Aspergillus conidia activate the complement by the mannan-binding lectin C2 bypass mechanism

Innate immunity is the major host defense against invasive aspergillosis. To determine whether the collectin mannan-binding lectin (MBL) is involved in the initial protective immunity through complement activation against opportunistic fungal infections caused by Aspergillus, we performed in vitro studies on 29 different strains of Aspergillus conidia from five different species. Incubation of Aspergillus conidia in human normal serum leads to activation of the alternative pathway, whereas neither the classical nor the lectin pathways through C4 and C2 cleavage are activated. Complement response to conidia was investigated using a MBL-deficient serum and reconstitution experiments were conducted with MBL/MASPs complexes. We found that MBL can directly support C3 activation by a C2 bypass mechanism. Finally, a stronger activation of the alternative pathway was observed for the clinical strains isolated from patients with invasive aspergillosis, compared with the environmental strains.     

Isolation and characterization of a mannan-binding protein from rabbit serum

A binding protein which recognizes mannose and N-acetylglucosamine has been isolated from rabbit serum to apparent homogeneity. The serum binding protein was nearly identical to the mannan-binding protein isolated previously from rabbit liver [Kawasaki, T., Etoh, R. and Yamashina, I. (1978) Biochem. Biophys. Res. Commun. $\text{81}$, 1018–1024] in respect of immunochemical properties and subunit profiles, but could be differentiated from the liver protein in its larger molecular size and inferior sensitivity to monosaccharides as haptenic inhibitors of the binding to ¹²⁵I-mannan. A postulation was made that the plasma was, comparable with the liver, a major locus of mannan-binding protein in the rabbit.     

Structural basis of mannan-binding lectin recognition by its associated serine protease MASP-1: implications for complement activation

Complement activation contributes directly to health and disease. It neutralizes pathogens and stimulates immune processes. Defects lead to immunodeficiency and autoimmune diseases, whereas inappropriate activation causes self-damage. In the lectin and classical pathways, complement is triggered upon recognition of a pathogen by an activating complex. Here we present the first structure of such a complex in the form of the collagen-like domain of mannan-binding lectin (MBL) and the binding domain of its associated protease (MASP-1/-3). The collagen binds within a groove using a pivotal lysine side chain that interacts with Ca(2+)-coordinating residues, revealing the essential role of Ca(2+). This mode of binding is prototypic for all activating complexes of the lectin and classical pathways, and suggests a general mechanism for the global changes that drive activation. The structural insights reveal a new focus for inhibitors and we have validated this concept by targeting the binding pocket of the MASP.     

The activation of the alternative complement pathway by fetal calf serum and mouse thymocytes.

Mouse thymocytes activated the alternative complement pathway of mouse serum in the presence of heated fetal calf serum. The activation required C3 from the fetal calf serum but was independent of antibody either in the murine or bovine serum. No other murine cells tested, including erythrocytes, peripheral blood lymphocytes, lymph node cells, spleen cells, and various cultured cell lines, activated the alternative complement pathway as effectively as thymocytes. In addition, sera from species other than cows could not substitute for fetal calf serum. The C3 deposited on thymocytes was in the form of both C3b (immune adherence positive) and C3bi (conglutinable). We propose that the basis of activation in this system is the specific protection of bovine C3b on mouse thymocyte surface.     

Two constituents of the initiation complex of the mannan-binding lectin activation pathway of complement are encoded by a single structural gene

Mannan-binding lectin (MBL) forms a multimolecular complex with at least two MBL-associated serine proteases, MASP-1 and MASP-2. This complex initiates the MBL pathway of complement activation by binding to carbohydrate structures present on bacteria, yeast, and viruses. MASP-1 and MASP-2 are composed of modular structural motifs similar to those of the C1q-associated serine proteases C1r and C1s. Another protein of 19 kDa with the same N-terminal sequence as the 76-kDa MASP-2 protein is consistently detected as part of the MBL/MASP complex. In this study, we present the primary structure of this novel MBL-associated plasma protein of 19 kDa, MAp19, and demonstrate that MAp19 and MASP-2 are encoded by two different mRNA species generated by alternative splicing/polyadenylation from one structural gene.     

