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Properdin acts as an essential positive regulator of the alternative pathway of complement by stabilizing enzymatic convertases. Identical properdin monomers form head-to-tail associations of oligomers in a reported 20:54:26 ratio (most often described as an approximate 1:2:1 ratio) of tetramers (P 4 ), trimers (P 3 ), and dimers (P 2 ), in blood, under normal physiological conditions. Oligomeric size is proportional to properdin function with tetramers being more active, followed by trimers and dimers. Neutrophils are the most abundant granulocyte, are recruited to inflammatory microenvironments, and are a significant source of properdin, yet the ratio of properdin oligomers released from neutrophils is unknown. The oligomer ratio of neutrophil-derived properdin could have functional consequences in local microenvironments where neutrophils are abundant and complement drives inflammation. We investigated the oligomer properties of neutrophil-derived properdin, as compared to that of normal human sera, using a novel ELISA-based method that detects function of properdin in a way that was proportional to the oligomeric size of properdin (i.e., the larger the oligomer, the higher the detected function). Unexpectedly, neutrophil-derived properdin had 5-fold lower function than donor-matched serum-derived properdin. The lower function was due to a lower percentage of tetramers/trimers and more dimers, indicating a significantly different P 4 :P 3 :P 2 ratio in neutrophil-derived properdin (18:34:48) as compared to donor-matched serum (29:43:29). Release of lower-order oligomers by neutrophils may constitute a novel regulatory mechanism to control the rate of complement activation in cellular microenvironments. Further studies to determine the factors that affect properdin oligomerization and whether, or how, the predominant dimers in neutrophil-derived properdin, assimilate to the ~1:2:1 ratio found in serum are warranted., Competing Interests: During part of this work, VF served as a consultant for, and received grant funding from, Apellis Pharmaceuticals. These relationships no longer exist. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest., (Copyright © 2023 Moore, Menon, Galwankar, Khuder, Pangburn and Ferreira.)

The evolutionary history of complement suggests that the alternative pathway arose prior to the arrival of the classical and lectin pathways. In these pathways, target specificity is provided by antibodies and sugar specific lectins. While these efficient initiation systems dominate activation on most targets, the alternative pathway produces most of the C3b and 80%-90% of the C5b-9. While the tickover process, originally proposed by Peter Lachmann, provided ancient hosts with a crude self/non-self-discriminatory system that initiated complement attack on everything foreign, tickover clearly plays a more minor role in complement activation in modern organisms possessing classical and lectin pathways. Spontaneous activation of the alternative pathway via tickover may play a major role in human pathologies where tissue damage is complement-mediated. The molecular mechanism of tickover is still not convincingly proven. Prevailing hypotheses include (a) spontaneous hydrolysis of the thioester in C3 forming the C3b-like C3(H 2 O) in solution and (b) "enhanced tickover" in which surfaces cause specific or non-specific contact activated conformational changes in C3. Theoretical considerations, including computer simulations, suggest that the latter mechanism is more likely and that more research needs to be devoted to understanding interactions between biological surfaces and C3., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Cobra venom factor (CVF) is the complement-activating protein in cobra venom. CVF is a structural and functional analog of complement component C3. CVF, like C3b, forms a convertase with factor B. This bimolecular complex CVF, Bb is an enzyme that cleaves C3 and C5. However, CVF, Bb exhibits significantly different functional properties from C3b,Bb. Whereas both, CVF, Bb and C3b, Bb exhibit spontaneous decay-dissociation into the respective subunits, thereby eliminating the enzymatic activity, the CVF, Bb convertase is physico-chemically far more stable, decaying with a half-life that is more than two orders of magnitude slower than that of C3b,Bb. In addition, CVF, Bb is completely resistant to inactivation by Factors H and I. These two properties of CVF, Bb allow continuous activation of C3 and C5, and complement depletion in serum. In order to understand the structural basis for the physico-chemical stability of CVF,Bb, we have created recombinant hybrid proteins of CVF and human C3, based on structural differences between CVF and human C3b in the C-terminal C345C domain. Here we describe three human C3/CVF hybrid proteins which differ in only one, two, or five amino acid residues from earlier described hybrid proteins. In all three cases, the hybrid proteins containing CVF residues form more stable convertases, and exhibit stronger complement-depletion activity than hybrid proteins with human C3 residues. Three bonds between CVF residues and Factor Bb residues could be identified by crystallographic modeling that contribute to the greater stability of the convertases., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Factor H exists as a 155,000 dalton, extended protein composed of twenty small domains which is flexible enough that it folds back on itself. Factor H regulates complement activation through its interactions with C3b and polyanions. Three binding sites for C3b and multiple polyanion binding sites have been identified on Factor H. In intact Factor H these sites appear to act synergistically making their individual contributions difficult to distinguish. Recombinantly expressed fragments of human Factor H were examined using surface plasmon resonance (SPR) for interactions with C3, C3b, iC3b, C3c, and C3d. Eleven recombinant proteins of lengths from one to twenty domains were used to show that the three C3b-binding sites exhibit 100-fold different affinities for C3b. The N-terminal site [complement control protein (CCP) domains 1-6] bound C3b with a K d of 0.08 μM and this interaction was not influenced by the presence or absence of domains 7 and 8. Full length Factor H similarly exhibited a K d for C3b of 0.1 μM. Unexpectedly, the N-terminal site (CCP 1-6) bound native C3 with a K d of 0.4 μM. The C-terminal domains (CCP 19-20) exhibited a K d of 1.7 μM for C3b. We localized a weak third C3b binding site in the CCP 13-15 region with a K d estimated to be ~15 μM. The C-terminal site (CCP 19-20) bound C3b, iC3b, and C3d equally well with a K d of 1 to 2 μM. In order to identify and compare regions of Factor H that interact with polyanions a family of 18 overlapping three domain recombinant proteins spanning the entire length of Factor H were expressed and purified. Immobilized heparin was used as a model polyanion and SPR confirmed the presence of heparin binding sites in CCP 6-8 ( K d 1.2 μM) and in CCP 19-20 (4.9 μM) and suggested the existence of a weak third polyanion binding site in the center of Factor H (CCP 11-13). Our results unveil the relative contributions of different regions of Factor H to its regulation of complement, and may contribute to the understanding of how defects in certain Factor H domains lead to disease., (Copyright © 2020 Haque, Cortes, Alam, Sreedhar, Ferreira and Pangburn.)

