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; C D55). On SDS-PAGE under reducing conditions the major form, DAF-1, mig rates as a 70-kDa protein and the minor form, DAF-2, present at <10% t he amount of DAF-1, migrates as a 140-kDa protein (Kinoshita, T., S. I . Rosenfeld, and V. Nussenzweig. 1987. J. Immunol. 138.2994). Both for ms possess decay-accelerating activity and, after purification from so lubilized E, reinsert into sheep E, indicating a glycosylphosphatidyli nositol anchor. In contrast to human cells, these two forms of DAF fro m 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 chara cterization of DAF-2. Orangutan and human DAF-1 were 98% and 95% homol ogous 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. Aft er treatment with phosphatidylinositol-specific phospholipase C and gl ycosidases, the change in M(r) of DAF-2 was consistent with it possess ing two glycosylphosphatidylinositol anchors and twice as much oligosa ccharide 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 o f human DAF deletion mutants localized the cross-link(s) within the sh ort consensus repeat domains.