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J. Biol. Chem., Vol. 280, Issue 51, 41811-41818, December 23, 2005

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Functional Characterization of Complement Proteases C1s/Mannan-binding Lectin-associated Serine Protease-2 (MASP-2) Chimeras Reveals the Higher C4 Recognition Efficacy of the MASP-2 Complement Control Protein Modules^*

From the Laboratoire d'Enzymologie Moléculaire, Institut de Biologie Structurale Jean-Pierre Ebel, 38027 Grenoble Cedex 1, France

C1s and mannan-binding lectin-associated serine protease-2 (MASP-2) are the proteases that trigger the classical and lectin pathways of complement, respectively. They have identical modular architectures and cleave the same substrates, C2 and C4, but show markedly different efficiencies toward C4. Multisite-directed mutagenesis was used to engineer hybrid C1s/MASP-2 molecules where either the complement control protein (CCP) modules or the serine protease (SP) domain of C1s were swapped for their MASP-2 counterparts. The resulting chimeras (C1s_{(MASP-2 CCP1/2)} and C1s_{(MASP-2 SP)}, respectively) were expressed and characterized chemically and functionally. Whereas C1s_{(MASP-2 SP)} was recovered as an active enzyme, C1s_{(MASP-2 CCP1/2)} was produced in a proenzyme form and was susceptible to activation by C1r, indicating that the activation properties of the chimeras were dictated by the nature of their SP domain. Similarly, each activated chimera had an esterolytic activity characteristic of its own SP domain and cleaved C2 with an efficiency comparable with that of their parent C1s and MASP-2 proteases. Both chimeras cleaved C4, but whereas C1s_{(MASP-2 SP)} and C1s had K_m values in the micromolar range, C1s_{(MASP-2 CCP1/2)} and MASP-2 had K_m values in the nanomolar range, resulting in 21–27-fold higher k_cat/K_m ratios. Thus, the higher C4 cleavage efficiency of MASP-2 arises from a higher substrate recognition efficacy of its CCP modules. Remarkably, C1s_{(MASP-2 CCP1/2)} retained C1s ability to associate with C1r and C1q to form a pseudo-C1 complex and to undergo activation within this complex, indicating that the C1s-CCP modules have no direct implication in either function.

Received for publication, April 8, 2005 , and in revised form, October 11, 2005.

^* This work was supported by the Commissariat à l'Energie Atomique, the CNRS, and the Université Joseph Fourier, Grenoble. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 33-4-38-78-49-81; Fax: 33-4-38-78-54-94; E-mail: arlaud{at}ibs.fr.

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