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PubMed1984

C-reactive protein inhibits pneumococcal activation of the alternative pathway by increasing the interaction between factor H and C3b.

We previously studied two alternative pathway activators, Streptococcus pneumoniae and positively charged liposomes, which react with C-reactive protein (CRP). Binding of CRP to these surfaces initiates classical pathway but blocks alternative pathway activation. In this study, we investigated the mechanism of this inhibition using S. pneumoniae, R36a. R36a were pretreated with CRP (CRP-R36a) or buffer and incubated with C2-deficient human serum to which 125I-labeled C3 had been added. The amount of specific 125I-C3 binding was decreased from 8200 mol/CFU on R36a to 2200 mol/CFU on CRP-R36a. In contrast, when the same experiment was performed with purified factors B, D, P, and C3, in the absence of regulatory proteins, specific 125I-C3 uptake was slightly lower on R36a (6100 mol/CFU) than on CRP-R36a (8100 mol/CFU). The ability of the fixed C3b to inactivate factor B in the presence of factor D was equivalent on the two surfaces. The binding of the regulatory factor H to C3b fixed to R36a and CRP-R36a was compared by using purified 125I-labeled factor H. The ratio of factor H bound to C3 bound was twofold greater on CRP-R36a than on R36a. This increase was found by using C2-deficient serum or purified factors B, D, P, and C3 to fix C3b to the surfaces. The ability of CRP to inhibit C3 binding to R36a was restored by the addition of factors H and I to factors B, D, P, and C3. These results indicate that CRP inhibits alternative pathway activation by increasing regulation of bound C3.

Binding, Competitive↗

PubMed1981

beta 1H-hemagglutination assay: a highly sensitive test for human beta 1H and its uses.

Particle-bound human C3b (EAC14oxy23b) serves as the carrier for the detection of biologically active human beta 1 H by IgG-anti-beta 1H. This hemagglutination assay is highly sensitive (greater than or equal to 2 p of beta 1H/40 microliters), easy to perform, rapid, highly reproducible, and needs no expensive laboratory equipment. All reagents are commercially available. The correlation of the beta 1H-hemagglutination assay is applicable for the detection of beta 1H in various buffer systems and may be used for the detection of beta 1H in serum or fractions thereof. It is a very useful tool for the detection of trace contamination of beta 1H in serum fractions, particularly in complement preparations.

Animals↗

PubMed2005

Complement protein isoforms in CSF as possible biomarkers for neurodegenerative disease.

It has been suggested that the activation of the complement system is involved in the pathogenesis of several neurodegenerative diseases including Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis (MS). Here, the CSF expression levels of complement proteins C3b, C4b, factor B, and factor H were compared between normal subjects and patients diagnosed with AD, PD, MS, and neurosyphilis. The CSF proteins were initially separated using two-dimensional gel electrophoresis, which allowed the comparison of some of the individual complement isoforms. Patients with AD, PD, and MS all showed more than one complement isoform with a significant change (p < 0.05) in CSF expression level compared to normal subjects. PD patients were found to have the greatest number of significantly changed isoforms, all showing a decreased expression level in PD CSF. The complement isoforms examined were able to distinguish between some, but not all, of the diseases studied. The data suggest that when investigating a protein as a possible biomarker, it may be useful to compare individual protein isoform expression levels in addition to the more commonly measured total protein expression level.

Alzheimer Disease↗

PubMed1981

Inhibitory effect of K-76 monocarboxylic acid, an anticomplementary agent, on the C3b inactivator system.

K-76COOH caused dose-dependent inhibition of the degradation, by C3b inactivator (C3bINA) and beta 1H, of membrane-bound C3b on EAC1-3b and of free C3b in the fluid phase, i.e., cleavage of the C3b alpha' peptide chain. K-76COOH primarily attacked C3bINA but not beta 1H or C3b. K-76COOH inhibited the suppression of immune adherence reactivity and the manifestation of conglutination reactivity of EAC1-3b cells by C3bINA and beta 1H. The drug also inhibited the reaction between conglutinin and EAC1-3b' cells derived from EAC1-3b by treatment with C3bINA and beta 1H. EAC1-3b cells did not form rosettes with either Daudi or Raji lymphoblastoid cells. Treatment with C3bINA and beta 1H rendered EAC1-3b cells reactive with Daudi cells, and this change was inhibited by K-76COOH. EAC1-3b cells became able to form rosettes with Raji cells after addition of beta 1H. This rosette formation was enhanced by further addition of C3bINA, and this enhancement was also suppressed by K-76COOH.

