A novel C3 mutation causing increased formation of the C3 convertase in familial atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome has been associated with dysregulation of the alternative complement pathway. In this study, a novel heterozygous C3 mutation was identified in a factor B-binding region in exon 41, V1636A (4973 T > C). The mutation was found in three family members affected with late-onset atypical hemolytic uremic syndrome and symptoms of glomerulonephritis. All three patients exhibited increased complement activation detected by decreased C3 levels and glomerular C3 deposits. Platelets from two of the patients had C3 and C9 deposits on the cell surface. Patient sera exhibited more C3 cleavage and higher levels of C3a. The C3 mutation resulted in increased C3 binding to factor B and increased net formation of the C3 convertase, even after decay induced by decay-accelerating factor and factor H, as assayed by surface plasmon resonance. Patient sera incubated with washed human platelets induced more C3 and C9 deposition on the cell surface in comparison with normal sera. More C3a was released into serum over time when washed platelets were exposed to patient sera. Results regarding C3 and C9 deposition on washed platelets were confirmed using purified patient C3 in C3-depleted serum. The results indicated enhanced convertase formation leading to increased complement activation on cell surfaces. Previously described C3 mutations showed loss of function with regard to C3 binding to complement regulators. To our knowledge, this study presents the first known C3 mutation inducing increased formation of the C3 convertase, thus explaining enhanced activation of the alternative pathway of complement.

Relation of putative thioester bond in C3 to activation of the alternative pathway and the binding of C3b to biological targets of complement.

The reaction of [14C]methylamine with native human C3 led to the stoichiometric incorporation of methylamine, loss of hemolytic activity, and the concomitant exposure of a sulfhydryl group that could be labeled with [14C]iodoacetamide. Both labeled sites were located in the C3d portion of the alpha-chain, which is known to contain the metastable binding of C3b. The methylamine-modified C3 [C3(CH3NH2)] was shown to exhibit many of the functional properties of C3b, although the C3a portion of the molecule remained covalently attached. C3(CH3NH2) bound Factor B and beta 1H, and could be cleaved by C3b inactivator in the presence of beta 1H. C3(CH3NH2) added to human serum caused activation of the alternative pathway and consumption of C3. In presence of Factors B and D and Mg++, C3(CH2NH2) formed a C3 convertase. The convertase-forming material could be removed from solution by anti-C3a Sepharose and the preformed convertase was completely inhibited by purified antibody to C3a. This antibody did not affect the function of the C3 convertase that contained C3b. Similar functional properties were exhibited by C3 exposed for short periods of time to relatively low concentrations of chaotropic reagents, such as KSCN or guanidine. These results suggest that the initial C3 convertase of the alternative pathway may be formed from native C3, without proteolysis, by the attack of a variety of nucleophiles including water. The C3 convertase formed from this altered C3 then generates by proteolytic cleavage the initial metastable C3b that is capable of attaching to receptive surfaces. Conversion of C3 to C3b exposes one sulfhydryl residue as does modification of C3 with methylamine. When the C3d portion of C3b bound to zymosan particles via the metastable binding site was treated with radiolabeled methylamine, the fragment was released from the particles in radiolabeled form. These findings are consistent with the concept that native C3 contains an active carbonyl group, probably in the form of a thioester, which can either react with water to form functionally C3b-l;ike C3 or, upon enzymatic conversion of C3 to C3b, allows C3b to form an ester bond with hydroxyl groups on the target surface.

The complement inhibitor FUT-175 suppresses T cell autoreactivity in experimental autoimmune encephalomyelitis.

