Nonspecific resistance in germ-free and E. coli monocontaminated miniature piglets]

Phagocytic activity of leukocytes, as well as the complement, properdin, and lysozyme levels in the blood serum of miniature piglets, germfree and monocontaminated with E. coli 055 and E. coli 083, were studied. E. coli 055 phagocytosis was decreased in the presence of autologous serum and complement and increased under the effect of specific opsonins (antibodies to E. coli 055). Complement, properdin, and lysozyme levels were decreased in the germfree, in comparison with conventional animals. In the E. coli contaminated piglets properdin and complement production was stimulated most, and lysozyme formation--less. No antibodies to E. coli 055 were revealed in monocontaminated piglets. The highest lysozyme levels were found in the ex-germfree animals, this indicating the participation of factors other than E. coli contamination in lysozyme stimulation. It is concluded that microbial contamination played an important role in the development of cellular and humoral factors of the organism resistance.     

Stimulating effect of prodigiozan on nonspecific reactivity in typhoid fever, dysentery and staphylococcal infection]

A number of factors of nonspecific reactivity, i.e. complement, lysozyme, properdin, blood serum bactericidal activity, leucocyte phagocytal activity, phagocytal index and completed phagocytosis were studied on 160 guinea pigs with experimental typhoid fever, dysentery and staphylococcal infections. The data of the study showed that with the use of prodigiozan the non-specific reactivity of the host increased even within a short period of time. At the same time the pathological changes in the internal organs in their histochemical investigation decreased.     

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.     

A Novel Antibody against Human Properdin Inhibits the Alternative Complement System and Specifically Detects Properdin from Blood Samples

The complement system is an essential part of the innate immune system by acting as a first line of defense which is stabilized by properdin, the sole known positive regulator of the alternative complement pathway. Dysregulation of complement can promote a diversity of human inflammatory diseases which are treated by complement inhibitors. Here, we generated a novel blocking monoclonal antibody (mAb) against properdin and devised a new diagnostic assay for this important complement regulator. Mouse mAb 1340 specifically detected native properdin from human samples with high avidity. MAb 1340 inhibited specifically the alternative complement mediated cell lysis within a concentration range of 1–10 µg/mL. Thus, in vitro anti-properdin mAb 1340 was up to fifteen times more efficient in blocking the complement system as compared to anti-C5 or anti-Ba antibodies. Computer-assisted modelling suggested a three-dimensional binding epitope in a properdin-C3(H₂O)-clusterin complex to be responsible for the inhibition. Recovery of properdin in a newly established sandwich ELISA using mAb 1340 was determined at 80–125% for blood sample dilutions above 1∶50. Reproducibility assays showed a variation below 25% at dilutions less than 1∶1,000. Systemic properdin concentrations of healthy controls and patients with age-related macular degeneration or rheumatic diseases were all in the range of 13–30 µg/mL and did not reveal significant differences. These initial results encourage further investigation into the functional role of properdin in the development, progression and treatment of diseases related to the alternative complement pathway. Thus, mAb 1340 represents a potent properdin inhibitor suitable for further research to understand the exact mechanisms how properdin activates the complement C3-convertase and to determine quantitative levels of properdin in biological samples.     

Cellular and humoral immunity and nonspecific resistance factors in chronic opisthorchiasis patients during dispensary observation

In 116 patients with opisthorchiasis running a cholecystocholangitic variant of the disease course, the characteristics of nonspecific resistance (complement, lysozyme, properdin), cell-mediated and humoral immunity (T- and B-lymphocytes, T gamma-, T mu-, O-, D-, A-cells and auto-rosette-forming cells, IgG, IgA and IgM) have been studied. Essential changes in these characteristics before and after treatment, as well as at the remote periods of dispensary observation, have been established.     

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.     

