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Mapping of regions within the vaccinia virus complement control protein involved in dose-dependent binding to key complement components and heparin using surface plasmon resonance

Posted by on in 2003
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 Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics Volume 1650, Issues 1-2 , 21 August 2003, Pages 30-39
Scott A. Smith (a)(b), R. Sreenivasan (c), Gunasekaran Krishnasamy (d), Ken W. Judge (d), Krishna H. Murthy (d), Shrihari J. Arjunwadkar (a), David R. Pugh (e) and Girish J. Kotwal (a)(b)
(a) Division of Medical Virology, University of Cape Town Medical School, Anzio Road, Observatory, Cape Town, South Africa
(b) Department of Microbiology and Immunology, University of Louisville School of Medicine, Louisville, KY 40202, USA
(c) Department of Human Biology, MRC/UCT Medical Imaging Research Unit, University of Cape Town, Observatory, 7925, Cape Town, South Africa
(d) Center for Biophysical Science and Engineering, University of Alabama at Birmingham, Birmingham, AL 35294-0005, USA
(e) Department of Biotechnology, University of Western Cape, Cape Town, South Africa
Received 3 December 2002; revised 22 May 2003; accepted 23 May 2003; Available online 18 June 2003.


The vaccinia virus complement control protein (VCP) is involved in modulating the host inflammatory response by blocking both pathways of complement activity through its ability to bind C3b and C4b. Other activities arise from VCP's ability to strongly bind heparin. To map regions within VCP involved in binding complement and heparin experimentally, surface plasmon resonance (SPR) and recombinantly expressed VCP (rVCP) constructs were employed. Using C3b or heparin as the immobilized ligand, various rVCP constructs were tested for their ability to bind. Results suggest that VCP is the smallest functional unit able to bind C3b, thereby blocking complement activity, and only a single site, the large basic region near the C-terminus, is involved in heparin binding. Kinetic analysis was also performed to determine the relative binding affinities between rVCP and complement (C3-MA and C4b), as well as rVCP and heparin. rVCP was found to possess a significantly greater affinity for C3-MA than C4b, as indicated by the 1.50e3-fold greater association rate constant (ka). This study provides insights for the design of new therapeutic proteins capable of blocking complement activation.

Author Keywords: Poxvirus; Complement; Inflammation; Surface plasmon resonance

Abbreviations: C3b, activated form of C3; C3-MA, methylamine-treated C3; C4b-BP, complement 4b-binding protein; C4-MA, methylamine-treated C4; CCP, complement control protein; CPV, cowpox virus; CR1, complement receptor type 1; DAF, decay-accelerating factor; EDC, N-ethyl-N¢-(dimethylaminopropyl)-carbodiimine; fH, factor H; IMP, inflammation modulatory protein; MCP, membrane cofactor protein; MIP-1, macrophage inflammatory protein 1; NHS, N-hydroxysuccinimide; rVCP, recombinant vaccinia virus complement control protein; RCA, regulators of complement activation; RU, response units; SCR, short consensus repeats; SBTI, soybean trypsin inhibitor; SPR, surface plasmon resonance; VCP, vaccinia virus complement control protein

Corresponding author. Division of Medical Virology, University of Cape Town Medical School, Anzio Road, Observatory, , Cape Town, , South Africa. Tel.: +27-21-406-6676; fax: +27-21-448-4110.

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