title: Differential substrate and inhibitor profiles for human MASP-1 and MASP-2.
abstract: The mannan-binding lectin (MBL)-associated serine proteases (MASPs) circulate in serum complexed with mannan-binding lectin, a recognition molecule of the complement system. MASP-2 cleaves the complement components C4 and C2 to form the C3 convertase C4b2a. A definitive natural substrate for MASP-1 has not yet been described. We investigated the substrate specifities of MASP-1 and MASP-2 using cleavage of fluorescent amide substrates by recombinant and serum-derived MASPs. Recombinant MASP-1 cleaved Phe-Gly-Arg-aminomethylcoumarin (AMC) most rapidly at a rate of 16.8 nmol min(-1) microg(-1) rMASP-1. Recombinant MASP-2 barely cleaved any of 14 substrates used. This provides means of measuring MASP-1 activity in the absence of a known natural substrate. An assay for MBL-bound MASP-1 was established using the substrate Val-Pro-Arg-AMC. Assay of MBL-bound MASP-2 was done by cleavage of a natural protein substrate, C4. The condition of the serum used for the assays is important; simulated aging showed decreased detectable MASP-1 and MASP-2 activity. The inhibitors Z-D-Phe-Pro-methoxy-propylboroglycinepinanediol ester (boroMpg), anti-thrombin III in the presence and absence of heparin, hirudin and C1 inhibitor were tested against the MASPs. C1 inhibitor inhibits both enzymes, but the protease-serpin complex is unusually unstable at alkaline pH. The thrombin inhibitor boroMpg inhibited MASP-1 but not MASP-2 while hirudin did not inhibit either protease. Anti-thrombin III alone was not inhibitory, but in the presence of heparin inhibited both MASP-1 and MASP-2. The ancient origin of MASP-1 and its thrombin-like activity suggests its involvement in a coagulation-based defense mechanism in the early evolution of innate immunity.
authors.affiliation: MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. julia.presanis@green.ox.ac.uk
authors.forename: Julia S
authors.initials: JS
authors.lastname: Presanis
authors.forename: Krishnan
authors.initials: K
authors.lastname: Hajela
authors.forename: Géza
authors.initials: G
authors.lastname: Ambrus
authors.forename: Péter
authors.initials: P
authors.lastname: Gál
authors.forename: Robert B
authors.initials: RB
authors.lastname: Sim
mesh_terms: D004791:Enzyme Inhibitors / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D050606:Mannose-Binding Protein-Associated Serine Proteases; D010455:Peptides / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D010455:Peptides; C084067:MASP1 protein, human; C105296:MASP2 protein, human; D050606:Mannose-Binding Protein-Associated Serine Proteases; C491338:Masp2 protein, rat; D012697:Serine Endopeptidases