Revised mechanism of complement lectin-pathway activation revealing the role of serine protease MASP-1 as the exclusive activator of MASP-2

Abstract

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.

Fig. 1.

Inhibitory effect of SGMI-1 and SGMI-2 on the complement pathways. (A), (B) The formation of the membrane attack complex was detected by using the Wieslab kit. The activity of the classical (■), lectin (●), and alternative (▲) pathways were measured by separate experiments. (A) Inhibition by SGMI-2. (B) Inhibition by SGMI-1. (C), (D) Inhibition of the lectin pathway mediated C3 deposition in lepirudin-treated whole blood. (C) Inhibition by SGMI-2. (D) Inhibition by SGMI-1. (E) The time course of the inhibitory effect of the MASP-1–specific SGMI-1 (●) and the MASP-2–specific SGMI-2 (■) on the MBL–lectin pathway was monitored by measuring C4 deposition. As control experiments, lectin-pathway activity was monitored in the absence of inhibitors (▲) and in the presence of a strong broad-spectrum serine protease inhibitor FUT-175 (▼).

Fig. 2.

C3 deposition assay. (A) The MASP-1–specific SGMI-1 inhibits the lectin-pathway-mediated C3 deposition both on mannan-coated plates (●), as well as on acBSA-coated plates (○). (B) The MASP-2–specific SGMI-2 also inhibits the lectin pathway both on mannan-coated plates (●) and on acBSA-coated plates (○).

Fig. 3.

C4 deposition assays. (A) MASP-1–selective SGMI-1 prevented deposition of C4 efficiently when the MBL–MASP (●) or H-ficolin–MASP (○) complexes from the serum were immobilized in the presence of the inhibitor and the direct C4 cleavage was detected. (B) SGMI-1 was totally ineffective when the immobilized MBL–MASP (●) or H-ficolin–MASP (○) complexes were let to activate before purified C4 and SGMI-1 were added. The MASP-2–specific inhibitor SGMI-2 efficiently prevents C4 deposition from both normal (C) and preactivated (D) serum in the case of both MBL–MASP (●) or H-ficolin–MASP (○) complexes.

Fig. 4.

Effect of SGMI-1 and SGMI-2 on the autoactivation of isolated MASP-2. The time course of autoactivation of isolated zymogen catalytic fragment of MASP-2 was studied in the presence or absence of inhibitors. At 1-μM inhibitor concentration, the MASP-2–specific SGMI-2 (▲) completely inhibited the autoactivation process, and the MASP-1–specific SGMI-1 (■) had no effect at all compared with the noninhibited sample (●).

Fig. 5.

Contribution of MASP-1 and MASP-2 to C3–convertase formation through C2 cleavage. The C2 cleaving ability of MASP-1 and MASP-2 was determined through measuring C3 deposition on C4b-saturated plate using the MASP-1– and MASP-2–specific inhibitors. When MASP-1 was inhibited by SGMI-1, the level of C3 deposition dropped to 43.5% of the noninhibited experiment. In accordance with this result, when MASP-2 was inhibited by SGMI-2, the level of C3 deposition dropped to 61%. The two independent measurements unequivocally show that MASP-1 is the major contributor of the C2 cleavage. When applying both inhibitors at the same time the level of C3 deposition is reduced to 3.3%.

Fig. 6.

Comparison of the old model and our corrected (new) model of the lectin-pathway activation. According to the old model, MASP-2 is the autonomous activator of the lectin pathway, because it can autoactivate and cleave C4 and C2, while MASP-1 has only supportive role. In contrast to this, our model states that MASP-1 controls the entire activation process. MASP-1 autoactivates and cleaves zymogen MASP-2. The active MASP-2 then cleaves C4 and the deposited C4b binds C2, which is then cleaved primarily (60%) by MASP-1 and to a lesser extent (40%) by MASP-2. This results in the formation of the C4b2a complex. Red arrows indicate proteolytic cleavage.