Cobra venom factor (CVF) is the complement-activating protein in cobra venom. CVF is a structural and functional analog of complement component C3. CVF, like C3b, forms a convertase with factor B. This bimolecular complex CVF,Bb is an enzyme that cleaves C3 and C5. However, CVF,Bb exhibits significantly different functional properties from C3b,Bb. The CVF,Bb convertase is physico-chemically very stable, and completely resistant to an activation by Factors H and I. These two properties, in contrast to C3b,Bb, allow continuous activation of C3 and C5, and complement depletion in serum. In order to understand the structural basis for the functional differences between CVF and C3, we have created several hybrid proteins of CVF and human C3. Here we report that replacing the C-terminal 168 amino acid residues of human C3 with the corresponding residues from CVF results in a hybrid protein (HC3-1496) which is essentially a human C3 derivative exhibiting the functional properties of CVF. This result demonstrates that the important structures for the CVF-specific functions reside within the C-terminal 168 amino acid residues of CVF. We further demonstrate that reverting the 46 C-terminal CVF residues of HC3-1496 to human C3 sequence results in a hybrid protein (HC3-1496/1617) that exhibits a physico-chemically unstable convertase with only residual complement depleting activity. This result demonstrates that most, but not all, structural requirements for CVF activity reside within the 46 C-terminal amino acid residues. We also investigated the potential role of position 1633, which is an acidic residue in human C3 (glutamic acid) but a basic amino acid residue (histidine) in CVF. However, the charge at position 1633 appears to be of no functional relevance. Exchanging the neutral amino acids present in CVF at positions 1499 and 1501 with the two charged amino acids at these positions in human C3 (aspartic acid and lysine) resulted in a hybrid protein that exhibited significantly slower convertase formation although both binding to Factor B and C3 cleavage was not affected, demonstrating that the charged amino acid residues at these two positions interfere with the formation of the convertase. In conclusion, our work demonstrates that hybrid proteins of human C3 and CVF present valuable tools to identify functionally important amino acid residues in CVF., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Blood-feeding insects inject potent salivary components including complement inhibitors into their host's skin to acquire a blood meal. Sand fly saliva was shown to inhibit the classical pathway of complement; however, the molecular identity of the inhibitor remains unknown. Here, we identified SALO as the classical pathway complement inhibitor. SALO, an 11 kDa protein, has no homology to proteins of any other organism apart from New World sand flies. rSALO anti-complement activity has the same chromatographic properties as the Lu. longipalpis salivary gland homogenate (SGH)counterparts and anti-rSALO antibodies blocked the classical pathway complement activity of rSALO and SGH. Both rSALO and SGH inhibited C4b deposition and cleavage of C4. rSALO, however, did not inhibit the protease activity of C1s nor the enzymatic activity of factor Xa, uPA, thrombin, kallikrein, trypsin and plasmin. Importantly, rSALO did not inhibit the alternative or the lectin pathway of complement. In conclusion our data shows that SALO is a specific classical pathway complement inhibitor present in the saliva of Lu. longipalpis. Importantly, due to its small size and specificity, SALO may offer a therapeutic alternative for complement classical pathway-mediated pathogenic effects in human diseases.

Differences in sensitivity of monkeys and humans to antisense oligonucleotide (ASO)-induced complement alternative pathway (AP) activation were evaluated in monkeys, humans, and in serum using biochemical assays. Transient AP activation was evident in monkeys at higher doses of two 2'-O-methoxyethyl (2'-MOE) ASOs (ISIS 426115 and ISIS 183750). No evidence of AP activation was observed in humans for either ASO, even with plasma ASO concentrations that reached the threshold for activation in monkeys. The absence of complement activation in humans is consistent with a query of the Isis Clinical Safety Database containing 767 subjects. The in vivo difference in sensitivity was confirmed in vitro, as monkey and human serum exposed to increasing concentrations of ASO indicated that monkeys were more sensitive to AP activation with this class of compounds. The mechanistic basis for the greater sensitivity of monkeys to AP activation by 2'-MOE ASO was evaluated using purified human or monkey factor H protein. The binding affinities between a representative 2'-MOE ASO and either purified protein are similar. However, the IC50 of fluid-phase complement inhibition for monkey factor H is about 3-fold greater than that for human protein using either monkey serum or factor H-depleted human serum. Interestingly, there is a sequence variant in the monkey complement factor H gene similar to a single nucleotide polymorphism in humans that is correlated with decreased factor H protein function. These findings show that monkeys are more sensitive to 2'-MOE ASO-mediated complement activation than humans likely because of differences in factor H inhibitory capacity., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

The first update since 1981 of the nomenclature used in the field of complement has been completed by the Complement Nomenclature Committee established under the auspices of the International Complement Society (ICS) and by the boards of the ICS and the European Complement Network (ECN). Recommended names of complement pathways, proteins, protein complexes, protein fragments and receptors are listed. Authors are urged to use these names in their published and presented works., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Factor H (fH) is an endogenous negative regulator of the alternative pathway (AP) that binds polyanions as well as complement activation fragments C3b and C3d. The AP is both necessary and sufficient to develop collagen Ab-induced arthritis (CAIA) in mice; the mechanisms whereby normal control of the AP is overcome and injury develops are unknown. Although primarily a soluble circulating protein, fH can also bind to tissues in a manner dependent on the carboxyl-terminal domain containing short consensus repeats 19 and 20. We examined the role of fH in CAIA by blocking its binding to tissues through administration of a recombinant negative inhibitor containing short consensus repeats 19 and 20 (rfH19-20), which impairs fH function and amplifies surface AP activation in vitro. Administration of rfH19-20, but not control rfH3-5, significantly worsened clinical disease activity, histopathologic injury, and C3 deposition in the synovium and cartilage in wild-type and fH(+/-) mice. In vitro studies demonstrated that rfH19-20 increased complement activation on cartilage extracts and injured fibroblast-like synoviocytes, two major targets of complement deposition in the joint. We conclude that endogenous fH makes a significant contribution to inhibition of the AP in CAIA through binding to sites of immune complex formation and complement activation.

Acinetobacter baumannii is an important nosocomial pathogen. Infections are often preceded by intubation or catheter use, promoting the formation of biofilm, and some strains are able to cause severe cases of bacteremia because of their ability to resist killing by complement. We identified a secreted serine protease, termed "PKF," that provided resistance to complement killing and suppressed biofilm formation. Serum resistance was abrogated in A. baumannii treated with protease inhibitors, as well as in a PKF-negative mutant. Serum resistance could be restored by recombinant PKF, which was shown to reduce the complement activity of normal human serum by almost 50%. PKF was shown to inhibit biofilm formation, because the PKF-negative mutant and wild-type A. baumannii treated with protease inhibitors produced biofilm that could be inhibited by addition of recombinant PKF. Our data indicate that PKF is required for serum resistance and that it suppresses biofilm formation in A. baumannii.

Activation of the complement system results in formation of membrane attack complexes (MACs), pores that disrupt lipid bilayers and lyse bacteria and other pathogens. Here, we present the crystal structure of the first assembly intermediate, C5b6, together with a cryo-electron microscopy reconstruction of a soluble, regulated form of the pore, sC5b9. Cleavage of C5 to C5b results in marked conformational changes, distinct from those observed in the homologous C3-to-C3b transition. C6 captures this conformation, which is preserved in the larger sC5b9 assembly. Together with antibody labeling, these structures reveal that complement components associate through sideways alignment of the central MAC-perforin (MACPF) domains, resulting in a C5b6-C7-C8β-C8α-C9 arc. Soluble regulatory proteins below the arc indicate a potential dual mechanism in protection from pore formation. These results provide a structural framework for understanding MAC pore formation and regulation, processes important for fighting infections and preventing complement-mediated tissue damage., (Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.)

