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PubMed1988

Molecular mapping of the HLA-linked complement genes and the RCA linkage group.

Phenotypic genetics have established linkage of the genes encoding proteins involved in the activation of the complement component C3. C2, factor B and C4, three of the structural components of the classical and alternative pathway C3 convertases, are encoded by genes which have been mapped to the class III region of the major histocompatibility complex (MHC) on human chromosome 6. The regulatory proteins factor H, C4BP, CR1, CR2 and DAF, which are involved in the control of C3 convertase activity, are encoded by closely linked genes, termed the regulators of complement activation (RCA) linkage group, that have been mapped to human chromosome 1. cDNA clones for all these proteins have been isolated, and this has made it possible to investigate the organization and structure of the MHC class III genes and the genes in the RCA linkage group. This short review summarizes some of the main features which have emerged from recent cloning work.

Animals↗
PubMed1998

Two diverged complement factor B/C2-like cDNA sequences from a teleost, the common carp (Cyprinus carpio).

Mammalian complement components factor B and C2 act as proteolytic subunits of the C3 convertases in the alternative and the classical activation pathways, respectively, and are believed to have diverged from a common ancestor by gene duplication. However, it is unclear when the B/C2 duplication occurred. Here, we describe two diverged B/C2-like cDNA clones (B/C2-A and B/C2-B) isolated from a bony fish, the common carp (Cyprinus carpio). B/C2-A shares the same domain structure as the factor B and C2 complement components of vertebrates reported so far and shows a close similarity to zebrafish B and medaka fish B/C2. These teleost sequences show almost the same degree of similarity to C2 and B of higher vertebrates. In contrast, B/C2-B has a novel structural feature in that it contains four short consensus repeat modules and does not have a close relative upon phylogenetic analysis. Northern blotting revealed the presence of two transcripts with different sizes for both the B/C2-A and B/C2-B in the hepatopancreas of the carp. Southern blotting suggested the presence of multiple genes for B/C2-A and a single gene for B/C2-B. Although structural features of B/C2-B are slightly more C2-like than B-like, B/C2-B has a crucial amino acid substitution in the serine protease domain, which makes it unlikely that B/C2-B functions as a C3 convertase. A possible phylogenetic relationship between the two carp sequences and mammalian C2 and B is discussed.

Amino Acid Sequence↗
PubMed1989

The erythrocyte as instigator of inflammation. Generation of amidated C3 by erythrocyte adenosine deaminase.

Myocardial ischemia is characterized by the liberation of adenosine and by complement-mediated inflammation. We have reported that amidated C3, formed when ammonia (NH3) disrupts the thiolester bond of C3, serves as an alternative pathway convertase, generates C5b-9, and stimulates phagocytic oxidative metabolism. We investigated whether the deamination of adenosine by adenosine deaminase in hematopoietic cells might liberate sufficient ammonia to form amidated C3 and thereby trigger complement-mediated inflammation at ischemic sites. In the presence of 4 mM adenosine, NH3 production per erythrocyte (RBC) was equal to that per neutrophil (PMN) (3.3 X 10(-15) mol/cell per h). Because RBC outnumber PMN in normal blood by a thousandfold, RBC are the major source of NH3 production in the presence of adenosine. NH3 production derived only from the deamination of adenosine by the enzyme adenosine deaminase and was abolished by 0.4 microM 2'-deoxycoformycin, a specific inhibitor of adenosine deaminase. When purified human C3 was incubated with 5 X 10(8) human RBC in the presence of adenosine, disruption of the C3 thiolester increased more than twofold over that measured in C3 incubated with buffer, or in C3 incubated with RBC (P less than 0.05). The formation of amidated C3 was abolished by the preincubation of RBC with 2'-deoxycoformycin (P less than 0.001). Amidated C3 elicited statistically significant release of superoxide, myeloperoxidase, and lactoferrin from PMN. Thus, the formation of amidated C3 by RBC deamination of adenosine triggers a cascade of complement-mediated inflammatory reactions.

Adenosine↗
PubMed1999

Pneumococcal surface protein A inhibits complement activation by Streptococcus pneumoniae.