Several recent studies have shown that interacting antigen presenting cells and/or T cells produced complement activation products C5a and C3a, are integrally involved in T-cell activation, and promote the generation of myelin oligodendrocyte glycoprotein (MOG(35-55))-specific interferon-gamma and interleukin-17-producing T cells in experimental autoimmune encephalomyelitis, a rodent model of multiple sclerosis. In this study, we tested whether FUT-175, a clinical pharmaceutical that has been shown to inhibit the formation of C3/C5 convertases, can attenuate myelin-specific T-cell responses, as well as disease severity in experimental autoimmune encephalomyelitis. In vitro, FUT-175 inhibited local C5a/C3a production by antigen presenting cell-T-cell complexes and attenuated MOG(35-55)-specific Th1 and Th17 responses with little nonspecific cytotoxicity. In vivo administration of FUT-175 delayed experimental autoimmune encephalomyelitis disease onset, lowered clinical scores, decreased central nervous system inflammation, and reduced demyelination. The FUT-175-treated mice exhibited decreased numbers of MOG(35-55)-specific interferon-gamma- and interleukin-17-producing T cells. In addition, results from the FUT-175 treatment of naive recipients of adoptively transferred splenocytes from MOG(35-55)-immunized mice suggested that the effect of FUT-175 was on MOG-specific cellular responses and not on anti-MOG antibodies. These results argue that complement regulators, which inhibit C5a/C3a production, may have therapeutic efficacy in multiple sclerosis and in other clinical conditions in which T cells drive disease pathogenesis.

Structural insights into the central complement component C3.

C3 is a central protein of the complement system, which is important to immune defense and provides a link between innate and adaptive immunity. Three pathways of complement activation converge at the activation of C3 yielding a diverse set of biological responses. This versatile and flexible molecule interacts with various proteins to fulfill its functions. Here we review recent insights gained from the crystal structure determinations of human, native C3 and its physiological down-regulation product C3c. The data provided, for the first time, a complete and detailed view of the composition and arrangement of the domains in C3. Comparison of C3 with C3c indicates marked flexibility of the molecule, particularly in the alpha-chain. We discuss the observed domain rearrangements, conformational changes and the location of various protein binding sites. These detailed, and structural, insights are important for developing models of the molecular mechanisms underlying the diverse biological activities of this large and complex molecule.

Structural determinants of the capacity of heparin to inhibit the formation of the human amplification C3 convertase.

The ability of heparin glycosaminoglycan to prevent formation of the properdin-stabilized amplification C3 convertase is independent of antithrombin binding activity and requires substitution of the amino sugar and a degree of oxygen (O)-sulfation which could be on the uronic acid or the amino sugar. Preparations of heparin glycosaminoglycan isolated by different techniques from different species (rat, human, and porcine) exhibited an equivalent capacity to inhibit generation of the amplification C3 convertase. Hyaluronic acid, which is devoid of O-sulfation, had no inhibitory activity; chondroitin 4-sulfate of rat and whale origins, chondroitin 6-sulfate of rat and shark origins, and dermatan sulfate from porcine skin are O-sulfated on the galactosamine and had minimal activity. Porcine heparin glycosaminoglycan, isolated on the basis of affinity for antithrombin III, had no greater anticomplementary activity than porcine glycosaminoglycan, which failed to bind antithrombin III and had essentially no anticoagulant activity. Nitrogen (N)-desulfation of porcine heparin reduced anticomplementary activity to the level of the other sulfated mucopolysaccharides, and both N-resulfation and N-acetylation restored the original activities, thereby indicating a requirement for N-substitution, but not N-sulfation. N-resulfation of N-desulfated and O-desulfated heparin did not restore any activity, thus indicating that O-sulfation and N-substitution represent independent, critical structural requirements for the anticomplementary activity of heparin glycosaminoglycan. Inasmuch as N-desulfated-N-acetylated heparin had no anticoagulant activity, the nature of the N-substitution completely distinguishes the plasma-protein effector pathway that is inhibited.

Increased enzymatic activity of the alternative pathway convertase when bound to the erythrocytes of paroxysmal nocturnal hemoglobinuria.

To investigate the greater fixation of C3 to the erythrocytes of patients with paroxysmal nocturnal hemoglobinuria (PNH) upon activation of complement, we have examined the formation and the reaction of the C3 nephritic factor-stabilized alternative pathway convertase made with purified components on normal and PNH erythrocytes. Each convertase complex converts four to five times more fluid-phase C3 to C3b when affixed to a PNH cell than when affixed to a normal cell. The greater activity of the convertase on PNH cells is not due to differences in the intrinsic or extrinsic stability of the convertase complex. The excessive binding of C3 to PNH cell si due to this increased conversion of fluid-phase C3, because the efficiency of binding of nascent C3b was identical for the two cell types. This is the first instance in which the enzyme activity of a complement complex has been shown to be increased by being affixed to an abnormal surface.

