Oroszlán Gábor, Dani Ráhel, Szilágyi András, Závodszky Péter, Thiel Steffen, Gál Péter, Dobó József
Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.
Department of Biomedicine, Aarhus University, Aarhus, Denmark.
Front Immunol. 2017 Dec 18;8:1821. doi: 10.3389/fimmu.2017.01821. eCollection 2017.
Serine proteases (SPs) are typically synthesized as precursors, termed proenzymes or zymogens, and the fully active form is produced limited proteolysis by another protease or by autoactivation. The lectin pathway of the complement system is initiated by mannose-binding lectin (MBL)-associated SPs (MASP)-1, and MASP-2, which are known to be present as proenzymes in blood. The third SP of the lectin pathway, MASP-3, was recently shown to be the major activator, and the exclusive "resting blood" activator of profactor D, producing factor D, the initiator protease of the alternative pathway. Because only activated MASP-3 is capable of carrying out this cleavage, it was presumed that a significant fraction of MASP-3 must be present in the active form in resting blood. Here, we aimed to detect active MASP-3 in the blood by a more direct technique and to quantitate the active to zymogen ratio. First, MASPs were partially purified (enriched) from human plasma samples by affinity chromatography using immobilized MBL in the presence of inhibitors. Using this MASP pool, only the zymogen form of MASP-1 was detected by Western blot, whereas over 70% MASP-3 was in an activated form in the same samples. Furthermore, the active to zymogen ratio of MASP-3 showed little individual variation. It is enigmatic how MASP-3, which is not able to autoactivate, is present mostly as an active enzyme, whereas MASP-1, which has a potent autoactivation capability, is predominantly proenzymic in resting blood. In an attempt to explain this phenomenon, we modeled the basal level fluid-phase activation of lectin pathway proteases and their subsequent inactivation by C1 inhibitor and antithrombin using available and newly determined kinetic constants. The model can explain extensive MASP-3 activation only if we assume efficient intracomplex activation of MASP-3 by zymogen MASP-1. On the other hand, the model is in good agreement with the fact that MASP-1 and -2 are predominantly proenzymic and some of them is present in the form of inactive serpin-protease complexes. As an alternative hypothesis, MASP-3 activation by proprotein convertases is also discussed.
丝氨酸蛋白酶（SPs）通常以前体形式合成，称为酶原或前酶，其完全活性形式是通过另一种蛋白酶的有限蛋白水解作用或自身激活产生的。补体系统的凝集素途径由与甘露糖结合凝集素（MBL）相关的丝氨酸蛋白酶（MASP）-1和MASP-2启动，已知它们在血液中以前酶形式存在。凝集素途径的第三种丝氨酸蛋白酶MASP-3最近被证明是主要激活剂，也是前因子D的唯一“静息血液”激活剂，可产生因子D，即替代途径的起始蛋白酶。由于只有活化的MASP-3能够进行这种切割，因此推测在静息血液中一定有相当一部分MASP-3以活性形式存在。在此，我们旨在通过更直接的技术检测血液中的活性MASP-3，并定量活性与酶原的比例。首先，在存在抑制剂的情况下，使用固定化的MBL通过亲和色谱从人血浆样品中部分纯化（富集）MASP。使用这个MASP池，通过蛋白质印迹法仅检测到MASP-1的酶原形式，而在相同样品中超过70%的MASP-3处于活化形式。此外，MASP-3的活性与酶原比例个体差异很小。令人费解的是，不能自身激活的MASP-3大多以活性酶形式存在，而具有强大自身激活能力的MASP-1在静息血液中主要是酶原形式。为了解释这一现象，我们利用现有的和新测定的动力学常数，对凝集素途径蛋白酶的基础水平液相激活及其随后被C1抑制剂和抗凝血酶灭活进行了建模。只有当我们假设酶原MASP-1对MASP-3进行有效的复合物内激活时，该模型才能解释广泛的MASP-3激活。另一方面，该模型与MASP-1和-2主要是酶原形式且其中一些以无活性的丝氨酸蛋白酶抑制剂 - 蛋白酶复合物形式存在这一事实高度一致。作为另一种假设，还讨论了前蛋白转化酶对MASP-3的激活作用。
