József Dobó

Revised mechanism of complement lectin-pathway activation revealing the role of serine protease MASP-1 as the exclusive activator of MASP-2

The lectin pathway of complement activation is an important component of the innate immune defense. The initiation complexes of the lectin pathway consist of a recognition molecule and associated serine proteases. Until now the autoactivating mannose-binding lectin-associated serine protease (MASP)-2 has been considered the autonomous initiator of the proteolytic cascade. The role of the much more abundant MASP-1 protease was controversial. Using unique, monospecific inhibitors against MASP-1 and MASP-2, we corrected the mechanism of lectin-pathway activation. In normal human serum, MASP-2 activation strictly depends on MASP-1. MASP-1 activates MASP-2 and, moreover, inhibition of MASP-1 prevents autoactivation of MASP-2. Furthermore we demonstrated that MASP-1 produces 60% of C2a responsible for C3 convertase formation.

Active Site Distortion Is Sufficient for Proteinase Inhibition by Serpins STRUCTURE OF THE COVALENT COMPLEX OF α1-PROTEINASE INHIBITOR WITH PORCINE PANCREATIC ELASTASE

We report here the x-ray structure of a covalent serpin-proteinase complex, α1-proteinase inhibitor (α1PI) with porcine pancreatic elastase (PPE), which differs from the only other x-ray structure of such a complex, that of α1PI with trypsin, in showing nearly complete definition of the proteinase. α1PI complexes with trypsin, PPE, and human neutrophil elastase (HNE) showed similar rates of deacylation and enhanced susceptibility to proteolysis by exogenous proteinases in solution. The differences between the two x-ray structures therefore cannot arise from intrinsic differences in the inhibition mechanism. However, self-proteolysis of purified complex resulted in rapid cleavage of the trypsin complex, slower cleavage of the PPE complex, and only minimal cleavage of the HNE complex. This suggests that the earlier α1 PI-trypsin complex may have been proteolyzed and that the present structure is more likely to be representative of serpin-proteinase complexes. The present structure shows that active site distortion alone is sufficient for inhibition and suggests that enhanced proteolysis is not necessarily exploited in vivo.

MASP-1, a promiscuous complement protease: structure of its catalytic region reveals the basis of its broad specificity.

Mannose-binding lectin (MBL)-associated serine protease (MASP)-1 is an abundant component of the lectin pathway of complement. The related enzyme, MASP-2 is capable of activating the complement cascade alone. Though the concentration of MASP-1 far exceeds that of MASP-2, only a supporting role of MASP-1 has been identified regarding lectin pathway activation. Several non-complement substrates, like fibrinogen and factor XIII, have also been reported. MASP-1 belongs to the C1r/C1s/MASP family of modular serine proteases; however, its serine protease domain is evolutionary different. We have determined the crystal structure of the catalytic region of active MASP-1 and refined it to 2.55 Å resolution. Unusual features of the structure are an internal salt bridge (similar to one in factor D) between the S1 Asp189 and Arg224, and a very long 60-loop. The functional and evolutionary differences between MASP-1 and the other members of the C1r/C1s/MASP family are reflected in the crystal structure. Structural comparison of the protease domains revealed that the substrate binding groove of MASP-1 is wide and resembles that of trypsin rather than early complement proteases explaining its relaxed specificity. Also, MASP-1’s multifunctional behavior as both a complement and a coagulation enzyme is in accordance with our observation that antithrombin in the presence of heparin is a more potent inhibitor of MASP-1 than C1 inhibitor. Overall, MASP-1 behaves as a promiscuous protease. The structure shows that its substrate binding groove is accessible; however, its reactivity could be modulated by an unusually large 60-loop and an internal salt bridge involving the S1 Asp.

Early complement proteases: C1r, C1s and MASPs. A structural insight into activation and functions

C1r, C1s and the mannose-binding lectin-associated serine proteases (MASPs) are responsible for the initiation of the classical- and lectin pathway activation of the complement system. These enzymes do not act alone, but form supramolecular complexes with pattern recognition molecules such as C1q, MBL, and ficolins. They share the same domain organization but have different substrate specificities and fulfill different physiological functions. In the recent years the rapid progress of structural biology facilitated the understanding of the molecular mechanism of complement activation at atomic level. In this review we summarize our current knowledge about the structure and function of the early complement proteases, delineate the latest models of the multimolecular complexes and present the functional consequences inferred from the structural studies. We also discuss some open questions and debated issues that need to be resolved in the future.