IgG naturally occurring antibodies stabilize and promote the generation of the alternative complement pathway C3 convertase.

Normal human IgG contains naturally occurring anti-C3 antibodies (anti-C3 NAbs) that have been proposed to regulate complement amplification. Here, we report a novel procedure for anti-C3 NAb purification. Pooled human IgG was fractionated on a DEAE column prior to affinity chromatography on IgG and then on C3. Anti-C3 NAbs co-purified with anti-F(ab')2 NAbs. In a refined protocol, IgG fractions were absorbed on Fc, F(ab')2, and C3, which allowed to isolate the directly accessible NAbs and to remove IgG hinge-region-specific NAbs. Since a substantial fraction of total anti-C3 NAbs in whole IgG pre-existed as complexes, IgG that did not bind to the three affinity columns was treated with urea and the affinity chromatography repeated to collect the dissociated NAbs. The urea-accessible anti-F(ab')2 NAbs were rather pure but anti-C3 NAbs yet contained substantial amounts of anti-F(ab')2 NAbs. Anti-C3 NAbs showed up to 400-fold and anti-F(ab')2 NAbs, up to 30-fold enrichment as compared to pooled normal human IgG. Anti-C3 NAb preparations exhibited nephritic factor activity that was up to 60 times stronger than that of total IgG from a patient with membranoproliferative glomerulonephritis type 2. In addition, anti-C3 NAbs promoted C3 convertase generation, when added to the convertase precursor or during convertase assembly, suggesting a non-nephritic-factor mechanism. Factors H and I reduced the overall level of activity but had no influence on the NAb dose-response curve meaning that NAbs did not interfere with factor H binding. Convertase promoting activity during assembly correlated with the content of anti-C3 NAbs in NAb complexes. In conclusion, anti-C3 NAbs associated with framework-specific anti-idiotypic NAbs stabilize C3 convertase and promote its generation but their activity is compensated for in whole IgG.

Immune complex independent activation of complement, C1s secreted from hamster embryo malignant fibroblasts, Nil2C2 in serum free culture medium.

Antibody independent activation of complement C1s was examined by immunoblot analysis using an antibody against a synthetic peptide of hamster C1s L chain. Approx. 50% of C1s secreted from hamster embryo malignant fibroblasts Nil2C2 was functionally active in its two-chain form in the serum free culture medium. In contrast, no active C1s was found in a culture medium of hamster embryo fibroblasts (HEF). Active C1s was detectable, however, in the culture medium after HEF became a cell line. The immune complex independent activation of C1s was also observed in rat cell lines but not in secondary rat embryo fibroblasts. C1s in a membrane fraction of Nil2C2 was a proenzyme form and was not activated by incubation of the membrane itself suggesting that C1s was activated after secretion. The activation of C1s was not inhibited by human C1 inhibitor (C1-INH), benzamidine or soy bean trypsin inhibitor (SBTI) but was inhibited by leupeptin, nitrophenyl guanidinobenzoate and DFP. Our results suggest that C1s is activated either by a serine proteinase(s) other than those reported to cleave C1s or by an activator which directly stimulates autoactivation of C1s.

Binding of human C4 to C-reactive protein-pneumococcal C-polysaccharide complexes during activation of the classical complement pathway.

Sequential interaction of CRP-PnC aggregates, made at slight CRP excess, with purified human C1, C4 and C2oxy resulted in formation of an effective C3-convertase, indicating the binding of C1, C4 and C2 on the aggregates. Immunoprecipitation experiments demonstrated that, following cleavage of 125I-C4 by CRP-PnC-C1 complexes, approximately 3% of the 125I-C4 was bound to CRP while a lower percentage was bound to PnC, CRP-C4 complexes could also be demonstrated by substituting 125I-CRP for 125I-C4. The nature of the CRP-C4 bond was examined by electrophoretic analysis. Complexes of 125I-C4-CRP prepared as earlier were incubated at 100 degrees C for 2 min in buffer containing 2% SDS and 5% beta-mercaptoethanol and subjected to electrophoresis in SDS-containing polyacrylamide gradient slab gels. Autoradiography of the dried gels revealed the presence of high mol. wt bands containing the alpha'-chain of C4b. CRP could also be demonstrated in these high mol. wt bands which apparently represented covalent complexes between the alpha'-chain of C4b and CRP monomers. Since CRP contains no detectable carbohydrate, it seems likely that an amide bond is formed between the two proteins.

