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.     

The structure of MBL-associated serine protease-2 reveals that identical substrate specificities of C1s and MASP-2 are realized through different sets of enzyme-substrate interactions

A family of serine proteases mediates the proteolytic cascades of several defense mechanisms in vertebrates, such as the complement system, blood coagulation and fibrinolysis. These proteases usually form large complexes with other glycoproteins. Their common features are their modular structures and restricted substrate specificities. The lectin pathway of complement, where mannose-binding lectin (MBL) recognizes the carbohydrate structures on pathogens, is activated by mannose-binding lectin-associated serine protease-2 (MASP-2). We present the 2.25A resolution structure of the catalytic fragment of MASP-2 encompassing the second complement control protein module (CCP2) and the serine protease (SP) domain. The CCP2 module stabilizes the structure of the SP domain as demonstrated by differential scanning calorimetry measurements. The asymmetric unit contains two molecules with different CCP-SP domain orientations, reflecting increased modular flexibility at the CCP2/SP joint. This flexibility may partly explain the ability of the MASP-2 dimer to perform all of its functions alone, whereas the same functions are mediated by the much larger C1r2-C1s2 tetramer in the C1 complex of the classical pathway. The main scaffold of the MASP-2 SP domain is chymotrypsin-like. Eight surface loops determine the S1 and other subsite specificities. Surprisingly, some surface loops of MASP-2, e.g. loop 1 and loop 2, which form the S1 pocket are similar to those of trypsin, and show significant differences if compared with those of C1s, indicating that the nearly identical substrate specificities of C1s and MASP-2 are realized through different sets of enzyme-substrate interactions.     

Functional characterization of complement proteases C1s/mannan-binding lectin-associated serine protease-2 (MASP-2) chimeras reveals the higher C4 recognition efficacy of the MASP-2 complement control protein modules

C1s and mannan-binding lectin-associated serine protease-2 (MASP-2) are the proteases that trigger the classical and lectin pathways of complement, respectively. They have identical modular architectures and cleave the same substrates, C2 and C4, but show markedly different efficiencies toward C4. Multisite-directed mutagenesis was used to engineer hybrid C1s/MASP-2 molecules where either the complement control protein (CCP) modules or the serine protease (SP) domain of C1s were swapped for their MASP-2 counterparts. The resulting chimeras (C1s(MASP-2 CCP1/2) and C1s(MASP-2 SP), respectively) were expressed and characterized chemically and functionally. Whereas C1s(MASP-2 SP) was recovered as an active enzyme, C1s(MASP-2 CCP1/2) was produced in a proenzyme form and was susceptible to activation by C1r, indicating that the activation properties of the chimeras were dictated by the nature of their SP domain. Similarly, each activated chimera had an esterolytic activity characteristic of its own SP domain and cleaved C2 with an efficiency comparable with that of their parent C1s and MASP-2 proteases. Both chimeras cleaved C4, but whereas C1s(MASP-2 SP) and C1s had Km values in the micromolar range, C1s(MASP-2 CCP1/2) and MASP-2 had Km values in the nanomolar range, resulting in 21-27-fold higher kcat/Km ratios. Thus, the higher C4 cleavage efficiency of MASP-2 arises from a higher substrate recognition efficacy of its CCP modules. Remarkably, C1s(MASP-2 CCP1/2) retained C1s ability to associate with C1r and C1q to form a pseudo-C1 complex and to undergo activation within this complex, indicating that the C1s-CCP modules have no direct implication in either function.     

The Serine Protease Domain of MASP-3: Enzymatic Properties and Crystal Structure in Complex with Ecotin

Mannan-binding lectin (MBL), ficolins and collectin-11 are known to associate with three homologous modular proteases, the MBL-Associated Serine Proteases (MASPs). The crystal structures of the catalytic domains of MASP-1 and MASP-2 have been solved, but the structure of the corresponding domain of MASP-3 remains unknown. A link between mutations in the MASP1/3 gene and the rare autosomal recessive 3MC (Mingarelli, Malpuech, Michels and Carnevale,) syndrome, characterized by various developmental disorders, was discovered recently, revealing an unexpected important role of MASP-3 in early developmental processes. To gain a first insight into the enzymatic and structural properties of MASP-3, a recombinant form of its serine protease (SP) domain was produced and characterized. The amidolytic activity of this domain on fluorescent peptidyl-aminomethylcoumarin substrates was shown to be considerably lower than that of other members of the C1r/C1s/MASP family. The E. coli protease inhibitor ecotin bound to the SP domains of MASP-3 and MASP-2, whereas no significant interaction was detected with MASP-1, C1r and C1s. A tetrameric complex comprising an ecotin dimer and two MASP-3 SP domains was isolated and its crystal structure was solved and refined to 3.2 Å. Analysis of the ecotin/MASP-3 interfaces allows a better understanding of the differential reactivity of the C1r/C1s/MASP protease family members towards ecotin, and comparison of the MASP-3 SP domain structure with those of other trypsin-like proteases yields novel hypotheses accounting for its zymogen-like properties in vitro.     

