Quantitative characterization of the activation steps of mannan-binding lectin (MBL)-associated serine proteases (MASPs) points to the central role of MASP-1 in the initiation of the complement lectin pathway.

Abstract Mannan-binding lectin (MBL)-associated serine proteases, MASP-1 and MASP-2, have been thought to autoactivate when MBL/ficolin-MASP complexes bind to pathogens triggering the complement lectin pathway. Autoactivation of MASPs occurs in two steps: (1) zymogen autoactivation, when one proenzyme cleaves another proenzyme molecule, (2) autocatalytic activation, when the activated protease cleaves its own zymogen. Using recombinant catalytic fragments we demonstrated that a stable proenzyme MASP-1 variant (R448Q) cleaved the inactive, catalytic site Ser-to-Ala variant (S646A). MASP-1's autoactivation steps were separately quantified using these mutants and the wild-type enzyme. Analogous mutants were made for MASP-2, and rate constants of the autoactivation steps, and also the possible cross-activation steps between MASP-1 and MASP-2 were determined. Based on the rate constants a kinetic model of lectin pathway activation was outlined. The zymogen autoactivation rate of MASP-1 is approximately 3000-fold higher, and the autocatalytic activation of MASP-1 is about 140-fold faster than those of MASP-2. Moreover both activated and proenzyme MASP-1 can effectively cleave proenzyme MASP-2. MASP-3, which does not autoactivate, is also cleaved by MASP-1 quite efficiently. Structure of the catalytic region of proenzyme MASP-1 R448Q was solved at 2.5A. Proenzyme MASP-1 R448Q readily cleaves synthetic substrates, and it is inhibited by a specific canonical inhibitor developed against active MASP-1, indicating that zymogen MASP-1 fluctuates between an inactive and an active-like conformation. The determined structure provides a feasible explanation for this phenomenon. In summary, autoactivation of MASP-1 is crucial for the activation of MBL/ficolin-MASP complexes, and in the proenzymic phase zymogen MASP-1 controls the process.