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Correcting the Model of Lectin Pathway Activation Via in Vitro Evolved Monospecific MASP Inhibitors

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Correcting the Model of Lectin Pathway Activation Via in Vitro Evolved Monospecific MASP Inhibitors Correcting the model of lectin pathway activation via in vitro evolved monospecific MASP inhibitors PhD thesis Héja Dávid Structural Biochemistry Program, Doctoral School of Biology, Eötvös Loránd University, Faculty of Natural Sciences Thesis advisor: Dr. Gábor Pál Head of the Program: Dr. Nyitray László Head of the PhD School: Prof. Erdei Anna Department of Biochemistry, Faculty of Natural Sciences of Eötvös Loránd University 2012, Budapest BACKGROUND The complement system is an ancient pillar of the innate immunity. Its major function is to recognize and remove pathogen microbes and dangerously altered self cells. It comprises a network of about 35‐40 soluble and surface‐bound proteins in the blood. A proteolytic cascade provides the central module of the complement system. The cascade can be initiated via three different activation pathways triggered by pathway specific molecular events. The classical pathway is activated by antigen‐antibody complexes. When the body encounters a novel antigen the adaptive immune response requires weeks to develop the specific antibodies. Until this happens the classical pathway is unable to remove the novel intruder. On the contrary, the lectin and the alternative pathways represent an antibody‐ independent mechanism that provides an immediate line of defense against pathogens and altered self cells. The lectin and the alternative pathways have been adapted through natural evolution to be triggered byt ancien molecular patterns. The mannan‐containig cell wall of various bacteria, for example, is such a pattern for the lectin pathway. In the case of the classical and the lectin pathways, target recognition is followed by the activation of recognition molecule‐associated zymogen serine proteases. Activated proteases cleave, and activate the C4 and C2 complement components that results in the formation of the C4b2a C3 convertase. C2a, the protease component of the C3 convertase in turn, cleaves C3. C3b fragment of the cleaved C3 component is deposited on the surface inducing further molecular events. On one hand, the alternative pathway is triggered by C3b deposition, while on the other hand, deposited C3b allows for the cleavage of additional complement proteins. These events result in the assembly of the membrane‐attack complex (C5b‐9). The pore‐forming C5b‐9 itself, without any contribution of immune cells, causes the lysis of the target cell. The soluble and deposited complement fragments, on the other hand, induce various cellular immune responses. The complement system is vital, but its misregulation leads to various diseases. Severe tissue damage is caused by the overreaction of the complement system upon heart attack and stroke. Major contribution of the lectin pathway to this deleterious process was recently verified. Increasing general interest towards the lectin pathway might partly be due to this pathological involvement. Individual roles of the lectin pathway proteases (the MASPs), has been the most debated aspect of the activation mechanism. Active MASP‐2 is 2 unquestionably the major effector of the pathway, as it is the only MASP that can cleave both C4 and C2. This way MASP‐2 produces a C3 convertase, which once formed, will initiate complete complement activation. Moreover, autoactivating ability of zymogen MASP‐2 was confirmed by several in vitro experiments. These properties together seemed to qualify MASP‐2 to be the autonomous activator of the lectin pathway. The role of the other protease MASP‐1 was deduced mostly from experiments that involved either MASP‐1 knockout mice or human sera depleted of MASP‐1 by affinity chromatography. Lectin pathway activity could be clearly observed in the absence of MASP‐1, although it was diminished compared with the activity of the intact serum. It suggested that MASP‐1 has some role in lectin pathway activation, but it also clearly suggested that MASP‐1 is not as important as MASP‐2. Andrea Kocsis in her PhD work developed the first lectin pathway specific inhibitors. The SFMI‐2 peptide selectively inhibited MASP‐2, while SFMI‐1 inhibited both MASP‐1 and MASP‐2 although it was 15 times weaker on MASP‐2. Experiments with these SFMI inhibitors suggested that MASP‐1 is more important than previously thought. However, without a genuine MASP‐1 specific inhibitor, the quantitative contribution and exact mechanistic role of MASP‐1 in the lectin pathway activation could not be assessed. Consequently, the original lectin pathway activation model claiming MASP‐2 to be autonomous activator remained widely accepted. In my PhD work I aimed to elucidate the exact roles of MASP‐1 and MASP‐2 in the lectin pathway activation process. 3 RESEARCH AIMS The present work comprises three subprojects to reach three consecutive experimental goals: 1. To develop high‐affinity monospecific inhibitors against both MASP‐1 and MASP‐2. 