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.     

L-ficolin is a pattern recognition molecule specific for acetyl groups

L-ficolin and H-ficolin are molecules of the innate immune system. Upon recognition of a suitable target they activate the complement system. The ligand recognition structure of ficolins is contained within a fibrinogen-like domain. We examined the selectivity of the ficolins through inhibiting the binding to bacteria or to beads coupled with N-acetylglucosamine. The binding of L-ficolin to Streptococcus pneumoniae 11F and the beads was inhibited by N-acetylated sugars and not by non-acetylated sugars. However, it was also inhibited by other acetylated compounds. Based on this selectivity L-ficolin is not easily defined as a lectin. The binding of H-ficolin to Aerococcus viridans was not inhibited by any of the sugars or other compounds examined. Based on the selectivity of L-ficolin we developed a new purification procedure involving affinity chromatography on N-acetylcysteine-derivatized Sepharose. The column was loaded in the presence of EDTA and high salt, and L-ficolin was eluted by decreasing the salt concentration. Further purification was achieved by ion exchange chromatography.     

Stoichiometry of complexes between mannose-binding protein and its associated serine proteases. Defining functional units for complement activation

Serum mannose-binding protein (MBP) initiates the lectin branch of the complement cascade by binding to sugars on the surfaces of microorganisms and activating two MBP-associated serine proteases (MASP-1 and MASP-2). Rat serum MBP consists of oligomers containing up to four copies of a subunit that is composed of three identical polypeptide chains. Biophysical analysis of intact and truncated MASPs indicates that each MASP is a homodimer that is stabilized through interactions involving an N-terminal CUB domain. The binding sites for MBP are formed from the three N-terminal MASP domains, in which two CUB modules interact with MBP. Each MASP dimer contains binding sites for two MBP subunits. Both sites must be occupied by subunits from a single MBP oligomer to form a stable complex. Thus, the smallest functional unit for complement activation consists of MBP dimers bound to MASP-1 or MASP-2 homodimers. Trimers and tetramers of MBP form complexes containing up to two MASPs. The results reveal how MASP-1 and MASP-2 can function independently to activate the complement cascade.     

Monospecific inhibitors show that both mannan-binding lectin-associated serine protease-1 (MASP-1) and -2 Are essential for lectin pathway activation and reveal structural plasticity of MASP-2

The lectin pathway is an antibody-independent activation route of the complement system. It provides immediate defense against pathogens and altered self-cells, but it also causes severe tissue damage after stroke, heart attack, and other ischemia reperfusion injuries. The pathway is triggered by target binding of pattern recognition molecules leading to the activation of zymogen mannan-binding lectin-associated serine proteases (MASPs). MASP-2 is considered as the autonomous pathway-activator, while MASP-1 is considered as an auxiliary component. We evolved a pair of monospecific MASP inhibitors. In accordance with the key role of MASP-2, the MASP-2 inhibitor completely blocks the lectin pathway activation. Importantly, the MASP-1 inhibitor does the same, demonstrating that MASP-1 is not an auxiliary but an essential pathway component. We report the first Michaelis-like complex structures of MASP-1 and MASP-2 formed with substrate-like inhibitors. The 1.28 Å resolution MASP-2 structure reveals significant plasticity of the protease, suggesting that either an induced fit or a conformational selection mechanism should contribute to the extreme specificity of the enzyme.     

Mannose-binding lectin serine proteases and associated proteins of the lectin pathway of complement: Two genes, five proteins and many functions?