Role of interaction of mannan-binding protein with meprins at the initial step of complement activation in ischemia/reperfusion injury to mouse kidney

Ischemia/reperfusion (I/R) is an important cause of acute renal failure. Recent studies have shown that the complement system mediated by the mannan-binding protein (MBP), which is a C-type serum lectin recognizing mannose, fucose and N-acetylglucosamine residues, plays a critical role in the pathogenesis of ischemic acute renal failure. MBP causes complement activation through the MBP lectin pathway and a resulting complement component, C3b, is accumulated on the brush borders of kidney proximal tubules in a renal I/R-operated mouse kidney. However, the initial step of the complement activation has not been studied extensively. We previously identified both meprins α and β, highly glycosylated zinc metalloproteases, localized on kidney proximal tubules as endogenous MBP ligands. In the present study, we demonstrated that serum-type MBP (S-MBP) and C3b were co-localized with meprins on both the cortex and the medulla in the renal I/R-operated mouse kidney. S-MBP was indicated to interact with meprins in vivo in the I/R-operated mouse kidney and was shown to initiate the complement activation through the interaction with meprins in vitro. Taken together, the present study strongly suggested that the binding of S-MBP to meprins triggers the complement activation through the lectin pathway and may cause the acute renal failure due to I/R on kidney transplantation and hemorrhagic shock.     

The mechanism of inhibition of the activation of the complement first component by polyanions and polycations

Polyethyleneimine (PEI, 50 kDa) and polymethacrylic acid (PMA, 200 kDa) were shown to inhibit the lysis of sheep erythrocytes induced by the guinea pig complement. They twofold suppress the hemolysis at the concentrations of 0.47 and 0.89 microgram/ml, respectively. The inhibitory effect on the binding of the C1q subunit of human complement to the sensitized sheep erythrocytes (EA) was found to depend on the component of the reaction with which the inhibitors were preliminarily incubated. When an inhibitor, C1q, and EA were simultaneously incubated, the inhibition constants for PEI and PMA were 17 +/- 6 and 8.1 +/- 0.1 micrograms/ml, respectively. The preincubation of EA with PEI and the subsequent washing out of the inhibitor resulted in the inhibition constant of 22 +/- 3 micrograms/ml. No inhibitory effect was observed after a similar preincubation of EA with PMA. No inhibition was also detected when the inhibitors were added after the formation of the C1q complex with antibodies. These observations suggest that the binding of antibodies to cationic PEI prevents the C1q-antibody complex formation, while the binding of anionic PMA to the active site of C1q impedes the interaction of this subunit with immunoglobulins. Moreover, within the range of concentrations studied, the studied inhibitors did not affect the subsequent C1q binding to the C1r and C1s enzymes.     

The complement control protein homolog of herpesvirus saimiri regulates serum complement by inhibiting C3 convertase activity.

The herpesvirus saimiri genome encodes a complement control protein homolog (CCPH). Stable mammalian cell transfectants expressing a recombinant transmembrane form of CCPH (mCCPH) or a 5'FLAG epitope-tagged mCCPH (5'FLAGmCCPH) conferred resistance to complement-mediated cell damage by inhibiting the lytic activity of human serum complement. The function of CCPH was further defined by showing that the mCCPH and the 5'FLAGmCCPH transfectants inhibited C3 convertase activity and effectively reduced cell surface deposition of the activated complement component, C3d.     

The activation mechanism of human complement system by immune precipitate formed with rabbit IgG antibody

Immune precipitate (Ippt) formed between egg albumin and rabbit IgG antibody activated both pathways of the human complement system. On incubation with diluted serum, Ippt combined with several factors in the serum to form a complex which acquired C3- and C5-cleaving activities. In serum chelated with ethyleneglycol tetraacetic acid (EGTA), C3- and C5-cleaving activities of properdin system enzymes were formed on Ippt. Kinetic studies on the formation and the decay of C3- and C5-cleaving enzymes on Ippt revealed that C3- and C5-cleaving activites were almost dependent on the properdin system enzymes. The experiments in which C3-cleaving activity formed on Ippt was inhibited by anti-properdin or anti-B but not by anti-C4 supported the above results. The participation of the classical pathway was considered to accelerate the assembly of the properdin system enzymes.     

