Origin of mannose-binding lectin-associated serine protease (MASP)-1 and MASP-3 involved in the lectin complement pathway traced back to the invertebrate,amphioxus

Mannose-binding lectin-associated serine proteases (MASPs) are involved in complement activation through the lectin pathway. To elucidate the phylogenetic origin of MASP and a primordial complement system, we cloned two MASP cDNAs from amphioxus (Branchiostoma belcheri) of the cephalochordates, considered to be the closest relative of vertebrates. The two sequences, orthologues of mammalian MASP-1 and MASP-3, were produced by alternative processing of RNA from a single gene consisting of a common H chain-encoding region and two L chain-encoding regions, a structure which is similar to that of the human MASP1/3 gene. We also isolated two MASP genes from the ascidian Halocynthia roretzi (urochordates) and found that each of them consists simply of an H chain-encoding region and a single L chain-encoding region. The difference in structure between the ascidian MASP genes and the amphioxus/mammalian MASP genes suggests that a prototype gene was converted to the MASP1/3-type gene possessing two L chain-encoding regions at an early stage of evolution before the divergence of amphioxus. This conclusion is supported by the presence of MASP-1 and MASP-3 homologues in almost all vertebrates, as demonstrated by the cloning of novel cDNA sequences representing lamprey (cyclostomes) MASP-1 and Xenopus MASP-3. The ancient origin of MASP-1 and MASP-3 suggests that they have crucial functions common to all species which emerged after cephalochordates.     

Interaction properties of human mannan-binding lectin (MBL)-associated serine proteases-1 and -2, MBL-associated protein 19, and MBL

The mannan-binding lectin (MBL) activation pathway of complement plays an important role in the innate immune defense against pathogenic microorganisms. In human serum, two MBL-associated serine proteases (MASP-1, MASP-2) and MBL-associated protein 19 (MAp19) were found to be associated with MBL. With a view to investigate the interaction properties of these proteins, human MASP-1, MASP-2, MAp19, as well as the N-terminal complement subcomponents C1r/C1s, Uegf, and bone morphogenetic protein-1-epidermal growth factor (CUB-EGF) segments of MASP-1 and MASP-2, were expressed in insect or human kidney cells, and MBL was isolated from human serum. Sedimentation velocity analysis indicated that the MASP-1 and MASP-2 CUB-EGF segments and the homologous protein MAp19 all behaved as homodimers (2.8-3.2 S) in the presence of Ca(2+). Although the latter two dimers were not dissociated by EDTA, their physical properties were affected. In contrast, the MASP-1 CUB-EGF homodimer was not sensitive to EDTA. The three proteins and full-length MASP-1 and MASP-2 showed no interaction with each other as judged by gel filtration and surface plasmon resonance spectroscopy. Using the latter technique, MASP-1, MASP-2, their CUB-EGF segments, and MAp19 were each shown to bind to immobilized MBL, with K:(D) values of 0.8 nM (MASP-2), 1.4 nM (MASP-1), 13.0 nM (MAp19 and MASP-2 CUB-EGF), and 25.7 nM (MASP-1 CUB-EGF). The binding was Ca(2+)-dependent and fully sensitive to EDTA in all cases. These data indicate that MASP-1, MASP-2, and MAp19 each associate as homodimers, and individually form Ca(2+)-dependent complexes with MBL through the CUB-EGF pair of each protein. This suggests that distinct MBL/MASP complexes may be involved in the activation or regulation of the MBL pathway.     

