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1.
The low C5 convertase activity of the C4A6 allotype of human complement component C4. 总被引:1,自引:0,他引:1
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We have compared the C5-convertase-forming ability of different C4 allotypes, including the C4A6 allotype, which has low haemolytic activity and which has previously been shown to be defective in C5-convertase formation. Recent studies suggest that C4 plays two roles in the formation of the C5 convertase from the C3 convertase. Firstly, C4b acts as the binding site for C3 which, upon cleavage by C2, forms a covalent linkage with the C4b. Secondly, C4b with covalently attached C3b serves to form a high-affinity binding site for C5. Purified allotypes C4A3, C4B1 and C4A6 were used to compare these two activities of C4. Covalently linked C4b-C3b complexes were formed on sheep erythrocytes with similar efficiency by using C4A3 and C4B1, indicating that the two isotypes behave similarly as acceptors for covalent attachment of C3b. C4A6 showed normal efficiency in this function. However, cells bearing C4b-C3b complexes made from C4A6 contained only a small number of high-affinity binding sites for C5. Therefore a lack of binding of C5 to the C4b C3b complexes is the reason for the inefficient formation of C5 convertase by C4A6. The small number of high-affinity binding sites created, when C4A6 was used, were tested for inhibition by anti-C3 and anti-C4. Anti-C4 did not inhibit C5 binding, whereas anti-C3 did. This suggests that the sites created when C4A6 is used to make C3 convertase may be C3b-C3b dimers, and hence the low haemolytic activity of C4A6 results from the creation of low numbers of alternative-pathway C5-convertase sites. 相似文献
2.
M J Anderson C M Milner R G Cotton R D Campbell 《Journal of immunology (Baltimore, Md. : 1950)》1992,148(9):2795-2802
The C4A6 allotype of the human complement component C4 is known to be defective in C5 binding within the C5 convertase. To characterize the position and nature of the molecular defect in the C4A6 allotype we have isolated the C4A6 gene from a cosmid genomic DNA library. Direct sequencing of a 4.4-kb region of the gene covering exons 17 to 31 and encoding the C4d fragment and most of the rest of the alpha chain of C4 revealed that the C4A6 allele encodes the A isotypic residues Pro Cys-Leu Asp at positions 1101, 1102, 1105, and 1106 and the same residues as the C4A3 alpha gene at the polymorphic positions 1054 (Asp), 1157 (Asn), 1182 (Thr), 1188 (Val), 1191 (Leu) and 1267 (Ala). In addition the C4A6 allele was shown to encode a Pro at the previously characterized polymorphic position 707 in the C4a peptide where the C4A3 alpha allele encodes a Leu. The remaining 26 exons of the C4A6 gene were analyzed by detecting nucleotide mismatches in C4A6/C4A3 and C4A6/C4B1 DNA heteroduplexes using the chemical cleavage of mismatch technique. The regions around detected mismatches were sequenced. In total seven nucleotide differences were defined on comparison of the C4A6 and other C4 sequences, of which three were present in exons. Two of these resulted in amino acid changes. One of the amino acid differences is a known polymorphism in C4, a Tyr/Ser substitution at position 328 in the beta-chain. The second amino acid difference caused by a C to T transition in the first base of the codon for amino acid residue 458 was the only one shown to be specific to the C4A6 allotype. The C4A6 allotype contains a Trp residue at this position in the beta-chain instead of the Arg residue found in all other C4A and C4B allotypes so far characterized. We propose that this Arg to Trp substitution at beta-chain residue 458 is responsible for the inability of C4A6 to bind C5 in the C5 convertase. 相似文献
3.
The origin of the very variable haemolytic activities of the common human complement component C4 allotypes including C4-A6. 总被引:7,自引:0,他引:7
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The human complement component C4 occurs in many different forms which show big differences in their haemolytic activities. This phenomenon seems likely to be of considerable importance both physiologically and pathologically. C4 is coded by duplicated genes between HLA-D and HLA-B loci in the major histocompatibility complex in man. Several fold differences in haemolytic activity between products of the two loci C4-A and C4-B have been correlated with changes of six amino acid residues in this large protein of 1722 residues and with differences of several fold in the covalent binding of C4 to antibody-antigen aggregates. Some allotypes of one locus also differ markedly, notably C4-A6 which has 1/10th the haemolytic activity of other C4-A allotypes. A monoclonal antibody affinity column has been prepared which is able to separate C4-A from C4-B proteins and, using serum from an individual expressing only the C4-A6 allele at the C4-A locus, C4-A6 protein has been prepared. Investigation has shown C4-A6 to have the same reactivity as other C4-A allotypes except in the formation of the complex protease, C5 convertase. This protease is formed from C4, C2 and C3 and if C4-A6 is used it has approximately 1/5th the catalytic activity compared with other C4-A allotype. Allelic differences in sequence identified in C4 proteins so far are few and it is probable that the big difference in catalytic activity of C5 convertase is caused by very small changes in structure. 相似文献
4.