C-reactive protein (CRP) is a cyclic pentameric protein whose major binding specificity, at physiological pH, is for substances bearing exposed phosphocholine moieties. Another pentameric form of CRP, which exists at acidic pH, displays binding activity for oxidized LDL (ox-LDL). The ox-LDL-binding site in CRP, which is hidden at physiological pH, is exposed by acidic pH-induced structural changes in pentameric CRP. The aim of this study was to expose the hidden ox-LDL-binding site of CRP by site-directed mutagenesis and to generate a CRP mutant that can bind to ox-LDL without the requirement of acidic pH. Mutation of Glu(42), an amino acid that participates in intersubunit interactions in the CRP pentamer and is buried, to Gln resulted in a CRP mutant (E42Q) that showed significant binding activity for ox-LDL at physiological pH. For maximal binding to ox-LDL, E42Q CRP required a pH much less acidic than that required by wild-type CRP. At any given pH, E42Q CRP was more efficient than wild-type CRP in binding to ox-LDL. Like wild-type CRP, E42Q CRP remained pentameric at acidic pH. Also, E42Q CRP was more efficient than wild-type CRP in binding to several other deposited, conformationally altered proteins. The E42Q CRP mutant provides a tool to investigate the functions of CRP in defined animal models of inflammatory diseases including atherosclerosis because wild-type CRP requires acidic pH to bind to deposited, conformationally altered proteins, including ox-LDL, and available animal models may not have sufficient acidosis or other possible modifiers of the pentameric structure of CRP at the sites of inflammation.

Activation of the alternative pathway of complement plays a critical role in the development of allergen-induced airway hyperresponsiveness (AHR) and inflammation in mice. Endogenous factor H, a potent inhibitor of the alternative pathway, is increased in the airways of sensitized and challenged mice, but its role in regulating inflammation or AHR has been unknown. We found that blocking the tissue-binding function of factor H with a competitive antagonist increased complement activation and tissue inflammation after allergen challenge of sensitized mice. Conversely, administration of a fusion protein that contains the iC3b/C3d binding region of complement receptor 2 linked to the inhibitory region of factor H, a molecule directly targeting complement-activating surfaces, protected mice in both primary and secondary challenge models of AHR and lung inflammation. Thus, although endogenous factor H does play a role in limiting the development of AHR, strategies to deliver the complement-regulatory region of factor H specifically to the site of inflammation provide greater protection than that afforded by endogenous regulators. Such an agent may be an effective therapy for the treatment of asthma.

Factor H is a regulator of the alternative pathway of complement, and genetic studies have shown that patients with mutations in factor H are at increased risk for several types of renal disease. Pathogenic activation of the alternative pathway in acquired diseases, such as ischemic acute kidney injury, suggests that native factor H has a limited capacity to control the alternative pathway in the kidney. Here we found that an absolute deficiency of factor H produced by gene deletion prevented complement activation on tubulointerstitial cells after ischemia/reperfusion (I/R) injury, likely because alternative pathway proteins were consumed in the fluid phase. In contrast, when fluid-phase regulation by factor H was maintained while the interaction of factor H with cell surfaces was blocked by a recombinant inhibitor protein, complement activation after renal I/R increased. Finally, a recombinant form of factor H, specifically targeted to sites of C3 deposition, reduced complement activation in the tubulointerstitium after ischemic injury. Thus, although factor H does not fully prevent activation of the alternative pathway of complement on ischemic tubules, its interaction with the tubule epithelial cell surface is critical for limiting complement activation and attenuating renal injury after ischemia.

Activation of complement represents one means of natural resistance to infection from a wide variety of potential pathogens. Recently, properdin, a positive regulator of the alternative pathway of complement, has been shown to bind to surfaces and promote complement activation. Here we studied whether properdin-mediated complement activation occurs on the surface of Chlamydia pneumoniae, an obligate intracellular Gram-negative bacterium that causes 10 to 20% of community-acquired pneumonia. We have determined for the first time that the physiological P₂, P₃, and P₄ forms of human properdin bind to the surface of Chlamydia pneumoniae directly. The binding of these physiological forms accelerates complement activation on the Chlamydia pneumoniae surface, as measured by C3b and C9 deposition. Finally, properdin-depleted serum could not control Chlamydia pneumoniae infection of HEp-2 cells compared with normal human serum. However, after addition of native properdin, the properdin-depleted serum recovered the ability to control the infection. Altogether, our data suggest that properdin is a pattern recognition molecule that plays a role in resistance to Chlamydia infection.

C-reactive protein (CRP) is a phylogenetically conserved protein; in humans, it is present in the plasma and at sites of inflammation. At physiological pH, native pentameric CRP exhibits calcium-dependent binding specificity for phosphocholine. In this study, we determined the binding specificities of CRP at acidic pH, a characteristic of inflammatory sites. We investigated the binding of fluid-phase CRP to six immobilized proteins: complement factor H, oxidized low-density lipoprotein, complement C3b, IgG, amyloid β, and BSA immobilized on microtiter plates. At pH 7.0, CRP did not bind to any of these proteins, but, at pH ranging from 5.2 to 4.6, CRP bound to all six proteins. Acidic pH did not monomerize CRP but modified the pentameric structure, as determined by gel filtration, 1-anilinonaphthalene-8-sulfonic acid-binding fluorescence, and phosphocholine-binding assays. Some modifications in CRP were reversible at pH 7.0, for example, the phosphocholine-binding activity of CRP, which was reduced at acidic pH, was restored after pH neutralization. For efficient binding of acidic pH-treated CRP to immobilized proteins, it was necessary that the immobilized proteins, except factor H, were also exposed to acidic pH. Because immobilization of proteins on microtiter plates and exposure of immobilized proteins to acidic pH alter the conformation of immobilized proteins, our findings suggest that conformationally altered proteins form a CRP-ligand in acidic environment, regardless of the identity of the protein. This ligand binding specificity of CRP in its acidic pH-induced pentameric state has implications for toxic conditions involving protein misfolding in acidic environments and favors the conservation of CRP throughout evolution.

