The Kinetic Regulation of the Complement Protease MASP-2
thesisposted on 2017-03-01, 23:47 authored by Drentin, Nicole Catherine
Complement is an ancient component of immunity, playing a primary role in defending against pathogens as part of the innate immune system. Mannose-binding lectin-associated serine protease-2 (MASP-2) is a critical component of the lectin complement pathway and cleavage of its substrates C2 and C4 contributes to the formation of a key complement component, the C3 convertase. MASP-2 has been implicated in a number of inflammatory diseases, thus the development of inhibitors of MASP-2 presents a potential route for prevention and/or treatment of these diseases. The structure of MASP-2 in complex with C4 confirmed the location of an accessory binding site (exosite) on the complement control protein (CCP) domains and revealed a potential exosite on the serine protease (SP) domain. This study sought to quantitate the contribution of these MASP-2 exosites to the kinetic mechanism of interaction between the enzyme and C4. This was done by mutating selected residues from the CCP and SP domain exosites and measuring the effects on C4 binding and cleavage efficacy using a variety of methods. The role of the CCP domain exosite in high affinity binding and efficient cleavage of C4 was confirmed and it was shown to play a major role in the initial interaction with C4. A naturally occurring mutation in the CCP2 domain that has been linked to a number of chronic infectious diseases, D371Y, did not impair efficient cleavage of C2 and C4, nor was the activation by MASP-1 or MASP-2 affected. The effect of the mutation in the context of the disease linkages is thus unknown. The potential SP domain exosite was shown to be a functional exosite, with residues R578 and R583 shown to be necessary for efficient C4 binding and cleavage. The SP domain exosite was also shown to play the major role in a secondary conformational change between MASP-2 and C4 required to form a high affinity complex. The serine protease inhibitor (serpin), C1 inhibitor (C1-INH) is known to be the main endogenous inhibitor of MASP-2. C1-INH activity can be enhanced through the binding of polyanionic molecules, such as the naturally occurring glycosaminoglycan, heparin. The polyanionic molecule, polyphosphate (polyP), has recently been found to reduce complement activity and may represent a more potent enhancer than heparin of C1-INH activity. Heparin and long chain polyP (>1000 units) were both shown to bind to MASP-2, with the SP domain exosite shown to play a key role in this process. Platelet-length polyP (70-120 units) was found to have no effect on C1-INH activity against MASP-2 at physiological concentrations and had an inhibitory effect on C1-INH activity at higher concentrations. Heparin had an inhibitory effect on C1-INH activity, even at lower concentrations. Further study into the effects of polyP size on C1-INH activity is required to determine if polyP represents a more potent enhancer of C1-INH activity against MASP-2 than heparin. The results from this study provide information on the regulation of MASP-2 activity that may be used to design potential therapeutics to regulate the enzyme in inflammatory diseases.