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1.
We have previously demonstrated that the alpha'-chain of human activated form of the fourth (C4b) and third (C3b) component of C are cleaved by plasma or serum from vertebrate species spanning through 300,000,000 yr of evolution yielding fragments identical with those obtained with human plasma. In this study, we investigated the molecular basis of this reaction. We chose barred sand bass plasma because this is the most primitive species analyzed possessing these activities. Barred sand bass plasma proteins were separated on a Sephadex G-200 column and the eluted samples analyzed for C4b and C3b cleavage. Individual fractions were inactive, but degradation was obtained when proteins of 380 and 155 kDa were combined. In contrast to the human regulatory proteins, the sand bass proteins require Ca2+ ions. K76COOH, an inhibitor of human factor I, inhibited the function of the 155-kDa but not of the 380 kDa-fraction. Thus it appears that the 155-kDa fraction functions as the C4b/C3b cleaving enzyme (I) and the 380-kDa material as its cofactor. Further purification of the 380-kDa fraction yielded a protein that by SDS-PAGE consisted of two noncovalently linked subunits of 110 and 42 kDa at a molecular ratio of 2:1. These two chains were antigenically distinct, and constitute domains of the same protein. The 110-kDa peptide binds C4b and not C3b but it fully expresses the cofactor function for the 155-kDa fraction on the cleavage of both C4b and C3b. Limited tryptic digestion of the 110-kDa domain demonstrated C4b binding activity in fragments of 34, 25, and 23 kDa. The activity of the 34-kDa fragment was the same as that of the undigested protein. Comparison of the amino acid composition of the barred sand bass cofactor and of human C4bp shows similar high content of cysteine and proline but not of tryptophan. It differs from human factor H in cysteine, serine, proline, and tryptophan. These studies indicate that regulatory proteins for the C4b and C3b C fragments may have appeared very early phylogenetically.  相似文献   

2.
Functional properties of membrane cofactor protein of complement.   总被引:11,自引:0,他引:11       下载免费PDF全文
Membrane cofactor protein (MCP or gp45-70) of the complement system is a cofactor for factor I-mediated cleavage of fluid-phase C3b and C3b-like C3, which opens the thioester bond. In the present study the activity of MCP was further characterized. Unexpectedly, in the absence of factor I, MCP stabilized the alternative- and, to a lesser extent, the classical-pathway cell-bound C3 convertases and thereby enhanced C3b deposition. Soluble MCP, if added exogenously, hardly functioned as cofactor for the cleavage of erythrocyte-bound C3b to iC3b; i.e. its activity, compared with the cofactor activity of factor H, was inefficient, since less than 10% of the bound C3b was MCP-sensitive. Further, exogenously added soluble MCP was also a weak cofactor for the cleavage of C3b bound to zymosan. Likewise, factor I, in the presence of cells bearing MCP, cleaved fluid-phase C3b inefficiently. These results imply that MCP has very little extrinsic cofactor activity for factor I. In contrast, exogenously added MCP and factor I mediated efficient cleavage of erythrocyte-bound C3b if the concentration of Nonidet P40 was sufficient to solubilize the cells. Interestingly, soluble MCP and factor I degraded C3b attached to certain solubilized acceptor membrane molecules more readily than others. The cleavage reaction of fluid-phase and cell-bound C3b by soluble MCP and factor I produced iC3b, but no C3c and C3dg. These and prior data indicate that soluble MCP has potent cofactor activity for fluid-phase C3b or C3b bound to solubilized molecules, but acts inefficiently towards C3b on other cells. This functional profile is unique for a C3b/C4b binding protein and, taken together with its wide tissue distribution, suggests an important role for MCP in the regulation of the complement system.  相似文献   