Cleavage of kininogen and subsequent bradykinin release by the complement component: mannose-binding lectin-associated serine protease (MASP)-1

Bradykinin (BK), generated from high-molecular-weight kininogen (HK) is the major mediator of swelling attacks in hereditary angioedema (HAE), a disease associated with C1-inhibitor deficiency. Plasma kallikrein, activated by factor XIIa, is responsible for most of HK cleavage. However other proteases, which activate during episodes of angioedema, might also contribute to BK production. The lectin pathway of the complement system activates after infection and oxidative stress on endothelial cells generating active serine proteases: MASP-1 and MASP-2. Our aim was to study whether activated MASPs are able to digest HK to release BK. Initially we were trying to find potential new substrates of MASP-1 in human plasma by differential gel electrophoresis, and we identified kininogen cleavage products by this proteomic approach. As a control, MASP-2 was included in the study in addition to MASP-1 and kallikrein. The proteolytic cleavage of HK by MASPs was followed by SDS-PAGE, and BK release was detected by HPLC. We showed that MASP-1 was able to cleave HK resulting in BK production. MASP-2 could also cleave HK but could not release BK. The cleavage pattern of MASPs is similar but not strictly identical to that of kallikrein. The catalytic efficiency of HK cleavage by a recombinant version of MASP-1 and MASP-2 was about 4.0×10(2) and 2.7×10(2) M(-1) s(-1), respectively. C1-inhibitor, the major inhibitor of factor XIIa and kallikrein, also prevented the cleavage of HK by MASPs. In all, a new factor XII- and kallikrein-independent mechanism of bradykinin production by MASP-1 was demonstrated, which may contribute to the pro-inflammatory effect of the lectin pathway of complement and to the elevated bradykinin levels in HAE patients.     

Moraxella catarrhalis is only a weak activator of the mannose-binding lectin (MBL) pathway of complement activation

A hemolytic bystander assay was used to assess the functional serum mannose-binding lectin (MBL) activating capacity of five isolates of Moraxella catarrhalis obtained from children who suffered recurrent acute otitis media episodes. Results showed that this organism is only a poor activator of the lectin pathway of complement activation, with subsequent consequences for the etiology of otitis media by this organism.     

Mannose-binding lectin (MBL) facilitates opsonophagocytosis of yeasts but not of bacteria despite MBL binding

Mannose-binding lectin (MBL) is a serum protein of the innate immune system. After binding to a microorganism, MBL in complex with MBL-associated serine proteases activates the complement system, resulting in cleavage of complement factor C3. Cleaved C3 on the surface of the microorganism mediates opsonization for clearance, but the impact of MBL on subsequent phagocytosis has not been widely studied. We investigated the role of MBL in complement activation and phagocytosis of various bacteria and yeast species by flow cytometry. We measured both the C3 deposition during serum opsonization of fluorescent-labeled microorganisms as well as subsequent uptake of these microorganisms by human neutrophils. In MBL-deficient sera, a consistently decreased C3 deposition on both zymosan and Candida albicans was found and a reduced phagocytosis by neutrophils that was restored by exogenous MBL. This indicates that the lectin pathway of complement activation is important for the opsonophagocytosis of yeasts. In contrast, the C1q-dependent classical pathway dominated in the opsonization and phagocytosis of Staphylococcus aureus, Streptococcus pneumoniae, and Escherichia coli, whereas no effect of MBL was found. Both the lectin and the classical pathway of complement activation were highly amplified by the alternative route for opsonophagocytosis by neutrophils of yeast as well as microbial species. In summary, our data demonstrate that yeast species are preferentially opsonized and subsequently phagocytosed via activation of the lectin pathway of complement, whereas the uptake of bacterial strains was found to be largely MBL independent.     

Mannose-binding lectin (MBL) mutants are susceptible to matrix metalloproteinase proteolysis: potential role in human MBL deficiency