Y U Kim M C Carroll D E Isenman M Nonaka P Pramoonjago J Takeda K Inoue T Kinoshita 《The Journal of biological chemistry》1992,267(6):4171-4176
C5 convertase of the classical complement pathway is a protein complex consisting of C4b, C2a, and C3b. Within this complex C3b binds to C4b via an ester linkage. We now present evidence that the covalent C3b-binding site on human C4b is Ser at position 1217 of C4. We also show that formation of the covalently linked C4b.C3b complex occurs in the mouse complement system and that the C3b-binding site on mouse C4b is Ser at position 1213 which is homologous to Ser-1217 of human C4. Therefore, covalent binding of C3b to a single specific site on C4b within the classical pathway C5 convertase is likely a common phenomenon in the mammalian complement system. Specific noncovalent association of metastable C3b with C4b would occur first, leading to reaction of the thioester with a specific hydroxy group. This is supported by two lines of experimental evidence, one which shows that a mutant C4 that does not make a covalent linkage with C3b is still capable of forming C5 convertase and a second in which the C4b.C3b complex has been demonstrated by cross-linking erythrocytes bearing this C5 convertase. 相似文献
5.
A covalent dimer of complement C4b serves as a subunit of a novel C5 convertase that involves no C3 derivatives 总被引:1,自引:0,他引:1
T Masaki M Matsumoto R Yasuda R P Levine H Kitamura T Seya 《Journal of immunology (Baltimore, Md. : 1950)》1991,147(3):927-932
A C intermediate, LAC14, was prepared from TNP-aminocaproyl liposomes sensitized with anti-TNP antibody (Ab) and purified human C1 and C4. LAC14, containing radiolabeled C4, was analyzed by SDS-PAGE followed by autoradiography, and yielded a 210-kDa band and a predominant 400-kDa band. The 210-kDa band consisted of monomeric C4b bound to low molecular mass acceptors. The 400-kDa band was comprised of a 200-kDa moiety, as well as beta- and gamma-chains of C4. The 200-kDa moiety contained neither C1 nor sensitizing Ab, but it was largely decreased by treatment with NH2OH to the 90-kDa moiety with the mobility corresponding to the alpha'-chain of C4b. A covalent dimer of C4b, therefore, is the predominant form of C4b deposited on liposomes sensitized with antibody. The C4b-C4b dimer formed rapidly (within 5 min) followed by slow dissociation into monomers. The LAC14 bearing the C4b dimer but not the monomer was lysed, although with relatively low efficiency, by the addition of oxyC2 and EDTA-supplemented C3-deficient serum (C3DS), and, furthermore, LAC142 possessed the ability to convert C5 into C5a and C5b. Moreover, lysis was inhibited not by anti-C3 Ab but by anti-C4 Ab. In other experiments, the dimer served as an element of C3 convertase, as well. These findings imply that the C4b dimer, when complexed with C2, expresses C3/C5 convertase activity without participation of C3, and may provide a molecular mechanism whereby sera from patients with complete C3 deficiency retain the ability to induce C-mediated cytolysis. 相似文献
6.
