Functional role of the linker between the complement control protein modules of complement protease C1s.
C1s is the modular serine protease responsible for cleavage of C4 and C2, the protein substrates of the first component of C (C1). Its catalytic domain comprises two complement control protein (CCP) modules connected by a four-residue linker Gln340-Pro-Val-Asp343 and a serine protease domain. To assess the functional role of the linker, a series of mutations were performed at positions 340-343 of human C1s, and the resulting mutants were produced using a baculovirus-mediated expression system and characterized functionally. All mutants were secreted in a proenzyme form and had a mass of 77,203-77,716 Da comparable to that of wild-type C1s, except Q340E, which had a mass of 82,008 Da, due to overglycosylation at Asn391. None of the mutations significantly altered C1s ability to assemble with C1r and C1q within C1. Whereas the other mutations had no effect on C1s activation, the Q340E mutant was totally resistant to C1r-mediated activation, both in the fluid phase and within the C1 complex. Once activated, all mutants cleaved C2 with an efficiency comparable to that of wild-type C1s. In contrast, most of the mutations resulted in a decreased C4-cleaving activity, with particularly pronounced inhibitory effects for point mutants Q340K, P341I, V342K, and D343N. Comparable effects were observed when the C4-cleaving activity of the mutants was measured inside C1. Thus, flexibility of the C1s CCP1-CCP2 linker plays no significant role in C1 assembly or C1s activation by C1r inside C1 but plays a critical role in C4 cleavage by adjusting positioning of this substrate for optimal cleavage by the C1s active site.
Bally
Isabelle
I
Laboratoire d'Enzymologie Moléculaire, Institut de Biologie Structurale Jean-Pierre Ebel, Grenoble, France.
Rossi
Véronique
V
Thielens
Nicole M
NM
Gaboriaud
Christine
C
Arlaud
Gérard J
GJ
D020816:Amino Acid Motifs; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001216:Asparagine / Q000378:metabolism; D016367:Baculoviridae; D003173:Complement C1s / Q000235:genetics / Q000502:physiology*; D003175:Complement C2 / Q000378:metabolism; D003181:Complement C4 / Q000378:metabolism; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D018411:Spodoptera
D003175:Complement C2; D003181:Complement C4; D001216:Asparagine; D003173:Complement C1s