The lectin pathway of the complement system is activated following the binding of carbohydrate-based ligands by recognition molecules such as mannose-binding lectin (MBL) or ficolins. Engagement of the recognition molecules causes activation of associated MBL-associated serine proteases or MASPs, which in turn activate downstream complement molecules to activate the system. Two MASP genes are alternatively spliced during expression to yield 5 proteins, including three proteases (MASP-1, -2 and -3) and two truncated proteins, MAp19 and MAp44. Here we discuss what is currently known about these proteins in terms of their structure and function. MASP-2 is autoactivated following the initial binding events of the pathway and is able to subsequently activate the C4 and C2 substrates required to activate the rest of the pathway. MASP-1 is able to augment MASP-2 activation, but also appears to play other roles, although the physiological significance of these is not yet clear. The roles of the truncated Map19 and Map44 proteins and the MASP-3 protease are currently unknown. The proteases form an interesting sub-family of proteins that clearly should be the focus of future research in order to establish their biological roles.This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Interaction of C1q and mannan-binding lectin (MBL) with C1r, C1s, MBL-associated serine proteases 1 and 2, and the MBL-associated protein MAp19

Mannan-binding lectin (MBL) and C1q activate the complement cascade via attached serine proteases. The proteases C1r and C1s were initially discovered in a complex with C1q, whereas the MBL-associated serine proteases 1 and 2 (MASP-1 and -2) were discovered in a complex with MBL. There is controversy as to whether MBL can utilize C1r and C1s or, inversely, whether C1q can utilize MASP-1 and 2. Serum deficient in C1r produced no complement activation in IgG-coated microwells, whereas activation was seen in mannan-coated microwells. In serum, C1r and C1s were found to be associated only with C1q, whereas MASP-1, MASP-2, and a third protein, MAp19 (19-kDa MBL-associated protein), were found to be associated only with MBL. The bulk of MASP-1 and MAp19 was found in association with each other and was not bound to MBL or MASP-2. The interactions of MASP-1, MASP-2, and MAp19 with MBL differ from those of C1r and C1s with C1q in that both high salt concentrations and calcium chelation (EDTA) are required to fully dissociate the MASPs or MAp19 from MBL. In the presence of calcium, most of the MASP-1, MASP-2, and MAp19 emerged on gel-permeation chromatography as large complexes that were not associated with MBL, whereas in the presence of EDTA most of these components formed smaller complexes. Over 95% of the total MASPs and MAp19 found in serum are not complexed with MBL.     

Localization of the serine protease-binding sites in the collagen-like domain of mannose-binding protein: indirect effects of naturally occurring mutations on protease binding and activation

Mutations in the collagen-like domain of serum mannose-binding protein (MBP) interfere with the ability of the protein to initiate complement fixation through the MBP-associated serine proteases (MASPs). The resulting deficiency in the innate immune response leads to susceptibility to infections. Studies have been undertaken to define the region of MBP that interacts with MASPs and to determine how the naturally occurring mutations affect this interaction. Truncated and modified MBPs and synthetic peptides that represent segments of the collagen-like domain of MBP have been used to demonstrate that MASPs bind on the C-terminal side of the hinge region formed by an interruption in the Gly-X-Y repeat pattern of the collagen-like domain. The binding sites for MASP-2 and for MASP-1 and -3 overlap but are not identical. The two most common naturally occurring mutations in MBP result in substitution of acidic amino acids for glycine residues in Gly-X-Y triplets on the N-terminal side of the hinge. Circular dichroism analysis and differential scanning calorimetry demonstrate that the triple helical structure of the collagen-like domain is largely intact in the mutant proteins, but it is more easily unfolded than in wild-type MBP. Thus, the effect of the mutations is to destabilize the collagen-like domain, indirectly disrupting the binding sites for MASPs. In addition, at least one of the mutations has a further effect on the ability of MBP to activate MASPs.     

Characterization of the interaction between L-ficolin/p35 and mannan-binding lectin-associated serine proteases-1 and -2