Complement C2 receptor inhibitor trispanning (CRIT) of the Schistosoma parasite binds human C2 via the C2a segment. The receptor in vivo functions as C2 decoy receptor by directly competing with C4b for binding to C2. As a result, CRIT is able to limit the extent of classical pathway (CP) C3 convertase formation. We report that the CRIT-extracellular domain 1 (ed1) peptide inhibits CP-mediated complement activation with an ICH(50) of approximately 0.1 microM, the C-terminal 11 aa of CRIT-ed1, named H17, even more effectively. The beta-chain region F222-Y232 of C4 shares 55% identity and 73% similarity with H17. Peptides based on this region also inhibit CP in a dose-dependent manner. As further evidence of C2 binding we showed CRIT-ed1 peptides and homologous C4 beta-chain peptides to inhibit complement in C2 hemolytic assays. We have predicted C4 beta-c F222-Y232 as a C2 binding site which we have termed the CRIT-ed1 domain, and the sequence [F/H]EVKX(4/5)P as a consensus C2-binding sequence. Anti-CRIT-ed1 cross-reacts with the C4 beta-chain and F222EVKITPGKPY232 appears to be the key epitope recognized by this Ab. Furthermore, anti-CRIT-ed1 was found to inhibit CP activation in a total hemolytic assay. We believe that Schistosoma CRIT-ed1, as well as C4 beta-chain peptides based on the CRIT-ed1 domain, function as interface peptides. These peptides, based on C2-binding sequences in CRIT, or C4, competitively inhibit the binding of C2 to C4b and thus limit the activation of C. The C4 peptides, unlike CRIT-ed1, did not inhibit the cleavage of C2 by C1s. 相似文献
7.
Nunn MA Sharma A Paesen GC Adamson S Lissina O Willis AC Nuttall PA 《Journal of immunology (Baltimore, Md. : 1950)》2005,174(4):2084-2091
Blood-feeding ticks must control C activation or be damaged by the host inflammatory response. We report the characterization and expression of a novel, relatively small, broad-acting C inhibitory protein (termed OmCI) from the soft tick Ornithodoros moubata. The native 17-kDa nonglycosylated protein inhibits both human and guinea pig classical and alternative C activation pathways. The IC50 values for each pathway were 12 and 27 nM, respectively, in hemolytic assays using human serum diluted 40-fold. The cDNA encodes a protein of 168 aa, including an 18-aa secretion signal sequence that is absent in the mature form. The inhibitor has 46% amino acid identity with moubatin, a platelet aggregation inhibitor also from O. moubata that is an outlying member of the lipocalin family. Native OmCI had no inhibitory effect on the addition of C8 and C9 to preformed C5b-C7 and C5b-C8 to form the membrane attack complex and no effect on the rate of C3a production by the C3 convertase enzymes C4bC2a, C3(H2O)Bb, or C3bBb. Both recombinant and native OmCI abolish production of C5a by human classical (C4bC3bC2a) and alternative (C3bC3bBb) C5 convertases. Addition of excess C5 but not C3 competes away the inhibitory activity of OmCI, indicating that OmCI targets C5 itself rather than inhibiting the C5 convertase C4bC3bC2a itself. Direct binding of OmCI to C5 was demonstrated by Western blotting and gel filtration chromatography using 125I-labeled proteins. OmCI is the first lipocalin family member shown to inhibit C and also the first natural inhibitor that specifically targets the C5 activation step. 相似文献
8.
The conversion of human complement component C5 into fragment C5b by the alternative-pathway C5 convertase. 总被引:1,自引:1,他引:0
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R G DiScipio 《The Biochemical journal》1981,199(3):497-504
The cleavage of human complement component C5 to fragment C5b by the alternative pathway C5 convertase was studied. The alternative-pathway C5 convertase on zymosan can be represented by the empirical formula zymosan--C3b2BbP. Both properdin-stabilized C3 and C5 convertase activities decay with a half life of 34 min correlating with the loss of the Bb subunit. The C5 convertase functions in a stepwise fashion: first, C5 binds to C3b and this is followed by cleavage of C5 to C5b. The capacity to bind C3b is a stable feature of component C5, as C5b also has this binding capacity. Component C5, unlike component C3, does not form covalent bonds with zymosan after activation, and C5 is not inhibited by amines. Therefore C5, although similar in structure to C3, does not appear to contain the internal thioester group reported for C3 and C4. 相似文献
9.