Properdin, a positive regulator of the complement system, has recently been reported to bind to certain pathogenic microorganisms, to early or late apoptotic and necrotic cells, and to particular live human cell lines, thus providing a platform for de novo convertase assembly and complement activation. These studies, with some contradictory results, have been carried out with purified properdin, which forms a series of oligomers of a ∼53,000 Mr subunit, assembling into dimers (P₂), trimers (P₃), tetramers (P₄) and higher forms (P(n)). The P(n) forms have been shown to likely be an artefact of purification that results from procedures including freeze-thawing of properdin. In this study we isolated the individual natural forms of properdin (P₂, P₃, and P₄) and separated them from the P(n) forms present in purified frozen properdin using ion exchange and/or size exclusion chromatography. We analyzed the ability of each form to bind to live or necrotic Jurkat and Raji cells, rabbit erythrocytes (E(R)), and zymosan by FACS analysis. While the unseparated properdin and the purified P(n) forms bound to all the surfaces except E(R), the physiological P₂-P₄ forms specifically bound only to zymosan and to necrotic nucleated cells. Our results indicate that aggregated P(n) present in unseparated properdin may bind non-specifically to some surfaces and should be separated before analysis in order to obtain meaningful results. Finally, we have determined for the first time that the physiological forms of human properdin can selectively recognize surfaces and enhance or promote complement activation, which is in agreement with the reported role for properdin as a complement initiator., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Congenital and acquired deficiencies of complement regulatory proteins are associated with pathologic complement activation in several renal diseases. To elucidate the mechanisms by which renal tubular epithelial cells (TECs) control the complement system, we examined the expression of complement regulatory proteins by the cells. We found that Crry is the only membrane-bound complement regulator expressed by murine TECs, and its expression is concentrated on the basolateral surface. Consistent with the polarized localization of Crry, less complement activation was observed when the basolateral surface of TECs was exposed to serum than when the apical surface was exposed. Furthermore, greater complement activation occurred when the basolateral surface of TECs from Crry(-/-)fB(-/-) mice was exposed to normal serum compared with TECs from wild-type mice. Complement activation on the apical and basolateral surfaces was also greater when factor H, an alternative pathway regulatory protein found in serum, was blocked from interacting with the cells. Finally, we injected Crry(-/-)fB(-/-) and Crry(+/+)fB(-/-) mice with purified factor B (an essential protein of the alternative pathway). Spontaneous complement activation was seen on the tubules of Crry(-/-)fB(-/-) mice after injection with factor B, and the mice developed acute tubular injury. These studies indicate that factor H and Crry regulate complement activation on the basolateral surface of TECs and that factor H regulates complement activation on the apical surface. However, congenital deficiency of Crry or reduced expression of the protein on the basolateral surface of injured cells permits spontaneous complement activation and tubular injury.

The complement system is an essential component of the innate immune system that participates in elimination of pathogens and altered host cells and comprises an essential link between the innate and adaptive immune system. Soluble and membrane-bound complement regulators protect cells and tissues from unintended complement-mediated injury. Complement factor H is a soluble complement regulator essential for controlling the alternative pathway in blood and on cell surfaces. Normal recognition of self-cell markers (i.e. polyanions) and C3b/C3d fragments is necessary for factor H function. Inadequate recognition of host cell surfaces by factor H due to mutations and polymorphisms have been associated with complement-mediated tissue damage and disease. On the other hand, unwanted recognition of pathogens and altered self-cells (i.e. cancer) by factor H is used as an immune evasion strategy. This review will focus on the current knowledge related to these versatile recognition properties of factor H., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Properdin, a positive regulator of the alternative pathway (AP) of complement is important in innate immune defenses against invasive neisserial infections. Recently, commercially available unfractionated properdin was shown to bind to certain biological surfaces, including Neisseria gonorrhoeae, which facilitated C3 deposition. Unfractionated properdin contains aggregates or high-order oligomers, in addition to its physiological "native" (dimeric, trimeric, and tetrameric) forms. We examined the role of properdin in AP activation on diverse strains of Neisseria meningitidis and N. gonorrhoeae specifically using native versus unfractionated properdin. C3 deposition on Neisseria decreased markedly when properdin function was blocked using an anti-properdin mAb or when properdin was depleted from serum. Maximal AP-mediated C3 deposition on Neisseriae even at high (80%) serum concentrations required properdin. Consistent with prior observations, preincubation of bacteria with unfractionated properdin, followed by the addition of properdin-depleted serum resulted in higher C3 deposition than when bacteria were incubated with properdin-depleted serum alone. Unexpectedly, none of 10 Neisserial strains tested bound native properdin. Consistent with its inability to bind to Neisseriae, preincubating bacteria with native properdin followed by the addition of properdin-depleted serum did not cause detectable increases in C3 deposition. However, reconstituting properdin-depleted serum with native properdin a priori enhanced C3 deposition on all strains of Neisseria tested. In conclusion, the physiological forms of properdin do not bind directly to either N. meningitidis or N. gonorrhoeae but play a crucial role in augmenting AP-dependent C3 deposition on the bacteria through the "conventional" mechanism of stabilizing AP C3 convertases.

Genetic variations in complement factor H (fH), an inhibitor of the complement alternative pathway (CAP), and oxidative stress are associated with age-related macular degeneration (AMD). Recently, novel complement therapeutics have been created with the capacity to be "targeted" to sites of complement activation. One example is our recombinant form of fH, CR2-fH, which consists of the N-terminus of mouse fH that contains the CAP-inhibitory domain, linked to a complement receptor 2 (CR2) targeting fragment that binds complement activation products. CR2-fH was investigated in vivo in the mouse model of choroidal neovascularization (CNV) and in vitro in oxidatively stressed RPE cell monolayers. RPE deterioration and CNV development were found to require CAP activation, and specific CAP inhibition by CR2-fH reduced the loss of RPE integrity and angiogenesis in CNV. In both the in vivo and in vitro paradigm of RPE damage, a model requiring molecular events known to be involved in AMD, complement-dependent VEGF production, was confirmed. These data may open new avenues for AMD treatment strategies.

Uncontrolled activation of the alternative pathway of complement is thought to be associated with age-related macular degeneration (AMD). The alternative pathway is continuously activated in the fluid phase, and tissue surfaces require continuous complement inhibition to prevent spontaneous autologous tissue injury. Here, we examined the effects of oxidative stress on the ability of immortalized human retinal pigment epithelial cells (ARPE-19) to regulate complement activation on their cell surface. Combined treatment with H(2)O(2) (to induce oxidative stress) and complement-sufficient serum was found to disrupt the barrier function of stable ARPE-19 monolayers as determined by transepithelial resistance (TER) measurements. Neither treatment alone had any effect. TER reduction was correlated with increased cell surface deposition of C3, and could be prevented by using C7-depleted serum, an essential component of the terminal complement pathway. Treatment with H(2)O(2) reduced surface expression of the complement inhibitors DAF, CD55, and CD59, and impaired regulation at the cell surface by factor H present within the serum. Combined treatment of the monolayers with H(2)O(2) and serum elicited polarized secretion of vascular epidermal growth factor (VEGF). Both, secretion of VEGF and TER reduction could be attenuated using either an alternative pathway inhibitor or by blocking VEGF receptor-1/2 signaling. Regarded together, these studies demonstrate that oxidative stress reduces regulation of complement on the surface of ARPE-19 cells, increasing complement activation. This sublytic activation results in VEGF release, which mediates disruption of the cell monolayer. These findings link oxidative stress, complement activation, and apical VEGF release, which have all been associated with the pathogenesis of AMD.

