Association of activated properdin with complexes of properdin with C3

An ELISA using antibody to properdin (P), followed by antibody to C3 to detect complexes of P with C3 (P-C3), detected low levels of P-C3 complexes in human serum and plasma samples. Incubating serum for 1 h at 37 degrees C increased the amount of P-C3 and diminished factor B hemolytic activity without altering total alternative pathway activity or C3 activity in serum. When P and C3 in incubated serum were analyzed by size exclusion HPLC, complexes of P-C3 were detected at retention times corresponding to molecular mass measuring in excess of 2 x 10(6) Da. Activation of serum with zymosan or cobra venom factor greatly increased the level of P-C3 and decreased alternative pathway hemolytic activity. Chromatography of proteins eluted from serum-treated zymosan detected a peak of P at 9.7 x 10(5) Da and a peak of P-C3 at 1.5 x 10(6) Da. Functional assays for activated properdin also revealed a peak of activity at 1.5 x 10(6) Da, congruent with the peak of P-C3. Native properdin was detected at 3.9 x 10(5) Da. When native properdin was added to properdin-depleted serum and incubated for 1 h at 37 degrees C, activated properdin was detected at the same position in the chromatograph as were P-C3 complexes. We conclude that incubation of serum at 37 degrees C produces complexes of P with C3, that exposure of serum to alternative pathway activators increases the amount of P-C3, and that generation of P-C3 complexes is associated with the presence of activated P.     

Application of the C3-Binding Motif of Streptococcal Pyrogenic Exotoxin B to Protect Mice from Invasive Group A Streptococcal Infection

Group A streptococcus (GAS) is an important human pathogen that produces several extracellular exotoxins to facilitate invasion and infection. Streptococcal pyrogenic exotoxin B (SPE B) has been demonstrated to be an important virulence factor of GAS. Our previous studies indicate that SPE B cleaves complement 3 (C3) and inhibits the activation of complement pathways. In this study, we constructed and expressed recombinant fragments of SPE B to examine the C3-binding site of SPE B. Using enzyme-linked immunosorbent assays and pull-down assays, we found that the C-terminal domain, containing amino-acid residues 345–398, of SPE B was the major binding site of human serum C3. We further identified a major, Ala³⁷⁶-Pro³⁹⁸, and a minor C3-binding motif, Gly³⁴⁶-Gly³⁶⁰, that both mediated the binding of C3 complement. Immunization with the C3-binding motifs protected mice against challenge with a lethal dose of non-invasive M49 strain GAS but not invasive M1 strains. To achieve higher efficiency against invasive M1 GAS infection, a combination of synthetic peptides derived from C-terminal epitope of streptolysin S (SLSpp) and from the major C3-binding motif of SPE B (PP6, Ala³⁷⁶-Pro³⁹⁸) was used to elicit specific immune response to those two important streptococcal exotoxins. Death rates and the severity of skin lesions decreased significantly in PP6/SLSpp-immunized mice that were infected with invasive M1 strains of GAS. These results indicate a combination of the C3-binding motif of SPE B and the protective epitope of SLS could be used as a subunit vaccine against invasive M1 strains group A streptococcal infection.     

Assembly of the membrane attack complex promotes decay of the alternative pathway C3 convertase on Neisseria gonorrhoeae

C3, C4, factor B, properdin, and C2 binding to serum-sensitive and serum-resistant gonococci was quantitated in C8-deficient and normal human serum by using fluorescein-conjugated antibodies and 3H-labeled components. Organism and serum-specific differences were noted, the most striking of which involved factor B and properdin binding to the serum-sensitive strains in the different sera. C3 binding to these organisms was quantitatively and kinetically equivalent in C8-deficient and normal human serum. In contrast, factor B and properdin binding reached a plateau after 5 min in C8-deficient serum but peaked and fell to control values in normal human serum. Identical results were obtained with normal human serum immunochemically depleted of C8. Between 7 and 15% of the bound C3 participated in formation of the alternative pathway convertase C3bBb/P. Reconstitution of the C cascade by adding purified C8 to C8-deficient serum led to the loss of factor B previously bound to the organisms. Factor B loss occurred coincident with bacterial killing and membrane disruption as observed by electron microscopy. Prevention of membrane disruption by depleting normal human serum of lysozyme had no effect on killing and failed to prevent factor B loss. Stabilization of the C3bBb complex with Ni2+ prevented factor B loss as well as gross membrane disruption but not bacterial killing. C2 (the classical pathway analog of factor B) binding to gonococci was equivalent in C8-deficient and normal human serum peaking within 2.5 min and falling to control values in both sera thereafter. We conclude that the assembly of the membrane attack complex promotes decay of C3bBb/P with release of factor B and properdin but not C3 from the organism surface. Membrane disruption does not appear to be required for this effect. This activity may represent a mechanism to limit continued C consumption.     

