László Beinrohr

Complement Protease MASP-1 Activates Human Endothelial Cells: PAR4 Activation Is a Link between Complement and Endothelial Function

Activation of the complement system can induce and enhance inflammatory reaction. Mannose-binding lectin-associated serine protease-1 (MASP-1) is an abundant protease of the complement lectin pathway; however, its physiological function is unclear. In this study, we demonstrate for the first time that MASP-1 is able to activate Ca2+ signaling, NF-κB, and p38 MAPK pathways in cultured HUVECs. Activation was initiated by MASP-1 only; the related protease, MASP-2, had no such effect. The phenomenon was dependent on the proteolytic activity of MASP-1, suggesting modulation of endothelial cell function through a protease-activated receptor (PAR). Using synthetic peptide substrates representing the protease-sensitive regions of PARs, we were able to demonstrate that PAR4 is a target of MASP-1. The presence of functionally active PAR4 in HUVECs was demonstrated using PAR4 agonist peptide and mRNA quantification. Finally, we showed that the amount of membrane-bound intact PAR4 decreases after MASP-1 treatment. All of these results provide a novel link between the regulation of endothelial cell function and complement system activation, and they suggest that MASP-1-induced PAR4 activation could contribute to the development of the inflammatory reaction.

MASP-1, a promiscuous complement protease: structure of its catalytic region reveals the basis of its broad specificity.

Mannose-binding lectin (MBL)-associated serine protease (MASP)-1 is an abundant component of the lectin pathway of complement. The related enzyme, MASP-2 is capable of activating the complement cascade alone. Though the concentration of MASP-1 far exceeds that of MASP-2, only a supporting role of MASP-1 has been identified regarding lectin pathway activation. Several non-complement substrates, like fibrinogen and factor XIII, have also been reported. MASP-1 belongs to the C1r/C1s/MASP family of modular serine proteases; however, its serine protease domain is evolutionary different. We have determined the crystal structure of the catalytic region of active MASP-1 and refined it to 2.55 Å resolution. Unusual features of the structure are an internal salt bridge (similar to one in factor D) between the S1 Asp189 and Arg224, and a very long 60-loop. The functional and evolutionary differences between MASP-1 and the other members of the C1r/C1s/MASP family are reflected in the crystal structure. Structural comparison of the protease domains revealed that the substrate binding groove of MASP-1 is wide and resembles that of trypsin rather than early complement proteases explaining its relaxed specificity. Also, MASP-1’s multifunctional behavior as both a complement and a coagulation enzyme is in accordance with our observation that antithrombin in the presence of heparin is a more potent inhibitor of MASP-1 than C1 inhibitor. Overall, MASP-1 behaves as a promiscuous protease. The structure shows that its substrate binding groove is accessible; however, its reactivity could be modulated by an unusually large 60-loop and an internal salt bridge involving the S1 Asp.

C1 inhibitor serpin domain structure reveals the likely mechanism of heparin potentiation and conformational disease.

C1 inhibitor, a member of the serpin family, is a major down-regulator of inflammatory processes in blood. Genetic deficiency of C1 inhibitor results in hereditary angioedema, a dominantly inheritable, potentially lethal disease. Here we report the first crystal structure of the serpin domain of human C1 inhibitor, representing a previously unreported latent form, which explains functional consequences of several naturally occurring mutations, two of which are discussed in detail. The presented structure displays a novel conformation with a seven-stranded β-sheet A. The unique conformation of the C-terminal six residues suggests its potential role as a barrier in the active-latent transition. 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-proteinase encounter complex. We show how polyanions change the activity of the 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 hereditary angioedema, organ transplant rejection, and heart attack.

