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Segment spanning residues 727-768 of the complement C3 sequence contains a neoantigenic site and accommodates the
J D Becherer1, J Alsenz, I Esparza
1Basel Institute for Immunology, Switzerland.
Insights
Complement system proteins factor H and complement receptor 1 (CR1) bind to distinct sites on C3b. These binding sites are proximal, influencing each other and the binding of other complement proteins like factor B.
Area of Science:
- Immunology
- Complement System Biology
- Protein-Protein Interactions
Background:
- The complement system involves structurally related molecules, including factor H (fH), complement receptor 1 (CR1), and C3b.
- Many complement proteins share functional activities and compete for binding to C3b.
- Factor H is known to interact with multiple sites on C3.
Purpose of the Study:
- To investigate the relationship between factor H and CR1 binding sites on C3b.
- To determine if these binding sites are proximal and influence each other.
Main Methods:
- Utilized C3c and C3d fragments to assess inhibition of factor H binding to C3b.
- Employed a monoclonal antibody (anti-C3c, anti-C3-9) recognizing a neoantigenic epitope on C3b.
- Tested a synthetic peptide (C3(727-768)) and antibodies against it to map binding sites.
Main Results:
- Factor H binding to C3b was inhibited by C3c and C3d fragments, with combined fragments showing augmented inhibition.
- A monoclonal antibody targeting C3b inhibited binding of factor H, CR1, and factor B.
- A synthetic peptide and antibodies against it confirmed proximal binding sites for fH, CR1, and factor B on C3b.
Conclusions:
- Factor H binds to at least two distinct sites on C3b.
- One factor H binding site is within the CR1-binding domain in the C3c fragment.
- Another factor H binding site is located near the CR2-binding site in the C3d fragment.
Abstract:
CR1, CR2, DAF, MCP, factor H, C4bp, factor B, and C3 are members of a family of structurally related molecules, the majority of which belong to the complement system. Several of these molecules also share functional features such as cofactor and decay/dissociation activity and compete with one another in binding to C3b. Since factor H appears to bind to multiple sites in C3, we investigated the relationship between the factor H- and CR1-binding sites in C3b. Factor H binding to C3b is inhibited by either the C3c or C3d fragments, and addition of both fragments together augments this inhibition. One monoclonal anti-C3c antibody, anti-C3-9, which recognizes a neoantigenic epitope expressed upon cleavage to C3 to C3b, inhibited both factor H and CR1 binding to EC3b cells. This monoclonal antibody (MoAb) also inhibited factor B binding to EC3b. Two observations further supported our hypothesis that these molecules bind to proximal sites in C3b. First, a synthetic peptide spanning this region of C3b (C3(727-768)) inhibited factor H binding. Second, antibodies raised against this peptide inhibited binding to CR1, factor H, and factor B to C3b. These data show that H binds to at least two sites in C3b: the site in the C3c fragment is within the identified CR1-binding domain while the site in the C3d fragment surrounds the CR2-binding site.(ABSTRACT TRUNCATED AT 250 WORDS)