Nine serine proteases are involved in the three pathways that lead to complement activation (Table 1). The classical pathway, found only in vertebrates, is initiated by antibody-antigen complexes. The mannan-binding lectin (MBL) pathway is initiated by the binding of MBL to specific carbohydrate structures found on the surface of a variety of microorganisms. The alternative pathway is initiated by structures on microbial surfaces.
|Initiating Complex||C1q (A,B,C chains)||MBL||C3b|
|Serine Proteases||C1r, C1s, C2||MASP-1,2,3,C2||Factors D,B,I|
|Table 1. Proteins involved in complement activation.|
MBL-associated serine proteases (MASPs) are secreted as single-chain precursors and processed into two chains (A and B) linked by disulfide bonds.
|Peptide Sequence (P3--P2--P1-P1')||Activity (%)|
|Z-Phe-Arg-AMC (Catalog # ES009)||14|
|Z-Leu-Arg-AMC (Catalog # ES008)||11|
|Table 2. Fluorogenic peptide substrates at 100 µM were cleaved by R&D Systems' recombinant human MASP3 catalytic domain (Catalog # 1724-SE) at 10 µg/mL in 50 mM Tris, pH 8.5. Initial rates were measured by a fluorescence plate reader (380/460 nm).|
In contrast to MASP1 and MASP2, MASP3 protease activity has not been described previously. We show here, for the first time, its activity toward peptide and protein substrates. R&D Systems' recombinant human MASP3 catalytic domain (rhMASP3CD; Catalog # 1724-SE) specifically cleaves peptides with Arg at the P1 position and prefers substrates with two additional residues occupied at the P2 and P3 positions (Table 2). Among seven insulin-like growth factor binding proteins (IGFBPs), the active enzyme selectively cleaves IGFBP-5 (Figure 1). This peptidase activity is inhibited by serine protease inhibitors such as Ecotin (Catalog # 1328-PI) and AEBSF (Catalog # EI001).
|Figure 1. Cleavage of IGFBP-5, but not other IGFBPs, by R&D Systems' recombinant human MASP3 catalytic domain (Catalog # 1724-SE) as shown by silver stained SDS-PAGE gel. All recombinant human IGFBP proteins were incubated at 100 µg/mL either alone (-) or with rhMASP3 catalytic domain (CD; +) at 5 µg/mL for 16 hours at 37 °C.|