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Characterization of Recombinant Mannan-Binding Lectin-Associated Serine Protease (MASP)-3 Suggests an Activation Mechanism Different from That of MASP-1 and MASP-2^1,2

Stéphanie Zundel^*, Sandor Cseh^*, Monique Lacroix^*, Mads R. Dahl^,, Misao Matsushita, Jean-Pierre Andrieu^*, Wilhelm J. Schwaeble, Jens C. Jensenius, Teizo Fujita^¶, Gérard J. Arlaud^* and Nicole M. Thielens^3,*

^* Laboratoire d’Enzymologie Moléculaire, Institut de Biologie Structurale Jean-Pierre Ebel (Commissariat à l’Energie Atomique-Centre National de la Recherche Scientifique-Université Joseph Fourier), Grenoble, France; Department of Infection, Immunity, and Inflammation, University of Leicester, Leicester, England; Department of Medical Microbiology and Immunology, University of Aarhus, Aarhus, Denmark; Department of Applied Biochemisty and Institute of Glycobiology, Tokai University, Hiratsuka, Japan; and ^¶ Department of Biochemistry, Fukushima Medical University School of Medicine, Fukushima, Japan

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 Ca²⁺, 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°C, resulting from cleavage at the Arg⁴³⁰-Ile⁴³¹ 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 Ser⁶⁴⁵ of MASP-3, indicating extrinsic proteolysis. In contrast, the corresponding mutations Ser⁶²⁷Ala in MASP-1 and Ser⁶¹⁸Ala 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.

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