Extensive Basal Level Activation of Complement Mannose-Binding Lectin-Associated Serine Protease-3: Kinetic Modeling of Lectin Pathway Activation Provides Possible Mechanism

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.

Be on Target: Strategies of Targeting Alternative and Lectin Pathway Components in Complement-Mediated Diseases

“…Another conclusion of our studies was that active MASP-3 must be present in the blood, since only the activated form of MASP-3 can convert pro-FD to FD. Later, we provided direct evidence for the extensive basal-level activation of MASP-3 in human blood by an unknown mechanism ( 76 ). Finally, the debate seems to have settled.…”

“…It has also no natural inhibitor in the blood; therefore, control of its activity is probably achieved simply by its very restricted substrate specificity. MASP-3 is present in the blood as the mixture of the proenzymic and the activated forms; moreover, the activated form seems to be the more dominant variant ( 76 ). In this aspect, it also differs from MASP-1 and MASP-2, which are proenzymic.…”

Cutting Edge: Role of MASP-3 in the Physiological Activation of Factor D of the Alternative Complement Pathway

“…MASP-1 circulates as an inactive or proenzyme form, and it turns into an active form when the PRM/MASP-1 complexes bind to carbohydrates (2). Recently, Oroszlán et al (14) demonstrated that MASP-3 circulates as an active form in humans and proposed a mechanism for MASP-3 activation by MASP-1. However, it is unknown whether MASP-3 circulates as an active form in the absence of MASP-1 or how MASP-3 is activated in vivo.…”

Complement in the Initiation and Evolution of Rheumatoid Arthritis

“…A recent additional breakthrough has been the finding that MASP-3, which is an alternative spliced form of MASP-1/3 gene, is a positive regulator of the AP of the complement system ( 44 ) and MASP-3 exclusively enables FD maturation ( 45 ). It has been shown that both MASP-1 and MASP-2 can activate MASP-3, but MASP-3 in resting human blood is also present in an active form ( 46 ). In vitro , not in vivo studies, have shown that MASP-1 is essential for bacterial LPS but not Zymosan-induced AP activation ( 47 ), indicating that MASP-1 can regulate a specific AP activation mechanism but not the entire AP.…”