Animals↗

PubMed1998

Stable transfection of Trypanosoma cruzi epimastigotes with the trypomastigote-specific complement regulatory protein cDNA confers complement resistance.

Trypanosoma cruzi blood stage trypomastigotes are highly resistant to complement-mediated killing in normal serum. A previously described trypomastigote surface glycoprotein was shown to have binding affinity for human complement components C3b and C4b and restrict activation of the complement cascade, thus preventing lysis of the parasites. Insect stage epimastigotes do not produce detectable levels of this 160-kDa complement regulatory protein (CRP) and are highly sensitive to the lytic effects of complement. Epimastigotes were stably transfected with a T. cruzi expression vector carrying the trypomastigote CRP cDNA and produced fully functional recombinant CRP. The recombinant CRP had binding affinity for C3b, and the transfected epimastigotes were protected from complement-mediated lysis. These results demonstrate for the first time that a developmentally regulated gene of T. cruzi trypomastigotes can be expressed in noninfectious epimastigotes and that production of CRP by epimastigotes is sufficient to confer a virulence-associated trait. Furthermore, these studies demonstrate the critical role that trypomastigote CRP plays in the protection of parasites from the deleterious effects of complement, thus establishing the protein as a virulence factor of T. cruzi.

Animals↗

PubMed1999

Co-operativity between modules within a C3b-binding site of complement receptor type 1.

Complement receptor type 1 (CR1) has 30 modules in its extracellular portion. An understanding of structure-function relationships within CR1 is being assembled gradually from studies of overlapping protein fragments. A CR1 fragment corresponding to modules 16 and 17 was expressed recombinantly as a non-glycosylated protein and its stability and unfolding characteristics studied using biophysical techniques. The results were compared with data collected previously on a CR1 fragment encompassing modules 15, 16 and 17 which together constitute a C3b-binding site (Kirkitadze, M.D., Krych, M., Uhrin, D. , Dryden, D.T.F., Smith, B.O., Wang, X., Hauhart, R., Atkinson, J.P. and Barlow, P.N. (1999) Biochemistry 38, 7019-7031). Modules within CR1 were found to co-operate during unfolding. The folding, stability and flexibility of this protein is therefore likely to be a complex function, and not just the sum, of contributions from individual modules.

Binding Sites↗

PubMed1989

Analysis of recognition in the alternative pathway of complement. Effect of polysaccharide size.

Covalent attachment of the complement (C) protein C3b to polysaccharides on biologic particles which activate the alternative pathway leads to changes in the affinity of C3b for factor H, a regulatory protein of the C system. In this study the size of the site with which the polysaccharides interact and its special relationship to the thioester site were investigated using a fluorimetric assay and soluble C3b attached to low m.w. polysaccharides. Oligomers of alpha 1-6 and alpha 1-4 polyglucose and beta 1-2 polyfructose were prepared and attached to C3b at the thioester site. C3b bound to monomeric, dimeric, or trimeric sugars exhibited the same interaction with factor H as free C3b, i.e., there was no effect due to attachment alone. Beginning with tetrameric oligosaccharides a linear decrease in factor H binding was observed with increasing oligosaccharide size and the effect reached an apparent maximum with large polysaccharides. Maximum inhibition of factor H function was estimated to occur at a length of 16 saccharide units. The results suggest that this site, which regulates the inactivation rate of surface-bound C3b and thus the activation of the alternative pathway of C, spans a maximum of 13 sugar units (less than 65 A) starting four units (approximately 15 A) from the thioester site in C3b.

Binding Sites↗

PubMed1979

Substrate modulation as a control mechanism in the activation of plasma multienzyme systems.