The role of properdin in the assembly of the alternative pathway C3 convertases of complement

Complement is a powerful host defense system that contributes to both innate and acquired immunity. There are three pathways of complement activation, the classical pathway, lectin pathway, and alternative pathway. Each generates a C3 convertase, a serine protease that cleaves the central complement protein, C3. Nearly all the biological consequences of complement are dependent on the resulting cleavage products. Properdin is a positive regulator of complement activation that stabilizes the alternative pathway convertases (C3bBb). Properdin is composed of multiple identical protein subunits, with each subunit carrying a separate ligand-binding site. Previous reports suggest that properdin function depends on multiple interactions between its subunits with its ligands. In this study I used surface plasmon resonance assays to examine properdin interactions with C3b and factor B. I demonstrated that properdin promotes the association of C3b with factor B and provides a focal point for the assembly of C3bBb on a surface. I also found that properdin binds to preformed alternative pathway C3 convertases. These findings support a model in which properdin, bound to a target surface via C3b, iC3b, or other ligands, can use its unoccupied C3b-binding sites as receptors for nascent C3b, bystander C3b, or pre-formed C3bB and C3bBb complexes. New C3bP and C3bBP intermediates can lead to in situ assembly of C3bBbP. The full stabilizing effect of properdin on C3bBb would be attained as properdin binds more than one ligand at a time, forming a lattice of properdin: ligand interactions bound to a surface scaffold.     

A novel mechanism of complement inhibition unmasked by a tick salivary protein that binds to properdin

Ixodes scapularis salivary protein 20 (Salp20) is a member of the Ixodes scapularis anti-complement protein-like family of tick salivary proteins that inhibit the alternative complement pathway. In this study, we demonstrate that the target of Salp20 is properdin. Properdin is a natural, positive regulator of the alternative pathway that binds to the C3 convertase, stabilizing the molecule. Salp20 directly bound to and displaced properdin from the C3 convertase. Displacement of properdin accelerated the decay of the C3 convertase, leading to inhibition of the alternative pathway. S20NS is distinct from known decay accelerating factors, such as decay accelerating factor, complement receptor 1, and factor H, which directly interact with either C3b or cleaved factor B.     

Resolution and analysis of ''native'' and ''activated'' properdin.

A rapid and reproducible procedure for the resolution of 'native' and 'activated' forms of properdin (a component of the alternative activation pathway of complement), by gel filtration on the polyvinyl matrix Fractogel TSK HW-55(S), is reported. This fractionation permitted effective screening of samples for conditions that cause activation. Only 'native' properdin was detected in serum, even after activation of the alternative pathway by yeast cell walls. Transformation of 'native' into 'activated' properdin in vitro was produced by freeze-thawing of the protein, but not upon binding to and dissociation from the C3 convertase, C3bBb. Electron microscopy showed that only the 'native' population contained the discrete cyclic structures described previously by Smith, Pangburn, Vogel & Müller-Eberhard [(1984) J. Biol. Chem. 259, 4582-4588]. 'Activated' properdin, which was eluted from the gel-filtration column close to the breakthrough peak, was mainly composed of large amorphous aggregates. We therefore conclude that properdin 'activation' is not a physiological event that occurs in serum on complement activation, but is an artifact of isolation. Fractionation of properdin on Fractogel TSK HW-55(S) has, however, enabled detailed analysis of functional heterogeneity within the 'native' population.     