Covalently-bound human C4b dimers consisting of C4B isotype show higher hemolytic activity than those of C4A in the C3-bypass complement pathway.

The ability to form a covalent dimer of human C4b was investigated with purified isotypes C4A and C4B, and antibody-sensitized liposomes supplemented with C1. In this system, no C4A or C4B formed a complex with the antibody or C1. Whereas both C4A and C4B isotypes formed dimers to a similar extent, C4B formed an ester-linked dimer and C4A an amide-linked dimer. Both of these dimers served as a subunit for the C3-bypass pathway C5 convertase, since liposomes bearing Ab, C1 and a dimer of C4A or C4B, allowed the formation of C5 convertase by the addition of C2. The degree of complement-mediated liposome lysis however, was observed to be 2-3 times higher in the C4B-bearing particles than in those bearing C4A. These results indicate that the second C4b-binding site on the first C4b is different between C4A and C4B, and that in the C3-bypass pathway, C4B has a higher degree of hemolytic activity than C4A, as in the conventional classical complement pathway.

A reexamination of the role of magnesium in the human alternative pathway of complement.

The formation of the alternative-pathway C3 convertase has been previously suggested to have an absolute requirement for Mg2+, especially at the level of complex formation between C3b and factor B (B). In the course of defining spectral probes that could be used to monitor the C3b-B interaction (e.g. 1-anilino-8-naphthalene sulfonic acid fluorescence and near-u.v. circular dichroism) we observed that the signal change reporting on this binding was not completely reversed upon addition of excess ethylene-diaminetetraacetic acid (EDTA). Using sucrose gradient ultracentrifugation, we have directly demonstrated a Mg2+-independent C3b-B complex in the fluid phase. B thus bound was not only susceptible to specific proteolytic activation by factor D, but the resulting C3bBb enzyme was able to convert native C3 to C3b. Interestingly, we were unable to detect Mg2+-independent specific binding of 125I-B to C3b which was particle-bound. Using a sensitive hemolytic assay, however, we estimated that the functional activity of B with surface-bound C3b is 80-fold greater in the presence of physiological Mg2+ (0.5 mM) than in 2 mM EDTA. In contrast, the fluid-phase association is estimated to differ less than three-fold under the same conditions. These data demonstrate that the requirement for Mg2+ in the formation of the fluid-phase alternative-pathway C3 convertase is not absolute. Furthermore, they suggest a difference in the stable functional properties of fluid-phase and surface-bound C3b.

Activation of the alternative complement pathway by the immune precipitate formed with F(ab')2 fragment of human IgG antibody.

The complement fixing ability of the F(ab')2 fragment of human IgG was studied using an immune precipitate (Ippt) formed between tetanus toxoid and the F(ab')2 of high-titer IgG antibody against tetanus toxin. A major subclass of the specific IgG antibody against tetanus toxin, which was separated by affinity column chromatography, was identified as IgG1. On incubation of normal human serum (NHS) with the Ippt formed at equivalence, a dose-dependent consumption of CH50, C3 and C5 activities was observed without significant loss of the early acting complement components. A similar consumption of CH50, C3 and C5 activities was found in NHS reacted with Ippt formed at any antigen/antibody ratio. The Ippt formed at antibody excess was more efficient in complement consumption than the Ippt formed at antigen excess. An apparent consumption of C3 and C5 activities was also noted in C4-deficient guinea pig serum treated with Ippt. When Ippt was incubated with Mg2+--EGTA-treated NHS, both C3 and C5 convertases of the alternative pathway were generated on the Ippt. From these results, it was concluded the the F(ab')2 of human IgG antibody, especially IgG1 antibody, when it formed an Ippt with antigen, could activate the alternative complement pathway.

Binding and activation of C4 and C3 on the red cell surface by non-complement enzymes.

