Baculovirus-mediated Expression of Truncated Modular Fragments from the Catalytic Region of Human Complement Serine Protease C1s: EVIDENCE FOR THE INVOLVEMENT OF BOTH COMPLEMENT CONTROL PROTEIN MODULES IN THE RECOGNITION OF THE C4 PROTEIN SUBSTRATE

Véronique Rossi,Isabelle Bally,Nicole M Thielens,Alfred F Esser,Gérard J Arlaud

doi:10.1074/jbc.273.2.1232

Abstract

C1s is the modular serine protease responsible for cleavage of C4 and C2, the protein substrates of the first component of complement. Its catalytic region (gamma-B) comprises two complement control protein (CCP) modules, a short activation peptide (ap), and a serine protease domain (SP). A baculovirus-mediated expression system was used to produce recombinant truncated fragments from this region, deleted either from the first CCP module (CCP2-ap-SP) or from both CCP modules (ap-SP). The aglycosylated fragment CCP2-ap-SPag was also expressed by using tunicamycin. The fragments were produced at yields of 0.6-3 mg/liter of culture, isolated, and characterized chemically and then tested functionally by comparison with intact C1s and its proteolytic gamma-B fragment. All recombinant fragments were expressed in a proenzyme form and cleaved by C1r to generate active enzymes expressing esterolytic activity and reactivity toward C1 inhibitor comparable to those of intact C1s. Likewise, the activated fragments gamma-B, CCP2-ap-SP, and ap-SP retained C1s ability to cleave C2 in the fluid phase. In contrast, whereas fragment gamma-B cleaved C4 as efficiently as C1s, the C4-cleaving activity of CCP2-ap-SP was greatly reduced (about 70-fold) and that of ap-SP was abolished. It is concluded that C4 cleavage involves substrate recognition sites located in both CCP modules of C1s, whereas C2 cleavage is affected mainly by the serine protease domain. Evidence is also provided that the carbohydrate moiety linked to the second CCP module of C1s has no significant effect on catalytic activity.

Highlights

C1s is the modular serine protease responsible for cleavage of C4 and C2, the protein substrates of the first component of complement
We have provided evidence that the second complement control protein (CCP) module of C1 ̄ s is closely associated with the serine protease domain [12]
The observed slight decrease in the secretion yield of the aglycosylated fragment CCP2-ap-SPag is probably not linked to a reduced stability of the protein in solution due to the lack of the oligosaccharide chain but rather is an indirect consequence of the deleterious effect of tunicamycin on insect cells, which is known to affect secretion of some proteins [37]

Summary

Introduction

C1s is the modular serine protease responsible for cleavage of C4 and C2, the protein substrates of the first component of complement. Human C1s is synthesized as a 673-residue single chain zymogen which, upon activation by C1 ̄ r, is cleaved between Arg422 and Ile423 to yield two disulfide-linked polypeptides, the N-terminal A chain comprising a series of five protein modules and the serine protease B domain (6 – 8). Limited proteolysis of C1 ̄ s with plasmin yields a C-terminal fragment ␥-B, comprising two contiguous complement control protein (CCP) modules, a 15-residue intermediary segment homologous to the activation peptide in chymotrypsinogen, and the serine protease domain [9]. Based on chemical cross-linking and three-dimensional homology modeling, it was shown recently that this module closely interacts with the serine protease domain on the side opposite to both the active site and the Arg422-Ile423 bond cleaved upon activation [12]. N-terminal CCP module and both CCP modules, respectively; CCP2-apSPag, aglycosylated fragment CCP2-ap-SP; CCP, complement control protein; MALDI, matrix-assisted laser desorption ionization; PAGE, polyacrylamide gel electrophoresis

Methods

Results

Conclusion