Properdin is critical for antibody-dependent bactericidal activity against Neisseria gonorrhoeae that recruit C4b-binding protein

Gonorrhea, a sexually transmitted disease caused by Neisseria gonorrhoeae, is an important cause of morbidity worldwide. A safe and effective vaccine against gonorrhea is needed because of emerging resistance of gonococci to almost every class of antibiotic. A gonococcal lipooligosaccharide epitope defined by the mAb 2C7 is being evaluated as a candidate for development of an Ab-based vaccine. Immune Abs against N. gonorrhoeae need to overcome several subversive mechanisms whereby gonococcus evades complement, including binding to C4b-binding protein (C4BP; classical pathway inhibitor) and factor H (alternative pathway [AP] inhibitor). The role of AP recruitment and, in particular, properdin in assisting killing of gonococci by specific Abs is the subject of this study. We show that only those gonococcal strains that bind C4BP require properdin for killing by 2C7, whereas strains that do not bind C4BP are efficiently killed by 2C7 even when AP function is blocked. C3 deposition on bacteria mirrored killing. Recruitment of the AP by mAb 2C7, as measured by factor B binding, occurred in a properdin-dependent manner. These findings were confirmed using isogenic mutant strains that differed in their ability to bind to C4BP. Immune human serum that contained bactericidal Abs directed against the 2C7 lipooligosaccharide epitope as well as murine antigonococcal antiserum required functional properdin to kill C4BP-binding strains, but not C4BP-nonbinding strains. Collectively, these data point to an important role for properdin in facilitating immune Ab-mediated complement-dependent killing of gonococcal strains that inhibit the classical pathway by recruiting C4BP.     

The heterocytotoxicity of human serum. I. Activation of the alternative complement pathway by heterologous target cells.

Human serum induces cytolysis of mouse thymus and thymoma cells, and cytostasis of mouse bone marrow and spleen cells, and various methylcholanthrene-induced tumour cells. The latter was manifested by deficient metabolic activity when cultured in the presence of fresh human sera. Decomplementation procedures demonstrated that these heterocytotoxic effects are mediated in part via activation of the alternative complement pathway in human serum samples. The presence of properdin and C3 on the target cell surface was confirmed by immune adherence and indirect immunofluorescent tests. Activation of the alternative complement pathway was elicited by incubation of the human serum with the relevant target cells, resulting in the appearance of the cathodal migrating fragment of the factor B, denoting complement activation. The following publication will present evidence that activation of the alternative complement pathway takes place via an antibody-independent mechanism acting at the cell surface. These and other observations in the literature raise the possibility that activation of the alternative complement pathway by surface cell receptors on tumour cells represents a mechanism of natural immunity versus tumours.     

Serum opsonic deficiency produced by Streptococcus pneumoniae and by capsular polysaccharide antigens.

The opsonic requirements for phagocytosis of S. pneumoniae types 6, 7, 18, and 23 were determined in normal and C2 deficient serum, and in normal serum chelated with magnesium ethyleneglycoltetraacetic acid. All four strains were effectively opsonized via the alternative complement pathway, a finding suggesting that the capsular polysaccharides of these strains activated complement via the alternative pathway. Since bacteremic pneumococcal disease is often associated with circulating capsular polysaccharide, it was considered that this cellular component may activate complement in vivo and impair host defenses by producing an opsonic defect for pneumococci. To examine this hypothesis, serum was incubated with suspensions of whole S. pneumoniae types 6, 7, 18, or 23 or with purified capsular polysaccharide from each of these types, and residual complement activity and opsonic capacity were measured. Hemolytic C 3--9 complement activity and opsonic capacity for 3H-thymidine labeled Salmonella typhimurium, a species effectively opsonized via the alternative pathway, were reduced in serum following incubation. Polysaccharide concentrations as low as 1 microgram/ml inhibited serum opsonic capacity for salmonella. Whole pneumococci and pneumococcal capsular polysaccharide also inhibited the opsonic activity of human C2 deficient serum for salmonella, further evidence for activation of complement via the alternative pathway. Pneumococcal capsular polysaccharide markedly inhibited the opsonic capacity of normal serum for the homologous pneumoccal type. Thus, amounts of pneumococcal capsular polysaccharide, similar to those found in the serum of patients with pneumococcal disease, bring about decomplementation of serum via activation of the alternative pathway and inhibit pneumococcal opsonization.     