C1 inhibitor serpin domain structure reveals the likely mechanism of heparin potentiation and conformational disease.

C1 inhibitor, a member of the serpin family, is a major down-regulator of inflammatory processes in blood. Genetic deficiency of C1 inhibitor results in hereditary angioedema, a dominantly inheritable, potentially lethal disease. Here we report the first crystal structure of the serpin domain of human C1 inhibitor, representing a previously unreported latent form, which explains functional consequences of several naturally occurring mutations, two of which are discussed in detail. The presented structure displays a novel conformation with a seven-stranded β-sheet A. The unique conformation of the C-terminal six residues suggests its potential role as a barrier in the active-latent transition. On the basis of surface charge pattern, heparin affinity measurements, and docking of a heparin disaccharide, a heparin binding site is proposed in the contact area of the serpin-proteinase encounter complex. We show how polyanions change the activity of the C1 inhibitor by a novel “sandwich” mechanism, explaining earlier reaction kinetic and mutagenesis studies. These results may help to improve therapeutic C1 inhibitor preparations used in the treatment of hereditary angioedema, organ transplant rejection, and heart attack.

Structural basis for activation of the complement system by component C4 cleavage.

An essential aspect of innate immunity is recognition of molecular patterns on the surface of pathogens or altered self through the lectin and classical pathways, two of the three well-established activation pathways of the complement system. This recognition causes activation of the MASP-2 or the C1s serine proteases followed by cleavage of the protein C4. Here we present the crystal structures of the 203-kDa human C4 and the 245-kDa C4⋅MASP-2 substrate⋅enzyme complex. When C4 binds to MASP-2, substantial conformational changes in C4 are induced, and its scissile bond region becomes ordered and inserted into the protease catalytic site in a manner canonical to serine proteases. In MASP-2, an exosite located within the CCP domains recognizes the C4 C345C domain 60 Å from the scissile bond. Mutations in C4 and MASP-2 residues at the C345C–CCP interface inhibit the intermolecular interaction and C4 cleavage. The possible assembly of the huge in vivo enzyme–substrate complex consisting of glycan-bound mannan-binding lectin, MASP-2, and C4 is discussed. Our own and prior functional data suggest that C1s in the classical pathway of complement activated by, e.g., antigen–antibody complexes, also recognizes the C4 C345C domain through a CCP exosite. Our results provide a unified structural framework for understanding the early and essential step of C4 cleavage in the elimination of pathogens and altered self through two major pathways of complement activation.

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 4.0×102 and 2.7×102 M−1s−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.

Biological variations of MASP-3 and MAp44, two splice products of the MASP1 gene involved in regulation of the complement system

The lectin pathway of complement is part of the innate immune system. The complement-activating pattern-recognition molecules (for which we suggest the abbreviation CAPREMs) mannan-binding lectin (MBL) and the three ficolins (H-, L- and M-ficolin) circulate in complexes with MBL-associated serine proteases (MASP-1, -2 and -3) and two additional proteins (MAp19 and MAp44, also termed sMAP and MAP-1, respectively). When MBL or ficolins recognize a microorganism or altered self components, activation of the MASPs ensues, leading to the activation of the complement system. MASP-1, MASP-3 and MAp44 are all three encoded by the MASP1 gene. MASP-1 and -3 share five domains (constituting the so-called A-chain), but have unique protease domains (B-chains). MAp44 shares the first four domains with MASP-1 and MASP-3, followed by 17 unique C-terminal amino acid residues. Thus, assays for the protease domain of MASP-3 and for the 17 C-terminal amino acids of MAp44 are required to measure these proteins specifically and here we present such assays for MASP-3 and MAp44. MASP-3 was captured with a monoclonal antibody (5F5) reacting with a common domain of the three proteins (CCP1) and the assay was developed with a monoclonal antibody (38.12.3) specific for the C-terminal part of the MASP-3 protease domain. MAp44 was captured with a monoclonal antibody (2D5) reacting with the C-terminus of MAp44 followed by assay development with a monoclonal anti-CCP1 antibody (4H2). Using Superose 6 gel permeation chromatography of serum, MASP-3 and MAp44 were found in complexes, which eluted in positions corresponding to 600-800 kDa and 500-700 kDa, respectively. The level of MASP-3 in donor sera (N=200) was log-normally distributed with a median value of 5.0 μg/ml (range: 1.8-10.6 μg/ml), and the corresponding value for MAp44, also log-normally distributed, was 1.7 μg/ml (range: 0.8-3.2 μg/ml). For MASP-3, the inter-assay coefficients of variation of low, intermediate and high level internal controls were 4.9%, 6.9% and 3.9% (N=12). For MAp44, the corresponding inter-assay CVs were 7.6%, 6.2%, and 7.0% (N=12). MASP-3 levels were low at birth and reached adult levels within the first 6 months, whereas MAp44 levels fell slightly during the first 6 months. Concomitant with the acute phase response in patients undergoing major surgery, levels of both proteins fell slightly over 1-2 days, but whereas MASP-3 recovered to baseline values over another 2 days, MAp44 only reached baseline values at around day 30. Thus, neither of the two proteins behaves as a classical acute phase protein.