Localization of the covalent C3b-binding site on C4b within the complement classical pathway C5 convertase, C4b2a3b 总被引:3,自引:0,他引:3
H Kozono T Kinoshita Y U Kim Y Takata-Kozono S Tsunasawa F Sakiyama J Takeda K Hong K Inoue 《The Journal of biological chemistry》1990,265(24):14444-14449
C5 convertase of the classical complement pathway is a trimolecular protein complex consisting of C4b, C2a, and C3b. In the complex there is an ester bond between C3b and C4b. We analyzed the C5 convertase formed on erythrocytes and localized the covalent binding site of C3b to a small region on C4b. The covalently linked C4b.C3b complex was purified from a detergent extract of the erythrocytes and digested with lysyl endopeptidase. An Mr 17,000 fragment containing the ester linkage between C4b and C3b was purified and its amino-terminal sequence was examined. Two amino acids were obtained at each cycle and identified with those in the sequences of C3 and C4. The sequence derived from C3 corresponded to the thioester region. The sequence derived from C4 started at Ala-1186. Alkali treatment of the fragment yielded an Mr 7,000 peptide derived from C4, which thus appeared to span the region of C4 from Ala-1186 to Lys-1259. Therefore, the covalent C3b-binding site on C4b is located within a 74-residue region of the primary structure. This finding supports the notion that after cleavage of C3 by the C4b2a complex, the covalent binding of metastable C3b to C4b is a specific reaction to form a trimolecular complex with a defined quaternary structure. 相似文献
10.
The binding of human complement proteins C5, factor B, beta 1H and properdin to complement fragment C3b on zymosan. 总被引:8,自引:5,他引:3
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R G DiScipio 《The Biochemical journal》1981,199(3):485-496
The covalent binding of complement fragment C3b to zymosan by the action of the alternative-pathway C3 convertase and the reversible binding of several complement proteins (component C5, factor B, beta 1H and properdin) to C3b on zymosan have been investigated. When C3b is deposited on zymosan after activation by a surface-bound C3 convertase, the C3b molecules are deposited in foci around the C3 convertase site, with an average of 30 C3b molecules per site. The association constants of C5, factor B, beta 1H, and properdin for C3b bound to zymosan have been determined. The association constants ranged from 6.5 x 10(-5) M-1 for factor B to 2.9 x 10(7) M-1 for properdin. An approximate stoichiometry of 1 : 1 for C5, factor B, and properdin binding to C3b has been observed. Curvilinear Scatchard plots were observed for beta 1H binding to C3b, with the maximal extrapolated ratio of beta 1H to C3b of 0.32. Physiological amounts of properdin increase by 7-fold the affinity constant for factor B binding to C3b with no alteration in the stoichiometry. Similarly, physiological amounts of factor B increase the affinity constant of properdin to C3b about 4-fold with only a small measured difference in stoichiometry. Competition binding studies and protein modification suggest that C5, factor B, beta 1H, and properdin each bind to a distinct region on C3b. 相似文献
11.
C3/C5 convertase is a serine protease that cleaves C3 and C5. In the present study we examined the C5 cleaving properties of classical pathway C3/C5 convertase either bound to the surface of sheep erythrocytes or in its free soluble form. Kinetic parameters revealed that the soluble form of the enzyme (C4b,C2a) cleaved C5 at a catalytic rate similar to that of the surface-bound form (EAC1,C4b,C2a). However, both forms of the enzyme exhibited a poor affinity for the substrate, C5, as indicated by a high Km (6-9 microM). Increasing the density of C4b on the cell surface from 8,000 to 172,000 C4b/cell did not influence the Km. Very high affinity C5 convertases were generated only when the low affinity C3/C5 convertases (EAC1,C4b,C2a) were allowed to deposit C3b by cleaving native C3. These C3b-containing C3/C5 convertases exhibited Km (0.0051 microM) well below the normal concentration of C5 in blood (0.37 microM). The data suggest that C3/C5 convertase assembled with either monomeric C4b or C4b-C4b complexes are inefficient in capturing C5 but cleave C3 opsonizing the cell surface with C3b for phagocytosis. Deposition of C3b converts the enzymes to high affinity C5 convertases, which cleave C5 in blood at catalytic rates approaching Vmax, thereby switching from C3 to C5 cleavage. Comparison of the kinetic parameters with those of the alternative pathway convertase indicates that the 6-9-fold greater catalytic rate of the classical pathway C5 convertase may compensate for the fewer numbers of C5 convertase sites generated upon activation of this pathway. 相似文献
12.