Substrate specificities of recombinant mannan-binding lectin-associated serine proteases-1 and -2

Mannan-binding lectin (MBL)-associated serine proteases-1 and 2 (MASP-1 and MASP-2) are homologous modular proteases that each interact with MBL, an oligomeric serum lectin involved in innate immunity. To precisely determine their substrate specificity, human MASP-1 and MASP-2, and fragments from their catalytic regions were expressed using a baculovirus/insect cells system. Recombinant MASP-2 displayed a rather wide, C1s-like esterolytic activity, and specifically cleaved complement proteins C2 and C4, with relative efficiencies 3- and 23-fold higher, respectively, than human C1s. MASP-2 also showed very weak C3 cleaving activity. Recombinant MASP-1 had a lower and more restricted esterolytic activity. It showed marginal activity toward C2 and C3, and no activity on C4. The enzymic activity of both MASP-1 and MASP-2 was specifically titrated by C1 inhibitor, and abolished at a 1:1 C1 inhibitor:protease ratio. Taken together with previous findings, these and other data strongly support the hypothesis that MASP-2 is the protease that, in association with MBL, triggers complement activation via the MBL pathway, through combined self-activation and proteolytic properties devoted to C1r and C1s in the C1 complex. In view of the very low activity of MASP-1 on C3 and C2, our data raise questions about the implication of this protease in complement activation.     

A true autoactivating enzyme. Structural insight into mannose-binding lectin-associated serine protease-2 activations

Few reports have described in detail a true autoactivation process, where no extrinsic cleavage factors are required to initiate the autoactivation of a zymogen. Herein, we provide structural and mechanistic insight into the autoactivation of a multidomain serine protease: mannose-binding lectin-associated serine protease-2 (MASP-2), the first enzymatic component in the lectin pathway of complement activation. We characterized the proenzyme form of a MASP-2 catalytic fragment encompassing its C-terminal three domains and solved its crystal structure at 2.4 A resolution. Surprisingly, zymogen MASP-2 is capable of cleaving its natural substrate C4, with an efficiency about 10% that of active MASP-2. Comparison of the zymogen and active structures of MASP-2 reveals that, in addition to the activation domain, other loops of the serine protease domain undergo significant conformational changes. This additional flexibility could play a key role in the transition of zymogen MASP-2 into a proteolytically active form. Based on the three-dimensional structures of proenzyme and active MASP-2 catalytic fragments, we present model for the active zymogen MASP-2 complex and propose a mechanism for the autoactivation process.     

Quantitative Characterization of the Activation Steps of Mannan-binding Lectin (MBL)-associated Serine Proteases (MASPs) Points to the Central Role of MASP-1 in the Initiation of the Complement Lectin Pathway

Mannan-binding lectin (MBL)-associated serine proteases, MASP-1 and MASP-2, have been thought to autoactivate when MBL/ficolin·MASP complexes bind to pathogens triggering the complement lectin pathway. Autoactivation of MASPs occurs in two steps: 1) zymogen autoactivation, when one proenzyme cleaves another proenzyme molecule of the same protease, and 2) autocatalytic activation, when the activated protease cleaves its own zymogen. Using recombinant catalytic fragments, we demonstrated that a stable proenzyme MASP-1 variant (R448Q) cleaved the inactive, catalytic site Ser-to-Ala variant (S646A). The autoactivation steps of MASP-1 were separately quantified using these mutants and the wild type enzyme. Analogous mutants were made for MASP-2, and rate constants of the autoactivation steps as well as the possible cross-activation steps between MASP-1 and MASP-2 were determined. Based on the rate constants, a kinetic model of lectin pathway activation was outlined. The zymogen autoactivation rate of MASP-1 is ∼3000-fold higher, and the autocatalytic activation of MASP-1 is about 140-fold faster than those of MASP-2. Moreover, both activated and proenzyme MASP-1 can effectively cleave proenzyme MASP-2. MASP-3, which does not autoactivate, is also cleaved by MASP-1 quite efficiently. The structure of the catalytic region of proenzyme MASP-1 R448Q was solved at 2.5 Å. Proenzyme MASP-1 R448Q readily cleaves synthetic substrates, and it is inhibited by a specific canonical inhibitor developed against active MASP-1, indicating that zymogen MASP-1 fluctuates between an inactive and an active-like conformation. The determined structure provides a feasible explanation for this phenomenon. In summary, autoactivation of MASP-1 is crucial for the activation of MBL/ficolin·MASP complexes, and in the proenzymic phase zymogen MASP-1 controls the process.     