2. To clarify individual roles of MASP‐1 and ‐2 in the lectin pathway activation providing a refined activation model. 3. To determine the crystal structure of both MASP enzymes in complex with the novel substrate‐like inhibitors. This work also aimed to provide lead molecules for a far reaching goal of the future, namely to lessen the tissue damage caused by the overreaction of the lectin pathway after heart attack and stroke. 4 METHODS Directed protein evolution Phage display was applied to evolve the monospecific MASP inhibitors. Basic recombinant DNA techniques Kunkel mutagenesis was applied to generate the inhibitor‐phage library. Amino acid sequence of the selected variants was deduced by DNA sequencing of the coding gene. PCR was applied to generate DNS cassettes to assembly a novel expression vector. PCR‐based mutagenesis was performed to clone the inhibitor variants into the novel vector. Expression and purification of the recombinant inhibitor variants I optimized a novel bacterial expression system to produce recombinant inhibitor variants in native form in E. coli BL21 Star cells. Protein was purified by crude fractionation method followed by several high performance liquid chromatography methods. Functional assays Equilibrium inhibitory constants were determined to assess the efficiency of the MASP inhibitor variants. Lectin pathway activation mechanism was explored using various complement deposition assays in ELISA formats. Structural studies X‐ray crystallography was applied to determine the SGMI/MASP complex structures. 5 RESULTS 1. SGPI‐2 reactive loop optimization to MASP binding resulted in the high affinity MASP‐1 and MASP‐2 inhibitors SGMI‐1 ad SGMI‐2, respectively. 2. SGMIs are monospecific inhibitors, in addition to their target enzymes they do not inhibit other complement or blood coagulation proteases. 3. As expected, MASP‐ 2 specific SGMI‐2 effectively inhibited C5b‐9 assembly, as well as, C3b and C4b deposition. More importantly and rather unexpectedly, the MASP‐1 specific SGMI‐1 did exactly the same. Inhibition of complement component deposition by SGMI‐1 could be observed only when SGMI‐1 was added before the activation of zymogen MASPs in the serum. MASP‐1 was totally inefficient if added to serum containing already activated MASP enzymes. 4. Under physiological conditions: in normal human serum or in whole blood, both SGMIs inhibited the lectin pathway. 5. Both SGMIs inhibited the lectin pathway regardless whether it was activated through MBL‐MASP or ficolin‐MASP complexes. 6. Both SGMI‐1/MASP‐1 (3,2 Å) and SGMI‐2/MASP‐2 (1,3 Å) complex structures were determined. Based on the results presented here a novel, corrected model of lectin pathway activation was constructed. This new model is in excellent agreement with others previous and my new results. 6 CONCLUSIONS Corrected model of the lectin pathway activation The results presented here led to the following, fundamentally new conclusions regarding the mechanism of lectin pathway activation: 1. MASP‐1 directly and exclusively activates zymogen MASP‐2 in normal human serum and in whole blood. 2. To this end, under physiological conditions, the inherent autoactivating capacity of zymogen MASP‐2 does not manifest. 3. Majority of the C2 cleavage leading to C3 convertase formation is contributed by MASP‐ 1. 4. The above conclusions are equally valid regardless whether the activation involves MBL/MASP or fikolin/MASP complexes. Activation of zymogen MASP‐2 by MASP‐1 in normal human serum requires close proximity of these components (e.g., inside the recognition complex or on the surface of the pathogen). As the concentration of MASP‐1 is about 20 times higher than that of MASP‐2, every zymogen MASP‐2 molecule should be surrounded by multiple copies of MASP‐1 molecules on the activator surface. This could explain our first conclusion, the efficient MASP‐1–mediated MASP‐2 activation in human blood. The same setup could also explain our second conclusion. If the more abundant MASP‐1 molecules topologically separate MASP‐2 zymogens from one another, inhibition of MASP‐1 should prevent autoactivation of MASP‐2. This clearly shows up when MASP‐1 is inhibited in situ as in this case zymogen MASP‐2 molecules are separated from each other by inactivated proteases. Nevertheless, let me consider what kind of functional consequences this simple model would predict for the entire removal of MASP‐1 from the system as opposed to its in situ inhibition. A complete removal of MASP‐1 would override the naturally isolated state of the MASP‐2 zymogens allowing their autoactivation. This way the lectin pathway could activate, and the central role of MASP‐1 would remain hidden. Note, however, that because MASP‐2 is 20‐fold less abundant than MASP‐1 and because MASP‐2 is a 20‐fold less efficient MASP‐2 activator than MASP‐1, a significantly diminished pathway activation would be expected. In 7 fact, as listed below, exactly these phenomena were observed previously when MASP‐1 was entirely removed from the serum. It was already suggested that MASP‐1 facilitates the activation of MASP‐2 but it was also stated that MASP‐1 is not essential because, in the absence of MASP‐1, MASP‐2 can initiate the lectin pathway.
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