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PubMed1986

C2 and factor B: structure and genetics.

Complement components C2 and factor B are novel types of serine protease that are encoded by single loci in the major histocompatibility complex on human chromosome 6. The two proteins share 39% homology, or 50% taking into account conservative amino acid replacements. The catalytic chains, C2a (509 residues) and Bb (505 residues) show homology in their C-terminal domains to the catalytic polypeptides of other serine proteases. The non-catalytic chains, C2b (223 residues) and Ba (234 residues) both contain three tandem repeats of approx. 60 amino acids each, which are homologous to the repeats in C4b-binding protein and factor H, and also the repeats in the non-complement protein beta 2-glycoprotein I. Molecular mapping and DNA sequence analysis has shown that the factor B gene is 6 kb in length and contains 18 exons, while the C2 gene is 18 kb in length; 425 bp separates the 3' end of the C2 gene from the 5' end of the factor B gene. C2 and factor B are polymorphic and structural variants have been detected at the protein level by differences in charge. The degree of polymorphism at the factor B locus has been defined by DNA sequence analysis of the two common alleles F and S. In addition restriction fragment length polymorphisms have been detected in the C2 gene. These DNA polymorphisms subdivide the common allelic variant of C2 (C2C) and reveal that there is much greater variability at the C2 locus than that detected by protein typing.

Alleles↗

PubMed1981

Linkage of total deficiency of the second component (C2) of the complement system and of genetic C2-polymorphism to the major histocompatibility complex of the guinea pig.

In the guinea pig six common phenotypes for hemolytically active C2 were detected. They resulted from three allotypic variants: a basic C2B and two more acidic variants C2A and C2A1. (The gene frequency for C2B in the outbred population was 0.36, 0.33 for C2A, and 0.31 for C2A1.) These variants were inherited as autosomal codominant traits. A strong linkage-disequilibrium between C2 and C4 allotypes was observed consisting of the pairs C2A1-C4F. C2A-C4S, whereas C2B was associated with either C4S or C4S1. To investigate the linkage of C2 to the MHC a family of strain 2 X strain OM3 was studied: the C2B allotype segregated with the strain haplotype of OM3 whereas C2A1 does the same with the strain 2 haplotype. During the C2-typing, an animal totally deficient in hemolytic C2 activity was discovered. Its C1, C4, C3, C5, factors B, D, and H were normal. Breeding yielded finally homozygous and heterozygous-deficient individuals. The latter ones exhibited 50 to 60% of the hemolytic activity of normal guinea pigs. Combined typing data of C4, C2 and GPLA showed that the deficient gene behaved like a rare, silent allele of C2, which is GPLA-linked and inherited like the normal C2-variants. An antiserum against C2 was raised in C2-deficient animals, which reacted with normal guinea pig serum but not with serum from C2-deficient animals. No cross-reactivity with human or mouse serum could be detected.

Alleles↗

PubMed1990

[Flow cytometric analysis of immunophagocytosis using sensitized fluorescent microspheres bearing C3b].

We analyzed the phagocytic activity of purified human monocytes using fluorescent latex beads sensitized with IgG or IgG.C3 by flow cytometry. To prepare IgG-sensitized latex beads (BA), BSA-coated latex beads (B) were incubated with diluted rabbit IgG anti-BSA. To bind complement components, BA were incubated with whole serum pretreated with K-76 monocarboxylic acid (K-76COOH). K-76COOH inhibits the activity of factor I and C5, resulting in deposition of C1, C4b, C2a, C3b on BA (BAC). Phagocytic activity was assessed by percent phagocytosis and phagocytic index (PI). To eliminate the effects of non-phagocytosed latex beads, subtraction of the data at 4 degrees C from 37 degrees C was performed. Percent phagocytosis for 60 min. was B 5.0%, BA 18.3%, and BAC 57.5%, and PI (ingested latex beads/100 cells) was B 7.9, BA 36.8, and BAC 152.7, respectively. In addition, K-76COOH caused dose dependent inhibition on IgG.C3 mediated phagocytosis. Comparison of inhibition pattern on BAC and BA indicated that K-76COOH directly inhibited C3.C3-receptor binding.