Molecular interactions between MASP-2, C4, and C2 and their activation fragments leading to complement activation via the lectin pathway

Activation of component C3 is central to the pathways of complement and leads directly to neutralization of pathogens and stimulation of adaptive immune responses. The convertases that catalyze this reaction assemble from fragments of complement components via multistep reactions. In the lectin pathway, mannose-binding lectin (MBL) and ficolins bind to pathogens and activate MBL-associated serine protease-2 (MASP-2). MASP-2 cleaves C4 releasing C4a and generating C4b, which attaches covalently to the pathogen surface upon exposure of its reactive thioester. C2 binds to C4b and is also cleaved by MASP-2 to form the C3 convertase (C4b2a). To understand how this complex process is coordinated, we have analyzed the interactions between MASP-2, C4, C2, and their activation fragments and have compared MASP-2-catalyzed cleavage of C4b2 and C2. The data show that C2 binds tightly to C4b but not to C4, implying that C4 and C2 do not circulate as preformed complexes but that C2 is recruited only after prior activation of C4. Following cleavage of C4, C4b still binds to MASP-2 (KD approximately 0.6 microM) and dissociates relatively slowly (koff approximately 0.06 s-1) compared with the half-life of the thioester (相似文献   

C1, MBL-MASPs and C1-inhibitor: novel approaches for targeting complement-mediated inflammation

Complement activation is initiated by the pattern-recognition molecules complement component C1q, mannose-binding lectin (MBL) and ficolins (H-, L-, M-ficolin), which typically recognize antibody-antigen complexes or foreign polysaccharides. The associated proteases (C1r, C1s, MASP-1 and MASP-2) then activate the complement system. The serpin C1-inhibitor (C1-inh) blocks activity of all these complexes and has been successfully used in models of disease. Many structures of these components became available recently, including that of C1-inh, facilitating the structure-guided design of drugs targeting complement activation. Here, we propose an approach in which therapeutic proteins are made up of natural protein domains and C1-inh to allow targeting to the site of inflammation and more specific inhibition of complement activation. In particular, engineering a fast-acting C1-inh or fusing it to an 'aiming module' has been shown to be feasible and economical using a humanized yeast expression system. Complement-mediated inflammation has been linked to ischemia-reperfusion injury, organ graft rejection and even neurodegeneration, so targeting this process has direct clinical implications.     

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.     

Assembly and enzymatic properties of the catalytic domain of human complement protease C1r.

The catalytic properties of C1r, the protease that mediates activation of the C1 complex of complement, are mediated by its C-terminal region, comprising two complement control protein (CCP) modules followed by a serine protease (SP) domain. Baculovirus-mediated expression was used to produce fragments containing the SP domain and either 2 CCP modules (CCP1/2-SP) or only the second CCP module (CCP2-SP). In each case, the wild-type species and two mutants stabilized in the proenzyme form by mutations at the cleavage site (R446Q) or at the active site serine residue (S637A), were produced. Both wild-type fragments were recovered as two-chain, activated proteases, whereas all mutants retained a single-chain, proenzyme structure, providing the first experimental evidence that C1r activation is an autolytic process. As shown by sedimentation velocity analysis, all CCP1/2-SP fragments were dimers (5.5-5.6 S), and all CCP2-SP fragments were monomers (3.2-3.4 S). Thus, CCP1 is essential to the assembly of the dimer, but formation of a stable dimer is not a prerequisite for self-activation. Activation of the R446Q mutants could be achieved by extrinsic cleavage by thermolysin, which cleaved the CCP2-SP species more efficiently than the CCP1/2-SP species and yielded enzymes with C1s-cleaving activities similar to their active wild-type counterparts. C1r and its activated fragments all cleaved C1s, with relative efficiencies in the order C1r < CCP1/2-SP < CCP2-SP, indicating that CCP1 is not involved in C1s recognition.     

