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, S. 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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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
Publication statusPublished - Apr 1 2004

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Mannose-Binding Protein-Associated Serine Proteases
Mannose-Binding Lectin
Enzyme Precursors
Peptide Hydrolases
Complement C2
Iodoacetamide
Surface Plasmon Resonance
Ultracentrifugation
Protease Inhibitors
Lectins
Innate Immunity
Edetic Acid
Proteolysis

ASJC Scopus subject areas

  • Immunology

Cite this

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. / Zundel, Stéphanie; Cseh, S.; Lacroix, Monique; Dahl, Mads R.; Matsushita, Misao; Andrieu, Jean Pierre; Schwaeble, Wilhelm J.; Jensenius, Jens C.; Fujita, Teizo; Arlaud, Gérard J.; Thielens, Nicole M.

In: Journal of Immunology, Vol. 172, No. 7, 01.04.2004, p. 4342-4350.

Research output: Contribution to journalArticle

Zundel, S, Cseh, S, Lacroix, M, Dahl, MR, Matsushita, M, Andrieu, JP, Schwaeble, WJ, Jensenius, JC, Fujita, T, Arlaud, GJ & Thielens, NM 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, vol. 172, no. 7, pp. 4342-4350.
Zundel, Stéphanie ; Cseh, S. ; Lacroix, Monique ; Dahl, Mads R. ; Matsushita, Misao ; Andrieu, Jean Pierre ; Schwaeble, Wilhelm J. ; Jensenius, Jens C. ; Fujita, Teizo ; Arlaud, Gérard J. ; Thielens, Nicole M. / 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. In: Journal of Immunology. 2004 ; Vol. 172, No. 7. pp. 4342-4350.
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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.",
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T1 - 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

AU - Zundel, Stéphanie

AU - Cseh, S.

AU - Lacroix, Monique

AU - Dahl, Mads R.

AU - Matsushita, Misao

AU - Andrieu, Jean Pierre

AU - Schwaeble, Wilhelm J.

AU - Jensenius, Jens C.

AU - Fujita, Teizo

AU - Arlaud, Gérard J.

AU - Thielens, Nicole M.

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N2 - 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.

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