scholarly journals Identification of Hot Spots in the Variola Virus Complement Inhibitor (SPICE) for Human Complement Regulation

2008 ◽  
Vol 82 (7) ◽  
pp. 3283-3294 ◽  
Author(s):  
Viveka Nand Yadav ◽  
Kalyani Pyaram ◽  
Jayati Mullick ◽  
Arvind Sahu
Keyword(s):  
Hot Spots ◽  
Classical Pathway ◽  
Variola Virus ◽  
Amino Acid Residues ◽  
Human Complement ◽  
Binding Ability ◽  
Complement Control Protein ◽  
Functional Advantage ◽  
Complement Regulator ◽  
Control Protein

ABSTRACT Variola virus, the causative agent of smallpox, encodes a soluble complement regulator named SPICE. Previously, SPICE has been shown to be much more potent in inactivating human complement than the vaccinia virus complement control protein (VCP), although they differ only in 11 amino acid residues. In the present study, we have expressed SPICE, VCP, and mutants of VCP by substituting each or more of the 11 non-variant VCP residues with the corresponding residue of SPICE to identify hot spots that impart functional advantage to SPICE over VCP. Our data indicate that (i) SPICE is ∼90-fold more potent than VCP in inactivating human C3b, and the residues Y98, Y103, K108 and K120 are predominantly responsible for its enhanced activity; (ii) SPICE is 5.4-fold more potent in inactivating human C4b, and residues Y98, Y103, K108, K120 and L193 mainly dictate this increase; (iii) the classical pathway decay-accelerating activity of activity is only twofold higher than that of VCP, and the 11 mutations in SPICE do not significantly affect this activity; (iv) SPICE possesses significantly greater binding ability to human C3b compared to VCP, although its binding to human C4b is lower than that of VCP; (v) residue N144 is largely responsible for the increased binding of SPICE to human C3b; and (vi) the human specificity of SPICE is dictated primarily by residues Y98, Y103, K108, and K120 since these are enough to formulate VCP as potent as SPICE. Together, these results suggest that principally 4 of the 11 residues that differ between SPICE and VCP partake in its enhanced function against human complement.

scholarly journals Species Specificity of Vaccinia Virus Complement Control Protein for the Bovine Classical Pathway Is Governed Primarily by Direct Interaction of Its Acidic Residues with Factor I

2017 ◽  
Vol 91 (19) ◽  
Author(s):  
Jitendra Kumar ◽  
Viveka Nand Yadav ◽  
Swastik Phulera ◽  
Ashish Kamble ◽  
Avneesh Kumar Gautam ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Classical Pathway ◽  
Variola Virus ◽  
Complement Control Protein ◽  
Factor I ◽  
Functional Assays ◽  
Alternative Complement ◽  
Cofactor Activity ◽  
Complement Regulator ◽  
Control Protein

ABSTRACTPoxviruses display species tropism—variola virus is a human-specific virus, while vaccinia virus causes repeated outbreaks in dairy cattle. Consistent with this, variola virus complement regulator SPICE (smallpox inhibitor of complement enzymes) exhibits selectivity in inhibiting the human alternative complement pathway and vaccinia virus complement regulator VCP (vaccinia virus complement control protein) displays selectivity in inhibiting the bovine alternative complement pathway. In the present study, we examined the species specificity of VCP and SPICE for the classical pathway (CP). We observed that VCP is ∼43-fold superior to SPICE in inhibiting bovine CP. Further, functional assays revealed that increased inhibitory activity of VCP for bovine CP is solely due to its enhanced cofactor activity, with no effect on decay of bovine CP C3-convertase. To probe the structural basis of this specificity, we utilized single- and multi-amino-acid substitution mutants wherein 1 or more of the 11 variant VCP residues were substituted in the SPICE template. Examination of these mutants for their ability to inhibit bovine CP revealed that E108, E120, and E144 are primarily responsible for imparting the specificity and contribute to the enhanced cofactor activity of VCP. Binding and functional assays suggested that these residues interact with bovine factor I but not with bovine C4(H2O) (a moiety conformationally similar to C4b). Mapping of these residues onto the modeled structure of bovine C4b-VCP-bovine factor I supported the mutagenesis data. Taken together, our data help explain why the vaccine strain of vaccinia virus was able to gain a foothold in domesticated animals.IMPORTANCEVaccinia virus was used for smallpox vaccination. The vaccine-derived virus is now circulating and causing outbreaks in dairy cattle in India and Brazil. However, the reason for this tropism is unknown. It is well recognized that the virus is susceptible to neutralization by the complement classical pathway (CP). Because the virus encodes a soluble complement regulator, VCP, we examined whether this protein displays selectivity in targeting bovine CP. Our data show that it does exhibit selectivity in inhibiting the bovine CP and that this is primarily determined by its amino acids E108, E120, and E144, which interact with bovine serine protease factor I to inactivate bovine C4b—one of the two subunits of CP C3-convertase. Of note, the variola complement regulator SPICE contains positively charged residues at these positions. Thus, these variant residues in VCP help enhance its potency against the bovine CP and thereby the fitness of the virus in cattle.