Mannose-binding lectin (MBL)-associated serine protease (MASP)-1 contributes to activation of the lectin complement pathway

The complement system plays an important role in innate immunity. In the lectin complement pathway, mannose-binding lectin (MBL) and ficolins act as recognition molecules, and MBL-associated serine protease (MASP) is a key enzyme. It has been suggested that MASP-2 is responsible for the activation of C4. Other serine proteases (MASP-1 and MASP-3) are also associated with MBL or ficolins; however, their functions are still controversial. In this study, a MASP-1- and MASP-3-deficient mouse model (MASP1/3(-/-)) was generated by a gene targeting strategy to investigate the roles of MASP-1 and MASP-3 in the lectin pathway. Serum derived from MASP1/3(-/-) mice showed significantly lower activity of both C4 and C3 deposition on mannan-agarose, and this low activity was restored by the addition of recombinant MASP-1. MASP-1/3-deficient serum showed a significant delay for activation of MASP-2 compared with normal serum. Reconstitution of recombinant MASP-1 in MASP-1/3-deficient serum was able to promote the activation of MASP-2. From these results, we propose that MASP-1 contributes to the activation of the lectin pathway, probably through the activation of MASP-2.     

The role of the individual domains in the structure and function of the catalytic region of a modular serine protease, C1r.

The first enzymatic event in the classical pathway of complement activation is autoactivation of the C1r subcomponent of the C1 complex. Activated C1r then cleaves and activates zymogen C1s. C1r is a multidomain serine protease consisting of N-terminal alpha region interacting with other subcomponents and C-terminal gammaB region mediating proteolytic activity. The gammaB region consists of two complement control protein modules (CCP1, CCP2) and a serine protease domain (SP). To clarify the role of the individual domains in the structural and functional properties of the gammaB region we produced the CCP1-CCP2-SP (gammaB), the CCP2-SP, and the SP fragments in recombinant form in Escherichia coli. We successfully renatured the inclusion body proteins. After renaturation all three fragments were obtained in activated form and showed esterolytic activity on synthetic substrates similar to each other. To study the self-activation process in detail zymogen mutant forms of the three fragments were constructed and expressed. Our major statement is that the ability of autoactivation and C1s cleavage is an inherent property of the SP domain. We observed that the CCP2 module significantly increases proteolytic activity of the SP domain on natural substrate, C1s. Therefore, we propose that CCP2 module provides accessory binding sites. Differential scanning calorimetric measurements demonstrated that CCP2 domain greatly stabilizes the structure of SP domain. Deletion of CCP1 domain from the CCP1-CCP2-SP fragment results in the loss of the dimeric structure. Our experiments also provided evidence that dimerization of C1r is not a prerequisite for autoactivation.     

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.     

Mannan-binding lectin-associated serine protease 3 cleaves synthetic peptides and insulin-like growth factor-binding protein 5

Mannan-binding lectin-associated serine proteases (MASPs) are secreted as single-chain precursors and processed into two disulfide bond-linked chains. MASP-3 and MASP-1, derived from the same gene, contain identical A chains, but entirely different catalytic domain-containing B chains. In contrast to MASP-1 and MASP-2, the proteinase activity of MASP-3 has not been described previously. We show here the proteolytic activity of the purified recombinant human MASP-3 catalytic domain toward peptides and protein substrates. Among the fluorogenic peptides tested, it specifically cleaved peptides with Arg at the P1 position. Among seven insulin-like growth factor-binding proteins, it selectively cleaved IGFBP-5, which is the first protein substrate identified for MASP-3. All three cleavage sites identified contained Arg or Lys at the P1 position and Pro at the P2 position. As compared to MASP-1 and MASP-2, MASP-3 has distinct substrate specificity and inhibitor profile. These results should be useful for further studies of the structure and function of human MASP-3.     

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.     

Composition of the lectin pathway of complement in Gallus gallus: absence of mannan-binding lectin-associated serine protease-1 in birds

The lectin pathway of complement is activated by multimolecular complexes that recognize and bind to microbial polysaccharides. These complexes comprise a multimeric carbohydrate recognition subunit (either mannan-binding lectin (MBL) or a ficolin), three MBL-associated serine proteases (MASP-1, -2, and -3), and MAp19 (a truncated product of the MASP-2 gene). In this study we report the cloning of chicken MASP-2, MASP-3, and MAp19 and the organization of their genes and those for chicken MBL and a novel ficolin. Mammals usually possess two MBL genes and two or three ficolin genes, but chickens have only one of each, both of which represent the undiversified ancestors of the mammalian genes. The primary structure of chicken MASP-2 is 54% identical with those of the human and mouse MASP-2, and the organization of its gene is the same as in mammals. MASP-3 is even more conserved; chicken MASP-3 shares approximately 75% of its residues with human and Xenopus MASP-3. It is more widely expressed than other lectin pathway components, suggesting a possible function of MASP-3 different from those of the other components. In mammals, MASP-1 and MASP-3 are alternatively spliced products of a single structural gene. We demonstrate the absence of MASP-1 in birds, possibly caused by the loss of MASP-1-specific exons during phylogeny. Despite the lack of MASP-1-like enzymatic activity in sera of chicken and other birds, avian lectin pathway complexes efficiently activate C4.     

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.     

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.