3.
Proteolytic inactivation of C4b is a crucial step for regulation of the classical complement pathway. A plasma protease factor I and membrane cofactors, C3b/C4b receptor (CR1) and membrane cofactor protein (MCP), participate in the regulation of cell-bound C4b although the physiological potency of these cofactors remains unknown. We have examined the optimal conditions of the factor I-mediated C4b regulatory system using purified cofactors. CR1 being a cofactor at a cofactor/C4b ratio less than 0.1 (w/w), fluid phase C4b, and methylamine-treated C4 (C4ma) were degraded by factor I into C4bi: minimal Cd4 was generated in the fluid phase. Liposome-bound C4b (LAC4b), on the other hand, was degraded into C4c and C4d. CR1 showed two optimal pHs (6.0 and 7.5) for fluid phase C4b, but one (6.0) for LAC4b, and in both cases low conductivity conditions enhanced the C4bi generation. CR1 cofactor activity was barely influenced by the NP-40 concentration. On the other hand, MCP degraded C4b and C4ma, as a factor I-cofactor, more efficiently into C4c and C4d. Though MCP cofactor activity, like that of CR1, was enhanced under low conductivity conditions, it has only one optimal pH, 6.0, in both fluid and solid phases. Furthermore, as in the case of C3b cleavage, a sufficient NP-40 concentration to solubilize membrane was needed for MCP to express full cofactor activity for C4b, in contrast to CR1. MCP was less potent for C4b inactivation than for C3b inactivation, while CR1 acted as a slightly more effective cofactor for C4b cleavage than for C3b cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

4.
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.  相似文献   

5.
The factor I-mediated cleavage of C3b, using factor H as a cofactor was completely inhibited by diisopropylfluorophosphate (DFP) when factor I and C3b were incubated with DFP before the addition of factor H. Inhibition, although to a lesser degree, was observed when factor H was present during DFP-exposure. No inhibition in factor I activity was seen when factor I and H were incubated with DFP either alone or together. It was also demonstrated that the 38-kDa subunit of factor I bound radiolabeled DFP when factor I and C3b together were exposed to DFP. These observations suggest that factor I interacts with C3b in a manner that exposes its catalytic site to DFP, an interaction that is independent of factor H. The inhibitory effect by DFP on factor I led us to further investigate the factor I cleavage products of iC3b, inasmuch as previous reports were ambiguous as to whether digestion occurs in the presence of DFP. Digestion of C3b bound to activated thiol Sepharose (ATS-C3b) in the presence of factor H at low pH and ionic strength and in serum by complement activation produced C3d,g-like fragments with apparent molecular mass of 41 and 43 kDa. These fragments were shown to have three different N-terminal and two different C-terminal ends. The major fragments had N-terminal sequences starting with Glu933, as shown by sequence determination. Traces of fragments extending beyond this point were also found, shown by Western blot analysis using a panel of mAb previously shown to bind to epitopes exposed within a region of C3 spanning residues 929 to 943, as well as a shorter fragment starting with Glu938. When digestion of C3b is carried out in the presence of DFP, the factor I level necessary for digestion is elevated and may explain how the first two cleavages producing iC3b but not the following giving C3d,g, can occur. The finding of several factor I cleavage sites in the C3d,g region of C3 demonstrates that factor I has a broad specificity, mainly for arginyl bonds. It has also been shown to digest a lysyl bond exposed in ATS-bound C3b.  相似文献   

6.
Trypsin treatment of human factor H (H160) [enzyme/substrate ratio 1:100 (w/w), 30 min, 37 degrees C] generated a 38 kDa (H38) and a 142 kDa (H142) fragment linked by disulphide bonds (H38/142). The fragments were purified by reduction with 2-mercapto-ethanol, gel filtration on a Sephadex G-200 column and affinity chromatography with monoclonal anti-(factor H) antibody coupled to Sepharose 4B. This monoclonal antibody bound to a site in the 38 kDa fragment. To localize the C3b binding site in factor H we used two enzyme-linked immunosorbent assays (e.l.i.s.a.). For the first test, e.l.i.s.a. plates were coated with C3b; H160, H38/142, H38 and H142 were added, and their binding was monitored by goat anti-(factor H) and peroxidase-labelled rabbit anti-goat antibodies. Only intact factor H bound to the C3b-coated plates. For the second test, e.l.i.s.a. plates were coated with comparable amounts of factor H or its fragments, and C3b was offered at several dilutions. In contrast with the results from the first assay, C3b bound to intact factor H, H38/142 and H38 but not to H142, thus characterizing H38 as the fragment carrying the C3b-binding site. To identify the fragment responsible for the cofactor activity of factor H (cleavage of fluid-phase C3b by factor I), 125I-C3b was incubated with either H38 or H142 and factor I. H142 had no cofactor activity, whereas H38 had the same cofactor function as intact H. To further investigate the relationship between the C3b-binding site and the site of factor H essential for its cofactor activity, we made use of monoclonal antibodies directed against the H38. Those antibodies inhibiting the binding of C3b to H160 also inhibited the cofactor function, whereas those without effect on the C3b binding also did not interfere with the cofactor activity. This suggests that the C3b-binding site and the site essential for the cofactor activity of factor H are both localized in the 38 kDa tryptic fragment of factor H in close proximity or are identical.  相似文献   