Mannose-binding lectin (MBL) plays a critical role in innate immunity. Point mutations in the collagen-like domain (R32C, G34D, or G37E) of MBL cause a serum deficiency, predisposing patients to infections and diseases such as rheumatoid arthritis. We examined whether MBL mutants show enhanced susceptibility to proteolysis by matrix metalloproteinases (MMPs), which are important mediators in inflammatory tissue destruction. Human and rat MBL were resistant to proteolysis in the native state but were cleaved selectively within the collagen-like domain by multiple MMPs after heat denaturation. In contrast, rat MBL with mutations homologous to those of the human variants (R23C, G25D, or G28E) was cleaved efficiently without denaturation in the collagen-like domain by MMP-2 and MMP-9 (gelatinases A and B) and MMP-14 (membrane type-1 MMP), as well as by MMP-1 (collagenase-1), MMP-8 (neutrophil collagenase), MMP-3 (stromelysin-1), neutrophil elastase, and bacterial collagenase. Sites and order of cleavage of the rat MBL mutants for MMP-2 and MMP-9 were: Gly(45)-Lys(46) --> Gly(51)-Ser(52) --> Gly(63)-Gln(64) --> Asn(80)-Met(81) which differed from that of MMP-14, Gly(39)-Leu(40) --> Asn(80)-Met(81), revealing that the MMPs were not functionally interchangeable. These sites were homologous to those cleaved in denatured human MBL. Hence, perturbation of the collagen-like structure of MBL by natural mutations or by denaturation renders MBL susceptible to MMP cleavage. MMPs are likely to contribute to MBL deficiency in individuals with variant alleles and may also be involved in clearance of MBL and modulation of the host response in normal individuals.     

The action of MBL-associated serine protease 1 (MASP1) on factor XIII and fibrinogen

The complement system is an important recognition and effector mechanism of the innate immune system that upon activation leads to the elimination of foreign bodies. It can be activated through three pathways of which the lectin pathway is one. The lectin pathway relies on the binding of mannan-binding lectin (MBL) or the ficolins and the subsequent activation of the MBL-associated serine proteases (MASPs), namely, MASP1, 2 and 3 which all form complexes with both MBL and the ficolins. Major substrates have only been identified for MASP2 i.e. C4 and C2. For MASP1 only a few protein substrates which are cleaved at a low rate have been identified while none are known for MASP3. Since chromogenic substrate screenings have shown that MASP1 has thrombin-like activity, we wanted to investigate the catalytic potential of MASP1 towards two major proteins involved in the clotting process, fibrinogen and factor XIII, and compare the activity directly with that of thrombin. We found that rMASP1 and thrombin cleave factor XIII A-chain and the fibrinogen beta-chain at identical sites, but differ in cleavage of the fibrinogen alpha-chain. The thrombin turnover rate of factor XIII is approximately 650 times faster than that of rMASP1 at 37 degrees C, pH 7.4. rMASP1 cleavage of fibrinogen leads to the release of the proinflammatory peptide fibrinopeptide B. Thus rMASP1 has similar, but not identical specificity to thrombin and its catalytic activity for factor XIII and fibrinogen cleavage is much lower than that of thrombin. Nevertheless, rMASP1 can drive the formation of cross-linked fibrinogen. Since MASP1 is activated on contact of MBL or the ficolins with microorganisms, fibrinogen and factor XIII may be involved in the elimination of invading pathogens.     

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.     

Characterization of recombinant mannan-binding lectin-associated serine protease (MASP)-3 suggests an activation mechanism different from that of MASP-1 and MASP-2

Mannan-binding lectin (MBL)-associated serine proteases (MASP-1, -2, and -3) are homologous modular proteases that each associate with MBL and L- and H-ficolins, which are oligomeric serum lectins involved in innate immunity. To investigate its physicochemical, interaction, and enzymatic properties, human MASP-3 was expressed in insect cells. Ultracentrifugation analysis indicated that rMASP-3 sedimented as a homodimer (s(20,w) = 6.2 +/- 0.1 S) in the presence of Ca(2+), and as a monomer (s(20,w) = 4.6 +/- 0.1 S) in EDTA. As shown by surface plasmon resonance spectroscopy, it associated with both MBL (K(D) = 2.6 nM) and L-ficolin (K(D) = 7.2 nM). The protease was produced in a single-chain, proenzyme form, but underwent slow activation upon prolonged storage at 4 degrees C, resulting from cleavage at the Arg(430)-Ile(431) activation site. Activation was prevented in the presence of protease inhibitors iodoacetamide and 1,10-phenanthroline but was not abolished upon substitution of Ala for the active site Ser(645) of MASP-3, indicating extrinsic proteolysis. In contrast, the corresponding mutations Ser(627)-->Ala in MASP-1 and Ser(618)-->Ala in MASP-2 stabilized the latter in their proenzyme form. Likewise, the MASP-1 and MASP-2 mutants were each activated by their active counterparts, but MASP-3 S645A was not. Activated MASP-3 did not react with C1 inhibitor; had no activity on complement proteins C2, C4, and C3; and only cleaved the N-carboxybenzyloxyglycine-L-arginine thiobenzyl ester substrate to a significant extent. Based on these observations, it is postulated that MASP-3 activation and control involve mechanisms that are different from those of MASP-1 and -2.     

