Characterization of DAF-2, a high molecular weight form of decay-accelerating factor (DAF; CD55), as a covalently cross-linked dimer of DAF-1.
Human E express two surface forms of decay-accelerating factor (DAF; CD55). On SDS-PAGE under reducing conditions the major form, DAF-1, migrates as a 70-kDa protein and the minor form, DAF-2, present at < 10% the amount of DAF-1, migrates as a 140-kDa protein (Kinoshita, T., S. I. Rosenfeld, and V. Nussenzweig. 1987. J. Immunol. 138:2994). Both forms possess decay-accelerating activity and, after purification from solubilized E, reinsert into sheep E, indicating a glycosylphosphatidylinositol anchor. In contrast to human cells, these two forms of DAF from orangutan E are expressed in approximately equal amounts (Nickells, M. W., and J. P. Atkinson. 1990. J. Immunol. 144:4262). An orangutan B lymphocyte cell line, CP81, also expresses similar quantities of both forms. These sources of orangutan DAF were utilized for further characterization of DAF-2. Orangutan and human DAF-1 were 98% and 95% homologous at the nucleotide and amino acid levels, respectively. Northern and Southern analyses of orangutan DAF were also similar to those for human DAF. Tryptic peptide maps of DAF-1 and DAF-2 were identical. After treatment with phosphatidylinositol-specific phospholipase C and glycosidases, the change in M(r) of DAF-2 was consistent with it possessing two glycosylphosphatidylinositol anchors and twice as much oligosaccharide as DAF-1. Biosynthetic analysis demonstrated a single 46-kDa precursor for both forms. Taken together, these data indicate that DAF-2 is a covalently cross-linked dimer of DAF-1. Analysis of a series of human DAF deletion mutants localized the cross-link(s) within the short consensus repeat domains.
Nickells
M W
MW
Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110.
Alvarez
J I
JI
Lublin
D M
DM
Atkinson
J P
JP
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry*; D001483:Base Sequence; D018958:CD55 Antigens; D017931:DNA Primers / Q000737:chemistry; D015870:Gene Expression; D005796:Genes; D017261:Glycosylphosphatidylinositols; D006801:Humans; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D010449:Peptide Mapping; D011148:Pongo pygmaeus; D012333:RNA, Messenger / Q000235:genetics; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
D015703:Antigens, CD; D018958:CD55 Antigens; D017931:DNA Primers; D017261:Glycosylphosphatidylinositols; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D012333:RNA, Messenger