Decay Accelerating Factor of Complement Is Anchored to Cells by a C-Terminal Glycolipid

Membrane-associated decay accelerating factor (DAF) of human erythrocytes (E^hu) was analyzed for a C-terminal glycolipid anchoring structure. Automated amino acid analysis of DAF following reductive radiomethylation revealed ethanolamine and glucosamine residues in proportions identical with those present in the E^hu acetylcholinesterase (AChE) anchor. Cleavage of radiomethylated 70-kilodalton (kDa) DAF with papain released the labeled ethanolamine and glucosamine and generated 61- and 55-kDa DAF products that retained all labeled Lys and labeled N-terminal Asp. Incubation of intact E^hu with phosphatidyl-inositol-specific phospholipase C (PI-PLC), which cleaves the anchors in trypanosome membrane form variant surface glycoproteins (mfVSGs) and murine thymocyte Thy-1 antigen, released 15% of the cell-associated DAF antigen. The released 67-kDa PI-PLC DAF derivative retained its ability to decay the classical C3 convertase C4b2a but was unable to membrane-incorporate and displayed physicochemical properties similar to urine DAF, a hydrophilic DAF form that can be isolated from urine. Nitrous acid deamination cleavage of E^hu DAF at glucosamine following labeling with the lipophilic photoreagent 3-(trifluoromethyl)-3-(m-[¹²⁵I]iodophenyl)diazirine ([¹²⁵I]TID) released the [¹²⁵I]TID label in a parallel fashion as from [¹²⁵I]TID-labeled AChE. Biosynthetic labeling of HeLa cells with [³H]ethanolamine resulted in rapid ³H incorporation into both 48-kDa pro-DAF and 72-kDa mature epithelial cell DAF. Our findings indicate that DAF and AChE are anchored in E^hu by the same or a similar glycolipid structure and that, like VSGs, this structure is incorporated into DAF early in DAF biosynthesis prior to processing of pro-DAF in the Golgi.