Mapping of epitopes, glycosylation sites, and complement regulatory domains in human decay accelerating factor.
Decay accelerating factor (DAF, CD55) is a glycophospholipid-anchored membrane protein that protects cells from complement-mediated damage by inhibiting the formation and accelerating the decay of C3/C5 convertases. DAF deletion mutants lacking each of the four short consensus repeats (SCR) or the serine/threonine-rich region (S/T) were created by site-directed mutagenesis. These deletion mutants were expressed by stable transfection in Chinese hamster ovary cells for the purpose of mapping important structural and functional sites in DAF. The epitopes on DAF for 16 murine mAb were mapped by immunoprecipitation studies as follows: SCR1, 6; SCR2, 3; SCR3, 3; SCR4, 3; S/T, 1. Testing of 13 mAb showed complete blocking of DAF function only by 1C6 and 1H4, both directed at SCR3. The single N-linked glycosylation site was confirmed at a location between SCR1 and SCR2, and the multiple O-linked oligosaccharides were localized to the S/T region. Functional activity of DAF mutants was assessed by the ability of these transfected constructs to protect Chinese hamster ovary cells from cytotoxicity induced by rabbit antibody plus human complement. Removal of SCR1 had no effect on DAF function, but individual deletion of SCR2, SCR3, or SCR4 totally abolished DAF function. Surprisingly, deletion of the S/T region totally abrogated DAF function, but this could be restored by a fusion construct placing the four SCR domains of DAF onto the HLA-B44 molecule, implying that the O-glycosylated S/T region serves as an important but nonspecific spacer projecting the DAF functional domains above the plasma membrane. Overall, the creation of DAF deletion mutants has elucidated important structure-function relations in the DAF molecule.
Coyne
K E
KE
Department of Pathology, Washington University School of Medicine, St. Louis 63110.
Hall
S E
SE
Thompson
S
S
Arce
M A
MA
Kinoshita
T
T
Fujita
T
T
Anstee
D J
DJ
Rosse
W
W
Lublin
D M
DM
D000818:Animals; D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000276:immunology*; D018958:CD55 Antigens; D002460:Cell Line; D003169:Complement Inactivator Proteins / Q000276:immunology*; D003165:Complement System Proteins / Q000378:metabolism*; D006224:Cricetinae; D003602:Cytotoxicity, Immunologic; D004306:Dose-Response Relationship, Immunologic; D000939:Epitopes / Q000235:genetics*; D006031:Glycosylation; D006801:Humans; D008562:Membrane Glycoproteins / Q000276:immunology*; D016297:Mutagenesis, Site-Directed; D014162:Transfection
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018958:CD55 Antigens; D003169:Complement Inactivator Proteins; D000939:Epitopes; D008562:Membrane Glycoproteins; D003165:Complement System Proteins