Ficolins are oligomeric lectins comprising a collagen-like and a fibrinogen-like domain, with a binding specificity for N-acetylglucosamine. It has been reported recently that L-ficolin/P35 associates with mannan-binding lectin (MBL)-associated serine proteases (MASP-1 and -2) and MBL-associated protein 19 (MAp19) in serum and forms complexes able to activate complement. Using surface plasmon resonance spectroscopy we have shown that recombinant MASP-1 and -2, their N-terminal CUB1 (module originally found in complement proteins C1r/C1s, Uegf, and bone morphogenetic protein-1)-epidermal growth factor (EGF)-CUB2 and CUB1-EGF segments, and MAp19 bind to immobilized L-ficolin/P35 in the presence of Ca(2+) ions. Comparable K(d) values were obtained for the full-length proteases and their CUB1-EGF-CUB2 segments (9.2 and 10 nM for MASP-1 and 4.6 and 5.4 nM for MASP-2, respectively), whereas higher values were obtained for the CUB1-EGF segments (26.7, 15.6, and 14.3 nM for MASP-1, MASP-2, and MAp19). These values are in the same range as those determined for the interaction of these proteins with MBL. Binding was Ca(2+) dependent and was only partly sensitive to EDTA for MASP-1, MASP-2, and MASP-2 CUB1-EGF-CUB2. Half-maximal binding was obtained at comparable Ca(2+) concentrations for MASP-1 and MASP-2 (0.45 and 0.47 micro M, respectively), their CUB1-EGF-CUB2 segments (0.37 and 0.72 micro M), and their CUB1-EGF segments (0.31 and 0.79 micro M). These values are lower than those determined in the case of MBL, indicating a difference between MBL and L-ficolin/P35 with respect to the Ca(2+) dependence of their interaction with the MASPs. Preincubation of the MASPs with soluble MBL inhibited subsequent binding to immobilized L-ficolin/P35 and, conversely, suggesting that these lectins compete with each other for binding to the MASPs in vivo.     

Two mechanisms for mannose-binding protein modulation of the activity of its associated serine proteases

Serum mannose-binding protein (MBP) neutralizes invading microorganisms by binding to cell surface carbohydrates and activating MBP-associated serine proteases-1, -2, and -3 (MASPs). MASP-2 subsequently cleaves complement components C2 and C4 to activate the complement cascade. To analyze the mechanisms of activation and substrate recognition by MASP-2, zymogen and activated forms have been produced, and MBP.MASP-2 complexes have been created. These preparations have been used to show that MBP modulates MASP-2 activity in two ways. First, MBP stimulates MASP-2 autoactivation by increasing the rate of autocatalysis when MBP.MASP-2 complexes bind to a glycan-coated surface. Second, MBP occludes accessory C4-binding sites on MASP-2 until activation occurs. Once these sites become exposed, MASP-2 binds to C4 while separate structural changes create a functional catalytic site able to cleave C4. Only activated MASP-2 binds to C2, suggesting that this substrate interacts only near the catalytic site and not at accessory sites. MASP-1 cleaves C2 almost as efficiently as MASP-2 does, but it does not cleave C4. Thus MASP-1 probably enhances complement activation triggered by MBP.MASP-2 complexes, but it cannot initiate activation itself.     

Interaction of mannose-binding protein with associated serine proteases: effects of naturally occurring mutations

Mannose-binding protein (MBP; mannose-binding lectin) forms part of the innate immune system. By binding directly to carbohydrates on the surfaces of potential microbial pathogens, MBP and MBP-associated serine proteases (MASPs) can replace antibodies and complement components C1q, C1r, and C1s of the classical complement pathway. In order to investigate the mechanisms of MASP activation by MBP, the cDNAs of rat MASP-1 and -2 have been isolated, and portions encompassing the N-terminal CUB and epidermal growth factor-like domains have been expressed and purified. Biophysical characterization of the purified proteins indicates that each truncated MASP is a Ca(2+)-independent homodimer in solution, in which the interacting modules include the N-terminal two domains. Binding studies reveal that both MASPs associate independently with rat MBP in a Ca(2+)-dependent manner through interactions involving the N-terminal three domains. The biophysical properties of the truncated MASPs indicate that the interactions with MBP leading to complement activation differ significantly from those between components C1q, C1r, and C1s of the classical pathway. Analysis of MASP binding by rat MBP containing naturally occurring mutations equivalent to those associated with human immunodeficiency indicates that binding to both truncated MASP-1 and MASP-2 proteins is defective in such mutants.     

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.     