Identification of tubular heparan sulfate as a docking platform for the alternative complement component properdin in proteinuric renal disease

Properdin binds to proximal tubular epithelial cells (PTEC) and activates the complement system via the alternative pathway in vitro. Cellular ligands for properdin in the kidney have not yet been identified. Because properdin interacts with solid-phase heparin, we investigated whether heparan sulfate proteoglycans (HSPG) could be the physiological ligands of properdin. Kidneys from proteinuric rats showed colocalization of syndecan-1, a major epithelial HSPG, and properdin in the apical membranes of PTEC, which was not seen in control renal tissue. In vitro, PTEC did not constitutively express properdin. However, exogenous properdin binds to these cells in a dose-dependent fashion. Properdin binding was prevented by heparitinase pretreatment of the cells and was dose-dependently inhibited by exogenous heparin. ELISA and surface plasmon resonance spectroscopy (BIAcore) showed a strong dose-dependent interaction between heparan sulfate (HS) and properdin (K(d) = 128 nm). Pretreatment of HSPG with heparitinase abolished this interaction in ELISA. Competition assays, using a library of HS-like polysaccharides, showed that sulfation pattern, chain length, and backbone composition determine the interaction of properdin with glycosaminoglycans. Interestingly, two nonanticoagulant heparin derivatives inhibited properdin-HS interaction in ELISA and BIAcore. Incubation of PTEC with human serum as complement source led to complement activation and deposition of C3 on the cells. This C3 deposition is dependent on the binding of properdin to HS as shown by heparitinase pretreatment of the cells. Our data identify tubular HS as a novel docking platform for alternative pathway activation via properdin, which might play a role in proteinuric renal damage. Our study also suggests nonanticoagulant heparinoids may provide renoprotection in complement-dependent renal diseases.     

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.     

Neutron and X-ray scattering studies on the human complement protein properdin provide an analysis of the thrombospondin repeat

Properdin is a regulatory glycoprotein of the alternative pathway of the complement system of immune defense. It is responsible for the stabilization of the C3 convertase complex formed between C3b and the Bb fragment of factor B. Neutron and X-ray solution scattering experiments were performed on the dimeric and trimeric forms of properdin. These have RG values of 9.1 and 10.7 nm, respectively. The scattering curves were compared with Debye sphere modeling simulations for properdin. Good agreements were obtained for models similar to published electron micrographs showing that the properdin trimer has a triangular structure with sides of 26 nm. Such a structure also accounted for sedimentation coefficient data on properdin. Primary structure analyses for mouse and human properdin have shown that this contains six homologous motifs known as the thrombospondin repeat (TSR), which is the second most abundant domain type found in the complement proteins. Sequences for these 12 TSRs were aligned with 19 others found in thrombospondin and the late complement components. Three distinct groups of TSRs were identified, namely, the TSRs found in thrombospondin and properdin, the TSRs mostly found at the N-terminus of the late complement components, and the TSRs found at the C-terminus of the late components. Averaged secondary structure predictions suggested that all three groups contain similar backbone structures with two amphipathic turn regions and one hydrophilic beta-strand region. The mean dimensions of the TSRs of properdin in solution were determined to be approximately 4 nm X 1.7 nm X 1.7 nm, showing that these are elongated in structure.     

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.     

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.     

Activation of the classical and properdin pathways of complement by bacterial lipopolysaccharides (LPS).

Bacterial lipopolysaccharides (LPS) have been demonstrated to activate both the classical and the properdin pathways of complement. The lipid A region of the LPS is responsible for classical pathway activation and the polysaccharide region responsible for properdin pathway activation. Classical pathway activation by lipid A does not depend upon antibody to the lipid A and properdin pathway activation proceeds by a lipid A-independent mechanism. The polysaccharide portion of the LPS molecule exerts a modifying influence on the potential anticomplementary activity of the lipid A.     

Properdin plays a protective role in polymicrobial septic peritonitis

Properdin is a positive regulator of complement activation so far known to be instrumental in the survival of infections with certain serotypes of Neisseria meningitidis. We have generated a fully backcrossed properdin-deficient mouse line by conventional gene-specific targeting. In vitro, properdin-deficient serum is impaired in alternative pathway-dependent generation of complement fragment C3b when activated by Escherichia coli DH5alpha. Properdin-deficient mice and wild-type littermates compare in their levels of C3 and IgM. In an in vivo model of polymicrobial septic peritonitis induced by sublethal cecal ligation and puncture, properdin-deficient mice appear immunocompromised, because they are significantly impaired in their survival compared with wild-type littermates. We further show that properdin localizes to mast cells and that properdin has the ability to directly associate with E. coli DH5alpha. We conclude that properdin plays a significant role in the outcome of polymicrobial sepsis.     