Complement C3b↗

PubMed2002

Structure and function of complement C5 convertase enzymes.

The multisubunit enzymes of the complement system that cleave C5 have many unusual properties, the most striking of which is that they acquire their specificity for C5 following cleavage of another substrate C3. C5 convertases are assemblies of two proteins C4b and C2a (classical or lectin pathways) or C3b and Bb (alternative pathway) and additional C3b molecules. The catalytic complexes (C4b, C2a or C3b, Bb) are intrinsically unstable ( t (1/2)=1-3 min) and the enzymes are controlled by numerous regulatory proteins that accelerate this natural decay rate. Immediately after assembly, the bi-molecular enzymes preferentially cleave the protein C3 and exhibit poor activity toward C5 (a K (m) of approx. 25 microM and a C5 cleavage rate of 0.3-1 C5/min at V (max)). Efficient C3 activation results in the covalent attachment of C3b to the cell surface and to the enzyme itself, resulting in formation of C3b-C3b and C4b-C3b complexes. Our studies have shown that deposition of C3b alters the specificity of the enzymes of both pathways by changing the K (m) for C5 more than 1000-fold from far above the physiological C5 concentration to far below it. Thus, after processing sufficient C3 at the surface of a microorganism, the enzymes switch to processing C5, which initiates the formation of the cytolytic membrane attack complex of complement.

Animals↗

PubMed2003

Formation of high affinity C5 convertase of the classical pathway of complement.

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.

Animals↗

PubMed1984

MHC Class III products: an electron microscopic study of the C3 convertases of human complement.

We have reported a transmission electron microscopic study of the two C3 convertases of human complement and their precursors. The corresponding proteins and complexes of the classical and alternative pathway appear very similar. Cofactors C3b and C4b are nearly indistinguishable and display a characteristic but highly irregular substructure. C2 and Factor B are globular with diameters of 85 +/- 8 A and 80 +/- 8 A and both consist of three discrete globular domains each approximately 40 A in diameter. Bb and C2a each contain two domains connected by a short linker segment. Both domains of Bb and one domain of C2a are 42 A in diameter (28 kd), while the second domain of C2 is 47 A in diameter (39 kd). Attachment of the enzymatic subunits to cofactors occurs through one domain only.

Complement Activating Enzymes↗

PubMed2001

Genetic selection in Saccharomyces of mutant mammalian adenylyl cyclases with elevated basal activities.

We show that co-expression of rat Galphas together with type I, II, IV, or VI mammalian adenylyl cyclase (AC) can suppress the growth defect of cyr1 strains of Saccharomyces cerevisiae, which lack a functional endogenous AC. Complemention of cvr1 is not observed in the absence of Galphas, indicating that the mammalian ACs retain their normal regulatory behavior in yeast. Selection for Galphas-independent growth of (cyr1 strains expressing type IV AC yielded several ACIV mutants with enhanced basal activity, each of which had a single amino acid substitution in the conserved C1a or C2a region of the protein. Expression of two of the mutant ACs in HEK293 cells resulted in increased levels of cAMP and elevated adenylyl cyclase activity. Further selection for reverting mutations in one of these constitutively active AC mutants yielded three independent intragenic suppressor mutations. The distribution of the activating and suppressor mutations throughout both C1a and C2a is consistent with a model in which the enhanced basal activity results from an increase in the affinity between C1a and C2a. These results demonstrate the utility of Saccharomyces as a tool for the identification of informative mutant forms of mammalian ACs.

Adenylyl Cyclases↗

PubMed1979

Limited proteolysis of complement components C2 and factor B. Structural analogy and limited sequence homology.

