Functional characterization of the complement control protein homolog of Herpesvirus saimiri: R118 is critical for factor I cofactor activities

Singh, Akhilesh K.; Mullick, Jayati; Bernet, John; Sahu, Arvind

doi:10.1016/j.molimm.2006.07.220

Cited by 11 publications

(31 citation statements)

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“…Both of these were then purified using a Mono Q column. Human factor B was purified according to the earlier established procedure (30), whereas bovine factors B (31) and I (32) were purified as described with few modifications. For bovine factor B purification, one part of inhibitor solution (1 M KH 2 PO 4 , 0.2 M Na 4 EDTA, 0.2 M benzamidine, and 1 mM PMSF) was mixed with 19 parts bovine plasma and sequentially precipitated with 12 and 26% polyethylene glycol at 0˚C.…”

Section: Purification Of Complement Proteinsmentioning

confidence: 99%

Species Selectivity in Poxviral Complement Regulators Is Dictated by the Charge Reversal in the Central Complement Control Protein Modules

Yadav

Pyaram

Ahmad

et al. 2012

The Journal of Immunology

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Variola and vaccinia viruses, the two most important members of the family Poxviridae, are known to encode homologs of the human complement regulators named smallpox inhibitor of complement enzymes (SPICE) and vaccinia virus complement control protein (VCP), respectively, to subvert the host complement system. Intriguingly, consistent with the host tropism of these viruses, SPICE has been shown to be more human complement-specific than VCP, and in this study we show that VCP is more bovine complement-specific than SPICE. Based on mutagenesis and mechanistic studies, we suggest that the major determinant for the switch in species selectivity of SPICE and VCP is the presence of oppositely charged residues in the central complement control modules, which help enhance their interaction with factor I and C3b, the proteolytically cleaved form of C3. Thus, our results provide a molecular basis for the species selectivity in poxviral complement regulators.

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Section: Purification Of Complement Proteinsmentioning

confidence: 99%

Species Selectivity in Poxviral Complement Regulators Is Dictated by the Charge Reversal in the Central Complement Control Protein Modules

Yadav

Pyaram

Ahmad

et al. 2012

The Journal of Immunology

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“…The full-length VCP and CCP modules 1-4 of DAF and MCP cloned in pPICZa (34,37) were used as a template for generation of VCP-DAF and VCP-MCP domain swap mutants. The construction of these mutants was achieved by gene splicing and overlap extension method (39).…”

Section: Construction Of Domain Swap Mutantsmentioning

confidence: 99%

“…Expression and purification of VCP, DAF, MCP, and the domain swap mutants were performed, as described (34,37). For purification, the supernatants containing the expressed mutants were concentrated by ultrafiltration, precipitated with 80% ammonium sulfate at 0˚C, and then dissolved and dialyzed in PBS.…”

Section: Expression and Purification Of Domain Swap Mutantsmentioning

confidence: 99%

“…In all of the above purifications, eluted fractions were analyzed by SDS-PAGE and Western blotting using anti-VCP and anti-MCP (Santa Cruz Biotechnology, Santa Cruz, CA)/anti-DAF (Santa Cruz Biotechnology) Abs. The fractions containing purified mutants were pooled, dialyzed into PBS, concentrated using ultrafiltration, and then subjected to SDS-PAGE and circular dichroism analyses (37,40).…”

Section: Expression and Purification Of Domain Swap Mutantsmentioning

confidence: 99%

“…In brief, C3b (∼1300 response units [RUs]) and C4b (∼1900 RUs), labeled through their free thiol group with biotin, were oriented on a streptavidin chip (Sensor Chip SA; Biacore AB). In case of C3b, additional molecules (∼4500 RUs) were coupled onto the C3b-immobilized flow cell by forming AP C3 convertase (C3b,Bb) onto the chip and then flowing native C3 (37). Binding studies for all the interactions were performed at 25˚C in PBS containing 0.05% Tween 20 at 50 ml/min flow rate to avoid mass transport effect.…”

Section: Surface Plasmon Resonance Measurementsmentioning

confidence: 99%

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Domain Swapping Reveals Complement Control Protein Modules Critical for Imparting Cofactor and Decay-Accelerating Activities in Vaccinia Virus Complement Control Protein

Ahmad

Raut

Pyaram

et al. 2010

The Journal of Immunology

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Vaccinia virus encodes a structural and functional homolog of human complement regulators named vaccinia virus complement control protein (VCP). This four-complement control protein domain containing secretory protein is known to inhibit complement activation by supporting the factor I-mediated inactivation of complement proteins, proteolytically cleaved form of C3 (C3b) and proteolytically cleaved form of C4 (C4b) (termed cofactor activity), and by accelerating the irreversible decay of the classical and to a limited extent of the alternative pathway C3 convertases (termed decay-accelerating activity [DAA]). In this study, we have mapped the VCP domains important for its cofactor activity and DAA by swapping its individual domains with those of human decay-accelerating factor (CD55) and membrane cofactor protein (MCP; CD46). Our data indicate the following: 1) swapping of VCP domain 2 or 3, but not 1, with homologous domains of decay-accelerating factor results in loss in its C3b and C4b cofactor activities; 2) swapping of VCP domain 1, but not 2, 3, or 4 with corresponding domains of MCP results in abrogation in its classical pathway DAA; and 3) swapping of VCP domain 1, 2, or 3, but not 4, with homologous MCP domains have marked effect on its alternative pathway DAA. These functional data together with binding studies with C3b and C4b suggest that in VCP, domains 2 and 3 provide binding surface for factor I interaction, whereas domain 1 mediates dissociation of C2a and Bb from the classical and alternative pathway C3 convertases, respectively.

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Complement and its role in protection and pathogenesis of flavivirus infections

Avirutnan

Mehlhop

Diamond

2008

Vaccine

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The complement system is a family of serum and cell surface proteins that recognize pathogenassociated molecular patterns, altered-self ligands, and immune complexes. Activation of the complement cascade triggers several antiviral functions including pathogen opsonization and/or lysis, and priming of adaptive immune responses. In this review, we will examine the role of complement activation in protection and/or pathogenesis against infection by Flaviviruses, with an emphasis on experiments with West Nile and Dengue viruses.

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Functional characterization of the complement control protein homolog of Herpesvirus saimiri: R118 is critical for factor I cofactor activities

Cited by 11 publications

References 0 publications

Species Selectivity in Poxviral Complement Regulators Is Dictated by the Charge Reversal in the Central Complement Control Protein Modules

Species Selectivity in Poxviral Complement Regulators Is Dictated by the Charge Reversal in the Central Complement Control Protein Modules

Domain Swapping Reveals Complement Control Protein Modules Critical for Imparting Cofactor and Decay-Accelerating Activities in Vaccinia Virus Complement Control Protein

Complement and its role in protection and pathogenesis of flavivirus infections

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