Factor H (fH) is essential for complement homeostasis in fluid-phase and on surfaces. Its two C-terminal domains (CCP 19-20) anchor fH to self-surfaces where it prevents C3b amplification in a process requiring its N-terminal four domains. In atypical hemolytic uremic syndrome (aHUS), mutations clustering toward the C terminus of fH may disrupt interactions with surface-associated C3b or polyanions and thereby diminish the ability of fH to regulate complement. To test this, we compared a recombinant protein encompassing CCP 19-20 with 16 mutants. The mutations had only very limited and localized effects on protein structure. Although we found four aHUS-linked fH mutations that decreased binding to C3b and/or to heparin (a model compound for cell surface polyanionic carbohydrates), we identified five aHUS-associated mutants with increased affinity for either or both ligands. Strikingly, these variable affinities for the individual ligands did not correlate with the extent to which all the aHUS-associated mutants were found to be impaired in a more physiological assay that measured their ability to inhibit cell surface complement functions of full-length fH. Taken together, our data suggest that disruption of a complex fH-self-surface recognition process, involving a balance of affinities for protein and physiological carbohydrate ligands, predisposes to aHUS.

Factor H is the primary soluble regulator of activation of the alternative pathway of complement. It prevents activation of complement on host cells and tissues upon association with C3b and surface polyanions such as sialic acids, heparin, and other glycosaminoglycans. Here we show that interaction with polyanions causes self-association forming tetramers of the 155,000 Da glycosylated protein. Monomeric human factor H is an extended flexible protein that exhibits an apparent size of 330,000 Da, relative to globular standards, during gel filtration chromatography in the absence of polyanions. In the presence of dextran sulfate (5000 Da) or heparin an intermediate species of apparent m.w. 700,000 and a limit species of m.w. 1,400,000 were observed by gel filtration. Sedimentation equilibrium analysis by analytical ultracentrifugation indicated a monomer Mr of 163,000 in the absence of polyanions and a Mr of 607,000, corresponding to a tetramer, in the presence of less than a 2-fold molar excess of dextran sulfate. Increasing concentrations of dextran sulfate increased binding of factor H to zymosan-C3b 4.5-fold. This result was accompanied by an increase in both the decay accelerating and cofactor activity of factor H on these cells. An expressed fragment encompassing the C-terminal polyanion binding site (complement control protein domains 18-20) also exhibited polyanion-induced self-association, suggesting that the C-terminal ends of factor H mediate self-association. The results suggest that recognition of polyanionic markers on host cells and tissues by factor H, and the resulting regulation of complement activation, may involve formation of dimers and tetramers of factor H.

Cobra venom factor (CVF) is a structural and functional analog of complement C3 isolated from cobra venom. Both CVF and C3b can bind factor B and subsequently form the bimolecular C3/C5 convertases CVF,Bb or C3b,Bb, respectively. The two homologous enzymes exhibit several differences of which the difference in physico-chemical stability is most important, allowing continuous activation of C3 and C5 by CVF,Bb, leading to serum complement depletion. Here we describe the detailed functional properties of two hybrid proteins in which the 113 or 315 C-terminal residues of C3 were replaced with corresponding CVF sequences. Both hybrid proteins formed stable convertases that exhibited C3-cleaving activity, although at different rates. Neither convertase cleaved C5. Both convertases showed partial resistance to inactivation by factors H and I, allowing them to deplete complement in human serum. These data demonstrate that functionally important structural differences between CVF and C3 are located in the very C-terminal region of both homologous proteins, and that small substitutions in human C3 with homologous CVF sequence result in C3 derivatives with CVF-like functions. Such hybrid proteins are important tools to study the structure/function relationships in both C3 and CVF, and these "humanized CVF" proteins may become reagents for therapeutic complement depletion.

Pathogen-specific complement activation requires direct recognition of pathogens and/or the absence of complement control mechanisms on their surfaces. Antibodies direct complement activation to potential pathogens recognized by the cellular innate and adaptive immune systems. Similarly, the plasma proteins MBL and ficolins direct activation to microorganisms expressing common carbohydrate structures. The absence of complement control proteins permits amplification of complement by the alternative pathway on any unprotected surface. The importance of complement recognition molecules (MBL, ficolins, factor H, C3, C1q, properdin, and others) to human disease are becoming clear as analysis of genetic data and knock out animals reveals links between complement proteins and specific diseases.

The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl](1/2) at 3.4-5M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150-1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys(36)-Cys(49) and two disulfide bonds formed by two pair of consecutive cysteines, Cys(22)-Cys(23) and Cys(56)-Cys(57), a unique disulfide structure of polypeptide that has not been documented previously.

Efficient protein digestion is a critical step for successful mass spectrometry analysis. Here we describe simultaneous tryptic digestion and gradual unfolding of native proteins by application of a temperature gradient using a single cycle of 5 min or less in a PCR thermocycler. Chemicals typically used for chromatographic techniques did not affect the digestion efficiency. Tryptic digestion was performed in a small volume (3 microL) with 1.5 microg of trypsin without denaturing agents. This rapid procedure yielded more peptides than conventional methods utilizing chemical denaturation for 18 proteins out of 20. Samples were directly spotted on the MALDI-TOF target plate, without additional purification, thus reducing losses on reversed-phase resins.

Factor H is a regulatory glycoprotein of the complement system. We expressed the three N-terminal complement control protein modules of human factor H (FH1-3) and confirmed FH1-3 to be the minimal unit with cofactor activity for C3b proteolysis by factor I. We reconstructed FH1-3 from NMR-derived structures of FH1-2 and FH2-3 revealing an approximately 105-A-long rod-like arrangement of the modules. In structural comparisons with other C3b-engaging proteins, factor H module 3 most closely resembles factor B module 3, consistent with factor H competing with factor B for binding C3b. Factor H modules 1, 2, and 3 each has a similar backbone structure to first, second, and third modules, respectively, of functional sites in decay accelerating factor and complement receptor type 1; the equivalent intermodular tilt and twist angles are also broadly similar. Resemblance between molecular surfaces is closest for first modules but absent in the case of second modules. Substitution of buried Val-62 with Ile (a factor H single nucleotide polymorphism potentially protective for age-related macular degeneration and dense deposit disease) causes rearrangements within the module 1 core and increases thermal stability but does not disturb the interface with module 2. Replacement of partially exposed (in module 1) Arg-53 by His (an atypical hemolytic uremic syndrome-linked mutation) did not impair structural integrity at 37 degrees C, but this FH1-2 mutant was less stable at higher temperatures; furthermore, chemical shift differences indicated potential for small structural changes at the module 1-2 interface.