A method is described for the simultaneous purification of milligram quantities of complement components C2 and Factor B. Both products are homogeneous by the criteria of polyacrylamide-gel electrophoresis and N-terminal sequence analysis. Component C2 is cleaved by serine proteinase C1s at an X-Lys bond to give fragment C2a (approx. mol.wt. 74000) and fragment C2b (approx. mol.wt. 34000). The two fragments can be separated by gel filtration without the need for reducing or denaturing agents. Fragment C2b represents the N-terminal end of the molecule. Similar results were seen on cleavage of Factor B by Factor D in the presence of component C3. Again two non-covalently linked fragments are formed. The smaller, fragment Ba (approx. mol.wt. 36,000),) has threonine as the N-terminal residue, as does Factor B; the larger, fragment Bb (approx. mol. wt. 58000), has lysine as the N-terminal residue. A similar cleavage pattern is obtained on limited proteolysis of Factor B by trypsin, suggesting an Arg-Lys-or Lys-Lys bond at the point of cleavage. Although component C2 and Factor B show no apparent N-terminal sequence homology, a limited degree of sequence homology is seen around the sites of proteolytic cleavage.

Amino Acid Sequence↗

PubMed1985

Regulation and deregulation of the fluid-phase classical pathway C3 convertase.

Three mechanisms that regulate the formation and function of the fluid-phase classical pathway C3 convertase (C4b2a) have been elucidated: a) a temperature-mediated intrinsic decay of the enzyme; b) an extrinsic accelerated decay mediated by the effect of the serum protein C4b-binding protein (C4-bp); and c) the inactivation of C4b in the C4b-C4b-p complex by the proteolytic action of C3b/C4b inactivator (I), which cleaves the alpha 1-chain of C4b yielding C4d (alpha 2-chain), and C4c (alpha 3-, alpha 4-, beta-, gamma-chains). A fourth mechanism is described based on the observation that the IgG fraction of the serum of certain patients with glomerulonephritis contains a protein that prevents the intrinsic and C4-bp-mediated decay of surface-bound C4b2a. This protein prolongs the half-life of fluid-phase C4b2a from 10 min to more than 5 hr, increasing the utilization of C3. It also inhibits the decay mediated by C4-bp by preventing the dissociation of C2a from the C4b, 2a complex. In addition, I alone or in the presence of C4-bp fails to cleave the alpha 1-chain of C4b in the stabilized C4b, 2a complex. This protective property of the stabilizing factor (NFc) requires the presence of C2a because C4b was not protected unless it was bound to C2a. Therefore, NFc provides a mechanism by which the serum regulatory proteins are bypassed.

Carrier Proteins↗

PubMed1991

Site-directed mutagenesis of the region around Cys-241 of complement component C2. Evidence for a C4b binding site.

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.

Amino Acid Sequence↗

PubMed1990

Effect of sodium chloride concentration on fluid-phase assembly and stability of the C3 convertase of the classical pathway of the complement system.

The assembly of the classical-pathway C3 convertase from C4 and I2-treated C2 by the action of C1s is an Mg2(+)-dependent reaction. The Mg2+ concentration necessary for the assembly of C3 convertase in the fluid phase was found to be dependent on NaCl concentration. In the absence of NaCl more than 5 mM-MgCl2 was found to be required, whereas 0.5 mM-MgCl2 was adequate for the assembly of C3 convertase in the presence of 150 mM-NaCl. The C3 convertase assembled in a low-ionic-strength buffer was extremely labile compared with that assembled in buffer of physiological ionic strength, and the stability of C3 convertase was improved with the increase in NaCl concentration. It was found that the stabilizing effect of NaCl on C3 convertase was due to inhibition of the dissociating activity of C2b, which was formed during the assembly of C3 convertase. In addition to the dissociation-accelerating effect, C2b inhibited the assembly of C3 convertase in low-ionic-strength buffer, and this effect also was diminished with increase in NaCl concentration. An increase in NaCl concentration to more than 200 mM resulted in a decrease in the assembly of C3 convertase. This effect was not due to the lability of the assembled C3 convertase but due rather to the inhibition of C2 cleavage by C1s. Purified C3 convertase itself is stable in dilute medium or high-ionic-strength medium such as 500 mM-NaCl, suggesting that the interactions between C4b and C2a are hydrophobic. In these respects C2b seemed to be functionally similar to C4bp, but C2b failed to act as a cofactor for the Factor I-catalysed C4b cleavage.