Pneumococcal surface protein A (PspA) is a surface-exposed protein virulence factor for Streptococcus pneumoniae. In this study, no significant depletion of serum complement was observed for the serum of mice infected with pneumococci that express PspA. In contrast, in mice infected with an isogenic strain of pneumococci lacking PspA, significant activation of serum complement was detected within 30 min after infection. Also, the PspA-deficient strain but not the PspA-expressing strain was cleared from the blood within 6 h. The contribution of PspA to pneumococcal virulence was further investigated by using mice deficient for C5, C3, or factor B. In mice deficient for C3 or factor B, PspA-negative pneumococci became fully virulent. In contrast, in C5-deficient mice as in wild-type mice, PspA-deficient pneumococci were avirulent. These in vivo data suggest that, in nonimmune mice infected with pneumococci, PspA interferes with complement-dependent host defense mechanisms mediated by factor B. Immunoblots of pneumococci opsonized in vitro suggested that more C3b was deposited on PspA-negative than on PspA-positive pneumococci. This was observed with and without anticapsular antibody. Furthermore, processing of the alpha chain of C3b was reduced in the presence of PspA. We propose that PspA exerts its virulence function by interfering with deposition of C3b onto pneumococci and/or by inhibiting formation of a fully functional alternative pathway C3 convertase. By blocking recruitment of the alternative pathway, PspA reduces the amount of C3b deposited onto pneumococci, thereby reducing the effectiveness of complement receptor-mediated pathways of clearance.

Animals↗
PubMed1990

Participation of C3 and its ligands in complement activation.

C3, the most abundant complement protein in blood, plays a central role in the activation sequence of the complement system as well as in host defense. Expression of the multiple functions of C3 requires its cleavage by highly specific enzymes termed C3 convertases. C3 in a conformationally altered form, C3H2O, resulting from the slow spontaneous hydrolysis of the internal thioester bond of native C3, initiates the assembly of a C3 convertase which continuously cleaves C3 in the blood at slow rates generating a constant supply of small amounts of C3b. When an activator of the alternative complement pathway is present, C3b becomes covalently attached to its surface via an ester or amide bond. Activator surface-bound C3b initiates the assembly of an "amplification" C3 convertase, C3bBb(P), which can efficiently activate C3 and generate additional convertase complexes on the surface of the activator. C3b generated by an amplification or classical pathway C3 convertase can also bind covalently to the noncatalytic subunit, C3b or C4b, respectively, resulting in the generation of a C5 convertase, an enzyme catalyzing the cleavage/activation of C5. In terms of participation in host defense, several fragments of C3, including C3a, C3b, iC3b, and C3dg, mediate a number of important functions such as increased vascular permeability, enhancement of phagocytosis, elimination of immune complexes, and perhaps also proliferative responses and/or differentiation of B cells.

Complement Activation↗
PubMed1984

C3 metabolism in acute glomerulonephritis: implications for sites of complement activation.

Immunochemical and metabolic studies of complement were performed in 11 patients with acute poststreptococcal glomerulonephritis (AGN) to determine the mechanism(s) of hypocomplementemia. Four patients with profound reduction in serum C3 showed metabolic changes comparable to those seen in hypocomplementemic mesangiocapillary GN (MCGN), that is, nonlinear plasma disappearance of 125I.C3 and a gross (that is, 30-fold) reduction in C3 synthesis (0.01 to 0.02 mg/kg/hr); fractional catabolic rate (FCR) and extravascular/intravascular distribution (EV/IV) ratio were also increased (3.02 to 6.99%/hr; 1.14 to 2.96, respectively). The remaining seven patients had less (or no) reduction in C3 and showed normal or elevated values for all three metabolic parameters; six had linear plasma disappearance curves. Metabolic data in five simultaneously studied control subjects were FCR: 1.56 to 2.12%/hr; EV/IV ratio: 0.12 to 0.41 and synthesis rate: 0.30 to 0.52 mg/kg/hr. C3 nephritic factor (NeF) could not be detected in any sera and a significant reduction in serum C5 accompanied the changes in C3 (r = 0.89; P less than 0.001). Previous studies of C3 NeF-associated MCGN show that the fluid phase alternative pathway convertase seen in this condition has little or no C5 cleaving ability. We propose, therefore, that complement activation in AGN occurs via a surface-bound convertase which is capable of cleaving both C3 and C5. The glomerular capillary could provide such a site for activation.

Adolescent↗
PubMed1995

A functional role for corpora amylacea based on evidence from complement studies.

