Isolation of a human erythrocyte membrane glycoprotein with decay-accelerating activity for C3 convertases of the complement system.

Abstract

Abstract An intrinsic human erythrocyte membrane glycoprotein has been purified to homogeneity based on an assay of its ability to accelerate decay of the functional activity of the classical complement C3 convertase, C4b,2a. The purification procedure utilized two sequential butanol extractions of erythrocyte stroma and chromatography on DEAE-Sephacel, hydroxylapatite, phenyl-Sepharose, and trypan blue coupled to Sepharose. The membrane glycoprotein, designated decay-accelerating factor of stroma (DAF-S), exhibited a 200-fold increase in specific activity (U/mg) from the initial butanol extract in augmenting the linear decay rate of C4b,2a. Purified DAF-S presented a single, stained protein band on analytic alkaline disc gel electrophoresis that corresponded to a single region of activity eluted from a sliced replicate gel. On electrophoresis in SDS-polyacrylamide gels, purified DAF-S demonstrated a single band with a m.w. of 70,000 under reduced and unreduced conditions that stained equally well with Coomassie Blue and periodic acid Schiff reagent. Specific decay-accelerating function of DAF-S was five times greater for the classical C3 convertase, C4b,2a, than for the properdin-stabilized alternative pathway C3 convertase, C3b,Bb,P. Conversely, purified C3b receptor of human erythrocyte, a single-chain membrane glycoprotein of 220,000 m.w., isolated by detergent extraction, was 10 times more active on a weight basis against C3b,Bb,P than against C4b,2a. Although anti-DAF-S and anti-C3b receptor each neutralized the decay-accelerating activity of the respective antigen, anti-DAFS did not affect the decay-accelerating activity of the purified C3b receptor, and anti-C3b receptor did not affect the decay-accelerating activity of purified DAF-S. Thus, although DAF-S and the C3b receptor are both integral membrane glycoproteins of human erythrocytes that can be isolated based on their capacity to accelerate the decay of C3 convertase function, they are physicochemically and immunochemically distinct.