7.
C3b/C4b receptor (CR1) and membrane cofactor protein (MCP) are integral membrane glycoproteins with factor I-dependent cofactor activity. They bind to C3b, allowing factor I to cleave C3b at two sites (first and second cleavage), which results in the generation of C3bi, a hemolytically inactive form which is a ligand for complement receptor type three (CR3). C3bi is further degraded by factor I and CR1 (third cleavage) to C3dg (a ligand for complement receptor type two, CR2) and C3c. Using two different substrates, fluid-phase C3b and cell-bound C3b, the cleavage of C3b by MCP and factor I was compared to that by CR1 and factor I under various conditions. The optimal pH for the first and second cleavage of either substrate was 6.0 for MCP and 7.5 for CR1. The third cleavage was mediated only by CR1 and factor I, the optimal pH being 8.0. Low ionic conditions enhanced the C3b binding and cofactor activity of both CR1 and MCP. The efficiency of binding C3b to CR1 or MCP was maximal at pH 6.2. The isoelectric point (pI) of MCP was acidic (approximately 4.0), while that of CR1 was 6.8. Therefore, compared to CR1, MCP possesses distinct functional profiles relative to C3b-binding and factor I-cofactor activity.  相似文献   

8.
The co-culture of Day-15 guinea-pig conceptuses or Day-15 pregnant guinea-pig endometrium with Day-15 non-pregnant guinea-pig endometrium had no inhibitory effect on PGF2 alpha output from the non-pregnant endometrium. Unpurified proteins secreted by the Day-15 guinea-pig conceptuses, or these proteins purified by Blue Sepharose CL-6B and ion-exchange column chromatography also had no inhibitory effect on PGF2 alpha output from Day-15 non-pregnant guinea-pig endometrium cultured in vitro. However, following the further purification of guinea-pig conceptus secreted proteins on Sephadex G-75SF, the proteins present in fraction F3:4 inhibited PGF2 alpha output from the Day-15 non-pregnant guinea-pig endometrium during the first 6 h of culture. The major protein present in F3:4 had a molecular weight of 38.2 kDa on SDS-PAGE. Proteins present in F3:4 formed only a minor proportion of the total proteins secreted. Nevertheless, the anti-luteolytic factor secreted by the guinea-pig conceptus may be this 38.2 kDa protein, but further study is required.  相似文献   

9.
1. Some properties of the triphosphoinositide phosphomonoesterase from the homogenates of guinea-pig brain were studied. The enzyme has an optimum pH range 6.7-7.3, is stimulated with KCl at a concentration of 0.1m, and under these conditions has K(m)1.43x10(-4)m. 2. A factor from the ;pH5 supernatant' of guinea-pig brain stimulates the enzyme activity over and above the stimulation produced by KCl. Subcellular fractions of guinea-pig brain varied in their response to the ;pH5 supernatant'. Maximum stimulation was observed with the P(1) fraction, containing myelin and nuclei. 3. An assay system for the enzyme was developed that contained optimum concentrations of both KCl and the ;pH5 supernatant'. Acid phosphatases were inhibited by NaF, but, in contrast with previous work, no EDTA was added to the assay system to inhibit the alkaline phosphatases. This reagent inhibited the triphosphoinositide phosphomonoesterase. It was estimated that the remaining fraction of non-specific phosphatases can account for only 14% of the observed triphosphoinositide phosphomonoesterase activity. 4. Subcellular fractions of guinea-pig brain were characterized by electron microscopy and subcellular markers. The triphosphoinositide phosphomonoesterase exhibited a distribution between the fractions similar to that of 5'-nucleotidase, but different from that of alkaline phosphatase.  相似文献   

