to Lectin Pathway Activation and -2: Significant Contribution of MASP-1 Lectin-Associated Serine Protease (MASP)-1 Peptides against Mannose-Binding Complement with Phage Display Selected Selective Inhibition of the Lectin Pathway of

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 four different enzymes: MASP-1, MASP-2, trypsin, and thrombin. A method developed for determining the equilibrium K I values of tight binding inhibitors was applied (36). A thioesther substrate, Z-L-LysSBzl hydrochloride (C3647; Sigma-Aldrich), was used in the photometric assay. MASP-1, MASP-2, or trypsin was incubated with serial dilutions of the inhibitors for 1 h at room temperature. The reactions were conducted in 1 ml final volume at room temperature in 20 mM HEPES, 145 mM NaCl, 5 mM CaCl 2 , and 0.05% Triton X-100 (pH 7.6). The sulfhydryl of the cleaved substrate reacted with the cosubstrate dithiopyridine (143057; Sigma-Aldrich) producing the product detected at 324 nm. Enzyme concentration of the stock prote- ase solutions was determined by active site titration. In the case of trypsin and thrombin 4-methylumbelliferyl-4-guanidinobenzoate (51010; Sigma- Aldrich), whereas in the case of MASP-1 and MASP-2, a C1 inhibitor was used for the titration. The concentration of the free enzyme in the inhibitory assay was determined by the ratio of the residual enzyme activity (in the presence of inhibitor) and the total enzyme hibitor). The K I values were determined by nonlinear regression analysis the following