Characterization of Recombinant Mannan-Binding Lectin-Associated Serine Protease (MASP)-3 Suggests an Activation Mechanism Different from That of MASP-1 and MASP-21,2

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, Nicole M. Thielens

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72 Citations (Scopus)

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 (S20,w = 6.2 ± 0.1 S) in the presence of Ca2+, and as a monomer (S20,w = 4.6 ± 0.1 S) in EDTA. As shown by surface plasmon resonance spectroscopy, it associated with both MBL (KD = 2.6 nM) and L-ficolin (KD = 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 Ser627→Ala in MASP-1 and Ser618→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.

Original languageEnglish
Pages (from-to)4342-4350
Number of pages9
JournalJournal of Immunology
Volume172
Issue number7
DOIs
Publication statusPublished - Apr 1 2004

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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    Zundel, S., Cseh, S., Lacroix, M., Dahl, M. R., Matsushita, M., Andrieu, J. P., Schwaeble, W. J., Jensenius, J. C., Fujita, T., Arlaud, G. J., & Thielens, N. M. (2004). Characterization of Recombinant Mannan-Binding Lectin-Associated Serine Protease (MASP)-3 Suggests an Activation Mechanism Different from That of MASP-1 and MASP-21,2. Journal of Immunology, 172(7), 4342-4350. https://doi.org/10.4049/jimmunol.172.7.4342