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 ...

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.