10.
We have identified and characterized C3b binding proteins of two primates, orangutan (Pongo pygmaeus) and gorilla (Gorilla gorilla). Detergent solubilized 125I surface-labeled E and PBMC were subjected to affinity chromatography with homologous or human iC3/C3b. These ligands bound a 225,000 single chain protein from orangutan E and PBMC and a 220,000 protein from gorilla E. Proteins of the same Mr were immunoprecipitated by a rabbit polyclonal and two murine mAb to the human CR1 (CD35). The C3b binding protein of gorilla E aligned with that of the common human CR1 polymorphic size variant. Human or orangutan iC3 was also a ligand for a surface-labeled protein doublet of 59,000 and 65,000 from orangutan E. The doublet pattern and mol wts are similar to membrane cofactor protein (or CD46). Further, this doublet was immunoprecipitated by a mAb to human MCP. The MCP-like protein doublet was not isolated from gorilla or human E. Decay accelerating factor (DAF) of orangutan E was also identified and was structurally and antigenically distinct from the MCP-like protein. Orangutan or gorilla E preparations were a cofactor for the cleavage of human iC3 by human factor I and produced the same cleavage fragments as human CR1. Cofactor activity of orangutan E was partially inhibited by preclearance of CR1 and more completely inhibited by preclearance of MCP. Cofactor activity of gorilla E was inhibited by coincubation with a monoclonal antibody to human CR1. These data indicate that the orangutan and gorilla high m.w. proteins are equivalent to human CR1. The orangutan E membrane protein doublet with m.w. of 59,000 and 65,000 possesses biochemical, antigenic, and functional properties of human membrane cofactor protein.  相似文献   

11.
Complement inhibition is to a large extent achieved by proteolytic degradation of activated complement factors C3b and C4b by factor I (FI). This reaction requires a cofactor protein that binds C3b/C4b. We found that the cofactor activity of C4b-binding protein towards C4b/C3b and factor H towards C3b increase at micromolar concentrations of Zn(2+) and are abolished at 2 mM Zn(2+) and above. 65Zn(2+) bound to C3b and C4b molecules but not the cofactors or FI when they were immobilized in a native form on a nitrocellulose membrane. Zn(2+) binding constants for C3met (0.2 microM) and C4met (0.1 microM) were determined using fluorescent chelator. It appears that higher cofactor activity at low zinc concentrations is due to an increase of affinity between C4b/C3b and cofactor proteins as assessed by surface plasmon resonance. Inhibition of the reaction seen at higher concentrations is due to aggregation of C4b/C3b.  相似文献   

12.
Digestion of methylamine-treated alpha 2-macroglobulin (alpha 2M X MA) with catalytic amounts of papain at pH 4.5 has been investigated. Cleavage of Lys(1313)-Glu resulted in two major products, which could be separated by gel chromatography: a large disulfide bridged fragment set nearly the size of intact alpha 2M X MA, and an 18 kDa fragment, constituting the carboxy-terminal domain of alpha 2M. This domain contained the receptor recognition site, exposed as a result of cleavage of the internal beta-cysteinyl-gamma-glutamyl thiol esters in alpha 2M. Compared with alpha 2M-trypsin complex the apparent affinity for binding to rat hepatocyte receptors was 0.1 and 2% at 4 and 37 degrees C, respectively. The receptor-binding domain presumably forms a compact globular beta-barrel-type structure, stable at pH 2.5-9.0. Chemical modification experiments suggest that receptor binding is contributed by a determinant formed by the precise folding of the polypeptide chain.  相似文献   

13.
Guinea-pig alveolar macrophages were harvested by bronchoalveolar lavage and purified by differential adhesion. They were labeled with 14C-Arachidonic acid and then exposed to platelet-activating factor or to the calcium ionophore A23187. The activity of cellular phospholipase A2 was considered as the release of free 14C-Arachidonic acid in the cell supernatant. The pretreatment of guinea-pig alveolar macrophages with two lipocortin-like proteins (36 kDa and 40 kDa) purified from mice lung induced a significant inhibition of their phospholipase A2 activity upon platelet-activating factor and calcium ionophore stimulation. These results indicate that lipocortin-like proteins can modulate the phospholipase A2 activity of isolated cells in vitro.  相似文献   

