Characterization of the interaction between L-ficolin/P35 and mannan-binding lectin-associated serine proteases-1 and-2
Characterization of the interaction between L-ficolin/P35 and mannan-binding lectin-associated serine proteases-1 and-2
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DOI：
10.4049/jimmunol.169.10.5735
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发表时间：
2002-11-15
影响因子：
4.4
通讯作者：
Thielens, NM
中科院分区：
文献类型：
--
作者：
Cseh, S;Vera, L;Thielens, NM
无花果蛋白酶是包含胶原蛋白样和纤维蛋白原样结构域的寡聚凝集素，对N-乙酰葡糖胺具有结合特异性。最近有报道，L-纤维胶凝蛋白/P35与血清中的甘露聚糖结合凝集素（MBL）相关丝氨酸蛋白酶（MASP-1和-2）和MBL相关蛋白19（MAp 19）缔合并形成能够激活补体的复合物。使用表面等离子体共振光谱，我们已经表明，重组MASP-1和-2，它们的N-末端CUB 1（最初在补体蛋白Clr/Cls、Uegf和骨形态发生蛋白-1中发现的模块）-表皮生长因子（EGF）-CUB 2和CUB 1-EGF片段，以及MAp 19在Ca 2+离子存在下与固定的L-纤维胶凝蛋白/P35结合。对于全长蛋白酶及其CUB 1-EGF-CUB 2区段获得了相当的Ki值（对于MASP-1分别为9.2和10 nM，对于MASP-2分别为4.6和5.4 nM），而对于CUB 1-EGF区段获得了更高的值（对于MASP-1、MASP-2和MAp 19分别为26.7、15.6和14.3 nM）。这些值与这些蛋白质与MBL相互作用的测定值范围相同。MASP-1、MASP-2和MASP-2 CUB 1-EGF-CUB 2的结合是Ca 2+依赖性的，并且仅对EDTA部分敏感。对于MASP-1和MASP-2（分别为0.45和0.47 μ M）、它们的CUB 1-EGF-CUB 2片段（0.37和0.72 μ M）和它们的CUB 1-EGF片段（0.31和0.79 μ M），在相当的Ca 2+浓度下获得半最大结合。这些值低于在NIBL的情况下测定的值，表明NIBL和L-纤维胶凝蛋白/P35之间在它们与MASP相互作用的Ca 2+依赖性方面存在差异。MASP与可溶性MBL的预孵育抑制了随后与固定化的L-纤维胶凝蛋白/P35的结合，并且相反地，表明这些凝集素在体内彼此竞争与MASP的结合。
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.