Selective inhibition of the lectin pathway of complement with phage display selected peptides against mannose-binding lectin-associated serine protease (MASP)-1 and -2: significant contribution of MASP-1 to lectin pathway activation.

The complement system, an essential part of the innate immune system, can be activated through three distinct routes: the classical, the alternative, and the lectin pathways. The contribution of individual activation pathways to different biological processes can be assessed by using pathway-selective inhibitors. In this paper, we report lectin pathway-specific short peptide inhibitors developed by phage display against mannose-binding lectin-associated serine proteases (MASPs), MASP-1 and MASP-2. On the basis of the selected peptide sequences, two 14-mer peptides, designated as sunflower MASP inhibitor (SFMI)-1 and SFMI-2, were produced and characterized. SFMI-1 inhibits both MASP-1 and MASP-2 with a KI of 65 and 1030 nM, respectively, whereas SFMI-2 inhibits only MASP-2 with a KI of 180 nM. Both peptides block the lectin pathway activation completely while leaving the classical and the alternative routes intact and fully functional, demonstrating that of all complement proteases only MASP-1 and/or MASP-2 are inhibited by these peptides. In a C4 deposition inhibitor assay using preactivated MASP-2, SFMI-2 is 10-fold more effective than SFMI-1 in accordance with the fact that SFMI-2 is a more potent inhibitor of MASP-2. Surprisingly, however, out of the two peptides, SFMI-1 is much more effective in preventing C3 and C4 deposition when normal human serum containing zymogen MASPs is used. This suggests that MASP-1 has a crucial role in the initiation steps of lectin pathway activation most probably by activating MASP-2. Because the lectin pathway has been implicated in several life-threatening pathological states, these inhibitors should be considered as lead compounds toward developing lectin pathway blocking therapeutics.

Mannan-binding lectin (MBL)-associated serine protease-1 (MASP-1), a serine protease associated with humoral pattern-recognition molecules: normal and acute-phase levels in serum and stoichiometry of lectin pathway components.

The pattern‐recognition molecules mannan‐binding lectin (MBL) and the three ficolins circulate in blood in complexes with MBL‐associated serine proteases (MASPs). When MBL or ficolin recognizes a microorganism, activation of the MASPs occurs leading to activation of the complement system, an important component of the innate immune system. Three proteins are produced from the MASP1 gene: MASP‐1 and MASP‐3 and MAp44. We present an assay specific for MASP‐1, which is based on inhibition of the binding of anti‐MASP‐1‐specific antibody to MASP‐1 domains coated onto microtitre wells. MASP‐1 was found in serum in large complexes eluting in a position corresponding to ∼600 kDa after gel permeation chromatography in calcium‐containing buffer and as monomers of ∼75 kDa in dissociating buffer. The concentration of MASP‐1 in donor sera (n = 105) was distributed log‐normally with a median value of 11 µg/ml (range 4–30 µg/ml). Serum and citrate plasma levels were similar, while the values in ethylenediamine tetraacetic acid plasma were slightly lower and in heparin plasma were 1·5 times higher than in serum. MASP‐1 was present at adult level at 1 year of age, while it was 60% at birth. In normal healthy individuals the level of MASP‐1 was stable throughout a 2‐month period. After induction of an acute‐phase reaction by operation we found an initial short decrease, concomitant with an increase in C‐reactive protein levels, followed by an increase, doubling the MASP‐1 concentration after 2 days. The present data prepare the ground for studies on the associations of MASP‐1 levels with disease.