Mutants of complement component C3 cleaved by the C4-specific C1-s protease.
To identify some of the structural features determining specific protease recognition of complement components C3 and C4, we used site-specific mutagenesis to construct mutants of murine C3 that are cleaved by the C4-specific C1-s protease. Insertion of three amino acid residues corresponding to residues at the C1-s cleavage site of human C4 into murine C3 at the analogous C3 convertase cleavage site was adequate to render the mutant protein susceptible to C1-s cleavage. In addition, insertion of C3-specific residues at the same site or introduction of the C4-specific residues as substitutions rather than as an insertion also rendered the site susceptible to cleavage, but with 10- to 50-fold lower efficiencies, and insertion of even a single amino acid residue affected recognition by C1-s. Finally, insertion of amino acid residues into mC3 partially inhibited cleavage by the alternative-pathway C3 convertase, with insertion of C3- or C4-specific residues giving about the same level of inhibition. A simple interpretation of these data is that C1-s cleavage is dependent primarily on steric accessibility and on recognition of specific amino acid residues at the cleavage site, whereas C3 convertase cleavage is dependent primarily on specific interactions distal to the cleavage site, with only relatively weak, non-C3-specific interactions at the cleavage site itself.
Mathias
P
P
Medical Biology Institute, La Jolla, CA 92037.
Carrillo
C J
CJ
Zepf
N E
NE
Cooper
N R
NR
Ogata
R T
RT
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D003173:Complement C1s / Q000378:metabolism*; D003176:Complement C3 / Q000235:genetics / Q000378:metabolism*; D050577:Complement C3-C5 Convertases / Q000378:metabolism; D003181:Complement C4 / Q000378:metabolism; D004247:DNA / Q000235:genetics; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment; D013045:Species Specificity; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D003176:Complement C3; D003181:Complement C4; D004247:DNA; D050577:Complement C3-C5 Convertases; D003173:Complement C1s