Surfactant protein A regulates complement activation.

Complement proteins aid in the recognition and clearance of pathogens from the body. C1, the first protein of the classical pathway of complement activation, is a calcium-dependent complex of one molecule of C1q and two molecules each of C1r and C1s, the serine proteases that cleave complement proteins. Upon binding of C1q to Ag-bound IgG or IgM, C1r and C1s are sequentially activated and initiate the classical pathway of complement. Because of structural and functional similarities between C1q and members of the collectin family of proteins, including pulmonary surfactant protein A (SP-A), we hypothesized that SP-A may interact with and regulate proteins of the complement system. Previously, SP-A was shown to bind to C1q, but the functional significance of this interaction has not been investigated. Binding studies confirmed that SP-A binds directly to C1q, but only weakly to intact C1. Further investigation revealed that the binding of SP-A to C1q prevents the association of C1q with C1r and C1s, and therefore the formation of the active C1 complex required for classical pathway activation. This finding suggests that SP-A may share a common binding site for C1r and C1s or Clq. SP-A also prevented C1q and C1 from binding to immune complexes. Furthermore, SP-A blocked the ability of C1q to restore classical pathway activity to C1q-depleted serum. SP-A may down-regulate complement activity through its association with C1q. We hypothesize that SP-A may serve a protective role in the lung by preventing C1q-mediated complement activation and inflammation along the delicate alveolar epithelium.     

Human IgA activates the complement system via the mannan-binding lectin pathway

The recently identified lectin pathway of the complement system, initiated by binding of mannan-binding lectin (MBL) to its ligands, is a key component of innate immunity. MBL-deficient individuals show an increased susceptibility for infections, especially of the mucosal system. We examined whether IgA, an important mediator of mucosal immunity, activates the complement system via the lectin pathway. Our results indicate a dose-dependent binding of MBL to polymeric, but not monomeric IgA coated in microtiter plates. This interaction involves the carbohydrate recognition domain of MBL, because it was calcium dependent and inhibited by mannose and by mAb against this domain of MBL. Binding of MBL to IgA induces complement activation, as demonstrated by a dose-dependent deposition of C4 and C3 upon addition of a complement source. The MBL concentrations required for IgA-induced C4 and C3 activation are well below the normal MBL plasma concentrations. In line with these experiments, serum from individuals having mutations in the MBL gene showed significantly less activation of C4 by IgA and mannan than serum from wild-type individuals. We conclude that MBL binding to IgA results in complement activation, which is proposed to lead to a synergistic action of MBL and IgA in antimicrobial defense. Furthermore, our results may explain glomerular complement deposition in IgA nephropathy.     

G蛋白偶联受体激活丝裂原活化蛋白激酶的机理

多种Ｇ蛋白偶联受体的均能激活丝裂原活化蛋白激酶。Ｇｉ蛋白偶联受体主要通过其βγ亚基,依赖Ｒａｓ蛋白途径;在大多数哺乳类细胞中Ｇｓ蛋白偶联受体通过ＰＫＡ途径抑制Ｒａｓ依赖的ＭＡＰＫ活化,但在ＣＯＳ－７细胞,Ｇｓ蛋白偶联受体通过ＰＫＡ途径使表达的ＭＡＰＫ活化;Ｇｑ蛋白偶联受体主要通过ＰＫＣ途径依赖或非依赖于Ｒａｓ使ＭＡＰＫ活化。ＭＡＰＫ信号途径中ＥＧＦ受体,酪氨酸激酶及调节蛋白Ｓｈｃ等联级反应蛋白可能     

Activation of the human complement system by cholesterol-rich and PEGylated liposomes-modulation of cholesterol-rich liposome-mediated complement activation by elevated serum LDL and HDL levels

