Distinct pathways of mannan-binding lectin (MBL)- and C1-complex autoactivation revealed by reconstitution of MBL with recombinant MBL-associated serine protease-2
Vorup-Jensen T, Petersen SV, Hansen AG, Poulsen K, Schwaeble W, Sim RB, Reid KB, Davis SJ, Thiel S, Jensenius JC. (2000), J Immunol. 165, 2093-100
Mannan-binding lectin (MBL) plays a pivotal role in innate immunity by activating complement after binding carbohydrate moieties on pathogenic bacteria and viruses. Structural similarities shared by MBL and C1 complexes and by the MBL- and C1q-associated serine proteases, MBL-associated serine protease (MASP)-1 and MASP-2, and C1r and C1s, respectively, have led to the expectation that the pathways of complement activation by MBL and C1 complexes are likely to be very similar. We have expressed rMASP-2 and show that, whereas C1 complex autoactivation proceeds via a two-step mechanism requiring proteolytic activation of both C1r and C1s, reconstitution with MASP-2 alone is sufficient for complement activation by MBL. The results suggest that the catalytic activities of MASP-2 split between the two proteases of the C1 complex during the course of vertebrate complement evolution.
Key figure: Complex formation between rMASP-2 and MBL analyzed by GPC and Western blotting
rMASP-2 alone or preincubated with MBL was chromatographed on Superose 6B. rMASP-2 was chromatographed in buffer with Ca2+ and Mg2+ (A) and in EDTA-containing buffer (B). Fractions of 0.45 ml were collected, and samples were subjected to SDS-PAGE/Western blotting to establish the elution volume of rMASP-2. The blots were developed with anti-MASP-2 mAb (1.3B7). C and D show the elution pattern of rMASP-2 when preincubated with MBL before chromatography in Ca2+– and Mg2+-containing buffer (C) or EDTA-containing buffer (D). The elution volumes of MBL, C1r2, and C1s were established in separate experiments.