Purification, crystallization and preliminary X-ray analysis of human mannose-binding lectin-associated serine protease-1 (MASP-1) catalytic region

MASP-1, a multidomain serine protease, is a component of the lectin pathway of complement. Its precise function is unknown, although it seems to enhance the complement-activating capacity of MASP-2, a related enzyme. MASP-1 has also been implicated as playing a role in blood coagulation. It is mostly found associated with mannose-binding lectin (MBL) and ficolins. Early attempts to crystallize MASP-1 failed because of the inhomogeneity of the purified material. MASP-1 was shown by acidic nondenaturing PAGE to be composed of differently charged species, which are most likely to be the products of deamidation occurring during the refolding procedure. Sequential cation-exchange and anion-exchange chromatography resulted in a homogeneous material, which was successfully crystallized. The best crystal diffracted to 2.55 A resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 68.4, b = 70.4, c = 121.4 A. The crystal structure of MASP-1 may help in understanding the function of this mysterious serine protease.

Inhibition of the Serine Proteases of the Complement System

Proteases play important roles in human physiology and pathology. The complement system is a proteolytic cascade, where serine proteases activate each other by limited proteolysis in a strictly ordered manner. Serine proteases are essential in both the initiation and the amplification of the cascade. Since uncontrolled complement activation contributes to the development of serious disease conditions, inhibition of the complement serine proteases could be an attractive therapeutic approach. In this chapter, we give a brief overview of the major types of natural serine protease inhibitors and their role in controlling the complement cascade. A special emphasis is laid on C1-inhibitor, a natural complement protease inhibitor, which is approved for clinical use in hereditary angioedema (HAE). We also examine the potential of developing artificial complement protease inhibitors. Synthetic small-molecule drugs can be very efficient serine protease inhibitors, but they usually lack sufficient specificity. A promising approach to yield more specific compounds is the alteration of natural protease inhibitors through engineering or directed evolution resulting in new variants with fine-tuned specificity and enhanced affinity.

Cutting Edge: A New Player in the Alternative Complement Pathway, MASP-1 Is Essential for LPS-Induced, but Not for Zymosan-Induced, Alternative Pathway Activation

The complement system is a sophisticated network of proteases. In this article, we describe an unexpected link between two linear activation routes of the complement system: the lectin pathway (LP) and the alternative pathway (AP). Mannose-lectin binding–associated serine protease (MASP)-1 is known to be the initiator protease of the LP. Using a specific and potent inhibitor of MASP-1, SGMI-1, as well as other MASP-1 inhibitors with different mechanisms of action, we demonstrated that, in addition to its functions in the LP, MASP-1 is essential for bacterial LPS-induced AP activation, whereas it has little effect on zymosan-induced AP activation. We have shown that MASP-1 inhibition prevents AP activation, as well as attenuates the already initiated AP activity on the LPS surface. This newly recognized function of MASP-1 can be important for the defense against certain bacterial infections. Our results also emphasize that the mechanism of AP activation depends on the activator surface.

Serpins and the complement system

C1-inhibitor (serpin G1) is a 105 kDa inhibitor which functions as a major antiinflammatory protein in the body. It has its effects via inhibition of the proteases of the complement system and contact system of coagulation, as well as several direct effects mediated by its unique highly glycosylated N-terminal domain. The serpin controls a number of different proteases very efficiently and for some of these the function is augmented by the cofactor, heparin. Here, we describe the preparation of human plasma and recombinant C1-inhibitor and the basic methods required for their characterization, using the complement enzyme C1s as an example of a target enzyme.

C1-inhibitor structure reveals a novel mechanism of heparin potentiation

24 European Crystallographic Meeting, ECM24, Marrakech, 2007 Page s129 Acta Cryst. (2007). A63, s129 motif, while the C-terminal domain shows a typical restriction endonuclease fold. By structural comparison and mutational analysis we showed that the active site of SdaI is located at the C-terminal domain and exhibits a new variation of the canonical PD...(D/E)XK active site motif. Mutational analysis of the residues from the predicted recognition helix of the wHTH motif suggests that SdaI determinants of sequence specificity are clustered at the N-terminal domain. The modular architecture of SdaI, wherein one domain mediates DNA binding while the other domain is predicted to catalyze hydrolysis, distinguishes SdaI from the previously characterized restriction enzymes interacting with symmetric recognition sequences.