Intravenously infused liposomes may induce cardiopulmonary distress in some human subjects, which is a manifestation of "complement activation-related pseudoallergy." We have now examined liposome-mediated complement activation in human sera with elevated lipoprotein (LDL and HDL) levels, since abnormal or racial differences in serum lipid profiles seem to modulate the extent of complement activation and associated adverse responses. In accordance with our earlier observations, cholesterol-rich (45 mol% cholesterol) liposomes activated human complement, as reflected by a significant rise in serum level of S-protein-bound form of the terminal complex (SC5b-9). However, liposome-induced rise of SC5b-9 was significantly suppressed when serum HDL cholesterol levels increased by 30%. Increase of serum LDL to levels similar to that observed in heterozygous familial hypercholesterolemia also suppressed liposome-mediated SC5b-9 generation considerably. While intravenous injection of cholesterol-rich liposomes into pigs was associated with an immediate circulatory collapse, the drop in systemic arterial pressure following injection of liposomes preincubated with human lipoproteins was slow and extended. Therefore, surface-associated lipoprotein particles (or apolipoproteins) seem to lessen liposome-induced adverse haemodynamic changes, possibly as a consequence of suppressed complement activation in vivo. PEGylated liposomes were also capable of activating the human complement system, and the presence of surface projected methoxypoly(ethylene glycol) chains did not interfere with generation of C3 opsonic fragments. We also show that poly(ethylene glycol) is not responsible for PEGylated liposome-mediated complement activation. The net anionic charge on the phosphate moiety of the phospholipid-mPEG conjugate seemed to play a critical role in activation of both the classical and alternative pathways of the complement system.     

Human astrovirus coat protein inhibits serum complement activation via C1, the first component of the classical pathway

Human astroviruses (HAstVs) belong to a family of nonenveloped, icosahedral RNA viruses that cause noninflammatory gastroenteritis, predominantly in infants. Eight HAstV serotypes have been identified, with a worldwide distribution. While the HAstVs represent a significant public health concern, very little is known about the pathogenesis of and host immune response to these viruses. Here we demonstrate that HAstV type 1 (HAstV-1) virions, specifically the viral coat protein (CP), suppress the complement system, a fundamental component of the innate immune response in vertebrates. HAstV-1 virions and purified CP both suppress hemolytic complement activity. Hemolytic assays utilizing sera depleted of individual complement factors as well as adding back purified factors demonstrated that HAstV CP suppresses classical pathway activation at the first component, C1. HAstV-1 CP bound the A chain of C1q and inhibited serum complement activation, resulting in decreased C4b, iC3b, and terminal C5b-9 formation. Inhibition of complement activation was also demonstrated for HAstV serotypes 2 to 4, suggesting that this phenomenon is a general feature of these human pathogens. Since complement is a major contributor to the initiation and amplification of inflammation, the observed CP-mediated inhibition of complement activity may contribute to the lack of inflammation associated with astrovirus-induced gastroenteritis. Although diverse mechanisms of inhibition of complement activation have been described for many enveloped animal viruses, this is the first report of a nonenveloped icosahedral virus CP inhibiting classical pathway activation at C1.     

Novel methods to determine complement activation in human serum induced by the complex of Dezamizumab and serum amyloid P

Lack of simple and robust methods to determine complement activation in human serum induced by antigen–antibody complexes is a major hurdle for monitoring therapeutic antibody drug quality and stability. Dezamizumab is a humanized IgG1 monoclonal antibody that binds to serum amyloid P component (SAP) for potential treatment of systemic amyloidosis. The mechanism of action of Dezamizumab includes the binding of SAP, complement activation through classical pathway, and phagocytosis; however, the steps in this process cannot be easily monitored. We developed two novel methods to determine Dezamizumab-SAP complex-induced complement activation. Complement component 3 (C3) depletion was detected by homogeneous time-resolved fluorescence (HTRF), and C3a desArg fragment, formed after the cleavage of C3 to yield C3a followed by removal of its C-terminal arginine residue, was determined using Meso Scale Discovery (MSD) technology. We found that the presence of both Dezamizumab and SAP was required for complement activation via both methods. The optimal molar ratio of Dezamizumab:SAP was 6:1 in order to obtain maximal complement activation. The relative potency from both methods showed a good correlation to Dezamizumab-SAP-dependent complement component 1q (C1q) binding activity in Dezamizumab thermal-stressed samples. Both SAP and C1q binding, as determined by surface plasmon resonance and the two complement activation potency methods described here, reflect the mechanism of action of Dezamizumab. We conclude that these methods can be used to monitor Dezamizumab quality for drug release and stability testing, and the novel potency methods reported here can be potentially used to evaluate complement activity induced by other antigen–antibody complexes.     