14.
Factor H (FH) is the predominant soluble inhibitor of the complement system. With a concentration of 200-800 microg/ml in human and rat plasma it acts as a cofactor for the soluble factor I (FI)-mediated cleavage of the component C3b to iC3b. Furthermore it competes with factor B for binding to C3b and C3(H2O) and promotes the dissociation of the C3bBb complex. FH is a monomer of about 155 kDa which comprises 20 short consensus repeats (SCR), each of which is composed of approximately 60 amino acid (aa) residues. Two functional fragments of FH comprising the SCR1-4 or SCR1-7 were generated using either the Baculovirus system or stably transfected human embryonal kidney cells, respectively. These fragments, as well as FH purified from rat serum, were first analyzed for their relative molecular weights (Mr) using non-reducing or reducing SDS-PAGE. The Mr of the FH variants differed by about 20% depending on the experimental conditions employed. Only the Mr of proteins separated under reducing conditions were in accordance with the MW calculated from the aa sequence. Analyses of the glycosylation patterns using PAS-staining showed a lack of staining of the recombinant variants (SCR1-4 and SCR1-7) in contrast to FH(SCR1-20) from serum. Using a complement hemolysis assay (CH50-assay) all three variants exhibited a molar complement inhibitory activity of FH(1-20)/FH(1-7)/FH(1-4) of about 3/1/1. These data support the postulated model of FH bearing three binding sites for its ligand C3b, from which one is located in the SCR1-4, whereas the other two are located in the SCR8-20.  相似文献   

15.
Human membrane cofactor protein (MCP, CD46) is a 45-70 kDa protein with genetic and tissue-specific heterogeneity, and is expressed on all nucleated cells. MCP consists from N-terminus of 4 short consensus repeats (SCRs), 1-3 serine/threonine-rich (ST) domains, a transmembrane domain (TM) and a cytoplasmic tail (CYT). More than 8 isoforms are generated secondary to alternative splicing due to combinations of various exons encoding the ST, TM and CYT domains. It serves as a cofactor of serine protease factor I for inactivation of complement C3b and C4b. Its primary role is to protect host cells from homologous complement attack by inactivating C3b/C4b deposited on the membrane. It also acts as receptors for measles virus (MV), some kinds of bacteria and for a putative ligand on oocytes. MV infection causes temporal host immune suppression, which may appear secondary to signaling events through MCP on macrophages and dendritic cells. These functional properties of human MCP may facilitate xenotransplantation and may be useful in the generation of animal models of measles by creating human MCP-expressing animals.  相似文献   

16.
Crystals of methylamine-treated alpha 2-macroglobulin (alpha 2M-MA), alpha 2-macroglobulin in complex with two molecules of trypsin, alpha 2M-T2, one molecule of plasmin, alpha 2M-PL, and one molecule of plasmin followed by methylamine-treatment, alpha 2M-PL(MA), have reproducibly been obtained using ammonium sulfate or magnesium sulfate as precipitants. The crystals are fragile tetragonal bipyramids of up to 1.5 mm in length. Crystals of alpha 2M-MA diffracted to at least 9 A resolution, crystals of alpha 2M-T2 diffracted to 10 A resolution and crystals of alpha 2M-PL and alpha 2M-PL(MA) diffracted to 11 A resolution. For alpha 2M-MA the cell parameters were determined as: a=b=257 A, c=555 A; and for alpha 2M-T2 as: a=b=247 A, c=559 A. For both preparations the space group was I4(1)22. As estimated from density measurements, the crystals of alpha 2M-MA and alpha 2M-T2 contain one 360 kDa alpha 2M dimer per asymmetric unit. The volume of the asymmetric unit/molecular weight, Vm, was estimated at 5.6 A3/Da. The crystal parameters of alpha 2M-PL and alpha 2M-PL(MA) were not determined.  相似文献   