Factor h and properdin recognize different epitopes on renal tubular epithelial heparan sulfate

During proteinuria, renal tubular epithelial cells become exposed to ultrafiltrate-derived serum proteins, including complement factors. Recently, we showed that properdin binds to tubular heparan sulfates (HS). We now document that factor H also binds to tubular HS, although to a different epitope than properdin. Factor H was present on the urinary side of renal tubular cells in proteinuric, but not in normal renal tissues and colocalized with properdin in proteinuric kidneys. Factor H dose-dependently bound to proximal tubular epithelial cells (PTEC) in vitro. Preincubation of factor H with exogenous heparin and pretreatment of PTECs with heparitinase abolished the binding to PTECs. Surface plasmon resonance experiments showed high affinity of factor H for heparin and HS (K(D) values of 32 and 93 nm, respectively). Using a library of HS-like polysaccharides, we showed that chain length and high sulfation density are the most important determinants for glycosaminoglycan-factor H interaction and clearly differ from properdin-heparinoid interaction. Coincubation of properdin and factor H did not hamper HS/heparin binding of one another, indicating recognition of different nonoverlapping epitopes on HS/heparin by factor H and properdin. Finally we showed that certain low anticoagulant heparinoids can inhibit properdin binding to tubular HS, with a minor effect on factor H binding to tubular HS. As a result, these heparinoids can control the alternative complement pathway. In conclusion, factor H and properdin interact with different HS epitopes of PTECs. These interactions can be manipulated with some low anticoagulant heparinoids, which can be important for preventing complement-derived tubular injury in proteinuric renal diseases.     

The binding of human complement proteins C5, factor B, beta 1H and properdin to complement fragment C3b on zymosan.

The covalent binding of complement fragment C3b to zymosan by the action of the alternative-pathway C3 convertase and the reversible binding of several complement proteins (component C5, factor B, beta 1H and properdin) to C3b on zymosan have been investigated. When C3b is deposited on zymosan after activation by a surface-bound C3 convertase, the C3b molecules are deposited in foci around the C3 convertase site, with an average of 30 C3b molecules per site. The association constants of C5, factor B, beta 1H, and properdin for C3b bound to zymosan have been determined. The association constants ranged from 6.5 x 10(-5) M-1 for factor B to 2.9 x 10(7) M-1 for properdin. An approximate stoichiometry of 1 : 1 for C5, factor B, and properdin binding to C3b has been observed. Curvilinear Scatchard plots were observed for beta 1H binding to C3b, with the maximal extrapolated ratio of beta 1H to C3b of 0.32. Physiological amounts of properdin increase by 7-fold the affinity constant for factor B binding to C3b with no alteration in the stoichiometry. Similarly, physiological amounts of factor B increase the affinity constant of properdin to C3b about 4-fold with only a small measured difference in stoichiometry. Competition binding studies and protein modification suggest that C5, factor B, beta 1H, and properdin each bind to a distinct region on C3b.     

Properdin can initiate complement activation by binding specific target surfaces and providing a platform for de novo convertase assembly

Complement promotes the rapid recognition and elimination of pathogens, infected cells, and immune complexes. The biochemical basis for its target specificity is incompletely understood. In this report, we demonstrate that properdin can directly bind to microbial targets and provide a platform for the in situ assembly and function of the alternative pathway C3 convertases. This mechanism differs from the standard model wherein nascent C3b generated in the fluid phase attaches nonspecifically to its targets. Properdin-directed complement activation occurred on yeast cell walls (zymosan) and Neisseria gonorrhoeae. Properdin did not bind wild-type Escherichia coli, but it readily bound E. coli LPS mutants, and the properdin-binding capacity of each strain correlated with its respective serum-dependent AP activation rate. Moreover, properdin:single-chain Ab constructs were used to direct serum-dependent complement activation to novel targets. We conclude properdin participates in two distinct complement activation pathways: one that occurs by the standard model and one that proceeds by the properdin-directed model. The properdin-directed model is consistent with a proposal made by Pillemer and his colleagues >50 years ago.     