Nonimmunologic complement activation in normal human serum induced by radiographic contrast media.

Two different radiographic contrast media (RCM), iothalamate and iodipamide, induced the activation of several complement (C) components in normal, genetically C2-deficient and agammaglobulinemic human sera in vitro. This activation was dose dependent and demonstrable by a reduction in whole C as well as C4, C2, C3, and C5 hemolytic activities. C6, C8, and C9 hemolytic activities were unaffected. Concommitant with the loss of C3 hemolytic activity was the appearance of C3 proteolytic cleavage products that were identified by immunoelectrophoresis. Both the loss of C3 hemolytic activity and the production of C3 fragments occurred in the presence of 10 mM EDTA, indicating RCM-induced C3 cleavage occurred without participation of the multicomponent C3/C5 convertases of either the classical or alternative C pathways. Furthermore, loss of C3 hemolytic activity was not due to the direct alteration of the C3 molecule by RCM because purified C3 was unaffected upon incubation with RCM at a concentration that induced 80% reduction in the C3 hemolytic activity in normal human serum. Serum samples obtained from 40 patients, before and 30 min after undergoing i.v. pyelography, revealed no significant change in total hemolytic C activity; 34 patients received sodium and methylglucamine diatrizoate and six received sodium iothalamate. Hemolytic C3 levels were also determined for the six patients before and 30 min after administration of sodium iothalamate and no significant change in activity was detectable.     

Identification of a major human serum DNA-binding protein as beta 1H of the alternative pathway of complement activation

The conformation of the molecule cyclo (Aha-$\text{Cys-Phe-D-Trp-Lys-Thr-Cys}$) was studied by empirical conformational energy calculations. The low-energy structure found contains a type II' bend centered at the D-Trp-Lys residues. The lowest energy conformer has the aromatic ring of DTrp positioned such that the γ-protons of the Lys side-chain are in the shielding region (i.e., perpendicular to the center of the aromatic ring). This is in agreement with the NMR results. A mechanism of action for the inhibition of GH release is presented which suggests a conformational change occurs in the D-Trp side-chain ring upon binding to the receptor. The resulting structure has the Phe-D-Trp ring-ring stacking suggested to be responsible for binding and agonist activity of model growth-hormone releasing peptides.     

Regulation of complement activation by C-reactive protein: targeting of the inhibitory activity of C4b-binding protein

C-reactive protein (CRP) is the major acute phase protein in humans. It has been shown that CRP interacts with factor H, an inhibitor of the alternative pathway of complement, and now we demonstrate binding of CRP to the fluid-phase inhibitor of the classical pathway, C4b-binding protein (C4BP). C4BP bound to directly immobilized recombinant CRP as well as CRP attached to phosphorylcholine. The binding was sensitive to ionic strength and was enhanced in the presence of calcium. C4BP lacking beta-chain and protein S, which is a form of C4BP increasing upon inflammation, bound CRP with higher affinity than the C4BP-protein S complex. The binding could not be blocked with mAbs directed against peripheral parts of the alpha-chains of C4BP while the isolated central core of C4BP obtained by partial proteolytic digestion bound CRP, indicating that the binding site for CRP is localized in the central core of the C4BP molecule. Furthermore, we found complexes in serum from a patient with an elevated CRP level and trace amounts of CRP were also identified in a plasma-derived C4BP preparation. We were also able to detect C4BP-CRP complexes in solution and established that C4BP retains full complement regulatory activity in the presence of CRP. In addition, we found that C4BP can compete with C1q for binding to immobilized CRP and that it inhibits complement activation locally. We hypothesize that CRP limits excessive complement activation on targets via its interactions with both factor H and C4BP.