Identification of mutations in rat CD59 that increase the complement regulatory activity.
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.
Hinchliffe
S J
SJ
Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XX, United Kingdom.
Morgan
B P
BP
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001216:Asparagine / Q000235:genetics; D001666:Binding Sites, Antibody; D018957:CD59 Antigens / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology / Q000502:physiology; D016466:CHO Cells; D002460:Cell Line; D003169:Complement Inactivator Proteins / Q000235:genetics / Q000494:pharmacology / Q000502:physiology; D015938:Complement Membrane Attack Complex / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003171:Complement Pathway, Classical / Q000235:genetics*; D006224:Cricetinae; D018698:Glutamic Acid / Q000235:genetics; D006801:Humans; D008239:Lysine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology; D014162:Transfection
D000911:Antibodies, Monoclonal; D018957:CD59 Antigens; D003169:Complement Inactivator Proteins; D015938:Complement Membrane Attack Complex; D011994:Recombinant Proteins; D018698:Glutamic Acid; D001216:Asparagine; D008239:Lysine