Activation of the alternative pathway of complement by malassezia ovalis (Pityrosporum ovale)

Activation of C3 and factor B in normal human serum by P. ovale was demonstrated using a standard unidirectional immunoelectrophoresis technique. Activation of complement by the alternative (properdin) pathway is a possible mechanism by which P. ovale may mediate an inflammatory response.     

Roles of the alternative complement pathway and C1q during innate immunity to Streptococcus pyogenes

Complement is important for innate immunity to the common bacterial pathogen Streptococcus pyogenes, but the relative importance of the alternative and classical pathways has not been investigated. Using mice and human serum deficient in either C1q, the first component of the classical pathway, or factor B, an important component of the alternative pathway, we have investigated the role of both pathways for innate immunity to S. pyogenes. C3b deposition on four different strains of S. pyogenes was mainly dependent on factor B. As a consequence opsonophagocytosis of S. pyogenes was reduced in serum from factor B-deficient mice, and these mice were very susceptible to S. pyogenes infection. In contrast, C3b deposition was not dependent on C1q for two of the strains investigated, H372 and H305, yet opsonophagocytosis of all four S. pyogenes strains was impaired in serum deficient in C1q. Furthermore, infection in C1q-deficient mice with strain H372 resulted in a rapidly progressive disease associated with large numbers of bacteria in target organs. These results demonstrate the important role of the alternative pathway and C1q for innate immunity to S. pyogenes and suggest that C1q-mediated innate immunity to at least some strains of S. pyogenes may involve mechanisms that are independent of C3b on the bacteria.     

Heavy chains of inter alpha inhibitor (IαI) inhibit the human complement system at early stages of the cascade

Inter alpha inhibitor (IαI) is an abundant serum protein consisting of three polypeptides: two heavy chains (HC1 and HC2) and bikunin, a broad-specificity Kunitz-type proteinase inhibitor. The complex is covalently held together by chondroitin sulfate but during inflammation IαI may interact with TNF-stimulated gene 6 protein (TSG-6), which supports transesterification of heavy chains to hyaluronan. Recently, IαI was shown to inhibit mouse complement in vivo and to protect from complement-mediated lung injury but the mechanism of such activity was not elucidated. Using human serum depleted from IαI, we found that IαI is not an essential human complement inhibitor as was reported for mice and that such serum has unaltered hemolytic activity. However, purified human IαI inhibited classical, lectin and alternative complement pathways in vitro when added in excess to human serum. The inhibitory activity was dependent on heavy chains but not bikunin and detected at the level of initiating molecules (MBL, properdin) in the lectin/alternative pathways or C4b in the classical pathway. Furthermore, IαI affected formation and assembly of the C1 complex and prevented assembly of the classical pathway C3-convertase. Presence and putative interactions with TSG-6 did not affect the ability of IαI to inhibit complement thus implicating IαI as a potentially important complement inhibitor once enriched onto hyaluronan moieties in the course of local inflammatory processes. In support of this, we found a correlation between IαI/HC-containing proteins and hemolytic activity of synovial fluid from patients suffering from rheumatoid arthritis.     

The Role of Properdin in Zymosan- and Escherichia coli-Induced Complement Activation

Properdin is well known as an enhancer of the alternative complement amplification loop when C3 is activated, whereas its role as a recognition molecule of exogenous pathogen-associated molecular patterns and initiator of complement activation is less understood. We therefore studied the role of properdin in activation of complement in normal human serum by zymosan and various Escherichia coli strains. In ELISA, microtiter plates coated with zymosan induced efficient complement activation with deposition of C4b and terminal complement complex on the solid phase. Virtually no deposition of C4b or terminal complement complex was observed with mannose-binding lectin (MBL)-deficient serum. Reconstitution with purified MBL showed distinct activation in both readouts. In ELISA, normal human serum-induced deposition of properdin by zymosan was abolished by the C3-inhibiting peptide compstatin. Flow cytometry was used to further explore whether properdin acts as an initial recognition molecule reacting directly with zymosan and three E. coli strains. Experiments reported by other authors were made with EGTA Mg(2+) buffer, permitting autoactivation of C3. We found inhibition by compstatin on these substrates, indicating that properdin deposition depended on initial C3b deposition followed by properdin in a second step. Properdin released from human polymorphonuclear cells stimulated with PMA did not bind to zymosan or E. coli, but when incubated in properdin-depleted serum this form of properdin bound efficiently to both substrates in a strictly C3-dependent manner, as the binding was abolished by compstatin. Collectively, these data indicate that properdin in serum as well as polymorphonuclear-released properdin is unable to bind and initiate direct alternative pathway activation on these substrates.     

