Structure-function studies of the vaccinia virus complement control protein.

Vaccinia virus;Complement control protein;Immunomodulation;Inflammatory response;Heparin

Scott Alan Smith

University:University of Louisville

Department:Microbiology and Immunology

关键词: Vaccinia virus;    Complement control protein;    Immunomodulation;    Inflammatory response;    Heparin;   

Others  :  https://ir.library.louisville.edu/cgi/viewcontent.cgi?article=2346&context=etd

美国|英语

来源: The Universite of Louisville's Institutional Repository

【 摘 要 】

The vaccinia virus complement control protein (VCP) is involved in the modulation of the host inflammatory response during vaccinia virus infection. It possesses the ability to inhibit both classical and alternative pathways of complement activation, as well as bind to heparin or heparan sulfate proteoglycans, making it a unique multifunctional protein with therapeutic potential. VCP is able to block complement activity through its ability to bind C3b and C4b. Other very novel activities arise from VCP's ability to bind heparin and heparan sulfate proteoglycans, allowing the protein to attach itself to cell surfaces. The goal of this research was to fully characterize the structure and various functions of VCP. The structural basis for VCP's ability to bind heparin was investigated using heparin affinity chromatography, surface plasmon resonance analysis, and homology modeling with well-characterized heparin binding proteins. VCP was found to possess two regions involved in heparin binding, one involved in weak binding located at the junction of complement control protein (CCP) modules 1 and 2, and a second involved in strong binding located at the extreme C-terminal tip of the protein. Additional functions, involved in blocking molecular interactions with cells, were also identified and determined to be a result of VCP's ability to bind heparin. The structural basis for VCP's ability to bind complement was investigated using the hemolysis assay, surface plasmon resonance analysis, and homology modeling with well-mapped complement regulatory proteins. VCP was found to be the smallest functional unit able to bind and cause factor I cleavage of C3b/C4b, thus inhibiting complement activity. VCP was also shown to possess the ability to simultaneously bind and inhibit complement activation, using its entire exposed surface, while remaining attached to heparin by its C-terminal heparin binding site. The structure of VCP and its structural and functional stability were determined using X-ray crystallography, NMR, DSC, and the hemolysis assay. VCP was found to be an elongated filamentous protein, with defined CCP modular regions with limited intermodular interfacing, existing as a monomer in solution. VCP was found to possess the ability to structurally withstand temperatures in excess of 90oC. In addition, VCP was able to retain functional integrity following exposure to many adverse physical conditions, which would result in irreversible denaturation of most other proteins. Lastly, the in vivo pharmacokinetics of VCP was investigated in rats. VCP was injected as a bolus at different routes and using various concentrations. Continuous and intermittent intravenous