Technical Notes: Proteases for Complement Research

Proteases for Complement Research

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.

  Complement Pathway
  Classical MBL Alternative
Initiating Complex C1q (A,B,C chains) MBL C3b
Serine Proteases C1r, C1s, C2 MASP-1,2,3,C2 Factors D,B,I
Non-enzymatic C1q,C4,C3,C3b MBL,C4,C3,C3b Properdin,C3,C3b
Table 1. Proteins involved in complement activation.

Human MASP3: An IGFBP-5-specific Protease

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 (%)
H-Pro-Phe-Arg-AMC 100
Boc-Val-Pro-Arg-AMC 82
Z-Gly-Gly-Arg-AMC 51
Z-Phe-Arg-AMC (Catalog # ES009) 14
Z-Leu-Arg-AMC (Catalog # ES008) 11
H-Gly-Arg-AMC 0
Suc-Ala-Phe-Lys-AMC 0
Suc-Ala-Pro-Phe-AMC 0
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.