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Serine proteases of the immune system: structure, function and physiological relevance Print E-mail
The complement system plays a major role in innate immunity. It is capable of recognizing and eliminating invading pathogens and altered host cells through different activation routes. Our research activity is focused on the serine proteases of the complement system, and on their physiological inhibitor: C1-inhibitor. We use molecular biology, enzymology, X-ray crystallography and other physico-chemical methods to characterize the individual serine proteases and the C1-inhibitor, in order to reveal the mechanism of their action and control and to estimate their physiological significance in health and disease. 

The complement system is an important mediator of the innate immune defence. The function of complement is to recognize, then opsonize or lyse invading microorganisms and altered host cells. The system consists of at least 30 protein components, both soluble and membrane bound ones. The activation of the system leads to the formation of enzyme complexes called C3 convertases and finally to the assembly of the membrane-attack complex, which causes cell lysis.

Figure 1. The complement system.
Fig 1.
Some proteolysis products (e.g. anaphylatoxins) trigger inflammatory reactions by activating the cellular elements of the immune system (e.g. leukocytes, endothelial cells). An intact complement system is essential in maintaining the internal inflammatory homeostasis, however its uncontrolled activation results in self-tissue damage and contributes to development of different diseases.

The major objective of our research is to characterize the activation and the control of the complement system, in order to prevent pathological activation. The central components of the complement system are serine proteases, which are present in zymogenic forms and activate each other in a cascade-like manner (Fig. 1.).These proteases have some unusual characteristics, which distinguish them from other serine protease enzymes. They have extremely restricted substrate specificity, i. e. they cleave only one or two natural substrates. These proteases do not act independently, but usually form large supramolecular complexes with other serine proteases and nonenzymatic proteins (e. g. recognition subunits, like C1q, MBL, ficolins). These enzymes have multidomain structure, which is similar to the enzymes of the other cascade systems found in the blood (e.g. blood clotting, fibrinolysis).

The complement system can be activated by three different routes.

  • The classical pathway is associated with the adaptive immune response, since it can be activated by immune complexes.

  • The lectin pathway

  • The alternative pathway

The lectin and the alternative pathways however are triggered directly by the surfaces of the pathogens. The first components of the classical and lectin pathways consist of recognition subunits (C1q and mannose-binding lectin=MBL) and closely related serine proteases (C1r, C1s and MASP-1, MASP-2, MASP-3) (Fig. 2.).

Figure 2. The domain structure
Fig 2.

These enzymes share similar domain organization, but have different enzymatic properties. In order to characterize these proteases, we expressed them in recombinant form using heterologous expression systems.

We expressed the entire molecules and smaller fragments, as well. In the case of the enzymes of the classical pathway one of our major goals is to reveal the role of the individual domains in the structural and functional properties of the proteases. We found that the serine protease (SP) domain of C1r alone is capable of autoactivation and can cleave C1s, the natural substrate of C1r. The complement control protein (CCP) modules however, which associate with the serine protease domain, increase significantly the proteolytic efficiency of the serine protease against C1s [1]. A similar role of the CCP modules was detected in the case of MASP-2, the C1s-like protease of the lectin pathway. According to our hypothesis the CCP modules bind the substrate and present it to the SP domain. We determined the 3D structure of the active catalytic fragment (CCP1-CCP2-SP) of human C1r by X-ray diffraction. In the crystal lattice there is an enzyme-product relationship between the neighboring C1r molecules, and the predicted CCP2-SP intermolecular interaction can be seen (Fig. 3.).