Few theories have been advanced for the production of corpora amylacea (CA) by the normal ageing brain and by the CNS under various neurological conditions. Proteins derived from neurons and oligodendrocytes are found in CA and to understand their origins brain tissue from patients with Alzheimer's disease (AD), multiple sclerosis (MS) and Pick's disease (PD) were tested for complement activity. All CA were immunopositive for antisera to classical pathway-specific components, the activation products C3d and the terminal complement complex (TCC), the C3 convertase regulator membrane cofactor protein (MCP) and the fluid phase regulators S-protein and clusterin. CA were immunonegative for the alternative complement pathway proteins and the complement regulators, decay accelerating factor (DAF) and CD59. Western immunoblotting of isolated solubilized CA from the same tissues demonstrated a weak band for MCP but TCC was more easily shown by immunoprecipitation. A filamentous fringe around CA, probably of astrocytic origin, was also immunopositive for complement factors. CA consist of an inert mucopolysaccharide matrix encasing ubiquitinated proteins, resulting from death of and damage to neurons, myelin and oligodendrocytes. A function of CA, therefore, could be to prevent the recognition of these immunogenic proteins by lymphocytes and microglia and thus protect the CNS from further injury.

Aging↗
PubMed1993

Mouse Crry/p65 is a regulator of the alternative pathway of complement activation.

Like man, mouse has evolved a unique set of regulatory proteins which provide protection from complement-mediated damage to self membranes. The recently described mouse protein Crry/p65 has been shown to inhibit classical complement pathway C3 deposition on cell membranes in which it is expressed. In two distinct experimental systems, we now further delineate the regulatory activity of Crry/p65 and demonstrate its inhibitory effect on alternative complement pathway C3 activation. First, significant inhibition of mouse alternative pathway C3 deposition was demonstrated on neuraminidase-treated human K562 cells expressing recombinant Crry/p65. Second, using a baculovirus technique, recombinant Crry/p65 was synthesized as a soluble molecule and then purified. This molecule was found to inhibit mouse C3 deposition on the surface of zymosan, a potent alternative complement pathway activator. These studies, combined with our earlier findings, demonstrate that Crry/p65 can regulate both the classical and alternative complement pathways. Crry/p65 must, therefore, exert its effects prior to, or at the level of, the C3 convertases, in a fashion similar to that of human membrane cofactor protein and/or decay-accelerating factor. These studies provide further proof of the hypothesis that Crry/p65 is an evolutionarily unique, complement regulatory protein which has developed in mouse.

Animals↗
PubMed1995

Processing of human factor I in COS-1 cells co-transfected with factor I and paired basic amino acid cleaving enzyme (PACE) cDNA.

Factor I is an active serine proteinase in plasma that regulates both the classical and alternative complement pathways by cleaving C3b and C4b thereby preventing the assembly of C3 and C5 convertase enzymes. In this study, a full-length human factor I cDNA was cloned into the pMT2 expression vector and the pMT2-fI construct was expressed transiently in COS-1 cells and stably in CHO-K1 cells. The transfected COS-1 cells secreted large amounts of recombinant pro-factor I (85 kD). Co-transfection of COS-1 cells with pMT2-fI and the cDNA expression plasmid for PACE (paired basic amino acid cleaving enzyme), resulted predominantly in the secretion of a proteolytically processed form of recombinant factor I (heavy chain, 47 kD; light chain, 35 kD). Following co-transfection of pMT2-fI and pSVNeo.1 into CHO-K1 cells and selection in medium containing G418, a stably transfected clone was isolated that secreted pro-factor I (85 kd) and proteolytically processed factor I (heavy chain, 48 kD; light chain, 37 kD) in approximately equal amounts. The molecular sizes of the subunit chains of the expressed factor I were generally slightly smaller than those of human plasma factor I. The activity of recombinant factor I present in the culture supernatants of transfected COS-1 and CHO-K1 cells was assayed by its ability to cleave 125I-C3b in the presence of factor H and was found to be low when compared with factor I purified from human plasma. However, since the functional activity of purified factor I was reduced approximately 50% in the presence of conditioned medium from non-transfected cells, it is suggested that the cold C3b present in the factor I-deficient serum used to supplement the culture medium probably competed with the 125I-C3b tracer, thereby decreasing the sensitivity of the assay for the recombinant factor I proteins.

Animals↗
PubMed1980

Partial sequence of human complement component factor B: novel type of serine protease.