Since plasma is a homogenous fluid its biochemical systems need special control mechanisms to prevent their unlimited and continuous activity. In general, enzymic activity is not preformed but generated by trigger processes. Activation is then enhanced by cascade reactions. It can be controlled by enzyme inhibitors, by enzymes which destroy co-factors, or by spontaneous decay or enzyme complexes involved in the system. In addition, in some reactions of the complement and Hageman factor-dependent systems the necessity of substrate modulation controls the extent of activation. Thus, factor B of the properdin system is activatable by factor D only when bound to the activated third component of complement, and C3b fragment; similarly C2 is activated and forms the C3 converting complex, C42, only when bound to C4b prior to its cleavage by C1; C5 can be activated by either of the two convertases only when bound to surface-fixed C3b; and the mutural activation of Hageman factor and pre-kallikrein on surfaces proceeds efficiently only when HMW-kininogen is present which complexes with pre-kallikrein and possibly with Hageman factor.

Complement C3↗

PubMed1983

Isolation of an iC3b forming enzyme from peritoneal polymorphonuclear leukocytes of guinea pigs.

We have investigated fluid phase cleavage of C3b by peritoneal polymorphonuclear leukocytes of guinea pigs and found that polymorphonuclear leukocytes expressed an iC3b forming enzyme as well as C3b receptor with maturation in peritoneal cavity. The iC3b forming enzyme was found to be distinct from C3bINA, a physiological iC3b forming enzyme in plasma, since the activity was inhibited by monoiodoacetic acid and did not require a cofactor plasma protein, beta 1H, for the cleavage of C3b into iC3b. The iC3b forming enzyme is gradually released upon incubation of PMN at 37 degrees C. The molecular weight of the iC3b forming enzyme was estimated to be 48,000 from gel filtration on Sephadex G-200.

Animals↗

PubMed1982

Isolation and characterization of the C3b-binding entity of C3b-receptor from human erythrocytes.

Applying 2 M KBr, membranes of Ehu were solubilized. By C3-affinity chromatography an activity could be isolated that inhibited the immune adherence reaction and C3b-dependent rosette formation. Since this material did not agglutinate EAC14oxy23b we termed it monovalent C3b receptor (mC3bR). PAGE and SDS-PAGE and staining with Coomassie brilliant blue and PAS reagent revealed a single glycoprotein band with a mol. wt. of 55,000-60,000 daltons and an electrophoretic mobility comparable to ovalbumin. This mC3bR proved to be antigenetically related to gp 205 [17]. The potential of mC3bR to react with C3b-carrying particles was not destroyed by heat and trypsin treatment but by neuraminidase or periodic acid treatment suggesting that mC3bR reacted by its carbohydrate moiety with C3b. As by mC3bR, immune adherence could be inhibited by D-glucose and D-galactose but not by their optical antipodes, L-glucose and L-galactose.

Animals↗

PubMed1997

Interactions between human complement components factor H, factor I and C3b.

Using a microtitre plate assay, direct binding between complement factors I and H was demonstrated, and ligand blotting indicated that factor H interacts with the heavy chain of factor I. Similarly, direct C3(NH3)-factor I and C3(NH3)-factor H binding was characterized [where C3(NH3) is a form of C3 that is cleaved by factor I in the presence of factor H]. Both factor H and factor I interacted with both chains of C3(NH3) in ligand blotting. Binding reactions between all three pairs of components were highly dependent on ionic strength, and showed similar pH optima. Binding assays with all three components present led to the following conclusions. (a) Binding sites for C3(NH3) and factor I on factor H do not overlap, and binding of factor I and C3(NH3) to soluble factor H promotes the weak factor I-C3(NH3) interaction. (b) Anomalies arise with immobilized factor H, which may be artefactual or may reflect the physiological situation. (c) Similarly, binding sites on factor I for C3(NH3) and for factor H do not overlap, and binding of factor H and C3(NH3) to factor I promotes direct factor H-C3(NH3) interactions. Based on these results, a model of the interactions between factor H, factor I and C3(NH3) leading to the processing of C3(NH3) is proposed.

Cations, Divalent↗

PubMed1981

Cleavage of membrane bound C3bi, an intermediate of the third component of complement, to C3c and C3d-like fragments by crude leucocyte lysosomal lysates and purified leucocyte elastase.