Activation of the alternative complement pathway by EBV and the viral envelope glycoprotein, gp350

The EBV-producing B lymphoblastoid cell line B95-8 was found to efficiently activate the alternative C pathway whether assessed with Mg-EGTA-treated human serum or with mixtures of the purified proteins of the pathway (PAP). The ability of the cells to activate was markedly increased after stimulation of EBV replication by treatment of the cells with a phorbol ester, and decreased by treatment of the cells with a viral polymerase inhibitor. Alternative pathway activation was dependent on the presence of either properdin or EBV-immune IgG; the addition of either alone to the PAP led to the deposition of 200,000 C3 molecules/cell. The addition of both properdin and immune IgG to the PAP markedly increased C3 binding to a level of 800,000 molecules/cell. Several lines of evidence indicate that the major external glycoprotein of EBV, gp350, mediates alternative pathway activation by B95-8 cells. First, the ability to activate C positively correlated with gp350 expression on the surface of the EBV-producing cells and gp350- cells failed to activate; second, the anti-EBV antibody in immune human sera which enhanced activation specifically immunoprecipitated gp350 from membranes of B95-8 cells; third, a significant proportion of the C3 which became bound to the cells during activation was attached either to gp350 or to the anti-gp350 antibody found in immune human sera; and fourth, purified gp350, as well as EBV, efficiently activated the alternative pathway. These results indicate that gp350, an EBV envelope glycoprotein, is an efficient alternative pathway activator and its expression on cell membranes is associated with the ability to activate C.     

Residual hemolytic and proteolytic activity expressed by Bb after decay-dissociation of C3b,Bb

Bb (Mr = 63,000) is the catalytic site-bearing subunit of the C3 convertase of the alternative complement pathway, C3b,Bb, which is dissociated from the complex upon decay of the enzyme. Because purified Bb induced certain leukocyte activities, we examined whether it expresses residual hemolytic or proteolytic activity. Hemolytic activity of Bb was tested by using Factor B- or Factor D-depleted normal human serum and rabbit or sheep erythrocytes. Proteolytic activity of Bb was assessed by using purified C3 or C5 as substrates and SDS-PAGE to detect protein cleavage. Bb expressed metal-dependent hemolytic activity that was approximately 100-fold lower than that of Factor B. This activity could be inhibited by Factor H and enhanced by properdin. Low but statistically significant binding of 125I-labeled Bb to C3b on erythrocytes was demonstrated. Monoclonal antibodies that bind to Bb but not to intact Factor B inhibited the Bb hemolytic activity. Purified Bb cleaved C3 to C3a and C3b, as evidenced by the appearance of the alpha'-chain of C3b. It also cleaved C5 to C5a and C5b when cobra venom factor was present in the reaction mixture. Metal ions were required for expression of proteolytic activity, and Ni supported the activity better than Mg. These results indicate that decayed Bb has residual C3 and C5 cleaving activity and hemolytic activity, expression of which appears to require its association with C3b, C3(H2O), or cobra venom factor. These observations may aid in explaining the mechanism of action of Bb on leukocytes.     

