Molecular characterization of the human red cell Rho(D) antigen.

Abstract

Human red cells of Rh blood groups ‐D‐/‐D‐ (‘super‐D’), ‐/‐ (Rhnull) and normal Rho(D)+ cells were radioactively surface‐labeled using the lactoperoxidase 125I method. Polyacrylamide gel electrophoresis in the presence of SDS followed by fluorography showed a strong enrichment of a polypeptide with an apparent mol. wt. of 28,0000‐33,000 in the 125I‐labeled ‐D‐/‐D‐ membranes. This polypeptide was specifically immune precipitated with anti‐Rho(D) antiserum. Treatment of intact cells with trypsin or Pronase did not digest the protein. The Rho polypeptide migrated identically on polyacrylamide gel electrophoresis under reducing and non‐reducing conditions. It was not phosphorylated after in vitro incubation of red cells with 32P. When whole labeled membranes were solubilized in neutral detergent and applied to lectin‐Sepharose columns the Rho(D) polypeptide adsorbed to Ricinus communis lectin but not to wheat germ lectin or Lens culinaris lectin. The purified molecule did not adsorb to R. communis lectin‐Sepharose. Treatment of the Rho(D) antigen with endo‐N‐acetyl glucosaminidase H, endo‐beta‐galactosidase or mild alkali did not lower its apparent mol. wt.