Cutting edge: complement-activating complex of ficolin and mannose-binding lectin-associated serine protease

Both ficolins and mannose-binding lectin (MBL) are lectins characterized by the presence of collagen-like and carbohydrate-binding domains in a subunit, although their carbohydrate-binding moieties are quite different. A fibrinogen-like domain is in ficolins, and a carbohydrate recognition domain is in MBL. On binding to pathogens, human MBL activates the complement system via the lectin pathway in association with two types of MBL-associated serine proteases (MASP), MASP-1 and MASP-2 and its truncated form, small MBL-associated protein (sMAP, also called MAp19). We report here that ficolin/P35, a human serum ficolin, was found to copurify with MASPs and sMAP. MASPs that were complexed with ficolin/P35 exhibited proteolytic activities against complement components C4, C2, and C3. The ficolin/P35-MASPs-sMAP complex that was bound to Salmonella typhimurium activated complement. These findings indicate that ficolin/P35 is a second collagenous lectin capable of activating the lectin pathway and thus plays a role in innate immunity.     

Decoupling of carbohydrate binding and MASP-2 autoactivation in variant mannose-binding lectins associated with immunodeficiency

Mannan-binding lectin (MBL) initiates complement activation by binding to arrays of carbohydrates on the surfaces of pathogenic microorganisms and activating MBL-associated serine proteases (MASPs). Separate point mutations to the collagenous domain of human MBL are associated with immunodeficiency, caused by reduced complement activation by the variant MBLs as well as by lower serum MBL concentrations. In the work reported here, we have used the well characterized rat lectin pathway to analyze the molecular and functional defects associated with two of the variant proteins. Mutations Gly25 --> Asp and Gly28 --> Glu create comparable structural changes in rat MBL but the G28E variant activates complement >10-fold less efficiently than the G25D variant, which in turn has approximately 7-fold lower activity than wild-type MBL. Analysis of mutant MBL . MASP-2 complexes assembled from recombinant components shows that reduced complement activation by both mutant MBLs is caused by failure to activate MASP-2 efficiently on binding to a mannan-coated surface. Disruption of MBL-MASP-2 interactions as well as to changes in oligomeric structure and reduced binding to carbohydrate ligands compared with wild-type MBL probably account for the intermediate phenotype of the G25D variant. However, carbohydrate binding and MASP-2 activation are ostensibly completely decoupled in complexes assembled from the G28E mutant, such that the rate of MASP-2 activation is no greater than the basal rate of zymogen MASP-2 autoactivation. Analogous molecular defects in human MBL probably combine to create the mutant phenotypes of immunodeficient individuals.     

Human M-ficolin is a secretory protein that activates the lectin complement pathway

Three types of ficolins have been identified in humans: L-ficolin, M-ficolin, and H-ficolin. Similar to mannose-binding lectin, L-ficolin and H-ficolin are the recognition molecules in the lectin complement pathway. Another human ficolin, M-ficolin, is a nonserum ficolin that is expressed in leukocytes and lung; however, little is known about its physiologic roles. In this study, we report the characterization of M-ficolin in terms of its protein localization and lectin activity. M-ficolin was localized in secretory granules in the cytoplasm of neutrophils, monocytes, and type II alveolar epithelial cells in lung. M-ficolin precipitated with mannose-binding lectin-associated serine proteases (MASP)-1 and MASP-2 in a co-immunoprecipitation assay, indicating that M-ficolin forms complexes with MASP-1 and MASP-2. M-ficolin-MASP complexes activated complement on N-acetylglucosamine (GlcNAc)-coated microplates in a C4 deposition assay. M-ficolin bound to several neoglycoproteins bearing GlcNAc, N-acetylgalactosamine, and sialyl-N-acetyllactosamine, suggesting that M-ficolin can recognize the common carbohydrate residues found in microbes. Indeed, M-ficolin bound to Staphylococcus aureus through GlcNAc. These results indicate that M-ficolin, like its family members, functions as a recognition molecule of the lectin complement pathway and plays an important role in innate immunity.     

