The complement system is the other major proteolytic cascade in the blood of vertebrates besides the coagulation–fibrinolytic system. Among the three main activation routes of complement, the lectin pathway (LP) has been discovered the latest, and it is still the subject of intense research. Mannose-binding lectin (MBL), other collectins, and ficolins are collectively termed as the pattern recognition molecules (PRMs) of the LP, and they are responsible for targeting LP activation to molecular patterns, e.g., on bacteria. MBL-associated serine proteases (MASPs) are the effectors, while MBL-associated proteins (MAps) have regulatory functions. Two serine protease components, MASP-1 and MASP-2, trigger the LP activation, while the third component, MASP-3, is involved in the function of the alternative pathway (AP) of complement. Besides their functions within the complement system, certain LP components have secondary (“moonlighting”) functions, e.g., in embryonic development. They also contribute to blood coagulation, and some might have tumor suppressing roles. Uncontrolled complement activation can contribute to the progression of many diseases (e.g., stroke, kidney diseases, thrombotic complications, and COVID-19). In most cases, the lectin pathway has also been implicated. In this review, we summarize the history of the lectin pathway, introduce their components, describe its activation and regulation, its roles within the complement cascade, its connections to blood coagulation, and its direct cellular effects. Special emphasis is placed on disease connections and the non-canonical functions of LP components.

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.

Renal ischemia–reperfusion injury (IRI) is a common complication in several clinical scenarios including kidney transplantation. Mannan-binding lectin-associated serine proteinase (MASP)-2 is essential for activation of the complement lectin pathway, which has been implicated in the pathogenesis of renal IRI and therefore represents a potential therapeutic target. We developed a new, affinity-enhanced MASP-2 inhibitor, EVO24, by directed evolution of the D2 domain of human tissue factor pathway inhibitor. EVO24 was fused with a human IgG1-Fc to create the homodimer EVO24L, which potently and selectively inhibited the lectin pathway in human and mouse serum in vitro. EVO24L was tested in a mouse model of unilateral warm renal IRI. EVO24L administered before and after ischemia significantly protected against IRI, with improved renal function as well as reduced tubular injury and inflammatory cell infiltration at 24 h compared to vehicle-treated mice. Immunofluorescence analyses showed reduced deposition of complement components (C3d, C4d, and C9) and reduced expression of VCAM-1, indicating a decrease in complement activation and endothelial cell activation. Additionally, EVO24L treatment lowered plasma levels of complement C5a, hyaluronan (a marker of endothelial glycocalyx shedding), and the proinflammatory cytokines IL-6 and TNF-α. Our findings indicate that EVO24L inhibits acute inflammatory responses in renal IRI by blocking the lectin pathway, confirming the important role of this pathway in acute ischemic kidney injury and warranting further investigation of EVO24L in clinical settings.

Ecotin is a homodimeric serine protease inhibitor produced by many commensal and pathogenic microbes. It functions as a virulence factor, enabling survival of various pathogens in the blood. The ecotin dimer binds two protease molecules, and each ecotin protomer has two protease-binding sites: site1 occupies the substrate-binding groove, whereas site2 engages a distinct secondary region. Owing to the twofold rotational symmetry within the ecotin dimer, sites 1 and 2 of a protomer bind to different protease molecules within the tetrameric complex. Escherichia coli ecotin inhibits trypsin-like, chymotrypsin-like, and elastase-like enzymes, including pancreatic proteases, leukocyte elastase, key enzymes of blood coagulation, the contact and complement systems, and other antimicrobial cascades. Here, we show that mannan-binding lectin-associated serine protease-1 (MASP-1) and MASP-2, essential activators of the complement lectin pathway, and MASP-3, an essential alternative pathway activator, are all inhibited by ecotin. We decipher in detail how the preorganization of site1 and site2 within the ecotin dimer contributes to the inhibition of each MASP enzyme. In addition, using mutated and monomeric ecotin variants, we show that site1, site2, and dimerization contribute to inhibition in a surprisingly target-dependent manner. We present the first ecotin:MASP-1 and ecotin:MASP-2 crystal structures, which provide additional insights and permit structural interpretation of the observed functional results. Importantly, we reveal that monomerization completely disables the MASP-2-inhibitory, MASP-3-inhibitory, and lectin pathway–inhibitory capacity of ecotin. These findings provide new opportunities to combat dangerous multidrug-resistant pathogens through development of compounds capable of blocking ecotin dimer formation.

The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a viral structural protein that is abundant in the circulation of infected individuals. Previous published studies reported controversial data about the role of the N protein in the activation of the complement system. It was suggested that the N protein directly interacts with mannose-binding lectin-associated serine protease-2 (MASP-2) and stimulates lectin pathway overactivation/activity. In order to check these data and to reveal the mechanism of activation, we examined the effect of the N protein on lectin pathway activation. We found that the N protein does not bind to MASP-2 and MASP-1 and it does not stimulate lectin pathway activity in normal human serum. Furthermore, the N protein does not facilitate the activation of zymogen MASP-2, which is MASP-1 dependent. Moreover, the N protein does not boost the enzymatic activity of MASP-2 either on synthetic or on protein substrates. In some of our experiments, we observed that MASP-2 digests the N protein. However, it is questionable, whether this activity is biologically relevant. Although surface-bound N protein did not activate the lectin pathway, it did trigger the alternative pathway in 10% human serum. Additionally, we detected some classical pathway activation by the N protein. Nevertheless, we demonstrated that this activation was induced by the bound nucleic acid, rather than by the N protein itself.

Transmembrane protease serine (TMPRSS)2 is a cell surface host protease, which plays a decisive role in viral infections. This trypsin-like enzyme cleaves the spike protein of coronaviruses [e.g. severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)] and the hemagglutinin protein of influenza viruses, enabling viral membrane fusion and subsequent viral cell entry. Consequently, TMPRSS2 is an attractive therapeutic target, and it is of utmost importance to produce wildtype, enzymatically active recombinant TMPRSS2 for drug development purposes. We present the first successful strategy for the expression of wildtype and proteolytically fully active ectodomain of human and Syrian hamster TMPRSS2 in mammalian cells. To achieve that, we co-expressed the TMPRSS2 ectodomain with its natural inhibitor, hepatocyte growth factor activator inhibitor-2, which yielded substantial amounts of secreted, native protease. Most of the recombinant TMPRSS2 was secreted as zymogen, which was activated during purification. The purified activated recombinant TMPRSS2 ectodomain cleaved synthetic and protein substrates with high efficiency (kcat/KM in the 104–106 M-1s-1 range). To study the mechanism of auto-activation, we expressed zymogen TMPRSS2 mutants as well. We found that the zymogen is an ideal substrate for the active protease as it cleaves it extremely efficiently. We also showed that zymogen TMPRSS2 itself has a weak proteolytic activity, which can initiate the auto-activation process. We demonstrated that a related cell surface protease, TMPRSS13, is also able to activate zymogen TMPRSS2. Our findings prove both a zymogen trans(auto)-activation as well as a TMPRSS13-based cross-activation mechanism, the latter supporting that type II transmembrane serine proteases can form a pericellular proteolytic cascade.