We investigated the binding of C4 and C3 to red cell surfaces by non-complement enzymes. Cell bound C components were quantitated by a radioimmunoassay, the chain structure of bound components was analyzed by Western blotting and the hemolytic activity of bound components was determined. Trypsin, chymotrypsin, plasmin, elastase, thrombin, kallikrein and enzymes from Bacillus subtilis, Staphylococcus aureus and Streptomyces griseus all were found capable of binding C4b and C3b to sheep red cells. C4b bound by any of these enzymes was hemolytically active; both classical and alternate pathway activity of C3 could be demonstrated for most enzymes except plasmin and thrombin. In addition, trypsin and the bacterial enzymes were also able to generate the classical pathway C3-convertase from C4b + C2. The hemolytic efficiency of enzyme bound C4b and C3b was about the same as for these molecules bound by complement enzymes. In contrast, the process of binding by the non-complement enzymes was several hundred-fold less efficient than by cell bound complement enzymes. The results demonstrate that several enzymes can replace the C1 and C42 enzymes in the classical pathway and are able to initiate the alternative pathway by activating C3 and binding C3b to the cell surface.

Anopheles midgut epithelium evades human complement activity by capturing factor H from the blood meal.

Hematophagous vectors strictly require ingesting blood from their hosts to complete their life cycles. Exposure of the alimentary canal of these vectors to the host immune effectors necessitates efficient counteractive measures by hematophagous vectors. The Anopheles mosquito transmitting the malaria parasite is an example of hematophagous vectors that within seconds can ingest human blood double its weight. The innate immune defense mechanisms, like the complement system, in the human blood should thereby immediately react against foreign cells in the mosquito midgut. A prerequisite for complement activation is that the target cells lack complement regulators on their surfaces. In this work, we analyzed whether human complement is active in the mosquito midgut, and how the mosquito midgut cells protect themselves against complement attack. We found that complement remained active for a considerable time and was able to kill microbes within the mosquito midgut. However, the Anopheles mosquito midgut cells were not injured. These cells were found to protect themselves by capturing factor H, the main soluble inhibitor of the alternative complement pathway. Factor H inhibited complement on the midgut cells by promoting inactivation of C3b to iC3b and preventing the activity of the alternative pathway amplification C3 convertase enzyme. An interference of the FH regulatory activity by monoclonal antibodies, carried to the midgut via blood, resulted in increased mosquito mortality and reduced fecundity. By using a ligand blotting assay, a putative mosquito midgut FH receptor could be detected. Thereby, we have identified a novel mechanism whereby mosquitoes can tolerate human blood.

Evidence that a nicked C4b, C4b', is a functionally active C4b derivative.

Factor I-catalyzed C4b cleavage is a regulatory reaction for the classical pathway of the complement system. Although the reaction was shown to be a two-step reaction, production of a nicked form of C4b, C4b', as an intermediate cleavage product and subsequent splitting of C4b' into C4c and C4d, it is not known which of the two steps represents the inactivation of the C4b function in the assembly of C3 convertase, C4b,2a. We have purified C4b' and assessed the ability of C4b' to assemble C3 convertase with C2 by utilizing size exclusion high performance liquid chromatography. Evidence was obtained demonstrating that C4b' still retains the function of C4b to assemble C3 convertase. Thus, the substantial step for the inactivation of the C4b function appears to be the second cleavage reaction, that is, the cleavage of C4b' into C4c and C4d.

Structural and functional similarities between the major hemolymph protein of fall armyworm and cat C4 binding protein of the complement system.

We have investigated the structural and functional similarities between insect (armyworm) major larval serum protein (LSP-I) and mammalian (Cat) C4 binding protein (i) by comparing the major chymotryptic peptide patterns using sodium dodecyl sulfate polyacrylamide gel electrophoresis and (ii) their ability to prevent assembly of classical C3 convertase (EAC142). Both Cat C4 bp and Spodoptera LSP-I are composed of identical subunits of Mr = 75,000 and Mr = 80,000, respectively and generate the characteristic cleavage product of Mr = 50,000 on limited proteolysis. These observations are interpreted as an indication that the major hemolymph protein (LSP-I) of fall armyworm is a homologue of the C4bp of the mammalian complement system.

A cobra venom factor (CVF)-induced C3 convertase activity in the hemolymph of Galleria mellonella.