Partial degradation of the biologically-active major fragment of the third complement component (C3b) to C3bi is catalysed by the endopeptidase C3b inactivator (I) and its co-factor, beta 1H globulin (H). Complete degradation to the fragments C3c and C3d requires an additional protease, which can be simulated in vitro by trypsin. This study was designed to identify the in vivo correlate of trypsin. Purified and 125I-labelled C3b bound to sheep erythrocytes was used as substrate. Release of label into the supernate served as an index of proteolysis. The chain structure of the peptides in the supernate or remaining bound to the erythrocytes was assessed by SDS polyacrylamide gel electrophoresis. Significant cleavage of cell-bound C3b was obtained by treatment with I, H and extracts from human peripheral blood leucocyte azurophil granules. Purified elastase also removed label in the presence of I and H. The peptide remaining on the cell had the characteristic 33K molecular weight of C3d. The activity of elastase in cleaving was blocked by alpha-1-anti-trypsin, the chloromethyl ketone, MeO-Suc-Ala-Pro-Val-Ch2Cl and by rabbit antibody to elastase. Thus, elastase purified from azurophil granules of human polymorphonuclear neutrophils (PMN) is a potent catalyst of the cleavage of C3bi to C3c- and C3d-like fragments and may contribute in vivo to the control of complement-mediated inflammation.

Animals↗

PubMedDate not supplied

C-type lectins and galectins mediate innate and adaptive immune functions: their roles in the complement activation pathway.

In recent years, a 'new' pathway for complement activation mediated by the mannose-binding lectin (MBL) has been described as a key mechanism for the mammalian acute phase response to infection. This complement activation pathway is initiated by a non-self recognition step: the binding of a humoral C-type lectin [mannose-binding lectin (MBL)] to microbial surfaces bearing 'foreign' carbohydrate determinants. The recognition factor, MBL, is associated with a serine protease [MBL-associated serine protease (MASP)] which, upon MBL binding to the microbial ligand, activates the complement component C3, leading to either (a) phagocytosis of the opsonized target via the complement receptor, or (b) humoral cell killing via assembly of the membrane attack complex. Galectins (formerly known as S-type lectins) modulate activity of the complement receptor 3 (CR3), the macrophage membrane receptor for complement components C3b and iC3b, downstream products of the MBL pathway which are covalently bound to 'target cells. Galectins also mediate macrophage- and dendrocyte-adhesion to lymphocytes activated by signaling through another C-type lectin, the L-selectin, leading to immunoglobulin-mediated responses. Thus, the functional interplay of MBL, galectins and L-selectin in the acute phase response neutralizes the microbial challenge, and lead to further adaptive immunity. Although the observation of various components of the lectin pathway in different invertebrate species demonstrates the high conservation and ancient roots of the components of innate immunity, there has previously been no evidence supporting the possibility that the integral lectin-mediated complement activation pathway is present in invertebrates. We now have evidence for the coexistence of homologs of all the pathway's key components (MBL, MASP, C3, and galectin) in the protochordate Clavelina picta, suggesting the lectin-mediated pathway of complement activation preceded the immunoglobulin pathway in evolution. Therefore, despite being 'new' to the textbooks, experimental evidence indicates that this pathway is ancient, and has been conserved intact throughout its evolution.

Amino Acid Sequence↗

PubMed1982

Behavior of soluble human 125I-labeled C3b, the third component of complement, after binding to human cells.

The behavior of 125I-labeled C3b incubated with two C3b receptor-positive cells (human erythrocytes and the B lymphoblastoid Raji line), one C3b receptor-negative cell (T lymphoblastoid CEM line) and solubilized membranes from each cell was analyzed by sucrose density gradient (SDG) and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Whichever whole cell was tested, the unbound 125I-labeled C3b recovered in the cell supernatant was not cleaved. When 125I-labeled C3b was bound to whole cells or incubated with solubilized membranes, three different activities were detected: (a) nonspecific C3b polymerization, induced on the membrane of C3b receptor-positive or C3b receptor-negative cells; (b) specific C3b receptor activity solubilized only from the membrane of the two C3b receptor-positive cells and (c) C3b hydrolytic activity, inhibited by 5 X 10(-4)M phenyl methyl sulfonyl fluoride, only extracted from human erythrocyte membranes and carried by a molecule different from that of C3b receptor. C3b receptor activity solubilized from Raji and human erythrocyte membranes was detected by a 12S peak complex formation on a 10-30% SDG and characterized by an affinity constant of 2 X 10(7) to 4 X 10(7) mol-1. Hydrolysis of labeled C3b (Mr = 175000) by solubilized human erythrocyte membranes led to the formation of a split product of Mr = 35000 consisting of two disulfide-linked polypeptide chains of Mr = 17000. This is the first report of a breakdown of C3b on cell membranes different from the physiological breakdown described in the fluid phase.