scholarly journals Functional Characterization of LcpA, a Surface-Exposed Protein of Leptospira spp. That Binds the Human Complement Regulator C4BP

2010 ◽  
Vol 78 (7) ◽  
pp. 3207-3216 ◽  
Author(s):  
Angela S. Barbosa ◽  
Denize Monaris ◽  
Ludmila B. Silva ◽  
Zenaide M. Morais ◽  
Sílvio A. Vasconcellos ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Functional Characterization ◽  
Lectin Pathway ◽  
Classical Pathway ◽  
Dependent Manner ◽  
Human Complement ◽  
Cofactor Activity ◽  
Complement Regulator

ABSTRACT We have previously shown that pathogenic leptospiral strains are able to bind C4b binding protein (C4BP). Surface-bound C4BP retains its cofactor activity, indicating that acquisition of this complement regulator may contribute to leptospiral serum resistance. In the present study, the abilities of seven recombinant putative leptospiral outer membrane proteins to interact with C4BP were evaluated. The protein encoded by LIC11947 interacted with this human complement regulator in a dose-dependent manner. The cofactor activity of C4BP bound to immobilized recombinant LIC11947 (rLIC11947) was confirmed by detecting factor I-mediated cleavage of C4b. rLIC11947 was therefore named LcpA (for leptospiral complement regulator-acquiring protein A). LcpA was shown to be an outer membrane protein by using immunoelectron microscopy, cell surface proteolysis, and Triton X-114 fractionation. The gene coding for LcpA is conserved among pathogenic leptospiral strains. This is the first characterization of a Leptospira surface protein that binds to the human complement regulator C4BP in a manner that allows this important regulator to control complement system activation mediated either by the classical pathway or by the lectin pathway. This newly identified protein may play a role in immune evasion by Leptospira spp. and may therefore represent a target for the development of a human vaccine against leptospirosis.

scholarly journals The Sez6 Family Inhibits Complement by Facilitating Factor I Cleavage of C3b and Accelerating the Decay of C3 Convertases

2021 ◽  
Vol 12 ◽  
Author(s):  
Wen Q. Qiu ◽  
Shaopeiwen Luo ◽  
Stefanie A. Ma ◽  
Priyanka Saminathan ◽  
Herman Li ◽  
...  
Keyword(s):  
Alternative Pathway ◽  
Family Members ◽  
Dendritic Morphology ◽  
Classical Pathway ◽  
Strong Inhibitor ◽  
Alternative Pathways ◽  
Complement Control Protein ◽  
Factor I ◽  
Complement Regulator ◽  
The Brain

The Sez6 family consists of Sez6, Sez6L, and Sez6L2. Its members are expressed throughout the brain and have been shown to influence synapse numbers and dendritic morphology. They are also linked to various neurological and psychiatric disorders. All Sez6 family members contain 2-3 CUB domains and 5 complement control protein (CCP) domains, suggesting that they may be involved in complement regulation. We show that Sez6 family members inhibit C3b/iC3b opsonization by the classical and alternative pathways with varying degrees of efficacy. For the classical pathway, Sez6 is a strong inhibitor, Sez6L2 is a moderate inhibitor, and Sez6L is a weak inhibitor. For the alternative pathway, the complement inhibitory activity of Sez6, Sez6L, and Sez6L2 all equaled or exceeded the activity of the known complement regulator MCP. Using Sez6L2 as the representative family member, we show that it specifically accelerates the dissociation of C3 convertases. Sez6L2 also functions as a cofactor for Factor I to facilitate the cleavage of C3b; however, Sez6L2 has no cofactor activity toward C4b. In summary, the Sez6 family are novel complement regulators that inhibit C3 convertases and promote C3b degradation.