Linkage analysis of the properdin deficiency gene: suggestion of a locus in the proximal part of the short arm of the X chromosome

Properdin is a component of the alternative pathway of complement activation. Inherited deficiency of the protein predisposes an individual to develop meningococcal disease. A family segregating for properdin deficiency (McKNo 31206), in a manner consistent with X-linked recessive inheritance, was studied by RFLP analysis using 24 X-chromosome-specific DNA probes of known regional assignments. Linkage was observed to the OTC locus and the DXS7 locus. These results suggest that the properdin gene is located on the short arm of the X chromosome in the region Xp21.1-Xcen.     

Localization of the properdin factor complement locus Pfc to band A3 on the mouse X chromosome

The locus for properdin (properdin factor complement, Pfc), a plasma glycoprotein, has been mapped to band A3 of the mouse X chromosome by in situ hybridization to metaphase spreads containing an X;2 Robertsonian translocation. The X-linkage of the locus has also been confirmed by analysis of Mus musculus x Mus spretus interspecific crosses. The XA3 localization for Pfc places it in the chromosomal segment conserved between man and mouse which is known to contain at least six other homologous loci (Cybb, Otc, Syn-1 Maoa, Araf, Timp).     

Streptococcal pyrogenic exotoxin B cleaves properdin and inhibits complement-mediated opsonophagocytosis

Streptococcal pyrogenic exotoxin B (SPE B), a cysteine protease, is an important virulence factor in group A streptococcal (GAS) infection. The reduction of phagocytic activity by SPE B may help prevent bacteria from being ingested. In this study, we investigated the mechanism SPE B uses to enable bacteria to resist opsonophagocytosis. Using Western blotting and an affinity column immobilized with SPE B, we found that both SPE B and C192S, an SPE B mutant lacking protease activity, bound to serum properdin, and that SPE B, but not C192S, degraded serum properdin. Further study showed that SPE B-treated, but not C192S-treated, serum blocked the alternative complement pathway. Reconstitution of properdin into SPE B-treated serum unblocked the alternative pathway. GAS opsonized with SPE B-treated serum was more resistant to neutrophil killing than GAS opsonized with C192S-treated or normal serum. These results suggest that a novel SPE B mechanism, one which degrades serum properdin, enables GAS to resist opsonophagocytosis.     

Variability and action mechanism of a family of anticomplement proteins in Ixodes ricinus

Background

Ticks are blood feeding arachnids that characteristically take a long blood meal. They must therefore counteract host defence mechanisms such as hemostasis, inflammation and the immune response. This is achieved by expressing batteries of salivary proteins coded by multigene families.

Methodology/Principal Findings

We report the in-depth analysis of a tick multigene family and describe five new anticomplement proteins in Ixodes ricinus. Compared to previously described Ixodes anticomplement proteins, these segregated into a new phylogenetic group or subfamily. These proteins have a novel action mechanism as they specifically bind to properdin, leading to the inhibition of C3 convertase and the alternative complement pathway. An excess of non-synonymous over synonymous changes indicated that coding sequences had undergone diversifying selection. Diversification was not associated with structural, biochemical or functional diversity, adaptation to host species or stage specificity but rather to differences in antigenicity.

Conclusions/Significance

Anticomplement proteins from I. ricinus are the first inhibitors that specifically target a positive regulator of complement, properdin. They may provide new tools for the investigation of role of properdin in physiological and pathophysiological mechanisms. They may also be useful in disorders affecting the alternative complement pathway. Looking for and detecting the different selection pressures involved will help in understanding the evolution of multigene families and hematophagy in arthropods.