Characterization of the interaction between L-ficolin/P35 and mannan-binding lectin-associated serine proteases-1 and-2

Ficolins are oligomeric lectins comprising a collagen-like and a fibrinogen-like domain, with a binding specificity for N-acetylglucosamine. It has been reported recently that L-ficolin/P35 associates with mannan-binding lectin (MBL)-associated serine proteases (MASP-1 and -2) and MBL-associated protein 19 (MAp19) in serum and forms complexes able to activate complement. Using surface plasmon resonance spectroscopy we have shown that recombinant MASP-1 and -2, their N-terminal CUB1 (module originally found in complement proteins Clr/Cls, Uegf, and bone morphogenetic protein-1)-epidermal growth factor (EGF)-CUB2 and CUB1-EGF segments, and MAp19 bind to immobilized L-ficolin/P35 in the presence of Ca2+ ions. Comparable K, values were obtained for the full-length proteases and their CUB1-EGF-CUB2 segments (9.2 and 10 nM for MASP-1 and 4.6 and 5.4 nM for MASP-2, respectively), whereas higher values were obtained for the CUB1-EGF segments (26.7, 15.6, and 14.3 nM for MASP-1, MASP-2, and MAp19). These values are in the same range as those determined for the interaction of these proteins with MBL. Binding was Ca2+ dependent and was only partly sensitive to EDTA for MASP-1, MASP-2, and MASP-2 CUB1-EGF-CUB2. Half-maximal binding was obtained at comparable Ca2+ concentrations for MASP-1 and MASP-2 (0.45 and 0.47 muM, respectively), their CUB1-EGF-CUB2 segments (0.37 and 0.72 muM), and their CUB1-EGF segments (0.31 and 0.79 muM). These values are lower than those determined in the case of NIBL, indicating a difference between NIBL and L-ficolin/P35 with respect to the Ca2+ dependence of their interaction with the MASPs. Preincubation of the MASPs 'with soluble MBL inhibited subsequent binding to immobilized L-ficolin/P35 and, conversely, suggesting that these lectins compete with each other for binding to the MASPs in vivo.