Loss of Rh Antigen Activity Following the Action of Phospholipase A₂ on Red Cell Stroma

Abstract

The degradation of red cell stroma phospholipids by phospholipase A2 is accompanied by a concomitant fall in the activity of the Rh antigens, c, D and e. The action of phospholipase C on stroma also brings about a fall in D antigen activity. Anti-D bound to the red cells protects the D antigen from inactivation by phospholipase A2.

Isolation and purification of Rh0(D) antigen of human erythrocyte membrane and its serological property.

“…The component carrying Rh-Hr antigens has been reported to be protein (Mollison 1979a) because antigenic activities were diminished by treatment by alkylating reagents or non-ionic detergents. On the other hand, it has been claimed that lipid is required for expression of the antigen activities (Green 1972;Weicker et al 1973;Hughes-Jones et al 1975;Mollison 1979b). Green (1972) reported that D activity which had disappeared by n-butanol treatment was restored by addition of phosphatidylcholine or phosphatidylethanolamine.…”

Immunochemical investigations of Rh0(D) activity detected in band 3 of red cell membrane.

“…It has been claimed that lipids are essential for expression of the antigen activity (Green 1972;Hughes-Jones et al 1975). Magnesium or calcium ion was also reported to be necessary for the blood type (Lorusso and Green 1975).…”

Phospholipid Dependence of Wr^b Antigen Expression in Human Erythrocyte Membranes

“…Erythrocyte membranes, prepared by the method of Dodge et al [6] using 5 mM phosphate buffer pH 8.0, were reacted with phospholipase A2 as described by Hughes-Jones et al [7], Membranes in buffer containing 0.15 M glycine, 80 mM NaCl, 2 mM CaCE, pH 6.7 were reacted with up to 100 units phospholipase A2 for 1 h at 37'C; most experiments used 50 units. Control membranes were reacted with buffer omitting phospholipase A2.…”

“…Antibody binding to erythro cyte membranes was assessed by differential absorp tion following the method of Hughes-Jones et al [7], Antibody was reacted with unmodified or phospholipase-A2-modified membranes for 30 min at 37 "C. Membranes were removed by centrifugation at 13,000# for 5 min in a microcentrifuge (Model 235, Fisher Scientific, Pittsburg, Pa.), and the amount of antibody remaining in the supernatant was assessed by hemagglutination titer. The minimal volume of un modified erythrocyte membrane needed to completely absorb each antibody was determined in preliminary experiments.…”

“…Rhesus blood group antibody, anti-D (human), was obtained from Ortho Diagnostic Systems, Raritan, N.J. Anti-D was used as a control because the D antigen is known to be phospholipid-dependent in erythrocyte membranes [7], Assessment of Antibody Binding by Hemagglutination Antibody binding to unmodified and phospholi pase A2-modified native erythrocytes was assessed by hemagglutination titer. Antibody binding to erythro cyte membranes was assessed by differential absorp tion following the method of Hughes-Jones et al [7], Antibody was reacted with unmodified or phospholipase-A2-modified membranes for 30 min at 37 "C. Membranes were removed by centrifugation at 13,000# for 5 min in a microcentrifuge (Model 235, Fisher Scientific, Pittsburg, Pa.), and the amount of antibody remaining in the supernatant was assessed by hemagglutination titer.…”