scholarly journals The Sez6 family inhibits complement at the level of the C3 convertase

2020 ◽  
Author(s):  
Wen Q. Qiu ◽  
Shaopeiwen Luo ◽  
Stefanie A. Ma ◽  
Priyanka Saminathan ◽  
Herman Li ◽  
...  
Keyword(s):  
Alternative Pathway ◽  
Family Members ◽  
Dendritic Morphology ◽  
Classical Pathway ◽  
Strong Inhibitor ◽  
Complement Control Protein ◽  
Factor I ◽  
The Brain ◽  
Representative Family ◽  
Control Protein

AbstractThe Sez6 family consists of Sez6, Sez6L, and Sez6L2. Its members are expressed throughout the brain and have been shown to influence synapse numbers and dendritic morphology. They are also linked to various neurological and psychiatric disorders. All Sez6 family members contain 2-3 CUB domains and 5 complement control protein (CCP) domains, suggesting that they may be involved in complement regulation. We show that all Sez6 family members inhibit C3 deposition by the classical and alterative pathways with varying degrees of efficacy. For the classical pathway, Sez6 is a strong inhibitor, Sez6L2 is a moderate inhibitor, and Sez6L is a weak inhibitor. Using Sez6L2 as the representative family member, we show that it specifically deactivates C3 convertases by accelerating the decay or dissociation of the C3 convertase components. Sez6L2 also deactivates C3 convertases of the alternative pathway by serving as a cofactor for Factor I to facilitate the cleavage of C3b. However, Sez6L2 has no cofactor activity toward C4b. In summary, the Sez6 family are novel complement regulators that inhibit C3 convertases.

scholarly journals Kinetic Analysis of the Interactions between Vaccinia Virus Complement Control Protein and Human Complement Proteins C3b and C4b

2004 ◽  
Vol 78 (17) ◽  
pp. 9446-9457 ◽  
Author(s):  
John Bernet ◽  
Jayati Mullick ◽  
Yogesh Panse ◽  
Pradeep B. Parab ◽  
Arvind Sahu
Keyword(s):  
Vaccinia Virus ◽  
Binding Site ◽  
Molecular Mechanisms ◽  
Accurate Determination ◽  
Factor H ◽  
Human Complement ◽  
Target Proteins ◽  
Complement Control Protein ◽  
Complement Proteins ◽  
Control Protein

ABSTRACT The vaccinia virus complement control protein (VCP) is an immune evasion protein of vaccinia virus. Previously, VCP has been shown to bind and support inactivation of host complement proteins C3b and C4b and to protect the vaccinia virions from antibody-dependent complement-enhanced neutralization. However, the molecular mechanisms involved in the interaction of VCP with its target proteins C3b and C4b have not yet been elucidated. We have utilized surface plasmon resonance technology to study the interaction of VCP with C3b and C4b. We measured the kinetics of binding of the viral protein to its target proteins and compared it with human complement regulators factor H and sCR1, assessed the influence of immobilization of ligand on the binding kinetics, examined the effect of ionic contacts on these interactions, and sublocalized the binding site on C3b and C4b. Our results indicate that (i) the orientation of the ligand is important for accurate determination of the binding constants, as well as the mechanism of binding; (ii) in contrast to factor H and sCR1, the binding of VCP to C3b and C4b follows a simple 1:1 binding model and does not involve multiple-site interactions as predicted earlier; (iii) VCP has a 4.6-fold higher affinity for C4b than that for C3b, which is also reflected in its factor I cofactor activity; (iv) ionic interactions are important for VCP-C3b and VCP-C4b complex formation; (v) VCP does not bind simultaneously to C3b and C4b; and (vi) the binding site of VCP on C3b and C4b is located in the C3dg and C4c regions, respectively.

