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.
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.
Rossi
V
V
Laboratoire d'Enzymologie Moléculaire, Institut de Biologie Structurale Jean-Pierre Ebel (CEA-CNRS), 41 avenue des Martyrs, 38027 Grenoble Cedex 1, France.
Bally
I
I
Thielens
N M
NM
Esser
A F
AF
Arlaud
G J
GJ
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002384:Catalysis; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D003173:Complement C1s / Q000235:genetics / Q000378:metabolism*; D003181:Complement C4 / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D010446:Peptide Fragments / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018411:Spodoptera; D013379:Substrate Specificity
D003181:Complement C4; D010446:Peptide Fragments; D011994:Recombinant Proteins; D003173:Complement C1s