17.
Herpesvirus saimiri (HVS) is a lymphotropic virus that causes T-cell lymphomas in New World primates. It encodes a structural homolog of complement control proteins named complement control protein homolog (CCPH). Previously, CCPH has been shown to inhibit C3d deposition on target cells exposed to complement. Here we have studied the mechanism by which it inactivates complement. We have expressed the soluble form of CCPH in Escherichia coli, purified to homogeneity and compared its activity to vaccinia virus complement control protein (VCP) and human complement regulators factor H and soluble complement receptor 1. The expressed soluble form of CCPH bound to C3b (KD = 19.2 microm) as well as to C4b (KD = 0.8 microm) and accelerated the decay of the classical/lectin as well as alternative pathway C3-convertases. In addition, it also served as factor I cofactor and supported factor I-mediated inactivation of both C3b and C4b. Time course analysis indicated that although its rate of inactivation of C4b is comparable with VCP, it is 14-fold more potent than VCP in inactivating C3b. Site-directed mutagenesis revealed that Arg-118, which corresponds to Lys-120 of variola virus complement regulator SPICE (a residue critical for its enhanced C3b cofactor activity), contributes significantly in enhancing this activity. Thus, our data indicate that HVS encodes a potent complement inhibitor that allows HVS to evade the host complement attack.  相似文献   

18.
A method has been developed for the purification to homogeneity of guinea-pig complement component C2. Contrary to previous reports, guinea-pig C2 is a single polypeptide chain with apparent mol.wt. of 102000, the same as human C2. It is cleaved by C1s to yield fragments C2a (apparent molwt. 74000) and C2b (apparent mol.wt. 34000). The amino acid composition and N-terminal sequences of these fragments are similar to those of human C2a and C2b. Human and guinea-pig C2 show more extensive sequence homology to Factor B than previously identified. The known homology around the sites of cleavage by C1s and Factor D has now been extended by a stretch of ten identical or conservatively substituted residues. Sequence homology has now been identified at the N-terminal of C2b and Factor Ba. The properties of the classical-pathway C3 convertases assembled from human C4b, C1s and human or guinea-pig C2 have been compared. The rates of cleavage of human and guinea-pig C2 by C1s (and therefore the rates of assembly of the C3 convertases) are similar. The rate of decay of the activity of the C3 convertase formed from guinea-pig C2 is 10-fold lower than for human C2. This greater stability reflects a higher affinity of guinea-pig C2a for human C4b. The presence of C2b is not necessary for C3 convertase activity.  相似文献   

19.
Human C4b-binding protein (C4bp) facilitates the factor I-mediated proteolytic cleavage of the active forms of complement effectors C3b and C4b into their inactive forms. C4bp comprises a disulfide-linked heptamer of alpha-chains with complement (C) regulatory activity and a beta-chain. Each alpha-chain contains 8 short consensus repeat (SCR) domains. Using SCR-deletion mutants of recombinant multimeric C4bp, we identified the domains responsible for the C3b/C4b-binding and C3b/C4b-inactivating cofactor activity. The C4bp mutant with deletion of SCR2 lost the C4b-binding ability, as judged on C3b/C4b-Sepharose binding assaying and ELISA. In contrast, the essential domains for C3b-binding extended more to the C-terminus, exceeding SCR4. Using fluid phase cofactor assaying and deletion mutants of C4bp, SCR2 and 3 were found to be indispensable for C4b cleavage by factor I, and SCR1 contributed to full expression of the factor I-mediated C4b cleaving activity. On the other hand, SCR1, 2, 3, 4, and 5 participated in the factor I-cofactor activity for C3b cleavage, and SCR2, 3, and 4 were absolutely required for C3b inactivation. Thus, different sets of SCRs participate in C3b and C4b inactivation, and the domain repertoire supporting C3b cofactor activity is broader than that supporting C4b inactivation by C4bp and factor I. Furthermore, the domains participating in C3b/C4b binding are not always identical to those responsible for cofactor activity. The necessity of the wide range of SCRs in C3b inactivation compared to C4b inactivation by C4bp and factor I may reflect the physiological properties of C4bp, which is mainly directed to C4b rather than C3b.  相似文献   

20.
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.  相似文献   

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