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The complement system is a sophisticated network of proteases. In this article, we describe an unexpected link between two linear activation routes of the complement system: the lectin pathway (LP) and the alternative pathway (AP). Mannose-lectin binding-associated serine protease (MASP)-1 is known to be the initiator protease of the LP. Using a specific and potent inhibitor of MASP-1, SGMI-1, as well as other MASP-1 inhibitors with different mechanisms of action, we demonstrated that, in addition to its functions in the LP, MASP-1 is essential for bacterial LPS-induced AP activation, whereas it has little effect on zymosan-induced AP activation. We have shown that MASP-1 inhibition prevents AP activation, as well as attenuates the already initiated AP activity on the LPS surface. This newly recognized function of MASP-1 can be important for the defense against certain bacterial infections. Our results also emphasize that the mechanism of AP activation depends on the activator surface., (Copyright © 2018 by The American Association of Immunologists, Inc.)

MASP-3 was discovered 15 years ago as the third mannan-binding lectin (MBL)-associated serine protease of the complement lectin pathway. Lacking any verified substrate its role remained ambiguous. MASP-3 was shown to compete with a key lectin pathway enzyme MASP-2 for MBL binding, and was therefore considered to be a negative complement regulator. Later, knock-out mice experiments suggested that MASP-1 and/or MASP-3 play important roles in complement pro-factor D (pro-FD) maturation. However, studies on a MASP-1/MASP-3-deficient human patient produced contradicting results. In normal resting blood unperturbed by ongoing coagulation or complement activation, factor D is present predominantly in its active form, suggesting that resting blood contains at least one pro-FD activating proteinase that is not a direct initiator of coagulation or complement activation. We have recently showed that all three MASPs can activate pro-FD in vitro. In resting blood, however, using our previously evolved MASP-1 and MASP-2 inhibitors we proved that neither MASP-1 nor MASP-2 activates pro-FD. Other plasma proteinases, particularly MASP-3, remained candidates for that function. For this study we evolved a specific MASP-3 inhibitor and unambiguously proved that activated MASP-3 is the exclusive pro-FD activator in resting blood, which demonstrates a fundamental link between the lectin and alternative pathways.

The complement system plays an important role in the induction of inflammation. In this study we demonstrate that the initiation complexes of the lectin pathway, consisting of mannose-binding lectin (MBL) and associated serine proteases (MASPs) elicit Ca(2+) signaling in cultured endothelial cells (HUVECs). This is in agreement with our previous results showing that the recombinant catalytic fragment of MASP-1 activates endothelial cells by cleaving protease activated receptor 4. Two other proteases, MASP-2 and MASP-3 are also associated with MBL. Earlier we showed that recombinant catalytic fragment of MASP-2 cannot activate HUVECs, and in this study we demonstrate that the same fragment of MASP-3 has also no effect. We find the same to be the case if we use recombinant forms of the N-terminal parts of MASP-1 and MASP-2 which only contain non-enzymatic domains. Moreover, stable zymogen mutant form of MASP-1 was also ineffective to stimulate endothelial cells, which suggests that in vivo MASP-1 have the ability to activate endothelial cells directly as well as to activate the lectin pathway simultaneously. We show that among the components of the MBL-MASPs complexes only MASP-1 is able to trigger response in HUVECs and the proteolytic activity of MASP-1 is essential. Our results strengthen the view that MASP-1 plays a central role in the early innate immune response., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The lectin pathway of complement is an important effector arm of innate immunity. It forms a first line of defense against invading pathogens and dangerously altered self structures. Pattern recognition molecules (mannose-binding lectin (MBL), ficolins) bind to the dangerous particles, which is followed by activation of MBL-associated serine proteases, MASP-1 and MASP-2, resulting in the initiation of the complement cascade. The activation of the lectin pathway is strictly controlled by natural inhibitors, since uncontrolled activation can lead to serious self-tissue damage. Recently we have shown that inhibition of either MASP-1 or MASP-2 by in vitro evolved specific inhibitors completely blocks the lectin pathway in human serum. In this study, we examined the inhibitory action of C1-inhibitor (C1-inh), antithrombin (AT) and α(2)-macroglobulin (α(2)M) on MASP-1 and MASP-2, and studied the inhibition of the lectin pathway in normal human serum in the presence and absence of heparin using C3 and C4 deposition assays. We measured the association rate constants for the serpin/protease reactions. We found that in the presence of heparin both C1-inh and AT are equally efficient inhibitors of the lectin pathway. Although α(2)M formed complex with MASP-1 in fluid phase, it could not abolish lectin pathway activation on activator surfaces., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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.

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.

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 K(I) of 65 and 1030 nM, respectively, whereas SFMI-2 inhibits only MASP-2 with a K(I) 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.