Distinct pathways of mannan-binding lectin (MBL)- and C1-complex autoactivation revealed by reconstitution of MBL with recombinant MBL-associated serine protease-2

Mannan-binding lectin (MBL) plays a pivotal role in innate immunity by activating complement after binding carbohydrate moieties on pathogenic bacteria and viruses. Structural similarities shared by MBL and C1 complexes and by the MBL- and C1q-associated serine proteases, MBL-associated serine protease (MASP)-1 and MASP-2, and C1r and C1s, respectively, have led to the expectation that the pathways of complement activation by MBL and C1 complexes are likely to be very similar. We have expressed rMASP-2 and show that, whereas C1 complex autoactivation proceeds via a two-step mechanism requiring proteolytic activation of both C1r and C1s, reconstitution with MASP-2 alone is sufficient for complement activation by MBL. The results suggest that the catalytic activities of MASP-2 split between the two proteases of the C1 complex during the course of vertebrate complement evolution.     

Detection of a processed pseudogene of the human MBL-associated serine protease,MASP1

Southern hybridization analysis of the MASP1 gene using an intron-specific probe detected a single band. An exon-specific probe detected several bands. PCR of genomic DNA using several exon-specific primer sets of MASP1 produced short and long products. Sequence of the shorter products corresponded to the processed pseudogene of MASP1. By fluorescence in situ hybridization, this pseudogene (MASP1P1) was mapped to 1p34.     

CD91 interacts with mannan-binding lectin (MBL) through the MBL-associated serine protease-binding site

CD91 plays an important role in the scavenging of apoptotic material, possibly through binding to soluble pattern-recognition molecules. In this study, we investigated the interaction of CD91 with mannan-binding lectin (MBL), ficolins and lung surfactant proteins. Both MBL and L-ficolin were found to bind CD91. The MBL-CD91 interaction was time- and concentration-dependent and could be inhibited by known ligands of CD91. MBL-associated serine protease 3 (MASP-3) also inhibited binding between MBL and CD91, suggesting that the site of interaction is located at or near the MASP-MBL interaction site. This was confirmed by using MBL mutants deficient for MASP binding that were unable to interact with CD91. These findings demonstrate that MBL and L-ficolin interact with CD91, strongly suggesting that they have the potential to function as soluble recognition molecules for scavenging microbial and apoptotic material by CD91.     

cDNA cloning of a mannose-binding lectin-associated serine protease (MASP) gene from hagfish (Eptatretus burgeri)

Hagfish, agnathan cyclostome, is the most primitive extant vertebrate and its complement (C) system seems to be a primordial system in comparison with a well-developed C system in gnathostome vertebrates. From a phylogenic perspective of defense mechanisms, we have isolated complement C3 from the serum of hagfish (Eptatretus burgeri). In this study, we first attempted to identify a hagfish Bf or C2 as a C3 convertase by RT-PCR using degenerative primers designed on the basis of the conserved amino acid stretches among the several kinds of serine proteases. Contrary to our expectation, homology search of cloned RT-PCR product suggested that there was a partial cDNA encoding the homologue of neither Bf nor C2 but a mannose-binding lectin-associated serine protease (MASP). Analyses of a full-length cDNA clone isolated from a hagfish liver cDNA library by using the partial cDNA as a probe indicated that this cDNA encoded hagfish MASP 1. This evidence strongly suggests that the hagfish defends itself against pathogens at least by the complement system composed of lectin pathway.     

