Related Experiment Videos
Identification of mutations in rat CD59 that increase the complement regulatory activity
1Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XX, United Kingdom.
Insights
Glycosylphosphatidylinositol- (GPI-) anchored CD59 protein inhibits membrane attack complex formation. Site-directed mutagenesis revealed key residues in the hydrophobic groove critical for CD59
Area of Science:
- Biochemistry
- Immunology
- Molecular Biology
Background:
- The glycosylphosphatidylinositol- (GPI-) anchored glycoprotein CD59 regulates complement system activation.
- CD59 inhibits the formation of the membrane attack complex (MAC) on host cells.
- Existing data on the active site of human CD59 are conflicting.
Purpose of the Study:
- To elucidate the active site of a nonprimate CD59 molecule using site-directed mutagenesis.
- To investigate regions of potential species selectivity in CD59 function.
- To clarify conflicting published data on human CD59's active site.
Main Methods:
- Site-directed mutagenesis of rat CD59.
- Functional assays of mutated CD59 variants against complement.
- Comparative analysis of rat and human CD59 mutations.
Main Results:
- Mutations Y36A, W40A, and L54A in rat CD59 ablated its complement inhibitory function, confirming the hydrophobic groove as the active site.
- Mutations I56E, D24A, and D24R reduced rat CD59 function.
- Mutation K48E significantly increased rat CD59 function against human, rat, and rabbit serum, while K48A increased function against human serum alone.
- A similar mutation (N48E) in human CD59 abolished activity against rabbit serum, suggesting species-specific interactions.
Conclusions:
- The major hydrophobic groove is the active site of CD59, essential for complement inhibition.
- Specific residues within CD59, particularly at position 48, mediate species selectivity in complement inhibition.
- The alpha-helix adjacent to the hydrophobic groove influences interactions with complement components C8 and C9, potentially explaining species-specific activity.
Abstract:
Formation of the membrane attack complex (MAC) of complement on host cells is inhibited by the glycosylphosphatidylinositol- (GPI-) anchored glycoprotein CD59. Published data on the active site of human CD59 are confusing. To clarify these data, we set out to elucidate the active site of a nonprimate CD59 molecule by site-directed mutagenesis. We also undertook to investigate a region of potential species selectivity, and to this end rat CD59 was chosen for all mutations. Our investigations confirmed the proposal that the active site of CD59 is the major hydrophobic groove, with mutations Y36A, W40A, and L54A ablating complement inhibitory function of CD59. Other mutations reducing the function of rat CD59 were I56E, D24A, and D24R. Importantly, mutations at one residue increased the function of rat CD59. The K48E mutation significantly increased function against human rat or rabbit serum, whereas the K48A mutation increased function against human serum alone. A similar mutation in human CD59 (N48E) had no effect on activity against human or rat serum but completely abolished all activity against rabbit serum. These findings suggest that the alpha-helix of human CD59, adjacent to the hydrophobic groove, influences the interaction between human CD59 and rabbit C8, C9, or both.