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.
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.
C1 inhibitor, a member of the serpin family, is a major downregulator 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 -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 serpinproteinase 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.C1 inhibitor belongs to the group of serpin-type proteinase inhibitors in blood plasma. Serpins act as pseudosubstrates of serine and cysteine proteinases (although there are noninhibitory serpins as well) (1). A conformational change is triggered in the serpin upon peptide bond cleavage, which distorts the active site of proteinase and traps it in an inactive, covalently linked serpin-enzyme complex (2, 3). Serpins are vital downregulator components of proteolytic signal amplification cascades. Human C1 inhibitor (C1-inh) 4 is the only inhibitor that acts on early components of the classical pathway (C1r and C1s) and on that of the lectin pathway (MASP-1 and MASP-2) of the complement system (4). Complement mediates host defense against pathogens and altered host cells, but its uncontrolled activation could be harmful. Other physiologically crucial targets include plasma kallikrein and activated factor XII (fXIIa) of the contact activation and activated factor XI (fXIa) of the intrinsic coagulation systems (4). Apart from proteinase inhibition, it was recently discovered that C1-inh binds the central component C3b of complement (5), endotoxins from bacteria (6) and E-, P-selectin adhesion proteins on endothelial cells (7). C1-inh is a single-chain glycoprotein that has atypical two-domain architecture (8) with the C-terminal serpin and the unique N-terminal domains (9). C1-inh is extensively modified post-translationally, bearing six N-linked carbohydrates. Sequencing analysis revealed 14 potential O-glycosylation sites (9), seven of which had been verified by carbohydrate analysis (10). Most of the sugars are present in the N-terminal domain and do not affect proteinase inhibition (11, 12), but affinity to endotoxins and selectins depends on the N-glycans.The importance of C1-inh is underlined by its def...
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