Aarhus University Seal / Aarhus Universitets segl

Jens Christian Jensenius

Characterization of recombinant mannan-binding lectin-associated serine protease (MASP)-3 suggests an activation mechanism different from that of MASP-1 and MASP-2

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

  • Stéphanie Zundel
  • ,
  • Sandor Cseh
  • ,
  • Monique Lacroix
  • ,
  • Mads R Dahl
  • Misao Matsushita
  • ,
  • Jean-Pierre Andrieu
  • ,
  • Wilhelm J Schwaeble
  • ,
  • Jens Christian Jensenius
  • Teizo Fujita
  • ,
  • Gérard J Arlaud
  • ,
  • Nicole M Thielens
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.
Original languageEnglish
JournalJournal of Immunology
Volume172
Issue7
Pages (from-to)4342-50
Number of pages9
ISSN0022-1767
Publication statusPublished - 2004

    Research areas

  • Alanine, Amino Acid Substitution, Animals, Baculoviridae, Carrier Proteins, Complement Activation, Complement C1 Inactivator Proteins, Enzyme Activation, Humans, Lectins, Mannose-Binding Lectin, Mannose-Binding Lectins, Mannose-Binding Protein-Associated Serine Proteases, Recombinant Proteins, Serine, Serine Endopeptidases, Serpins, Spodoptera

See relations at Aarhus University Citationformats

ID: 43921170