Complement C2↗

PubMed1985

Purification and characterization of the C3 convertase of the classical pathway of human complement system by size exclusion high-performance liquid chromatography.

The C3 convertase of the classical pathway of the complement system is a liable complex, C4b,2a, and is activated by limited proteolysis of two components, C4 and C2, by C1s. By utilizing iodine-treated C2 and size exclusion high-performance liquid chromatography (HPLC), we have succeeded in isolating for the first time the classical pathway C3 convertase. Size exclusion HPLC demonstrated that the apparent molecular mass of the C3 convertase was 280K daltons. The C3 convertase decay-dissociates spontaneously into C4b and C2a. The decay-dissociation is a temperature-dependent reaction and the half-lives of the C3 convertase at 24, 30, and 37 degrees C were estimated to be 400, 180, and 60 min, respectively. The decay-dissociation was also dependent on pH and was accelerated by increasing pH. In addition, the decay-dissociation of the C3 convertase was accelerated by C2b. This result suggests that C2b acts as a feedback inhibitor on the activation of the classical pathway of complement system.

Chemical Phenomena↗

PubMed1980

The human complement system: assembly of the classical pathway C3 convertase.

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.

Chemical Phenomena↗

PubMed1990

C2 reference typing report.

Thirty samples contributed by seven laboratories to the VIth Complement Genetics Workshop were analyzed by isoelectric focusing and immunoblotting with a specific antihuman C2 antibody for the study of the polymorphism of native, activated and desialated C2. This study allowed to compare almost all the C2 variants so far described and also several 'new variants'. According to our results, the C2 system consists of nine structural variants at the protein level which include the common C2 C, the less common C2 B (in Caucasoids), four rare acidic and three rare basic variants. The polymorphic site for the basic variants is carried by the C2a fragment. Typing of desialated C2 is necessary to identify rare acidic or basic variants, especially the C2 BH and C2 BJ variants which seem difficult to be recognized in the native protein.

Antibodies↗

PubMed1986

A model system for the study of the assembly and regulation of human complement C3 convertase (classical pathway).

The formation of classical C3 convertase of complement and its regulation by C4b-binding protein (C4bp) were studied using two different approaches: (a) the analysis was first carried out in fluid phase; a soluble stabilized C3 proconvertase could be assembled from C4b (or C4b-like C4) and iodine-treated C2 in the presence of Ni2+ ions. Upon activation of this complex by C1s, a C3 convertase C4b(C4b-like C4)-C2a was generated which was able to cleave purified C3. C4bp dissociated both C3 proconvertase and C3 convertase, but its effect was more important on C3 convertase. (b) A model system of phospholipid vesicles has been developed to study the assembly of the C3 convertase on a membrane. Among different phospholipid mixtures tested, P-glycerol/P-choline vesicles were found most effective for C4b binding. Optimal conditions were determined for C4b fixation on these vesicles; bound C4b participated in the formation of a functional membrane-associated C3 convertase. C4bp was found to bind to phospholipid vesicles with a higher affinity than C4b; it was able to dissociate the vesicle-associated C3 convertase.

Buffers↗

PubMed1971

An alternate complement pathway: C-3 cleaving activity, not due to C4,2a, on endotoxic lipopolysaccharide after treatment with guinea pig serum; relation to properdin.

