Mutants of complement component C3 cleaved by the C4-specific C1-s protease.

Abstract

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 Cis protease. Insertion of three amino acid residues corresponding to residues at the Cis cleavage site of human C4 into murine C3 at the analogous C3 convertase cleavage site was adequate to render the mutant protein susceptible to Cis 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 Cis. 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 Cis 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.Complement components C3, C4, and C5 constitute a family of proteins that play central roles in complement activation and regulation. They share similar subunit structures, biosynthetic pathways, gene structures, and about 25% amino acid sequence identity; nevertheless they are quite distinct functionally and biochemically (reviewed in refs. 1 and 2).For example, while C3, C4, and C5 are activated by proteolytic cleavage at structurally analogous sites in their a subunits, with accompanying release of anaphylatoxins C3a, C4a, and C5a, proteolysis is carried out by three distinct proteases: C3 convertase, Cis, and C5 convertase, respectively. The two convertases are complex proteases assembled from multiple complement components; they share the same catalytic subunit (structurally and evolutionarily related C2a and Bb in the classical and alternative pathways, respectively) but differ in their noncatalytic subunits. The CIs protease, on the other hand, is a subunit of component C1 and is structurally distinct from C2 and B.What is the molecular basis for specific recognition of C3, C4, and C5? For all three proteins, cleavage occurs immediately after the sequence -Leu-Xaa-Arg-, where Xaa is Ala, Gln, or Gly. These residues are doubtless necessary for cleavage (3), but because of their similarity are unlikely to determine cleavage specificity. In aligning the amino acid sequences of mouse and human C3 (4, 5), C4 (6-8), and C5 (9, 10) near their respective proteolytic activation sites, we noticed that the best alignment requires the introduction of a gap, a few amino acid residues in length, into both the C3 and C5 sequences just downstream of the cleavage ...

Identification of Residues within the 727–767 Segment of Human Complement Component C3 Important for Its Interaction with Factor H and with Complement Receptor 1 (CR1, CD35)

“…Collectively, these data suggest that the mutations have not introduced conformational alterations that extend beyond the target segment. The partial defect observed on cleavability by the classical pathway C3 convertase in virtually all of the mutants is in keeping with a previous study (50) showing that susceptibility of mouse C3 to cleavage by the alternative pathway C3 convertase was quite sensitive to sequence alterations in the segment immediately downstream of the cleavage site.…”

Distal Recognition Site for Classical Pathway Convertase Located in the C345C/Netrin Module of Complement Component C5

“…In previous work we suggested that recognition of C3 and C5 by their respective convertases requires interactions at sites distal to the activation cleavage sites in these proteins (4,5). To find these putative distal sites, we developed a strategy focused on indels in the C345 protein family.…”

“…However, in previous work, we found that the susceptibility of C4 to C1s activation can be almost completely eliminated by deletion of three residues near the activation site (3) and, conversely, that C3 and C5 can both be rendered susceptible to C1s by substituting C4-like sequences near their activation sites (4,5). These results indicated that recognition by C1s primarily involves interactions with residues flanking the cleavage site.…”

Similarities between trunk and spätzle, putative extracellular ligands specifying body pattern in Drosophila.

“…In summary, the molecular analysis of the trk mutant alleles supports the functional importance of the domains found the pre- (Fritzinger et al 1992) (m) mouse (Wetsel et al 1984); (r) rat (Misumi et al 1990); and (h) human (De Brujin et al 1985)l. Cleavage of C3 to generate C3a and C3b (arrow) appears to depend on three distinct sub-domains designated A, B, and S. It has been proposed that region A contains the cleavage site, region B constitutes a binding site for the protease, and region S functions as a spacer, the length and acidity of which appear important for the normal cleavage event to occur (Mathias et al 1992). All three of these subdomains appear to be conserved in the trk protein.…”