Figeure 3.
Fig. 3
Using our enzymological and structural data we built an improved model for the autoactivation of C1r in the C1 complex [2]. The enzymes of the lectin pathway, the MASPs have been recently discovered and are much less characterized than C1r and C1s. We examined the substrate specificities of MASP-1 and MASP-2. We found that MASP-2 is a C1s-like enzyme, since it can cleave C2 and C4, the subcomponents of the C3 convertase. MASP-1 however cannot cleave any complement components efficiently, but we showed that it can cleave fibrinogen and fXIII, the substrates of thrombin [3,4]. This phenomenon represents and ancient form of innate immunity, which has not been studied in vertebrates yet. We hypothesize that there was an ancient proteolytic cascade system in the early stage of evolution, which fulfilled immunological roles. This ancient cascade system was the ancestor of the modern complement and blood coagulation cascades. The MBL-MASP-1 complex may represent a key linkage in this evolutionary process [5]. We determined the crystal structure of the active and zymogen forms of the catalytic fragment of MASP-2 [6,7]. We characterized the enzymatic properties of both forms.
Figure 4.
Fig. 4
We found that zymogen (one-chain) MASP-2 has significant proteolytic activity on its protein substrates. This substrate induction-like phenomenon could be responsible for the autoactivation, as well. Based on the experimental and structural data we built a model that provides structural and mechanistic insight into the autoactivation of this multidomain serine protease (Fig. 4.). C1-inhibitor, a member of the serpin family, is a major downregulator of inflammatory processes in blood. It is the only inhibitor that acts on early components of the classical (C1r and C1s) and on that of the lectin pathway (MASP-1 and MASP-2) of the complement system. Other physiologically crucial targets include plasma kallikrein and activated factor XII (XIIa) of the contact activation and activated factor XI (fXIa) of the intrinsic coagulation system. Importance of C1-inhibitor is underlined by its deficiency resulting in hereditary angioedema (HAE), a potentially lethal disease if untreated. We solved the first crystal structure of the serpin domain of human C1-inhibitor [8]. The structure displays a novel latent conformation with a 7-stranded β-sheet A (Fig. 5.).
Figure 5.
Fig. 5
On the basis of surface charge pattern, heparin affinity measurements and docking of a heparin disaccharide, a heparin binding site is proposed in the contact area of the serpin-protease encounter complex. We showed how polyanions change activity of C1-inhibitor by a novel “sandwich” mechanism, explaining earlier reaction kinetic and mutagenesis studies. These results may help to improve therapeutic C1-inhibitor preparations used in the treatment of HAE, organ transplant rejection and heart att

 

 

References:

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    The role of the individual domains in the structure and function of the catalytic region of a modular serine protease, C1r.
    J Immunol 167, 5202-8 [PubMed]
  2. Kardos J, Harmat V, Palló A, Barabás O, Szilágyi K, Gráf L, Náray-Szabó G, Goto Y, Závodszky P and Gál P (2008)
    Revisiting the mechanism of the autoactivation of the complement protease C1r in the C1 complex: structure of the active catalytic region of C1r.
    Mol Immunol 45, 1752-60 [PubMed]
  3. Ambrus G, Gál P, Kojima M, Szilágyi K, Balczer J, Antal J, Gráf L, Laich A, Moffatt BE, Schwaeble W, Sim RB and Závodszky P (2003)
    Natural substrates and inhibitors of mannan-binding lectin-associated serine protease-1 and -2: a study on recombinant catalytic fragments.
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    The biological functions of MBL-associated serine proteases (MASPs).
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    Serine proteases of the classical and lectin pathways: similarities and differences.
    Immunobiology 212, 267-77 [PubMed]
  6. Harmat V, Gál P, Kardos J, Szilágyi K, Ambrus G, Végh B, Náray-Szabó G and Závodszky P (2004)
    The structure of MBL-associated serine protease-2 reveals that identical substrate specificities of C1s and MASP-2 are realized through different sets of enzyme-substrate interactions.
    J Mol Biol 342, 1533-46 [PubMed]
  7. Gál P, Harmat V, Kocsis A, Bián T, Barna L, Ambrus G, Végh B, Balczer J, Sim RB, Náray-Szabó G and Závodszky P (2005)
    A true autoactivating enzyme. Structural insight into mannose-binding lectin-associated serine protease-2 activations.
    J Biol Chem 280, 33435-44 [PubMed]
  8. Beinrohr L, Harmat V, Dobó J, Lörincz Z, Gál P and Závodszky P (2007)
    C1 inhibitor serpin domain structure reveals the likely mechanism of heparin potentiation and conformational disease.
    J Biol Chem 282, 21100-9 [PubMed]