Paroxysmal nocturnal hemoglobinuria (PNH) cells are partially (type II) or completely (type III) deficient in GPI-linked complement regulatory proteins CD59 and CD55. PNH III erythrocytes circulate 6 to 60 days in vivo. Why these cells are not lysed as rapidly by complement as unprotected foreign cells, which normally lyse within minutes, remains undetermined. Factor H plays a key role in the homeostasis of complement in fluid phase and on cell surfaces. We have recently shown that a recombinant protein encompassing the C-terminus of factor H (rH19-20) specifically blocks cell-surface complement regulatory functions of factor H without affecting fluid-phase control of complement. Here we show that PNH II and III cells become highly susceptible to complement-mediated lysis by nonacidified normal human serum in vitro, when the cell surface complement-regulatory functions of factor H are blocked. The results indicate that cells deficient in surface-bound regulators are protected for extended periods of time by factor H.

A common single nucleotide polymorphism in the factor H gene predisposes to age-related macular degeneration. Factor H blocks the alternative pathway of complement on self-surfaces bearing specific polyanions, including the glycosaminoglycan chains of proteoglycans. Factor H also binds C-reactive protein, potentially contributing to noninflammatory apoptotic processes. The at risk sequence contains His (rather than Tyr) at position 402 (384 in the mature protein), in the seventh of the 20 complement control protein (CCP) modules (CCP7) of factor H. We expressed both His(402) and Tyr(402) variants of CCP7, CCP7,8, and CCP6-8. We determined structures of His(402) and Tyr(402) CCP7 and showed them to be nearly identical. The side chains of His/Tyr(402) have similar, solvent-exposed orientations far from interfaces with CCP6 and -8. Tyr(402) CCP7 bound significantly more tightly than His(402) CCP7 to a heparin affinity column as well as to defined-length sulfated heparin oligosaccharides employed in gel mobility shift assays. This observation is consistent with the position of the 402 side chain on the edge of one of two glycosaminoglycan-binding surface patches on CCP7 that we inferred on the basis of chemical shift perturbation studies with a sulfated heparin tetrasaccharide. According to surface plasmon resonance measurements, Tyr(402) CCP6-8 binds significantly more tightly than His(402) CCP6-8 to immobilized C-reactive protein. The data support a causal link between H402Y and age-related macular degeneration in which variation at position 402 modulates the response of factor H to age-related changes in the glycosaminoglycan composition and apoptotic activity of the macula.

A high affinity C5 convertase is generated when a C3 convertase deposits additional C3b molecules on and around itself thereby switching the substrate specificity of C3 convertase from C3 to C5. In the present study the role of the additional C3b molecules in influencing the regulation of classical pathway C5 convertase by C4b-binding protein (C4BP) was examined and compared to its precursor, the C3 convertase. Determination of IC(50) for inhibiting formation of the high affinity C5 convertase and for enhancing its decay (72 and 20 nM) were found to be similar to those obtained for the surface-bound C3 convertase (35 and 11 nM). No difference was observed in the cofactor activity of C4BP for surface-bound C4b alone or when in complex with C3b. Analysis of binding interactions between C4BP and EAC1,C4b cells revealed an average apparent dissociation constant (12 nM) similar to that obtained with EAC1,C4b cells with C3b on them (11 nM). Increasing the C4b or C3b density on the cell surface did not alter the affinity of C4BP. The data suggest that C4BP regulates the C5 convertase by mechanisms similar to those observed for the C3 convertase. Since the IC(50) for inhibiting formation of the soluble C3 convertase (5 nM) is 50-80-fold below the normal serum concentration of C4BP (250-400 nM), C4BP in blood effectively prevents formation of classical pathway C3 convertase in the fluid phase. Although deposition of additional C3b molecules is necessary to convert a C3 convertase to a high affinity C5 convertase, the additional C3b molecules play no role in the regulation of C5 convertase by C4BP.

The plasma protein factor H primarily controls the activation of the alternative pathway of complement. The C-terminal of factor H is known to be involved in protection of host cells from complement attack. In the present study, we show that domains 19-20 alone are capable of discriminating between host-like and complement-activating cells. Furthermore, although factor H possesses three binding sites for C3b, binding to cell-bound C3b can be almost completely inhibited by the single site located in domains 19-20. All of the regulatory activities of factor H are expressed by the N-terminal four domains, but these activities toward cell-bound C3b are inhibited by isolated recombinant domains 19-20 (rH 19-20). Direct competition with the N-terminal site is unlikely to explain this because regulation of fluid phase C3b is unaffected by domains 19-20. Finally, we show that addition of isolated rH 19-20 to normal human serum leads to aggressive complement-mediated lysis of normally nonactivating sheep erythrocytes and moderate lysis of human erythrocytes, which possess membrane-bound regulators of complement. Taken together, the results highlight the importance of the cell surface protective functions exhibited by factor H compared with other complement regulatory proteins. The results may also explain why atypical hemolytic uremic syndrome patients with mutations affecting domains 19-20 can maintain complement homeostasis in plasma while their complement system attacks erythrocytes, platelets, endothelial cells, and kidney tissue.

Mutations and polymorphisms in the regulator of complement activation, factor H, have been linked to atypical hemolytic uremic syndrome (aHUS), membranoproliferative glomerulonephritis, and age-related macular degeneration. Many aHUS patients carry mutations in the two C-terminal modules of factor H, which normally confer upon this abundant 155-kDa plasma glycoprotein its ability to selectively bind self-surfaces and prevent them from inappropriately triggering the complement cascade via the alternative pathway. In the current study, the three-dimensional solution structure of the C-terminal module pair of factor H has been determined. A binding site for a fully sulfated heparin-derived tetrasaccharide has been delineated using chemical shift mapping and the C3d/C3b-binding site inferred from sequence comparisons and computational docking. The resultant information allows assessment of the likely consequences of aHUS-associated amino acid substitutions in this critical region of factor H. It is striking that, excepting those likely to perturb the three-dimensional structure, aHUS-associated missense mutations congregate in the polyanion-binding site delineated in this study, thus potentially disrupting a vital mechanism for control of complement on self-surfaces in the microvasculature of the kidney. It is intriguing that a single nucleotide polymorphism predisposing to age-related macular degeneration occupies another region of factor H that harbors a polyanion-binding site.

Glycoprotein C (gC) of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) binds complement component C3b and protects virus from complement-mediated neutralization. Differences in complement interacting domains exist between gC of HSV-1 (gC1) and HSV-2 (gC2), since the amino terminus of gC1 blocks complement C5 from binding to C3b, while gC2 fails to interfere with this activity. We previously reported that neutralization of HSV-1 gC-null virus by HSV antibody-negative human serum requires activation of C5 but not of downstream components of the classical complement pathway. In this report, we evaluated whether activation of C5 is sufficient to neutralize HSV-2 gC-null virus, or whether formation of the membrane attack complex by C6 to C9 is required for neutralization. We found that activation of the classical complement pathway up to C5 was sufficient to neutralize HSV-2 gC-null virus by HSV antibody-negative human serum. We evaluated the mechanisms by which complement activation occurred in seronegative human serum. Interestingly, natural immunoglobulin M antibodies bound to virus, which triggered activation of C1q and the classical complement pathway. HSV antibody-negative sera obtained from four individuals differed over an approximately 10-fold range in their potency for complement-mediated virus neutralization. These findings indicate that humans differ in the ability of their innate immune systems to neutralize HSV-1 or HSV-2 gC-null virus and that a critical function of gC1 and gC2 is to prevent C5 activation.