The reaction between endotoxic lipopolysaccharide (LPS) and the guinea pig complement system was shown to proceed by way of an intermediate complex, LPS-X, which contains at least six guinea pig serum proteins. LPS-X, like [unk] (sheep erythrocytes carrying antibody molecules and [unk] complexes), destroys the C3 molecule by cleavage. On incubation at 37 degrees C, LPS-X loses its capacity to destroy C3 at about the same rate as the decay of [unk], so that it has been assumed that LPS-X carries [unk] sites that are responsible for the destruction of C3. We have now shown that monospecific rabbit antiguinea pig C2, which effectively inhibits C3 cleavage by [unk], does not interfere with the destruction of C3 by LPS-X. Furthermore, not more than a trace of C2a(d) is released from LPS-X on incubation at 37 degrees C. These results indicate that LPS-X does not carry a significant quantity of [unk] and, hence, that its capacity to destroy C3 is due to another factor which is presumably a component of the properdin system.

Animals↗

PubMed1981

Genetic polymorphism of the complement C2 in Japanese.

Genetic polymorphism of the second component of human complement (C2) was investigated in 521 unrelated healthy adult Japanese using isoelectric focusing in polyacrylamide gel followed by a specific hemolytic overlay method. Besides the phenotypes reported previously (C, AC and BC), a relatively infrequent double-banded phenotype (tentatively named A'C) was observed. Moreover, a homozygous variant (A) and a heterozygous "double" variant (AB) were observed. The estimated frequencies for the common allele, C2c(= C2(1)), and the variant alleles, C2A, C2B(= C2(2)) and C2A' were 0.939, 0.034, 0.022, and 0.006, respectively. The results of further typing for HLA-A, -B, -C specificities indicated the presence of significant associations of C2A with HLA-B15 and with A26, and of C2B with HLA-Bw61. These findings support our previous observation that in Japanese there are allelic combinations showing linkage disequilibrium between C2 and HLA loci which are different from those in Caucasians, and that the C2 structural locus is more closely linked to HLA-B than to HLA-A. C2 hemolytic activities of each phenotypes were assayed. The mean activity of type AC sera was significantly higher than that of type C or type BC, while there were no differences in the activities among the types C, BC or A'C. Also presented are two pedigrees demonstrating the segregation of C2 with HLA alleles in which a homozygous C2 A or C2 B individual was observed.

Alleles↗

PubMed2003

A corresponding tyrosine residue in the C2/factor B type A domain is a hot spot in the decay acceleration of the complement C3 convertases.

The cleavage of C3 by the C3 convertases (C3bBb and C4b2a) determines whether complement activation proceeds. Dissociation (decay acceleration) of these central enzymes by the regulators decay-accelerating factor (DAF), complement receptor 1 (CR1), factor H, and C4-binding protein (C4BP) controls their function. In a previous investigation, we obtained evidence implicating the alpha4/5 region of the type A domain of Bb (especially Tyr338) in decay acceleration of C3bBb and proposed this site as a potential interaction point with DAF and long homologous repeat A of CR1. Because portions of only two DAF complement control protein domains (CCPs), CCP2 and CCP3, are necessary to mediate its decay of the CP C3 convertase (as opposed to portions of at least three CCPs in all other cases, e.g. CCPs 1-3 of CR1), DAF/C4b2a provides the simplest structural model for this reaction. Therefore, we examined the importance of the C2 alpha4/5 site on decay acceleration of C4b2a. Functional C4b2a complexes made with the C2 Y327A mutant, the C2 homolog to factor B Y338A, were highly resistant to DAF, C4BP, and long homologous repeat A of CR1, whereas C2 substitutions in two nearby residues (N324A and L328A) resulted in partial resistance. Our new findings indicate that the alpha4/5 region of C2a is critical to decay acceleration mediated by DAF, C4BP, and CR1 and suggest that decay acceleration of C4b2a and C3bBb requires interaction of the convertase alpha4/5 region with a CCP2/CCP3 site of DAF or structurally homologous sites of CR1 and C4BP.

Amino Acid Sequence↗