A cobra venom factor (CVF)-induced C3 convertase has been generated from the hemolymph of Galleria mellonella. CVF was immobilized on Sepharose 4B and treated with cell-free hemolymph obtained from either unvaccinated G. mellonella larvae or larvae immunized with formalized Pseudomonas aeruginosa. The C3-cleaving activity was detected by the ability to cleave the alpha-chain of bovine C3 in a manner analogous to the CVF-induced mammalian C3 convertase, CVF,Bb. The insect-derived C3 convertase formed at 28 degrees C but not at 37 degrees C, then once formed was active at both 28 degrees C and 37 degrees C. EDTA did not inhibit the formation and action of the insect derived C3 cleaving activity.

Haemocompatibility evaluation of DLC- and SiC-coated surfaces.

Diamond-like carbon (DLC) and silicon carbide (SiC) coatings are attractive because of low friction coefficient, high hardness, chemical inertness and smooth finish, which they provide to biomedical devices. Silicon wafers (Si(waf)) and silicone rubber (Si(rub)) plates were coated using plasma-enhanced chemical vapour deposition (PE-CVD) techniques. This article describes: 1- the characterization of modified surfaces using attenuated total reflection-Fourier transform infrared spectroscopy (ATR/FTIR) and contact angle measurements, 2- the results of three in-vitro haemocompatibility assays. Coated surfaces were compared to uncoated materials and various substrates such as polymethylmethacrylate (PMMA), polyethylene (LDPE), polydimethylsiloxane (PDMS) and medical steel (MS). Thrombin generation, blood platelet adhesion and complement convertase activity tests revealed the following classification, from the most to the least heamocompatible surface: Si(rub)/ DLC-Si(rub)/ DLC-Si(waf)/ LDPE/ PDMS/ SiC-Si(waf)/ Si(waf)/ PMMA/ MS. The DLC coating surfaces delayed the clotting time, tended to inhibit the platelet and complement convertase activation, whereas SiC-coated silicon wafer can be considered as thrombogenic. This study has taken into account three events of the blood activation: coagulation, platelet activation and inflammation. The response to those events is an indicator of the in vitro haemocompatibility of the different surfaces and it allows us to select biomaterials for further in vivo blood contacting investigations.

Factor D of the alternative pathway of bovine complement: isolation and characterization.

Factor D of the bovine alternative complement pathway has been purified by chromatography on CM-Sephadex C-50, Sephacryl S-200 and hydroxylapatite. The isolated factor D (0.25 mg from 1 litre of bovine serum) had an apparent molecular weight of 27,500 and a pI of 7.2. In whole bovine serum the pI of factor D was also 7.2. The isolated protein caused the Mg++-dependent cleavage of bovine factor B in the presence of cobra venom factor (CVF) to generate a haemolytically active C3-convertase as shown by SDS-polyacrylamide gel electrophoresis and haemolytic diffusion plate assays. Bovine factor D and human factor D were interchangeable in restoring the alternative pathway haemolytic activities of both bovine RD and human RD (factor D deficient sera). The haemolytic activity of bovine serum factor D was completely inhibited by 20 mM diisopropylfluorophosphate (DFP) but only 25% inhibited by 1 mM DFP. Serum heated at 56 degrees C for 10 min completely lost factor D activity but purified factor D was relatively more heat stable.

Evasion of Trypanosoma cruzi from complement lysis.

This review outlines: a) the main biochemical and biological properties of the complement system (C) components; b) the manner through which they interact in the two distinct routes of C activation, the classical and the alternative pathways, to generate the enzymes C3 and C5 convertases responsible for release of the peptides C4a, C3a and C5a endowed with the properties of mediating the early events of the inflammatory process or the potentially cytolytic complex C5b-C9; c) the main features of control of these activation processes; d) the identification of cell surface components present in the trypomastigote forms of Trypanosoma cruzi possibly involved in the mechanisms developed by this parasite to evade C lysis; e) the inactivation or removal of these cell surface components by enzymatic (trypsin or papain), chemical (periodate) or physical (heating at 45 degrees C) treatments; f) isolation of these components by chromatographic methods; and, g) demonstration that some of these cell surface components interfere with C3 convertase formation or action in a manner similar to the decay accelerating factor (DAF).