Pneumococcal surface protein C (PspC) binds to the complement regulatory protein factor H (FH), which inhibits alternative pathway activation. In the present study, using a mouse model of systemic infection and flow-cytometric analyses, we demonstrated an in vivo interaction between FH and pneumococci and showed differential FH binding during bacteremia. Flow-cytometric analyses of pneumococci harvested after intraperitoneal (ip) challenge demonstrated increased binding of FH, compared with that after intravenous (iv) challenge. Real-time polymerase chain reaction analyses of PspC mRNA showed that, relative to pneumococci grown in vitro, those recovered from the blood of mice 24 h after iv challenge exhibited 23-fold higher mRNA levels; however, after ip challenge, PspC mRNA induction was increased 870-fold. A subsequent increase in PspC expression was detected by flow cytometry using a monoclonal antibody against PspC. Furthermore, pneumococci with FH bound to complement before exposure had increased proliferation, compared with pneumococci not pretreated with FH. These results suggest that the interaction between PspC and FH contributes to pneumococcal virulence.

Exploitation of recombinant technology to study proteins containing strings of short consensus repeat (SCR) domains largely depends on expression vectors. In this paper, we describe a vector for cloning and constitutive expression of single or multiple SCR domains. The recombinant vector has unique additive features over commercially available vectors that make it a universal cloning vector for SCR domains as well as a vector suitable for expressing any protein fragment beginning and ending with cysteine residues. As a demonstration of its usefulness, the constitutive extracellular expression of five SCR-containing proteins derived from complement factor H is presented.

Streptococcus pneumoniae, also known as the pneumococcus, contains several surface proteins that along with the polysaccharide capsule function in antiphagocytic activities and evasion of the host immune system. These pneumococcal proteins interact with the host immune system in various ways and possess a wide range of biological activities that suggests that they may be involved at different stages of pneumococcal infection. PspC, also known as CbpA and SpsA, is one of several pneumococcal surface proteins that binds host proteins, including factor H (FH) and secretory IgA (sIgA) via the secretory component. Previous work by our laboratory has demonstrated that PspC on the surface of live pneumococcal cells binds FH. This paper provides evidence that FH activity is maintained in the presence of PspC and that the PspC binding site is located in the short consensus repeat 6-10 region of FH. We also report for the first time that although both FH and sIgA binding has been localized to the alpha-helical domain of PspC, the binding of FH to PspC is not inhibited by sIgA. ELISA, surface plasmon resonance, and flow cytometry indicate that the two host proteins do not compete for binding with PspC and likely do not share the same binding sites. We confirmed by Western analysis that the binding sites are separate using recombinant PspC proteins. These PspC variants bind FH yet fail to bind sIgA. Thus, we conclude that FH and sIgA can bind concurrently to the alpha-helical region of PspC.

Background & Objectives: Streptococcus pneumoniae has acquired virulence factors such as the polysaccharide capsule and various surface proteins, which prevent opsonization mediated by the complement system. PspC is one of the multi-functional pneumococcal surface proteins capable of eliciting an antibody response in mice. Our study further explores the role of pneumococcal surface proteins in resistance to complement mediated opsonophagocytosis by providing evidence that PspC binds human Factor H (FH), a regulatory protein of the alternative complement pathway. The present study was carried out to map the binding regions on PspC and FH, and to assess the functional activity of FH upon binding to PspC., Methods: FH binding to D39 and other pneumococcal strains was observed by flow cytometry. A series of FH truncated and deletion mutants and PspC mutants were used to localize binding regions within these molecules. The functional activity of FH upon binding to PspC was measured by a haemolysis assay., Results: FH binding to D39 and not to TRE108 (PspC-) cells was demonstrated by flow cytometry. Pneumococcal isolates of 14 different strains varied in their ability to bind FH. The binding region of FH within PspC to the first 225 amino acids of the alpha-helical domain was localized. The corresponding binding site for PspC is located within the SCR 6-10 region of FH. Haemolysis of rabbit red blood cells was inhibited by FH even in the presence of PspC., Interpretation & Conclusion: FH binding is specific to PspC on the pneumococcal cell surface. The binding region on PspC mapped to the non-conserved N-terminal region of the alpha-helical domain. The binding site on FH to PspC is different from the active site that functions in degradation of C3b. A haemolysis assay provided evidence that the functional activity of FH was maintained upon binding to PspC. Thus, binding of FH to PspC might be an important mechanism by which S. pneumoniae resist complement activation and opsonophagocytosis.

C3/C5 convertase is a serine protease that cleaves C3 and C5. In the present study we examined the C5 cleaving properties of classical pathway C3/C5 convertase either bound to the surface of sheep erythrocytes or in its free soluble form. Kinetic parameters revealed that the soluble form of the enzyme (C4b,C2a) cleaved C5 at a catalytic rate similar to that of the surface-bound form (EAC1,C4b,C2a). However, both forms of the enzyme exhibited a poor affinity for the substrate, C5, as indicated by a high Km (6-9 microM). Increasing the density of C4b on the cell surface from 8,000 to 172,000 C4b/cell did not influence the Km. Very high affinity C5 convertases were generated only when the low affinity C3/C5 convertases (EAC1,C4b,C2a) were allowed to deposit C3b by cleaving native C3. These C3b-containing C3/C5 convertases exhibited Km (0.0051 microM) well below the normal concentration of C5 in blood (0.37 microM). The data suggest that C3/C5 convertase assembled with either monomeric C4b or C4b-C4b complexes are inefficient in capturing C5 but cleave C3 opsonizing the cell surface with C3b for phagocytosis. Deposition of C3b converts the enzymes to high affinity C5 convertases, which cleave C5 in blood at catalytic rates approaching Vmax, thereby switching from C3 to C5 cleavage. Comparison of the kinetic parameters with those of the alternative pathway convertase indicates that the 6-9-fold greater catalytic rate of the classical pathway C5 convertase may compensate for the fewer numbers of C5 convertase sites generated upon activation of this pathway.

Incidents of hemolytic uremic syndrome (HUS) include a subset of patients that exhibit mutations in C factor H. These mutations cluster in the C-terminal domains of factor H where previous reports have identified polyanion and C3b-binding sites. In this study, we show that recombinant human factor H with deletions at the C-terminal end of the protein loses the ability to control the spontaneous activation of the alternative C pathway on host-like surfaces. For the pathology of HUS, the findings imply that mutations that disrupt the normal functions of the C-terminal domains prevent host polyanion recognition. The resulting uncontrolled activation of complement on susceptible host tissues appears to be the initiating event behind the acute renal failure of familial HUS patients.

