Natural substrates and inhibitors of mannan-binding lectin-associated serine protease-1 and-2:: A study on recombinant catalytic fragments
Natural substrates and inhibitors of mannan-binding lectin-associated serine protease-1 and-2:: A study on recombinant catalytic fragments
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DOI：
10.4049/jimmunol.170.3.1374
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发表时间：
2003-02-01
影响因子：
4.4
通讯作者：
Závodszky, P
中科院分区：
文献类型：
--
作者：
Ambrus, G;Gál, P;Závodszky, P
甘露聚糖结合凝集素相关丝氨酸蛋白酶(MASP)-1和MASP-2是模块化的SP，与甘露聚糖结合凝集素形成复合物，甘露聚糖结合凝集素是补体系统凝集素途径的识别分子。为了表征这些蛋白酶的酶学性质，我们在大肠杆菌中表达了它们的催化区域c端三结构域。这两种酶都能自动激活和裂解合成寡肽底物。在竞争性寡肽底物文库分析中，masp - 1表现出极高的精氨酸选择性，而MASP-2表现出较少受限的胰蛋白酶样特异性。补体成分的酶分析表明，可以检测到MASP-1和MASP-2对完整C3的切割，但其效率仅与之前报道的C3bBb （C3转化酶的替代途径）的效率相似。这两种酶裂解CM的速度都快了10到20倍，但仍然只有MASP-2裂解C2效率的1%。我们认为C3不是这两种酶的天然底物。MASP-2高速切割C2和C4。为了确定MASP-2催化区域中各个结构域的作用，我们还表达和表征了第二补体控制蛋白模块以及SP模块和SP模块。我们证明了SP结构域可以像整个催化区域一样有效地自动激活和切割C2，而第二个补体控制蛋白模块是有效切割C4所必需的。这种行为与C1s非常相似。每个MASP-1和MASP-2片段与cl -抑制剂反应，完全阻断。酶的酶作用。然而，与α -2巨球蛋白和c1抑制剂的相对反应率表明α -2巨球蛋白可能是MASP-1的重要生理抑制剂。
Mannan-binding lectin-associated serine protease (SP) (MASP)-1 and MASP-2 are modular SP and form complexes with mannan-binding lectin, the recognition molecule of the lectin pathway of the complement system. To characterize the enzymatic properties of these proteases we expressed their catalytic region, the C-terminal three domains, in Escherichia coli. Both enzymes autoactivated and cleaved synthetic oligopeptide substrates. In a competing oligopeptide substrate library assay, MASP-I showed extreme Arg selectivity, whereas MASP-2 exhibited a less restricted, trypsin-like specificity. The enzymatic assays with complement components showed that cleavage of intact C3 by MASP-1 and MASP-2 was detectable, but was only similar to0.1% of the previously reported efficiency of C3bBb, the alternative pathway C3-convertase. Both enzymes cleaved CM 10- to 20-fold faster, but still at only similar to1% of the efficiency of MASP-2 cleavage of C2. We believe that C3 is not the natural substrate of either enzyme. MASP-2 cleaved C2 and C4 at high rates. To determine the role of the individual domains in the catalytic region of MASP-2, the second complement control protein module together with the SP module and the SP module were also expressed and characterized. We demonstrated that the SP domain alone can autoactivate and cleave C2 as efficiently as the entire catalytic region, while the second complement control protein module is necessary for efficient C4 cleavage. This behavior strongly resembles C1s. Each MASP-1 and MASP-2 fragment reacted with Cl-inhibitor, which completely blocked the. enzymatic action of the enzymes. Nevertheless, relative rates of reaction with alpha-2-macroglobulin and C1-inhibitor suggest that alpha-2-macroglobulin may be a significant physiological inhibitor of MASP-1.