Characterization of recombinant mannan-binding lectin-associated serine protease (MASP)-3 suggests an activation mechanism different from that of MASP-1 and MASP-2. Journal of immunology (Baltimore, Md. : 1950) [J Immunol] Journal article | | 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. | | Author(s) | Zundel S, Cseh S, Lacroix M, Dahl MR, Matsushita M, Andrieu JP, Schwaeble WJ, Jensenius JC, Fujita T, Arlaud GJ, Thielens NM | | Institution | Laboratoire d'Enzymologie Moléculaire, Institut de Biologie Structurale Jean-Pierre Ebel, Grenoble, France. | | Source | J Immunol 2004 Apr 1; 172(7):4342-50. | | MeSH | Alanine
Amino Acid Substitution
Animals
Baculoviridae
Carrier Proteins
Comparative Study
Complement Activation
Complement C1 Inactivator Proteins
Enzyme Activation
Humans
Lectins
Mannose-Binding Lectin
Mannose-Binding Lectins
Mannose-Binding Protein-Associated Serine Proteases
Recombinant Proteins
Research Support, Non-U.S. Gov't
Serine
Serine Endopeptidases
Serpins
Spodoptera
| | 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. | | Language | eng | | Pub Type(s) | Journal Article
| | PubMed ID | 15034049 |
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