Abstract
Serine proteases (SPs) are typically synthesized as precursors, termed proenzymes or zymogens, and the fully active form is produced via 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.
Highlights
The complement system, as an essential part of the innate immune response, eliminates invading microorganisms and dangerous host cells [1, 2]
Under non-reducing conditions zymogen and active MBL-associated serine protease (MASP)-3 are well separated by SDS-PAGE, on the other hand the mobilities of the two forms are only slightly different, which is favorable for the quantification
For the quantification of the two forms of plasma MASP-3, we had several reasonable assumptions as follows: (I) the mannose-binding lectin (MBL)-Sepharose resin binds zymogen and active MASP-3 with the same affinity, and no further activation occurs during the isolation; (II) the transfer efficiencies during Western blotting are equivalent due to the small difference between the mobility of zymogen and active MASP-3 under non-reducing conditions; and (III) the commercial monoclonal MASP-3-specific antibody recognizes both forms of MASP-3 with the same sensitivity
Summary
The complement system, as an essential part of the innate immune response, eliminates invading microorganisms and dangerous host cells [1, 2]. The complement cascade is composed of more than 30 proteins. Key components of the system are serine proteases (SPs), which typically circulate in bloodstream in the zymogen form until their successive cleavage and activation [3]. Complement activation can be triggered via three different, interconnected routes: the classical, lectin, and alternative pathways, the three routes converge into the common terminal pathway. When the classical or lectin pathways are activated, it results in the formation of the C3 convertase, C4bC2a, composed of the cleaved forms of complement factors C4 and C2 [4]. The alternative pathway serves as an amplification loop, but it can be activated on its own by the “tick-over” mechanism [5]
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