van den Elsen JM Martin A Wong V Clemenza L Rose DR Isenman DE 《Journal of molecular biology》2002,322(5):1103-1115
C4 fulfills a vital role in the propagation of the classical and lectin pathways of the complement system. Although there are no reports to date of a C4 functional activity that is mediated solely by the C4d region, evidence clearly points to it having a vital role in a number of the properties of native C4 and its major activation fragment, C4b. Contained within the C4d region are the thioester-forming residues, the four isotype-specific residues controlling the C4A/C4B transacylation preferences, a binding site for nascent C3b important in assembling the classical pathway C5 convertase and determinants for the Chido/Rodgers (Ch/Rg) blood group antigens. In view of its functional importance, we undertook to determine the three-dimensional structure of C4d by X-ray crystallography. Here we report the 2.3A resolution structure of C4Ad, the C4d fragment derived from the human C4A isotype. Although the approximately 30% sequence identity between C4Ad and the corresponding fragment of C3 might be expected to establish a general fold similarity between the two molecules, C4Ad in fact displays a fold that is essentially superimposable on the structure of C3d. By contrast, the electrostatic characteristics of the various faces of the C4Ad molecule show marked differences from the corresponding faces of C3d, likely reflecting the differences in function between C3 and C4. Residues previously predicted to form the major Ch/Rg epitopes were proximately located and accessible on the concave surface of C4Ad. In addition to providing further insights on the current models for the covalent binding reaction, the C4Ad structure allows one to rationalize why C4d is not a ligand for complement receptor 2. Finally the structure allows for the visualization of the face of the molecule containing the binding site for C3b utilized in the assembly of classical pathway C5 convertase. 相似文献
13.
Glycoprotein C of herpes simplex virus 1 is an inhibitor of the complement cascade 总被引:20,自引:0,他引:20
L F Fries H M Friedman G H Cohen R J Eisenberg C H Hammer M M Frank 《Journal of immunology (Baltimore, Md. : 1950)》1986,137(5):1636-1641
Mammalian cells in culture express membrane receptors for C3b when infected with HSV-1. C3b binding is mediated by glycoprotein C (gC), a virus-specified membrane glycoprotein. In view of the inhibitory functions of other C3b-binding proteins, we studied the capacity of gC to modulate complement activation. Glycoprotein C was purified from HSV-1-infected cells by immunoaffinity chromatography. Glycoprotein C, but not a control viral glycoprotein, demonstrated dose-dependent acceleration of decay of C3bBb sites. In addition, gC produced a dose-dependent, time-independent depression of the overall hemolytic efficiency of C3bBb sites. Inhibition of C5b6-initiated reactive lysis of cells bearing C3b, but not cells bearing antibody alone, by gC suggests that the second effect represents interference with the C3b-C5/5b interaction. This hypothesis is supported by the failure of gC to inhibit reactive lysis when added after C5b67 insertion into target cells. Glycoprotein C does not accelerate C14b2a decay, nor does it impair classical pathway hemolytic efficiency when excess C5 is present. By limiting available C5/5b, some gC inhibition of C3b-C5/5b interactions can be unmasked in the classical pathway system. Glycoprotein C is devoid of factor I co-factor activity. HSV-1 gC is a modulator of complement activation, especially via the alternative pathway, and may represent a novel viral mechanism for evading host defense processes. 相似文献
14.
T Horiuchi K J Macon J A Engler J E Volanakis 《Journal of immunology (Baltimore, Md. : 1950)》1991,147(2):584-589
We probed the functional significance of the region around Cys-241 in human C2 by testing the hemolytic activity of a series of mutant rC2. Mutant C2 cDNA were constructed by oligonucleotide-directed site-specific mutagenesis and expressed transiently in COS cells. Wild-type rC2 had threefold higher specific hemolytic activity than native serum C2. Substitution of Gly, Ala, or Ser for Cys-241 resulted in a slightly, but significantly, increased activity. In addition, I2 had no effect on the activity of these mutant C2. Substitution of Lys for Gln-243 increased the hemolytic activity by more than two-fold. Increased activity in all cases was due to slower decay rates of the C3 convertase. Finally, substitution of Leu or Ala for Asp-240 or Ser-244, respectively, resulted in more than 100-fold decrease of hemolytic activity. The results suggest that residues 240 to 244 of human C2 represent an important structural determinant of the C4b binding site of C2a. They also confirm that Cys-241 is the residue responsible for the increased activity of C2 reacted with I2. 相似文献
15.