Small mannose-binding lectin-associated protein plays a regulatory role in the lectin complement pathway

Mannose-binding lectin (MBL) and ficolins are pattern recognition proteins acting in innate immunity, and they trigger the activation of the lectin complement pathway through MBL-associated serine proteases (MASPs). Upon activation of the lectin pathway, MASP-2 cleaves C4 and C2. A truncated form of MASP-2, named small MBL-associated protein (sMAP), is also associated with MBL/ficolin-MASP complexes. To clarify the role of sMAP, we have generated sMAP-deficient (sMAP(-/-)) mice by targeted disruption of the sMAP-specific exon. Because of the gene disruption, the expression level of MASP-2 was also decreased in sMAP(-/-) mice. When recombinant sMAP (rsMAP) and recombinant MASP-2 (rMASP-2) reconstituted the MBL-MASP-sMAP complex in deficient serum, the binding of these recombinant proteins to MBL was competitive, and the C4 cleavage activity of the MBL-MASP-sMAP complex was restored by the addition of rMASP-2, whereas the addition of rsMAP attenuated the activity. Therefore, MASP-2 is essential for the activation of C4 and sMAP plays a regulatory role in the activation of the lectin pathway.     

The role of mannose-binding lectin-associated serine protease-3 in activation of the alternative complement pathway

Mannose-binding lectin (MBL)-associated serine proteases (MASPs) are responsible for activation of the lectin complement pathway. Three types of MASPs (MASP-1, MASP-2, and MASP-3) are complexed with MBL and ficolins in serum. Although MASP-1 and MASP-2 are known to contribute to complement activation, the function of MASP-3 remains unclear. In this study, we investigated the mechanism of MASP-3 activation and its substrate using the recombinant mouse MASP-3 (rMASP-3) and several different types of MASP-deficient mice. A proenzyme rMASP-3 was obtained that was not autoactivated during preparation. The recombinant enzyme was activated by incubation with Staphylococcus aureus in the presence of MBL-A, but not MBL-C. In vivo studies revealed the phagocytic activities of MASP-1/3-deficient mice and all MASPs (MASP-1/2/3)-deficient mice against S. aureus and bacterial clearance in these mice were lower than those in wild-type and MASP-2-deficient mice. Sera from all MASPs-deficient mice showed significantly lower C3 deposition activity on the bacteria compared with that of wild-type serum, and addition of rMASP-3 to the deficient serum restored C3 deposition. The low C3 deposition in sera from all MASPs-deficient mice was probably caused by the low level factor B activation that was ameliorated by the addition of rMASP-3. Furthermore, rMASP-3 directly activated factors B and D in vitro. These results suggested that MASP-3 complexed with MBL is converted to an active form by incubation with bacterial targets, and that activated MASP-3 triggered the initial activation step of the alternative complement pathway.     

The role of ficolins in the lectin pathway of innate immunity

Ficolins are a family of oligomeric proteins consisting of an N-terminal collagen-like domain and a C-terminal globular fibrinogen-like domain. They are novel lectins that employ the fibrinogen-like domain as a functional domain. Ficolins specifically recognize N-acetyl compounds such as N-acetylglucosamine, components of bacterial and fungal cell walls, and certain bacteria. Like mannose-binding lectin (MBL), ficolins circulate in complexes with MBL-associated serine proteases (MASPs). MASP complexes form with ficolins and MBL, thereby activating the complement through the lectin pathway. Upon binding of ficolins and MBL to carbohydrates on pathogens, MASPs convert to active forms, and subsequently activate the complement. The activated complements lead to pathogen phagocytosis, aggregation and lysis. In humans, three ficolins (L-, M- and H-ficolins) have been identified, which exhibit differences in tissue expression, protein location site, ligand-binding and bacteria-recognition, suggesting a specific role of each ficolin. In addition, these ficolins form complexes with three MASPs (MASP-1, MASP-2 and MASP-3) and two nonenzymatic proteins (sMAP and MAP-1), suggesting a highly sophisticated organization and regulated activation of the ficolin-dependent lectin pathway. This review provides an overview of our current knowledge of ficolins, especially human ficolins and their mouse homologues. We also discuss their possible physiological roles in innate immunity, especially their defensive role against bacterial infection.     

