title: 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.
abstract: 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 (k(a)). This study provides insights for the design of new therapeutic proteins capable of blocking complement activation.
authors.affiliation: Division of Medical Virology, University of Cape Town Medical School, Anzio Road, Observatory, Cape Town, South Africa.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Smith
authors.forename: R
authors.initials: R
authors.lastname: Sreenivasan
authors.forename: Gunasekaran
authors.initials: G
authors.lastname: Krishnasamy
authors.forename: Ken W
authors.initials: KW
authors.lastname: Judge
authors.forename: Krishna H
authors.initials: KH
authors.lastname: Murthy
authors.forename: Shrihari J
authors.initials: SJ
authors.lastname: Arjunwadkar
authors.forename: David R
authors.initials: DR
authors.lastname: Pugh
authors.forename: Girish J
authors.initials: GJ
authors.lastname: Kotwal
mesh_terms: D001665:Binding Sites; D003165:Complement System Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D006493:Heparin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014616:Vaccinia virus / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*; D015003:Yeasts / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D014764:Viral Proteins; C066459:complement-control protein, Vaccinia virus; D006493:Heparin; D003165:Complement System Proteins