B-Lymphocytes↗

PubMed1996

Interaction between complement subcomponent C1q and the Klebsiella pneumoniae porin OmpK36.

The interaction between C1q, a subcomponent of the complement classical pathway component C1, and OmpK36, a porin protein from Klebsiella pneumoniae, was studied in a solid-phase direct-binding assay, inhibition assays with the purified globular and collagen-like regions of C1q, and cross-linking experiments. We have shown that the binding of C1q to the OmpK36 porin of the serum-sensitive strain K. pneumoniae KT707 occurs in an in vivo situation and that this binding leads to activation of the complement classical pathway and the subsequent deposition of complement components C3b and C5b-9 on the OmpK36 porin. Scatchard analysis of the binding of [125I]C1q to the OmpK36 porin showed two binding sites with dissociation constants of 1.5 and 75 nM. The decrease of [125I]C1q binding to the OmpK36 porin in buffer with increasing salt concentrations and the pIs of the C1q subcomponent (10.3) and OmpK36 porin (4.5) suggest that charged amino acids are involved in the binding phenomenon. In inhibition assays, only the globular regions of C1q inhibited the interaction between C1q and OmpK36 porin, demonstrating that C1q binds to porin through its globular region and not through the collagen-like stalks.

Bacterial Proteins↗

PubMed2005

Peripheral clearance of amyloid beta peptide by complement C3-dependent adherence to erythrocytes.

Brain deposits of amyloid beta peptide (Abeta) have been a diagnostic hallmark of Alzheimer's disease (AD) for nearly a century. Recent studies have demonstrated that Abeta is also present in peripheral blood. Here, we present evidence that circulating Abeta42 is subject to complement C3b-dependent adherence to complement receptor 1 (CR1) on erythrocytes, a classical set of mechanisms by which pathogens and proteins recognized as foreign are cleared from the bloodstream. Levels of Abeta42 targeted by this pathway differ significantly in AD compared to mild cognitive impairment and nondemented elderly controls.

Alzheimer Disease↗

PubMed1987

Naturally occurring anti-band-3 antibodies and complement together mediate phagocytosis of oxidatively stressed human erythrocytes.

Treatment of erythrocytes with the thiol-specific oxidant azodicarboxylic acid bis(dimethylamide) (diamide) enhances their phagocytosis by adherent monocytes. Phagocytosis of diamide-treated erythrocytes required that the cells were opsonized with whole serum, since complement inactivation abolished phagocytosis. Opsonization with whole serum containing 20-100 times the physiological concentration of naturally occurring anti-band-3 antibodies enhanced phagocytosis of diamide-treated erythrocytes. High inputs of anti-band-3 also restored phagocytosis of erythrocytes that had been incubated with complement-inactivated serum. Elevated concentrations of anti-spectrin antibodies were ineffective in whole and complement-inactivated serum. Specific recognition of diamide-treated erythrocytes by anti-band-3 antibodies may be due to generation of anti-band-3 reactive protein oligomers on intact diamide-treated erythrocytes. Generation of such oligomers was dose-dependent with respect to diamide. Bound anti-band-3 alone was not sufficient to mediate phagocytosis. It resulted in deposition of complement component C3b on the cells through activation of the alternative complement pathway in amounts exceeding that of bound antibodies by two orders of magnitude. Thus, anti-band-3 and complement together mediate phagocytosis of oxidatively stressed erythrocytes, which stimulate senescent erythrocytes with respect to bound antibody and complement.

Anion Exchange Protein 1, Erythrocyte↗

PubMed1985

Defective modulation of complement in Crohn's disease. C3b opsonization of zymosan by the alternative pathway.

Opsonization of zymosan by plasma complement was studied in 12 consecutive patients with untreated, well-established Crohn's disease and in 12 healthy volunteers. Binding of C3b to zymosan was markedly increased in patients with Crohn's disease during the first 30 min of the alternative pathway reaction, whereas consumption of the native C3 protein was within the normal range in all patients. Our results are consistent with a decreased capacity for degradation of activated C3,C3b by the I-H factor inactivator system. This immunologic abnormality may contribute to a chronic activation of the complement sequence, previously demonstrated in Crohn's disease, and to a release of split products of inflammation-promoting character.

Complement C3b↗