Mannan-binding lectin-associated serine protease (SP) (MASP)-1 and MASP-2 are modular SP and form complexes with mannan-binding lectin, the recognition molecule of the lectin pathway of the complement system. To characterize the enzymatic properties of these proteases we expressed their catalytic region, the C-terminal three domains, in Escherichia coli. Both enzymes autoactivated and cleaved synthetic oligopeptide substrates. In a competing oligopeptide substrate library assay, MASP-1 showed extreme Arg selectivity, whereas MASP-2 exhibited a less restricted, trypsin-like specificity. The enzymatic assays with complement components showed that cleavage of intact C3 by MASP-1 and MASP-2 was detectable, but was only ∼0.1% of the previously reported efficiency of C3bBb, the alternative pathway C3-convertase. Both enzymes cleaved C3i 10- to 20-fold faster, but still at only ∼1% of the efficiency of MASP-2 cleavage of C2. We believe that C3 is not the natural substrate of either enzyme. MASP-2 cleaved C2 and C4 at high rates. To determine the role of the individual domains in the catalytic region of MASP-2, the second complement control protein module together with the SP module and the SP module were also expressed and characterized. We demonstrated that the SP domain alone can autoactivate and cleave C2 as efficiently as the entire catalytic region, while the second complement control protein module is necessary for efficient C4 cleavage. This behavior strongly resembles C1s. Each MASP-1 and MASP-2 fragment reacted with C1-inhibitor, which completely blocked the enzymatic action of the enzymes. Nevertheless, relative rates of reaction with α-2-macroglobulin and C1-inhibitor suggest that α-2-macroglobulin may be a significant physiological inhibitor of MASP-1.

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.innate immunity | complement system | directed evolution | phage display | canonical inhibitor T he lectin pathway of the complement system serves as a first line of defense against microbial intruders. The innate immune system recognizes danger signals presented by the pathogens (pathogen-associated molecular patterns) or altered host cells (damage-associated molecular patterns) by means of germlineencoded cell-surface bound or soluble pattern recognition molecules (1, 2). These pattern recognition molecules have evolved against evolutionarily conserved structures of microorganisms, such as carbohydrates and acetylated compounds. The prompt action of the innate immune system provides sufficient time for the adaptive immune system to react with less conservative antigens (e.g., proteins) to build up a more specific response. In humans, five different humoral pattern recognition molecules have been identified that are able to initiate the lectin pathway: mannose-binding lectin (MBL) (3), three ficolins (M-, L-, and H-ficolin; also called ficolin-1, -2, and -3) (4), and collectin 11 (CL11 or CL-K1) (5). The pattern recognition molecules do not act alone; they are associated with other proteins, mainly serine proteases (6). These serine proteases [MBL-associated serine proteases (MASPs)] are present as proenzymes (zymogens) in the complexes and become activated to initiate the complement cascade when the recognition molecules bind to their target. Activation of the complement cascade culminates in the destruction and elimination of pathogens via opsonization or direct cell lysis. Although the lectin pathway was discovered some 20 years ago (7), the mechanism of the activation is still enigmatic. One of the most controversial issues is the role of the serine proteases. Up to now, three serine proteases have been discovered and designated as MASP-1, MASP-2, and MASP-3. In addition to the proteases, two nonenzymatic fragments of the MASPs, MAp44 (8, 9) and MAp19 (10), have also been found in the recognition complexes. MASP-1, MASP-3, and MAp44 are the alternative splice products of the MASP-1/3 gene, and MASP-2 and MAp19 are encoded by the MASP-2 gene. The only consensus point in the literature is that ...