Factor B (a component of the alternative pathway of complement) is believed to contain the proteolytic site of the complex enzymes C3 convertase (C3bB) and C5 convertase (C3bnB). Conflicting results have been obtained in regard to the inactivation of these enzymes by diisopropyl phosphorofluoridate but it has been suggested that activated Factor B (Factor B) is a serine protease with the active site in Bb, a COOH-terminal fragment of approximately 60,000 molecular weight. Partial amino acid sequence studies of Bb derived from human Factor B have shown that the NH2-terminal 40 residues have no homology with NH2-terminal sequences of other serine proteases. However, positioning of a further 170 residues out of approximately 290 residues in two continuous CNBr fragments from the COOH terminus has shown that there is a strong homology of sequence in this section. The active site residues histidine, aspartic acid, and serine all are present in positions corresponding with those of typical serine proteases. It is suggested that Factor B is a novel type of serine protease with a catalytic chain of molecular weight twice that of proteases previously studied and probably with a different activation mechanism.

Amino Acid Sequence↗
PubMed1995

Human factor H and C4b-binding protein serve as factor I-cofactors both encompassing inactivation of C3b and C4b.

Human factor H in the complement (C) system has been characterized as a decay-accelerator for the alternative C pathway C3 convertase and a cofactor for factor I-mediated inactivation of C3b. The current concept is that it does not serve as a C4b-inactivating cofactor. In the present study, we demonstrated that in fluid-phase, factor H and Factor I can cleave methylamine-treated C4(C4ma), a C4b analogue, to C4d, regardless of its isotype. The buffer pH and ionic strength were critical factors for the C4ma cleavage, which proceeded at around pH 6.0 and low conductivity around 3.0 mS. Similar results were obtained with fluid-phase C4b. Cell-bound C4b, however, did not undergo factor I-mediated inactivation by factor H. Hence, all of the human cofactors reported to date can mediate factor I-mediated cleavage of both C3b and C4b at least in the fluid-phase.

Carrier Proteins↗
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↗
PubMed2003

CCP1-4 of the C4b-binding protein alpha-chain are required for factor I mediated cleavage of complement factor C3b.

C4b-binding protein (C4BP) is a potent regulator of the complement system because it strongly inhibits the classical pathway of complement. Furthermore, C4BP serves as a cofactor to factor I (FI) in the cleavage of fluid phase C3b and can, therefore, influence the alternative pathway of complement. The major form of C4BP in plasma consists of seven identical alpha-chains and one beta-chain. Both types of subunits are composed of complement control protein (CCP) domains, eight such domains make up one alpha-chain. To elucidate the structural requirements for the interaction between C3b and the alpha-chain, nineteen recombinant C4BP variants were used: 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. We found that C4BP requires all four N-terminal CCPs of the alpha-chain, with CCP2 and 3 being the most important, to act as a cofactor in the cleavage of C3b. Also, a cluster of positively charged amino acids on the interface between CCP1 and 2 is involved in the binding. Compared to the interaction with C4b, we conclude that binding of C3b to C4BP requires larger molecular surface on C4BP. We found that C4BP was able to act as cofactor in degradation of surface bound C3b and to accelerate decay of alternative C3-convertase. However, in both cases 1,000-fold molar excess of C4BP over factor H (FH), well known inhibitor of the alternative pathway, was required to obtain the same effect.

Amino Acid Substitution↗
PubMed2003

Murine complement interactions with Pseudomonas aeruginosa and their consequences during pneumonia.

Complement is necessary for defense against lung infection with Pseudomonas aeruginosa in mice. We studied in vitro interactions between complement and P. aeruginosa and in vivo effects of complement depletion to better understand this relationship. In vitro, P. aeruginosa strain UI-18 was resistant to killing by mouse serum. However, C3 opsonized the organism (via the alternative and mannose binding lectin [MBL] pathways), and C5 convertase activity on the bacterial surface was demonstrated. In vivo, compared with normal mice, complement-deficient mice experienced higher mortality and failed to sterilize their bronchoalveolar space within 24 h of inoculation. These changes did not seem to be a result of decreased inflammation because complement-deficient mice had normal neutrophil recruitment, greater lung myeloperoxidase content, and, by 24 h, a 35-fold higher level of the CXC chemokine KC. Lung static pressure-volume curves were abnormal in infected animals but were significantly more so in complement deficient mice. These data indicate that although P. aeruginosa is resistant to serum killing, C3 opsonization and C5 convertase assembly occur on its surface. This interaction in vivo plays a central role in host survival beyond just recruitment and activation of phagocytes and may serve to limit the inflammatory response to and tissue injury resulting from bacterial infection.

