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

Abstract

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.

Monospecific Inhibitors Show That Both Mannan-binding Lectin-associated Serine Protease-1 (MASP-1) and -2 Are Essential for Lectin Pathway Activation and Reveal Structural Plasticity of MASP-2

“…Inhibitory Constant (K I ) Measurements-Equilibrium inhibitory constants of the SGMI variants listed in Table 1 were determined on MASP-1 and MASP-2 as described (8). Representative plots of K I measurements are shown in supplemental Fig.…”

“…All these could be studied by selective inhibitors. In a recent paper we reported the first MASP-inhibitors developed by directed evolution of the 14-mer sunflower trypsin inhibitor (SFTI) (8). That study led to SFMI-1 (sunflower MASP inhibitor-1) and SFMI-2.…”

Serum MASP-1 in complex with MBL activates endothelial cells

“…An Arg-Ile bond is cleaved in the activation loop, which results in conformational change in the serine protease domain. Recently, we have shown that MASP-1, the most abundant protease component of the lectin pathway, controls lectin pathway activation Héja et al, 2012a;2012b;Kocsis et al, 2010;Megyeri et al, 2013). MASP-1 autoactivates and cleaves zymogen MASP-2, which then cleaves C4 and C2 components (Kidmose et al, 2012).…”

Heterocomplex Formation between MBL/Ficolin/CL-11–Associated Serine Protease-1 and -3 and MBL/Ficolin/CL-11–Associated Protein-1

“…Combined, these data suggest that the MASPs have the ability to assemble in heterocomplexes and in that way contribute to a cross activation, as suggested by several recent papers (34)(35)(36)(37). In figure, we show a schematic overview of different combinations including both MASP homodimers (red/red or green/green protease domains in one dimer) and heterodimers (red/green).…”