scholarly journals Identification of Complement Regulatory Domains in Vaccinia Virus Complement Control Protein

2005 ◽  
Vol 79 (19) ◽  
pp. 12382-12393 ◽  
Author(s):  
Jayati Mullick ◽  
John Bernet ◽  
Yogesh Panse ◽  
Sharanabasava Hallihosur ◽  
Akhilesh K. Singh ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Alternative Pathway ◽  
Deletion Mutants ◽  
Classical Pathway ◽  
Target Proteins ◽  
Complement Control Protein ◽  
Functional Activities ◽  
Regulatory Domains ◽  
Optimal Activity ◽  
Control Protein

ABSTRACT Vaccinia virus encodes a homolog of the human complement regulators named vaccinia virus complement control protein (VCP). It is composed of four contiguous complement control protein (CCP) domains. Previously, VCP has been shown to bind to C3b and C4b and to inactivate the classical and alternative pathway C3 convertases by accelerating the decay of the classical pathway C3 convertase and (to a limited extent) the alternative pathway C3 convertase, as well as by supporting the factor I-mediated inactivation of C3b and C4b (the subunits of C3 convertases). In this study, we have mapped the CCP domains of VCP important for its cofactor activities, decay-accelerating activities, and binding to the target proteins by utilizing a series of deletion mutants. Our data indicate the following. (i) CCPs 1 to 3 are essential for cofactor activity for C3b and C4b; however, CCP 4 also contributes to the optimal activity. (ii) CCPs 1 to 2 are enough to mediate the classical pathway decay-accelerating activity but show very minimal activity, and all the four CCPs are necessary for its efficient activity. (iii) CCPs 2 to 4 mediate the alternative pathway decay-accelerating activity. (iv) CCPs 1 to 3 are required for binding to C3b and C4b, but the presence of CCP 4 enhances the affinity for both the target proteins. These results together demonstrate that the entire length of the protein is required for VCP's various functional activities and suggests why the four-domain structure of viral CCP is conserved in poxviruses.

scholarly journals Complete nucleotide and deduced amino acid sequence of human β2-glycoprotein I

1991 ◽  
Vol 277 (2) ◽  
pp. 387-391 ◽  
Author(s):  
A Steinkasserer ◽  
C Estaller ◽  
E H Weiss ◽  
R B Sim ◽  
A J Day
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Protein A ◽  
Untranslated Regions ◽  
Leader Peptide ◽  
Amino Acid Residues ◽  
Complement Control Protein ◽  
Glycoprotein I ◽  
Control Protein ◽  
Major Amino Acid

The nucleotide and complete amino acid sequence for the human beta 2-glycoprotein I (beta 2I) was derived by sequencing the cDNA clone pB2I-1. In addition to the 326 amino acid residues of the mature protein this clone codes for a putative leader peptide and contains sequence representing 5′ and 3′ untranslated regions. When this amino acid sequence was compared with the previously published primary sequence, three major amino acid substitutions were found, two involving cysteine residues. These substitutions lead to a new alignment of the complement control protein (CCP) repeats present in beta 2I and a prediction of the complete disulphide bond organization. Northern-blot analysis indicates that hepatocytes are a major site of biosynthesis for this protein. A transcription signal of about 1.5 kb was detected by using RNA from HepG2 cells.

scholarly journals Purification of human C3b inactivator by monoclonal-antibody affinity chromatography

1982 ◽  
Vol 203 (1) ◽  
pp. 293-298 ◽  
Author(s):  
Li-min Hsiung ◽  
A. Neil Barclay ◽  
Malcolm R. Brandon ◽  
Edith Sim ◽  
Rodney R. Porter
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Amino Acid Analysis ◽  
Chain Structure ◽  
Affinity Column ◽  
Human Complement ◽  
Antibody Affinity ◽  
Complement Control Protein ◽  
Specific Affinity ◽  
Control Protein

Monoclonal antibody has been obtained to the human complement control protein C3b inactivator after immunization of mice with the enzyme prepared by conventional methods. Antibody from ascitic fluid was purified and coupled to Sepharose-CL-4B to give a specific affinity column, which was used to isolate C3b inactivator from human serum in 70% yield. The product was characterized by size, chain structure, amino acid analysis and proteolytic activity.

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