Enhancement of complement activation and opsonophagocytosis by complexes of mannose-binding lectin with mannose-binding lectin-associated serine protease after binding to Staphylococcus aureus

Human mannose-binding lectin (MBL) is a serum protein of the innate immune system that circulates as a complex with a group of so-called MBL-associated serine proteases (MASP-1, MASP-2, and MASP-3). Complexes of MBL-MASP2 are able to activate the complement system in an Ab and C1-independent fashion after binding of the lectin to appropriate microbial sugar arrays. We have evaluated the additive effect of the lectin pathway relative to other complement activation pathways and the subsequent effect on neutrophil phagocytosis. Complement activation in the sera of MBL-deficient individuals was studied with and without the addition of exogenous MBL-MASP. Flow cytometry was used to measure the deposition of C4, factor B, C3b, and iC3b on Staphylococcus aureus. Deposition of the first cleavage product of the lectin pathway, C4b, was increased using the sera of three different MBL-deficient individuals when exogenous MBL-MASP was added. Factor B was deposited in association with C4, but there was no evidence of independent alternative pathway activation. Similar enhancement of C3b deposition was also observed, with evidence of elevated amounts of C3b processed to iC3b. The increase in opsonic C3 fragments mediated by MBL was associated with a significant increase in the uptake of organisms by neutrophils. We also observed significant increases in phagocytosis with MBL-MASPs that were independent of complement activation. We conclude that MBL-MASP makes a major contribution to complement-mediated host defense mechanisms.     

Fasciola hepatica is refractory to complement killing by preventing attachment of mannose binding lectin (MBL) and inhibiting MBL-associated serine proteases (MASPs) with serpins

The complement system is a first-line innate host immune defence against invading pathogens. It is activated via three pathways, termed Classical, Lectin and Alternative, which are mediated by antibodies, carbohydrate arrays or microbial liposaccharides, respectively. The three complement pathways converge in the formation of C3-convertase followed by the assembly of a lethal pore-like structure, the membrane attack complex (MAC), on the pathogen surface. We found that the infectious stage of the helminth parasite Fasciola hepatica, the newly excysted juvenile (NEJ), is resistant to the damaging effects of complement. Despite being coated with mannosylated proteins, the main initiator of the Lectin pathway, the mannose binding lectin (MBL), does not bind to the surface of live NEJ. In addition, we found that recombinantly expressed serine protease inhibitors secreted by NEJ (rFhSrp1 and rFhSrp2) selectively prevent activation of the complement via the Lectin pathway. Our experiments demonstrate that rFhSrp1 and rFhSrp2 inhibit native and recombinant MBL-associated serine proteases (MASPs), impairing the primary step that mediates C3b and C4b deposition on the NEJ surface. Indeed, immunofluorescence studies show that MBL, C3b, C4b or MAC are not deposited on the surface of NEJ incubated in normal human serum. Taken together, our findings uncover new means by which a helminth parasite prevents the activation of the Lectin complement pathway to become refractory to killing via this host response, in spite of presenting an assortment of glycans on their surface.     

Simultaneous activation of complement and coagulation by MBL-associated serine protease 2

The complement system is an important immune mechanism mediating both recognition and elimination of foreign bodies. The lectin pathway is one pathway of three by which the complement system is activated. The characteristic protease of this pathway is Mannan-binding lectin (MBL)-associated serine protease 2 (MASP2), which cleaves complement proteins C2 and C4. We present a novel and alternative role of MASP2 in the innate immune system. We have shown that MASP2 is capable of promoting fibrinogen turnover by cleavage of prothrombin, generating thrombin. By using a truncated active form of MASP2 as well as full-length MASP2 in complex with MBL, we have shown that the thrombin generated is active and can cleave both factor XIII and fibrinogen, forming cross-linked fibrin. To explore the biological significance of these findings we showed that fibrin was covalently bound on a bacterial surface to which MBL/MASP2 complexes were bound. These findings suggest that, as has been proposed for invertebrates, limited clotting may contribute to the innate immune response.     