The multisubunit enzymes of the complement system that cleave C5 have many unusual properties, the most striking of which is that they acquire their specificity for C5 following cleavage of another substrate C3. C5 convertases are assemblies of two proteins C4b and C2a (classical or lectin pathways) or C3b and Bb (alternative pathway) and additional C3b molecules. The catalytic complexes (C4b, C2a or C3b, Bb) are intrinsically unstable ( t (1/2)=1-3 min) and the enzymes are controlled by numerous regulatory proteins that accelerate this natural decay rate. Immediately after assembly, the bi-molecular enzymes preferentially cleave the protein C3 and exhibit poor activity toward C5 (a K (m) of approx. 25 microM and a C5 cleavage rate of 0.3-1 C5/min at V (max)). Efficient C3 activation results in the covalent attachment of C3b to the cell surface and to the enzyme itself, resulting in formation of C3b-C3b and C4b-C3b complexes. Our studies have shown that deposition of C3b alters the specificity of the enzymes of both pathways by changing the K (m) for C5 more than 1000-fold from far above the physiological C5 concentration to far below it. Thus, after processing sufficient C3 at the surface of a microorganism, the enzymes switch to processing C5, which initiates the formation of the cytolytic membrane attack complex of complement.

PspC was found to bind human complement factor H (FH) by Western blot analysis of D39 (pspC(+)) and an isogenic mutant TRE108 (pspC). We confirmed that PspA does not bind FH, while purified PspC binds FH very strongly. The binding of FH to exponentially growing pneumococci varied among different isolates when analyzed by fluorescence activated cell sorting analysis.

Screening of 29 strains of Neisseria gonorrhoeae revealed that 16/21 serum resistant strains and 0/8 serum sensitive strains bound C4bp, suggesting that C4bp binding to gonococci could contribute to serum resistance. C4bp bound to gonococci retained cofactor (C4b-degrading) function. Using allelic exchange to construct strains with hybrid Por1A/B molecules, we demonstrate that the N-terminal loop (loop 1) of Por1A is required for C4bp binding. Serum resistant Por1B gonococcal strains also bind C4bp via their Por molecule. Using allelic exchange and site-directed mutagenesis, we have shown that loops 5 and 7 together form a negatively charged C4bp binding domain. C4bp-Por1B interactions are ionic in nature (inhibited by high salt as well as by heparin), while the C4bp-Por1A bond is hydrophobic. mAbs directed against SCR1 of the alpha-chain of C4bp inhibit C4bp binding to both Por1A and Por1B. Furthermore, only recombinant C4bp mutant molecules that contain alpha-chain SCR1 bind both Por1A and Por1B gonococci, confirming that SCR1 contains Por binding sites. C4bp alpha-chain monomers do not bind strains with either Por molecule, suggesting that the polymeric form of C4bp is required for binding to gonococci. Inhibition of C4bp binding to serum resistant Por1A and Por1B strains in a serum bactericidal assay using fAb fragments against C4bp SCR1 results in complete killing at 30 min of otherwise fully serum resistant strains in only 10% normal serum, underscoring the role of C4bp in mediating gonococcal serum resistance.

C5 convertases are serine proteases that cleave both C3 and C5. Alternative pathway C3/C5 convertases formed with monomeric C3b (C3b,Bb) because of their weak interaction with C5 primarily cleave C3 thereby opsonizing the cell surface with C3b. In contrast, C3/C5 convertases formed with a high density of C3b/cell exhibit higher affinities for C5 as indicated by Km values well below the physiological concentration of C5 in blood. These C3/C5 convertases bind C5 efficiently and cleave it at a velocity approaching Vmax thereby switching the enzyme from C3 cleavage to production of the cytolytic C5b-9 complex. Studies of the structure of C3/C5 convertases have postulated that C4b-C3b and C3b-C3b dimers from high affinity C5 binding sites while indel studies have shown two binding sites in C5 for the convertase in addition to the C5 cleavage site. Together, these studies indicate that with increasing deposition of C3b on the surface, C3b complexes are formed which through multivalent attachment bind the substrate C5 with higher affinities, thereby converting the low affinity C3/C5 convertases to high affinity C5 convertases. The process underlying the formation of high affinity C5 convertases during complement activation is discussed.

We screened 29 strains of Neisseria gonorrhoeae and found 16/21 strains that resisted killing by normal human serum and 0/8 serum sensitive strains that bound the complement regulator, C4b-binding protein (C4bp). Microbial surface-bound C4bp demonstrated cofactor activity. We constructed gonococcal strains with hybrid porin (Por) molecules derived from each of the major serogroups (Por1A and Por1B) of N. gonorrhoeae, and showed that the loop 1 of Por1A is required for C4bp binding. Por1B loops 5 and 7 of serum-resistant gonococci together formed a negatively charged C4bp-binding domain. C4bp-Por1B interactions were ionic in nature (inhibited by high salt or by heparin), whereas the C4bp-Por1A bond was hydrophobic. Only recombinant C4bp mutant molecules containing the NH2-terminal alpha-chain short consensus repeat (SCR1) bound to both Por1A and Por1B gonococci, suggesting that SCR1 contained Por binding sites. C4bp alpha-chain monomers did not bind gonococci, indicating that the polymeric form of C4bp was required for binding. Using fAb fragments against C4bp SCR1, C4bp binding to Por1A and Por1B strains was inhibited in a complement-dependent serum bactericidal assay. This resulted in complete killing of these otherwise fully serum resistant strains in only 10% normal serum, underscoring the importance of C4bp in mediating gonococcal serum resistance.

The envelope surface glycoprotein C (gC) of HSV-1 interferes with the complement cascade by binding C3 and activation products C3b, iC3b, and C3c, and by blocking the interaction of C5 and properdin with C3b. Wild-type HSV-1 is resistant to Ab-independent complement neutralization; however, HSV-1 mutant virus lacking gC is highly susceptible to complement resulting in > or =100-fold reduction in virus titer. We evaluated the mechanisms by which complement inhibits HSV-1 gC null virus to better understand how gC protects against complement-mediated neutralization. C8-depleted serum prepared from an HSV-1 and -2 Ab-negative donor neutralized gC null virus comparable to complement-intact serum, indicating that C8 and terminal lytic activity are not required. In contrast, C5-depleted serum from the same donor failed to neutralize gC null virus, supporting a requirement for C5. EDTA-treated serum did not neutralize gC null virus, indicating that complement activation is required. Factor D-depleted and C6-depleted sera neutralized virus, suggesting that the alternative complement pathway and complement components beyond C5 are not required. Complement did not aggregate virus or block attachment to cells. However, complement inhibited infection before early viral gene expression, indicating that complement affects one or more of the following steps in virus replication: virus entry, uncoating, DNA transport to the nucleus, or immediate early gene expression. Therefore, in the absence of gC, HSV-1 is readily inhibited by complement by a C5-dependent mechanism that does not require viral lysis, aggregation, or blocking virus attachment.