Enzymatic evidence for differences in the placement of Rh antigens within the red cell membrane.
Intact erythrocytes of different Rh genotypes were subjected to various enzyme treatments, the effects of which were monitored by separating the membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and performing Western blotting using an antibody preparation that recognizes only Rh-related polypeptides. We found that treatment of intact cells with either phospholipase A2 or proteases such as papain did not alter the size of Rh antigen-containing polypeptides. In contrast, phospholipase A2 treatment followed by papain digestion cleaved a fraction of these polypeptides. This cleavage appears, from such digestions of Rh(D) positive and negative cells of different genotypes, to occur solely at the extracellular domain of Rh(D) polypeptide, while the extracellular domains of other Rh antigen-containing polypeptides are unaffected. Digestion of red blood cell ghosts and inside-out vesicles with trypsin showed that Rh(D), (C/c), and (E/e) antigen-containing polypeptides span the lipid bilayer having cytoplasmic domains susceptible to the action of proteases. The size of the cleavage products at the cytoplasmic domain of -D-/-D- cells was found to differ from that of other Rh(D) positive genotypes, due possibly to a difference in folding of Rh(D) polypeptide at its cytoplasmic domain and within the cellular membrane of these cells.
Suyama
K
K
Lindsley F. Kimball Research Institute of the New York Blood Center, NY 10021.
Goldstein
J
J
D015153:Blotting, Western; D004910:Erythrocyte Membrane / Q000276:immunology / Q000648:ultrastructure*; D006801:Humans; D008565:Membrane Proteins / Q000648:ultrastructure; D010206:Papain / Q000494:pharmacology; D010446:Peptide Fragments / Q000032:analysis; D010741:Phospholipases A / Q000494:pharmacology; D054467:Phospholipases A2; D012204:Rh-Hr Blood-Group System; D014357:Trypsin / Q000494:pharmacology
D008565:Membrane Proteins; D010446:Peptide Fragments; D012204:Rh-Hr Blood-Group System; D010741:Phospholipases A; D054467:Phospholipases A2; D014357:Trypsin; D010206:Papain