The chaperone and potential mannan-binding lectin (MBL) co-receptor calreticulin interacts with MBL through the binding site for MBL-associated serine proteases

The chaperone calreticulin has been suggested to function as a C1q and collectin receptor. The interaction of calreticulin with mannan-binding lectin (MBL) was investigated by solid-phase binding assays. Calreticulin showed saturable and time-dependent binding to recombinant MBL, provided that MBL was immobilized on a solid surface or bound to mannan on a surface. The binding was non-covalent and biphasic with an initial salt-sensitive phase followed by a more stable salt-insensitive interaction. For plasma-derived MBL, known to be complexed with MBL-associated serine proteases (MASPs), no binding was observed. Interaction of calreticulin with recombinant MBL was fully inhibited by recombinant MASP-2, MASP-3 and MAp19, but not by the MASP-2 D105G and MAp19 Y59A variants characterized by defective MBL binding ability. Furthermore, MBL point mutants with impaired MASP binding showed no interaction with calreticulin. Comparative analysis of MBL with complement component C1q, its counterpart of the classical pathway, revealed that they display similar binding characteristics for calreticulin, providing further indication that calreticulin is a common co-receptor/chaperone for both proteins. In conclusion, the potential MBL co-receptor calreticulin binds to MBL at the MASP binding site and the interaction may involve a conformational change in MBL.     

Activation of the lectin complement pathway by H-ficolin (Hakata antigen)

Ficolins are a group of proteins which consist of a collagen-like domain and a fibrinogen-like domain. In human serum, there are two types of ficolins named L-ficolin/P35 and H-ficolin (Hakata Ag), both of which have lectin activity. We recently demonstrated that L-ficolin/P35 is associated with mannose-binding lectin (MBL)-associated serine proteases (MASP) 1 and 2 and small MBL-associated protein (sMAP), and that the complex activates the lectin pathway. In this study, we report the characterization of H-ficolin in terms of its ability to activate complement. Western blotting analysis showed the presence of MASP-1, MASP-2, MASP-3, and sMAP in H-ficolin preparations isolated from Cohn Fraction III. The MASPs in the preparations had proteolytic activities against C4, C2, and C3 in the fluid phase. When H-ficolin preparations were bound to anti-H-ficolin Ab which had been coated on ELISA plates, they activated C4, although no C4 activation was noted when anti-MBL and anti-L-ficolin/P35 were used. H-ficolin binds to PSA, a polysaccharide produced by Aerococcus viridans. C4 was activated by H-ficolin preparations bound to PSA which had been coated on ELISA plates. These results indicate that H-ficolin is a second ficolin which is associated with MASPs and sMAP, and which activates the lectin pathway.     

Pseudomonas aeruginosa alkaline protease blocks complement activation via the classical and lectin pathways

The complement system rapidly detects and kills Gram-negative bacteria and supports bacterial killing by phagocytes. However, bacterial pathogens exploit several strategies to evade detection by the complement system. The alkaline protease (AprA) of Pseudomonas aeruginosa has been associated with bacterial virulence and is known to interfere with complement-mediated lysis of erythrocytes, but its exact role in bacterial complement escape is unknown. In this study, we analyzed how AprA interferes with complement activation and whether it could block complement-dependent neutrophil functions. We found that AprA potently blocked phagocytosis and killing of Pseudomonas by human neutrophils. Furthermore, AprA inhibited opsonization of bacteria with C3b and the formation of the chemotactic agent C5a. AprA specifically blocked C3b deposition via the classical and lectin pathways, whereas the alternative pathway was not affected. Serum degradation assays revealed that AprA degrades both human C1s and C2. However, repletion assays demonstrated that the mechanism of action for complement inhibition is cleavage of C2. In summary, we showed that P. aeruginosa AprA interferes with classical and lectin pathway-mediated complement activation via cleavage of C2.