Assembly and Enzymatic Properties of the Catalytic Domain of Human Complement Protease C1r

Monique Lacroix,Christine Ebel,József Kardos,József Dobó,Péter Gál,Péter Závodszky,Gérard J Arlaud,Nicole M Thielens

doi:10.1074/jbc.m105688200

Monique Lacroix, Christine Ebel + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m105688200

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Abstract

The catalytic properties of C1r, the protease that mediates activation of the C1 complex of complement, are mediated by its C-terminal region, comprising two complement control protein (CCP) modules followed by a serine protease (SP) domain. Baculovirus-mediated expression was used to produce fragments containing the SP domain and either 2 CCP modules (CCP1/2-SP) or only the second CCP module (CCP2-SP). In each case, the wild-type species and two mutants stabilized in the proenzyme form by mutations at the cleavage site (R446Q) or at the active site serine residue (S637A), were produced. Both wild-type fragments were recovered as two-chain, activated proteases, whereas all mutants retained a single-chain, proenzyme structure, providing the first experimental evidence that C1r activation is an autolytic process. As shown by sedimentation velocity analysis, all CCP1/2-SP fragments were dimers (5.5-5.6 S), and all CCP2-SP fragments were monomers (3.2-3.4 S). Thus, CCP1 is essential to the assembly of the dimer, but formation of a stable dimer is not a prerequisite for self-activation. Activation of the R446Q mutants could be achieved by extrinsic cleavage by thermolysin, which cleaved the CCP2-SP species more efficiently than the CCP1/2-SP species and yielded enzymes with C1s-cleaving activities similar to their active wild-type counterparts. C1r and its activated fragments all cleaved C1s, with relative efficiencies in the order C1r < CCP1/2-SP < CCP2-SP, indicating that CCP1 is not involved in C1s recognition.

Highlights

C11 is the multimolecular protease that triggers the classical pathway of complement, a system that plays a major role in innate immunity against pathogenic microorganisms [2,3,4]
In addition to C1r, a number of proteases, including C1s, feature a complement control protein (CCP) module pair preceding a C-terminal serine protease domain, and it has been proposed that the second CCP module and the protease domain form a rigid assembly in this protein family [13,14,15]
Under non-reducing conditions, the anti-C1r antibody labeled a band of apparent molecular mass 50 or 40 kDa in the supernatants containing the CCP1/2-serine protease (SP) or CCP2-SP fragments, respectively

Summary

Introduction

C11 is the multimolecular protease that triggers the classical pathway of complement, a system that plays a major role in innate immunity against pathogenic microorganisms [2,3,4]. The crystal structure of the C-terminal fragment from the catalytic domain of human C1s, comprising the second complement control protein module and the serine protease domain, has been solved recently, showing that the ellipsoidal CCP2 module is oriented perpendicularly to the surface of the SP domain and that this arrangement is maintained through a rigid module– domain interface [16]. Characterization of the Human C1r Catalytic Domain and active forms of the C1r catalytic region, to gain precise structural and functional information about each form For this purpose, fragments of this region, comprising either one or two CCP modules associated to the serine protease domain (CCP1/ 2-SP and CCP2-SP) were expressed in a baculovirus/insect cell system, either under the wild-type, activable form, or stabilized in the proenzyme form. These data point out the essential role of the CCP1 module in the dimerization process and indicate that formation of a stable dimer is not a prerequisite for activation

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