Scabies Mite Inactive Serine Proteases Are Potent Inhibitors of the Human Complement Lectin Pathway

Scabies is an infectious skin disease caused by the mite Sarcoptes scabiei and has been classified as one of the six most prevalent epidermal parasitic skin diseases infecting populations living in poverty by the World Health Organisation. The role of the complement system, a pivotal component of human innate immunity, as an important defence against invading pathogens has been well documented and many parasites have an arsenal of anti-complement defences. We previously reported on a family of scabies mite proteolytically inactive serine protease paralogues (SMIPP-Ss) thought to be implicated in host defence evasion. We have since shown that two family members, SMIPP-S D1 and I1 have the ability to bind the human complement components C1q, mannose binding lectin (MBL) and properdin and are capable of inhibiting all three human complement pathways. This investigation focused on inhibition of the lectin pathway of complement activation as it is likely to be the primary pathway affecting scabies mites. Activation of the lectin pathway relies on the activation of MBL, and as SMIPP-S D1 and I1 have previously been shown to bind MBL, the nature of this interaction was examined using binding and mutagenesis studies. SMIPP-S D1 bound MBL in complex with MBL-associated serine proteases (MASPs) and released the MASP-2 enzyme from the complex. SMIPP-S I1 was also able to bind MBL in complex with MASPs, but MASP-1 and MASP-2 remained in the complex. Despite these differences in mechanism, both molecules inhibited activation of complement components downstream of MBL. Mutagenesis studies revealed that both SMIPP-Ss used an alternative site of the molecule from the residual active site region to inhibit the lectin pathway. We propose that SMIPP-Ss are potent lectin pathway inhibitors and that this mechanism represents an important tool in the immune evasion repertoire of the parasitic mite and a potential target for therapeutics.     

Proteolytic activities of two types of mannose-binding lectin-associated serine protease

Mannose (or mannan)-binding lectin (MBL) is an oligomeric serum lectin that plays a role in innate immunity by activating the complement system. In human, two types of MBL-associated serine protease (MASP-1 and MASP-2) and a truncated protein of MASP-2 (small MBL-associated protein; sMAP or MAp19) are complexed with MBL. To clarify the proteolytic activities of MASP-1 and MASP-2 against C4, C2, and C3, we isolated these two types of MASP in activated forms from human serum by sequential affinity chromatography. On an anti-MASP-1 column, MASP-2 passed through the column in the presence of EDTA and high salt concentration, whereas MASP-1 was retained. Isolated MASP-1 and MASP-2 exhibited proteolytic activities against C3 and C4, respectively. C2 was activated by both MASPs. C1 inhibitor (C1 INH), an inhibitor for C1r and C1s, formed equimolar complexes with MASP-1 and MASP-2 and inhibited their proteolytic activities.     

L-ficolin specifically binds to lipoteichoic acid, a cell wall constituent of Gram-positive bacteria, and activates the lectin pathway of complement

The lectin pathway of complement is activated when a carbohydrate recognition complex and associated serine proteases binds to the surface of a pathogen. Three recognition subcomponents have been shown to form active initiation complexes: mannan-binding lectin (MBL), L-ficolin, and H-ficolin. The importance of MBL in antimicrobial host defense is well recognized, but the role of the ficolins remains largely undefined. This report shows that L-ficolin specifically binds to lipoteichoic acid (LTA), a cell wall component found in all Gram-positive bacteria. Immobilized LTA from Staphylococcus aureus binds L-ficolin complexes from sera, and these complexes initiate lectin pathway-dependent C4 turnover. C4 activation correlates with serum L-ficolin concentration, but not with serum MBL levels. L-ficolin binding and corresponding levels of C4 turnover were observed on LTA purified from other clinically important bacteria, including Streptococcus pyogenes and Streptococcus agalactiae. None of the LTA preparations bound MBL, H-ficolin, or the classical pathway recognition molecule, C1q.