothelial cells and can regulate lymphagiogenesis via binding with VEGFR-3, or regulate angiogenesis and vasculogenesis via binding with VEGFR-2. Recently studies found that VEGF-C, -D and VEGFR-3 are related with tumor growth and metastasis. In this study, recombinant proteins of VEGF-C, VEGF-D, the first three Ig-like domains of VEGFR-3 (VEGFR-3-D3), whole extracellular domain (VEGF-R3- D7) and three types mutant of whole extracellular domain were expressed by Drosophila Schneider 2 (S2) cell expression system for structural studies of ligands, receptors and ligand/receptor complex. When we started this project, no any structural information of VEGF-C, VEGF-D, or VEGFR-3 is available. However, recently the crystal structures of VEGF-C in complex with VEGFR-2-D2~D3 and VEGF-D have been published, but still no structural information of VEGFR-3 yet. For this thesis, we conducted small-angle X-ray scattering (SAXS) to investigate the structure of VEGFR-3 and its complex with VEGF-C or -D. By this method, protein structure of mono-disperse in solution is the observation target which believe can represent as the native conformation of proteins. We also tried to find the amino acids which were important for VEGFR-3 to bind VEGFs with ELISA. Recombinant proteins were purified from concentrated culture media of stable transfected S2 cells, through Ni-affinity and gel filtration chromatography. The yield of any types of VEGFR-3 domain is greater than 10 mg/liter culture media; for VEGF-C and -D is about 2-3 mg/liter culture media. However, only VEGF-C in complex with VEGFR-3-D3 has been successfully obtained. The SAXS data of VEGFR-3-D3, -D7, and VEGF-C in complex with VEGFR-D3 have been characterized by programs implant in ATSAS, ab-initio envelope of each protein was reconstructed by DAMMIF and Situs. Their homology models have been built according to crystal structures of VEGFR-2 or/and cKit and their models have been docked into average envelops and assessed. The results showed that the model of VEGFR-3-D3 can dock into SAXS envelope very well. According to ELISA data, in normal situation the effinity of VEGFR-3/VEGF-C was lower than VEGFR-1/VEGF-A. This may explain why so difficult to get the complex of VEGFR-3/VEGF-C. We have established the enzyme-linked immunosorbent assay (ELISA) for the study of VEGF ligand-receptor bindings. The result revealed that the binding affinity of VEGF-C with its coguate receptor VEGFR-3 is much lower than that of VEGF-A and VEGFR-1, and implied the difficulty to form VEGFR-3/VEGF-C complex. According to the crystal structure of VEGFR-2 (D2-D3)/VEGF-C complex and sequence alignment of VEGFRs, we constructured several chimeric VEGFR-3, by substitute C1 or/and C3 regions of VEGFR-3 with VEGFR-1 sequence. Interestingly, the chimeric VEGFR-3 receptor gains the binding affinity with VEGF-A, particularly for the C1C3 VEGFR-3 mutant, and seems do not lost their binding affinity with VEGF-C. Due to the important roles of VEGF-A and VEGF-C in turmor growth and metastasis, based on the above results, we will next design a decoy VEGF receptor which can bind both VEGF-A and VEGF-C. Mean while, according to ELISA, we will investigate the appropriate condition for the VEGF-C/VEGFR-3 complex formation, in order to form the ligand-receptor compex for structural studies by SAXS and/or X-ray crystallography in the near future.
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