C3 adsorbed to a polymer surface can form an initiating alternative pathway convertase

Contact between blood and a biomaterial surface induces an immediate complement-mediated inflammatory response. Under these conditions, the alternative pathway of complement is often initiated and amplified on the biomaterial surface. Adsorption of a protein such as C3 to a polymer surface induces conformational changes in the protein. Based on the expression on adsorbed C3 of conformational neoepitopes specific for bound C3 fragments, we have hypothesized that adsorbed C3 is able to bind factor B and form a functional C3,Bb convertase. Using a quartz crystal microbalance to monitor binding of proteins to a polymer surface, we have demonstrated that a functional C3-containing alternative pathway convertase can be formed, in particular, in the presence of properdin. These data indicate that adsorption of C3 induces conformational changes that turn C3 into a C3b-like molecule that is able to participate in the functioning of the alternative convertase, and they suggest a new mechanism for complement activation on a biomaterial surface.     

Anti-opsonic properties of staphylokinase

Recently we described a novel bacteriophage-encoded pathogenicity island in Staphylococcus aureus that harbors a number of virulence factors that are all involved in the evasion of innate immunity. Here we describe a mechanism by which staphylokinase (SAK), frequently present on this pathogenicity island, interferes with innate immune defenses: SAK is anti-opsonic. By activating human plasminogen (PLG) into plasmin (PL) at the bacterial surface, it creates bacterium-bound serine protease activity that leads to degradation of two major opsonins: human immunoglobulin G (IgG) and human C3b. Incubation of opsonized bacteria with PLG and SAK resulted in removal of anti-staphylococcal IgGs and C3b from the bacterial surface. In phagocytosis assays this proved to be a very efficient mechanism to reduce the opsonic activity of human IgG and serum. The fact that SAK activates human PLG at the bacterial surface and removes IgG as well as C3b makes this protein a unique anti-opsonic molecule.     

Changes in the immunochemical properties of highly purified properdin in human serum.

The fate of highly purified properdin (P) upon introduction into normal human serum or properdin-depleted serum (RP) was investigated. It was observed that, concomitant with the activation of the alternate pathway components, properdin underwent immunochemical alterations characterized by a shift in mobility from gamma2 to beta2 position and by an increase in the sedimentation rate from 5.1S to between 6.8 and 9.3S. The immunoelectrophoretic behavior of C3 was also altered with the appearance of a beta2 arc in addition to the beta1C arc. The immunochemical properties of altered P resemble those of "native" properdin in fresh serum. The principle in serum (designated factor F) mediating these changes is a euglobulin with an approximate sedimentation rate and molecular weight of 9.0S and 250,000 daltons, respectively. The alteration in the immunochemical properties of P may be due to aggregation of P molecules or a complex formation between P and a serum euglobulin (probably C3) mediated by factor F and it is associated with loss of ability of P in initiate the alternate pathway of complement activation upon interaction with serum.     

Properdin binds to late apoptotic and necrotic cells independently of C3b and regulates alternative pathway complement activation

Cells that undergo apoptosis or necrosis are promptly removed by phagocytes. Soluble opsonins such as complement can opsonize dying cells, thereby promoting their removal by phagocytes and modulating the immune response. The pivotal role of the complement system in the handling of dying cells has been demonstrated for the classical pathway (via C1q) and lectin pathway (via mannose-binding lectin and ficolin). Herein we report that the only known naturally occurring positive regulator of complement, properdin, binds predominantly to late apoptotic and necrotic cells, but not to early apoptotic cells. This binding occurs independently of C3b, which is additional to the standard model wherein properdin binds to preexisting clusters of C3b on targets and stabilizes the convertase C3bBb. By binding to late apoptotic or necrotic cells, properdin serves as a focal point for local amplification of alternative pathway complement activation. Furthermore, properdin exhibits a strong interaction with DNA that is exposed on the late stage of dying cells. Our data indicate that direct recognition of dying cells by properdin is essential to drive alternative pathway complement activation.     

Sensitivity of Shigella strains to bactericidal activity of human serum. I. Participation of two independently active mechanisms in bactericidal action against Shigella sonnei phase II.

Normal human serum is strongly bactericidal for all studied Shigella sonnei phase II (10 strains). The studied bacteria were sensitive to two alternative mechanisms of the bactericidal activity of serum factors. The first mechanism involves the action of serum in which complement (C) is activated by the studied bacteria via the classical pathway. Lysozyme did not participate in this reaction. The second mechanism involves the combined action of two factors: C activated via the alternative pathway and lysozyme.