Isolation of erythrocyte membrane inhibitor of reactive lysis type II. Identification as glycophorin A.

When incubated with activated CoF and normal human serum (as the C source), a portion of the E from most patients with PNH are hemolyzed. In contrast, normal E are completely resistant to this form of C-mediated cytolysis called reactive lysis. This observation implies that normal E express membrane proteins that inhibit reactive lysis. In previous studies, we have shown that when E proteins are subjected to anion exchange chromatography, two peaks of inhibitory activity are observed. We isolated the inhibitor from the first peak and identified it as an 18-kDa protein that we call MIRL (CD59). The purpose of the studies presented herein was to isolate the inhibitory factor (MIRL type II) from the second peak. After anion exchange and gel filtration chromatography, aliquots of the fractions containing MIRL II activity were subjected to preparative SDS-PAGE (nonreducing conditions), and protein was eluted from gel slices electrophoretically. Inhibitory activity was found primarily in two adjacent slices, corresponding to an M(r) range of 84-51 kDa. When MIRL II was analyzed by SDS-PAGE and silver staining, two prominent bands representing proteins with M(r) of 77 and 39 kDa were observed under both reducing and nonreducing conditions. This behavior in SDS-PAGE is characteristic of GP-A, which migrates primarily as a nondisulfide-linked homodimer in equilibrium with a monomeric form. Immunoblotting studies confirmed that MIRL II is GP-A. The protein caused a concentration-dependent inhibition of reactive lysis, with approximately 100 ng producing 50% inhibition. GP-A inhibited reactive lysis by blocking the formation or binding of C5b-7, and this inhibitory activity was immunoprecipitated by monoclonal anti-GP-A. Furthermore, GP-A that was isolated by affinity chromatography also inhibited reactive lysis. These studies demonstrate that the major E sialoglycoprotein functions as an inhibitor of the membrane attack complex of C.