Animals↗
PubMed1989

Monoclonal anti-human C3d antibodies: stabilization of the alternative pathway C3 convertase.

IgG mouse monoclonal antibody (mAb) was prepared by fusion of spleen cells from mice immunized with human C3d (mAb:C3d) using syngeneic thymocytes as feeder cells. mAb:C3d was assessed for its effect on the stabilization of the cell-bound alternative pathway C3 convertase EAC3bBb. It bound to cell-bound C3b and stabilized C3bBb at 30 degrees in the presence of EDTA-GVB. The plasma protein H reduced the stabilization effect of the stabilized C3 convertase. These results suggest that binding of antibody to C3d may stabilize C3bBb. It seems likely that such antibody induces in C3b conformational change, which increases the C3bBb complex stability.

Antibodies, Monoclonal↗
PubMed1990

A mechanism of activation of the alternative complement pathway by the classical pathway: protection of C3b from inactivation by covalent attachment to C4b.

In this work we studied the role of the classical pathway complement component C4b in the activation of the alternative pathway. It was found that nascent C3b attaches with high efficiency to C4b and that C3b in C4bC3b complexes is protected from inactivation by factors H and I. Activation of C3 by factors B and D in the presence of Mg2+ ions and excess C4b led to 35% incorporation of nascent C3b into C4bC3b complexes in the fluid phase. In comparison, when human IgG was tested as an acceptor under similar conditions, only 12% of generated C3b was incorporated into IgGC3b complexes. The half-life time of dissociation of C3b from purified C4bC3b complexes was approximately 2.3 h at 37 degrees C. C4b in these complexes protected C3b from inactivation as effectively as any known alternative pathway activator. Thus, C3b bound to C4b was tenfold more stable than free C3b or C3b bound to a nonactivating surface. In comparison, the protection provided by attachment to human IgG was only 67% of that of C4b. The results provide an explanation for observations of alternative pathway recruitment following classical pathway activation and for the stability of the classical pathway C5 convertase on surfaces which do not provide protection for C3b from factors H and I.

Complement C3-C5 Convertases↗
PubMed1989

Targeting of complement to tumor cells by heteroconjugates composed of antibodies and of the complement component C3b.

Tumor cells have adapted several strategies which permit them to grow in an immunologically hostile environment. The C system can potentially destroy these cells; however, its action needs to be specifically potentiated on the surface of the tumor cells. To this end, a heteroconjugate composed of a mouse mAb and of the human C3b C component has been generated by using the heterobifunctional reagent N-succinimidyl-3-(2-pyridyldithio)propionate. The two mAb which were used in this study are V1-10 and TIB219 which bind to the human and mouse transferrin receptors, respectively. The mAb-C3b conjugates were purified by gel filtration and were each composed of one mAb and one C3b. They bound to the human K562 and HL60 or mouse ALB1 cell lines and amplified the killing of these cells by C from 10 to 15% to 70 to 100%. Fresh normal human or mouse sera were used as a source of C. The mAb-C3b conjugates activated primarily the alternative pathway of C since only C3 and factor B but not C4 were cleaved in the sera. After disulfide-linking to the mAb, the C3b became highly resistant to inactivation by factors H and I, probably due to its reduced factor H binding capacity. On the other hand, the conjugated C3b bound factor B better than free C3b and produced more C3 convertases which expressed increased stability. These results suggest that mAb-C3b conjugates may serve as an effective tool for the specific activation of the cytolytic C system on selected cells. As such, they may be used in vitro or in vivo to target the autologous C to tumor cells or to lymphocytes and may promote tumor immunotherapy.

Animals↗
PubMed1985

Activation of factor B of the complement system by kallikrein and its light chain.

The cleavage of factor B, a protein of the alternative pathway of complement, by kallikrein was studied. Like factor D, kallikrein can cleave B to generate the alternative pathway C3 convertase C3bBb. When this convertase was formed on erythrocytes previously coated with C3b, lysis was observed indicating that a functionally active C3 convertase was formed. B was also cleaved by kallikrein in the presence of fluid phase C3b, and this resulted in B fragments comparable in size to those generated in the presence of D. The capacity of kallikrein to cleave B is localised in the light chain of the kallikrein molecule, which is the same chain of kallikrein that is responsible for its other enzymatic activities. Since on a molar basis D is much more active then kallikrein in cleaving B, a physiological role for B activation by kallikrein is only likely under certain conditions, and still has to be established.

Binding Sites↗