Surface-dependent modulation by H of C5 cleavage by the cell-bound alternative pathway C5 convertase of human complement 总被引:1,自引:0,他引:1
Regulation by H of formation of the C3 and C5 alternative pathway convertases of complement on cells is dependent on such chemical characteristics of the cell surfaces as their membrane content in sialic acid. Properdin-stabilized C5 convertase sites were assembled on the non-activating cells of the alternative pathway, sheep erythrocytes (Es), and on the activating cells, desialated Es and rabbit erythrocytes (Er). C5 hemolytic sites were revealed by incubation of the convertase-bearing cells with limiting C5 and excess C6-C9. H inhibited generation of C5 hemolytic sites in a dose-related fashion on Es, Er, and desialated Es at molar ratios of H/C5 of 0.03 to 0.5. H similarly inhibited C5 utilization by the cell-bound C5 convertase on Es and desialated Es regardless of the cell membrane sialic acid content; however, H was three to five times less effective on Er. Kinetic experiments also suggested that C5 hemolytic sites are generated more rapidly on Er than on Es and desialated Es. The inhibition effect of H was independent of the number of C5 convertase sites per cell on all cell types; two to three times more residual hemolytic sites were found on convertase-bearing Es that had been incubated with C5 and H as compared with cells that had been decayed by H before incubation with C5. Furthermore, H also inhibited C5 interaction with a preformed classical pathway C5 convertase. These results suggest that H interacts with C5 so as to alter C5 binding and/or cleavage by the cell-bound C5 alternative pathway convertase. Sialic acid-independent modulation by H of C5 cleavage by the C5 convertase represents an additional regulatory step in the activation of the human alternative complement pathway. 相似文献
16.
The human complement system: assembly of the classical pathway C3 convertase. 总被引:4,自引:3,他引:1
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M A Kerr 《The Biochemical journal》1980,189(1):173-181
The assembly of the classical pathway C3 convertase in the fluid phase has been studied. The enzyme is assembled from C2 and C4 on cleavage of these proteins by C1s. Once assembled, the enzyme activity decays rapidly. Kinetic evidence has been obtained that this decay is even more rapid than previously suggested (kdecay is 2.0 min-1 at 37 degrees C). As a result, optimal C3 convertase activity is only observed with high C1s levels, which result in rapid rates of cleavage of C2 and increased rates of formation of the C3 convertase. Using high concentrations of C1s at lower temperatures (22 degrees C) in the presence of excess substrate we have demonstrated kinetically that the enzyme comprises an equimolar complex of C4b and cleaved C2. We have obtained direct evidence from gel-filtration experiments for the role of C2a as the catalytic subunit of the enzyme. C2b appears to mediate the interaction between C4 (or C4b) and C2 at pH 8.5 and at low ionic strength where the interactions can easily be detected. It may therefore be important in the assembly of the enzyme, though it is not involved in the catalytic activity. The decay of the C3 convertase reflects the release of C2a from the C4b x (C2b) x C2a complex, and the stabilizing effect of iodine on the C3 convertase is therefore apparently one of stabilizing the C4b-C2z interaction, which is otherwise weak. C1s is not a part of the C3 convertase enzyme. 相似文献
17.
Isolation and characterization of a 33,000-dalton fragment of complement Factor B with catalytic and C3b binding activity 总被引:2,自引:0,他引:2
Factor B is the zymogen of the catalytic site bearing subunit Bb of the C3/C5 convertase of the alternative pathway of complement. In this study, the location of the C3b binding site and the catalytic site within the Bb subunit were investigated. When human Factor B was treated with porcine elastase, fragments with respective molecular weights of 36,000, 35,000, 33,000, 31,000, and 25,000 were generated. Binding studies showed that only the 33,000-dalton fragment was capable of binding to C3b. The 33,000-dalton fragment was purified using fast protein liquid chromatography and found to be part of the Bb fragment upon testing with monoclonal antibody 15-6-19-1. Amino-terminal amino acid sequence analysis of the 33,000-dalton fragment placed it in the C-terminal half of Bb. The fragment expressed esterolytic activity as evidenced by cleavage of the synthetic substrate N alpha-acetyl-glycyl-L-lysine methyl ester and restored alternative pathway activity in Factor B-depleted serum. Its hemolytic activity was approximately 60-fold lower than that of Factor B. Comparative binding studies in the presence of metal ions using zymosan-C3b showed that the 33,000-dalton fragment bound to C3b with higher affinity than Factor B. Addition of the fragment to human serum inhibited alternative pathway activation by rabbit erythrocytes due to its high affinity for C3b and its low hemolytic activity compared to Factor B. These results show that the C-terminal 33,000-dalton portion of Bb contains not only the enzymatic site of Bb but also a C3b binding site which confers hemolytic activity upon the fragment. The observation that the fragment inhibited alternative pathway activation suggests that a synthetic peptide may be constructed that exhibits negative regulator activity in the alternative pathway. 相似文献
18.
