title: Characterization of recombinant mannan-binding lectin-associated serine protease (MASP)-3 suggests an activation mechanism different from that of MASP-1 and MASP-2.
abstract: Mannan-binding lectin (MBL)-associated serine proteases (MASP-1, -2, and -3) are homologous modular proteases that each associate with MBL and L- and H-ficolins, which are oligomeric serum lectins involved in innate immunity. To investigate its physicochemical, interaction, and enzymatic properties, human MASP-3 was expressed in insect cells. Ultracentrifugation analysis indicated that rMASP-3 sedimented as a homodimer (s(20,w) = 6.2 +/- 0.1 S) in the presence of Ca(2+), and as a monomer (s(20,w) = 4.6 +/- 0.1 S) in EDTA. As shown by surface plasmon resonance spectroscopy, it associated with both MBL (K(D) = 2.6 nM) and L-ficolin (K(D) = 7.2 nM). The protease was produced in a single-chain, proenzyme form, but underwent slow activation upon prolonged storage at 4 degrees C, resulting from cleavage at the Arg(430)-Ile(431) activation site. Activation was prevented in the presence of protease inhibitors iodoacetamide and 1,10-phenanthroline but was not abolished upon substitution of Ala for the active site Ser(645) of MASP-3, indicating extrinsic proteolysis. In contrast, the corresponding mutations Ser(627)-->Ala in MASP-1 and Ser(618)-->Ala in MASP-2 stabilized the latter in their proenzyme form. Likewise, the MASP-1 and MASP-2 mutants were each activated by their active counterparts, but MASP-3 S645A was not. Activated MASP-3 did not react with C1 inhibitor; had no activity on complement proteins C2, C4, and C3; and only cleaved the N-carboxybenzyloxyglycine-L-arginine thiobenzyl ester substrate to a significant extent. Based on these observations, it is postulated that MASP-3 activation and control involve mechanisms that are different from those of MASP-1 and -2.
authors.affiliation: Laboratoire d'Enzymologie Moléculaire, Institut de Biologie Structurale Jean-Pierre Ebel, Grenoble, France.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Zundel
authors.forename: Sandor
authors.initials: S
authors.lastname: Cseh
authors.forename: Monique
authors.initials: M
authors.lastname: Lacroix
authors.forename: Mads R
authors.initials: MR
authors.lastname: Dahl
authors.forename: Misao
authors.initials: M
authors.lastname: Matsushita
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Andrieu
authors.forename: Wilhelm J
authors.initials: WJ
authors.lastname: Schwaeble
authors.forename: Jens C
authors.initials: JC
authors.lastname: Jensenius
authors.forename: Teizo
authors.initials: T
authors.lastname: Fujita
authors.forename: Gérard J
authors.initials: GJ
authors.lastname: Arlaud
authors.forename: Nicole M
authors.initials: NM
authors.lastname: Thielens
mesh_terms: D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002352:Carrier Proteins / Q000378:metabolism; D003167:Complement Activation; D003174:Complement C1 Inactivator Proteins; D050718:Complement C1 Inhibitor Protein; D004789:Enzyme Activation; D006801:Humans; D037102:Lectins / Q000378:metabolism; D037601:Mannose-Binding Lectin / Q000378:metabolism*; D037241:Mannose-Binding Lectins; D050606:Mannose-Binding Protein-Associated Serine Proteases; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D012694:Serine / Q000235:genetics; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015843:Serpins / Q000378:metabolism; D018411:Spodoptera / Q000235:genetics; D000097604:Ficolins
chemical_list: D000409:Alanine; D002352:Carrier Proteins; D003174:Complement C1 Inactivator Proteins; D050718:Complement C1 Inhibitor Protein; D037102:Lectins; D037601:Mannose-Binding Lectin; D037241:Mannose-Binding Lectins; D050606:Mannose-Binding Protein-Associated Serine Proteases; D011994:Recombinant Proteins; D012694:Serine; D012697:Serine Endopeptidases; D015843:Serpins; D000097604:Ficolins; C469952:SERPING1 protein, human; C084067:MASP1 protein, human; C105296:MASP2 protein, human