title: MASP-1 and MASP-2 Do Not Activate Pro-Factor D in Resting Human Blood, whereas MASP-3 Is a Potential Activator: Kinetic Analysis Involving Specific MASP-1 and MASP-2 Inhibitors.
abstract: It had been thought that complement factor D (FD) is activated at the site of synthesis, and only FD lacking a propeptide is present in blood. The serum of mannose-binding lectin-associated serine protease (MASP)-1/3(-/-) mice contains pro-FD and has markedly reduced alternative pathway activity. It was suggested that MASP-1 and MASP-3 directly activate pro-FD; however, other experiments contradicted this view. We decided to clarify the involvement of MASPs in pro-FD activation in normal, as opposed to deficient, human plasma and serum. Human pro-FD containing an APPRGR propeptide was produced in insect cells. We measured its activation kinetics using purified active MASP-1, MASP-2, MASP-3, as well as thrombin. We found all these enzymes to be efficient activators, whereas MASP proenzymes lacked such activity. Pro-FD cleavage in serum or plasma was quantified by a novel assay using fluorescently labeled pro-FD. Labeled pro-FD was processed with t1/2s of ∼ 3 and 5 h in serum and plasma, respectively, showing that proteolytic activity capable of activating pro-FD exists in blood even in the absence of active coagulation enzymes. Our previously developed selective MASP-1 and MASP-2 inhibitors did not reduce pro-FD activation at reasonable concentration. In contrast, at very high concentration, the MASP-2 inhibitor, which is also a poor MASP-3 inhibitor, slowed down the activation. When recombinant MASPs were added to plasma, only MASP-3 could reduce the half-life of pro-FD. Combining our quantitative data, MASP-1 and MASP-2 can be ruled out as direct pro-FD activators in resting blood; however, active MASP-3 is a very likely physiological activator.
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, H-1117 Budapest, Hungary;
authors.forename: Gábor
authors.identifier: http://orcid.org/0000-0003-3798-9097
authors.initials: G
authors.lastname: Oroszlán
authors.affiliation: Institute for Ophthalmic Research, University of Tübingen, 72076 Tübingen, Germany;
authors.forename: Elod
authors.identifier: http://orcid.org/0000-0003-1599-3116
authors.initials: E
authors.lastname: Kortvely
authors.affiliation: Department of Biochemistry, Eötvös Loránd University, H-1117 Budapest, Hungary;
authors.forename: Dávid
authors.identifier: http://orcid.org/0000-0002-8758-6387
authors.initials: D
authors.lastname: Szakács
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, H-1117 Budapest, Hungary;
authors.forename: Andrea
authors.initials: A
authors.lastname: Kocsis
authors.affiliation: Institute for Ophthalmic Research, Medical Proteome Center, University of Tübingen, 72074 Tübingen, Germany; and.
authors.forename: Sascha
authors.identifier: http://orcid.org/0000-0003-0714-9409
authors.initials: S
authors.lastname: Dammeier
authors.affiliation: Natural and Medical Sciences Institute at the University of Tübingen, Department of Bioanalytics, 72770 Reutlingen, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Zeck
authors.affiliation: Institute for Ophthalmic Research, University of Tübingen, 72076 Tübingen, Germany; Institute for Ophthalmic Research, Medical Proteome Center, University of Tübingen, 72074 Tübingen, Germany; and.
authors.forename: Marius
authors.initials: M
authors.lastname: Ueffing
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, H-1117 Budapest, Hungary;
authors.forename: Péter
authors.initials: P
authors.lastname: Závodszky
authors.affiliation: Department of Biochemistry, Eötvös Loránd University, H-1117 Budapest, Hungary;
authors.forename: Gábor
authors.initials: G
authors.lastname: Pál
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, H-1117 Budapest, Hungary; dobo.jozsef@ttk.mta.hu gal.peter@ttk.mta.hu.
authors.forename: Péter
authors.initials: P
authors.lastname: Gál
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, H-1117 Budapest, Hungary; dobo.jozsef@ttk.mta.hu gal.peter@ttk.mta.hu.
authors.forename: József
authors.initials: J
authors.lastname: Dobó
mesh_terms: D011416:Complement Factor D / Q000276:immunology; D003170:Complement Pathway, Alternative / Q000276:immunology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D050606:Mannose-Binding Protein-Associated Serine Proteases / Q000276:immunology* / Q000378:metabolism; D013058:Mass Spectrometry
chemical_list: D004791:Enzyme Inhibitors; C084067:MASP1 protein, human; C105296:MASP2 protein, human; D050606:Mannose-Binding Protein-Associated Serine Proteases; C493488:CFD protein, human; D011416:Complement Factor D