Dr(a-) polymorphism of decay accelerating factor. Biochemical, functional, and molecular characterization and production of allele-specific transfectants.
The Dra antigen belongs to the Cromer-related blood group system, a series of antigens on decay accelerating factor (DAF), a glycosyl-phosphatidylinositol-anchored membrane protein that protects host cells from complement-mediated damage. We studied the rare inherited Dr(a-) phenotype to ascertain the associated biochemical and functional changes in DAF and to characterize the basis for this polymorphism. Radioimmunoassay assay and flow cytometric analysis of Dr(a-) erythrocytes demonstrated 40% of normal surface expression of DAF but normal levels of several other glycosyl-phosphatidylinositol-anchored proteins, distinguishing this phenotype from that of paroxysmal nocturnal hemoglobinuria. Western blots confirmed this reduced DAF expression and indicated a slightly faster mobility of the molecule on SDS-PAGE. Despite the reduced DAF expression, Dr(a-) erythrocytes functioned normally in the complement lysis sensitivity assay. Utilization of the polymerase chain reaction to amplify mononuclear cell genomic DNA from three unrelated Dr(a-) individuals demonstrated that a point mutation underlies the Dr(a-) phenotype: a C to T change in nucleotide 649 resulting in a serine165 to leucine change. This defines the Drb allele of DAF, which can be distinguished from Dra by a Taq I restriction fragment length polymorphism. We created transfected Chinese hamster ovary cell lines expressing either the Dra or the Drb allelic form of DAF. These allele-specific transfectants were tested by inhibition of hemagglutination or flow cytometry and confirmed the specificity of anti-Dra alloantisera. The allele-specific transfectants could form the basis of a new serological approach to immunohematology.
Lublin
D M
DM
Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110.
Thompson
E S
ES
Green
A M
AM
Levene
C
C
Telen
M J
MJ
D000483:Alleles; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D001789:Blood Group Antigens / Q000235:genetics*; D015153:Blotting, Western; D018958:CD55 Antigens; D006017:Glycolipids / Q000737:chemistry; D017261:Glycosylphosphatidylinositols; D006457:Hemoglobinuria, Paroxysmal / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D010716:Phosphatidylinositols / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D014162:Transfection
D000911:Antibodies, Monoclonal; D001789:Blood Group Antigens; D018958:CD55 Antigens; D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D009841:Oligonucleotides; D010716:Phosphatidylinositols