Active sites in complement components C5 and C3 identified by proximity to indels in the C3/4/5 protein family. 总被引:2,自引:0,他引:2
We recently suggested that sites of length polymorphisms in protein families (indels) might serve as useful guides for locating protein:protein interaction sites. This report describes additional site-specific mutagenesis and synthetic peptide inhibition studies aimed at testing this idea for the paralogous complement C3, C4, and C5 proteins. A series of C5 mutants was constructed by altering the C5 sequence at each of the 27 indels in this protein family. Mutants were expressed in COS cells and were assayed for hemolytic activity and protease sensitivity. Mutants at five indels showed relatively normal expression but substantially reduced sp. act., indicating that the mutations damaged sites important for C5 function. Twenty-three synthetic peptides with C5 sequences and 10 with C3 sequences were also tested for the ability to inhibit C hemolytic activity. Three of the C5 peptides and one of the C3 peptides showed 50% inhibition of both C hemolytic and bactericidal activities at a concentration of 100 microM. In several cases both the mutational and peptide methods implicated the same indel site. Overall, the results suggest that regions important for function of both C3 and C5 lie proximal to residues 150-200 and 1600-1620 in the precursor sequences. Additional sites potentially important for C5 function are near residue 500 in the beta-chain and at two or three sites between the N-terminus of the alpha'-chain and the C5d fragment. One of the latter sites, near residue 865, appears to be important for proteolytic activation of C5. 相似文献
19.
Sandoval A Ai R Ostresh JM Ogata RT 《Journal of immunology (Baltimore, Md. : 1950)》2000,165(2):1066-1073
Previous studies focused on indels in the complement C345 protein family identified a number of potential protein-protein interaction sites in components C3 and C5. Here, one of these sites in C5, near the alpha-chain C terminus, was examined by alanine-scanning mutagenesis at 16 of the 18 non-alanine residues in the sequence KEALQIKYNFSF RYIYPLD. Alanine substitutions affected activities in the highly variable manner characteristic of binding sites. Substitutions at the lysine or either phenylalanine residue in the central KYNFSF sequence had the greatest effects, yielding mutants with <20% of the normal activity. These three mutants were also resistant to the classical pathway (CP) C5 convertase, with sensitivities roughly proportional to their hemolytic activities, but had normal susceptibilities to the cobra venom factor (CVF)-dependent convertase. Synthetic peptide MGKEALQIKYNFS-NH2 was found similarly to inhibit CP but not CVF convertase activation, and the effects of alanine substitutions in this peptide largely reflected those of the equivalent mutations in C5. These results indicate that residues KYNFSF form a novel, distal binding site for the CP, but not CVF convertase. This site lies approximately 880 residues downstream of the convertase cleavage site within a module that has been independently named C345C and NTR; this module is found in diverse proteins including netrins and tissue inhibitors of metalloproteinases. 相似文献
20.
C4b-binding protein (C4BP) is a regulator of the classical complement pathway C3 convertase (C4bC2a complex). It is a disulfide-linked polymer of seven alpha-chains and a unique beta-chain; the alpha- and beta-chains are composed of eight and three complement control protein (CCP) domains, respectively. To elucidate the importance of the polymeric nature of C4BP and the structural requirements for the interaction between C4b and the alpha-chain, 19 recombinant C4BP variants were created. Six truncated monomeric variants, nine polymeric variants in which individual CCPs were deleted, and finally, four variants in which double alanine residues were introduced between CCPs were functionally characterized. The smallest truncated C4BP variant still active in regulating fluid phase C4b comprised CCP1-3. The monomeric variants were less efficient than polymeric C4BP in degrading C4b on cell surfaces. All three N-terminal CCP domains contributed to the binding of C4b and were important for full functional activity; CCP2 and CCP3 were the most important. The spatial arrangements of the first CCPs were found to be important, as introduction of alanine residues between CCPs 1 and 2, CCPs 2 and 3, and CCPs 3 and 4 resulted in functional impairment. The results presented here elucidate the structural requirements of individual CCPs of C4BP, as well as their spatial arrangements within and between subunits for expression of full functional activity. 相似文献