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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.
Biochemistry
|May 10, 2000
Summary
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.