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Mutational analysis of the active site and antibody epitopes of the complement-inhibitory glycoprotein, CD59
D L Bodian1, S J Davis, B P Morgan
1Laboratory of Molecular Biophysics, Oxford, United Kingdom.
Insights
CD59 protects cells from complement-mediated damage by preventing membrane attack complex assembly. This study identifies key residues on CD59
Area of Science:
- Immunology and Molecular Biology
- Complement System Regulation
- Cell Surface Molecule Function
Background:
- CD59 is a crucial regulator within the Ly-6 superfamily, protecting host cells from the complement system's membrane attack complex (MAC).
- The precise mechanism by which CD59 inhibits MAC assembly, likely through interaction with C8 and C9 proteins, remains incompletely understood.
Purpose of the Study:
- To systematically identify the specific regions and amino acid residues of CD59 essential for its complement-inhibitory activity.
- To elucidate the structure-function relationship of CD59's active site through a structure-based mutational analysis.
Main Methods:
- A structure-based mutational approach was employed, generating 16 CD59 mutants with single, nonconservative amino acid substitutions.
- Functional analysis of mutants, including competition assays and epitope mapping using monoclonal antibodies, was performed.
- Glycosylation independence of CD59 expression and function was investigated.
Main Results:
- A single active site on CD59 was identified, comprising residues Trp-40, Arg-53, and Glu-56 on the membrane-distal face, potentially including Asp-24.
- These identified residues are conserved across species, aligning with previous observations of broad CD59 functionality.
- Monoclonal antibody epitope mapping corroborated the location of the active site and revealed glycosylation-independent function.
Conclusions:
- The study precisely maps the active site of CD59, revealing key residues critical for its function in regulating the complement system.
- Findings provide a molecular basis for CD59's protective role against MAC-induced cell lysis and suggest conserved functional mechanisms.
- CD59's activity and expression are independent of glycosylation, simplifying its functional understanding and potential therapeutic targeting.
Abstract:
The Ly-6 superfamily of cell surface molecules includes CD59, a potent regulator of the complement system that protects host cells from the cytolytic action of the membrane attack complex (MAC). Although its mechanism of action is not well understood, CD59 is thought to prevent assembly of the MAC by binding to the C8 and/or C9 proteins of the nascent complex. Here a systematic, structure-based mutational approach has been used to determine the region(s) of CD59 required for its protective activity. Analysis of 16 CD59 mutants with single, highly nonconservative substitutions suggests that CD59 has a single active site that includes Trp-40, Arg-53, and Glu-56 of the glycosylated, membrane-distal face of the disk-like extra-cellular domain and, possibly, Asp-24 positioned at the edge of the domain. The putative active site includes residues conserved across species, consistent with the lack of strict homologous restriction previously observed in studies of CD59 function. Competition and mutational analyses of the epitopes of eight CD59-blocking and non-blocking monoclonal antibodies confirmed the location of the active site. Additional experiments showed that the expression and function of CD59 are both glycosylation independent.