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

Véronique Rossi,Florence Teillet,Nicole M Thielens,Isabelle Bally,Gérard J Arlaud

doi:10.1074/jbc.m503813200

Abstract

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 Km values in the micromolar range, C1s(MASP-2 CCP1/2) and MASP-2 had Km values in the nanomolar range, resulting in 21-27-fold higher kcat/Km 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.

Highlights

IntroductionThe lectin pathway can be triggered by three different oligomeric lectins (mannan-binding lectin, L-ficolin and H-ficolin), which exhibit an overall structure reminiscent of that of C1q and share the ability to form a complex with mannan-binding lectin-associated serine protease-2 (MASP-2) (4 – 6)
The lectin pathway can be triggered by three different oligomeric lectins, which exhibit an overall structure reminiscent of that of C1q and share the ability to form a complex with mannan-binding lectin-associated serine protease-2 (MASP-2)2 (4 – 6)
The objective of this study was to evaluate the respective contribution of the SP domain and complement control protein (CCP) modules of C1s and MASP-2 to their catalytic activity, notably with respect to their differential efficiency toward C4

Summary

Introduction

The lectin pathway can be triggered by three different oligomeric lectins (mannan-binding lectin, L-ficolin and H-ficolin), which exhibit an overall structure reminiscent of that of C1q and share the ability to form a complex with mannan-binding lectin-associated serine protease-2 (MASP-2) (4 – 6). The present study was undertaken to assess the relative contribution of the SP domain and the CCP modules of C1s and MASP-2 to their C4 cleaving activity and investigate what determines the higher efficiency of MASP-2 toward this substrate. For this purpose, we took advantage of the fact that C1s and MASP-2 share homologous modular structures to engineer chimeric C1s molecules comprising either the CCP modules or the SP domain of MASP-2. This domain swapping strategy allows us to demonstrate that the higher C4-cleavage efficiency of MASP-2 arises from the higher C4 recognition efficacy of its CCP modules and reveals that the SP domain of each protease is able to cooperate with the CCP modules of the other

Objectives

Results

Conclusion