title: Localization of the C termini of the Rh (rhesus) polypeptides to the cytoplasmic face of the human erythrocyte membrane.
abstract: We have raised a rabbit antiserum to a synthetic peptide corresponding to the C terminus (residues 400-416) of the Rh30A polypeptide. The rabbit antiserum reacted with the Rh30B (D30) polypeptide in addition to the Rh30A (C/c and/or E/e) polypeptide(s), indicating that these proteins share homology at their C termini. The antiserum did not react with erythrocyte membranes from an individual with Rh(null) syndrome. The rabbit antiserum immunoprecipitated Rh polypeptides from erythrocyte membranes and alkali-stripped membranes, but not from intact erythrocytes. Treatment of intact red cells with carboxypeptidase Y did not affect the reactivity of the antiserum, whereas treatment of alkali-stripped and unsealed erythrocyte ghost membranes resulted in the loss of antibody binding. Carboxypeptidase A treatment of intact erythrocytes and alkali-stripped membranes had no effect on antibody binding, indicating that the C-terminal domains of the Rh polypeptides contain lysine, arginine, proline, or histidine residues. These results show that the C termini of the Rh polypeptides are located toward the cytoplasmic face of the erythrocyte membrane. Treatment of intact radioiodinated erythrocytes with bromelain followed by immunoprecipitation with monoclonal anti-D gave a band of M(r) 24,000-25,000, indicating that the Rh30B (D30) polypeptide is cleaved at an extracellular domain close to the N or C terminus, with loss of the major radioiodinated domain. Immunoblotting of bromelain treated D-positive erythrocyte membranes with the rabbit antiserum to the C-terminal peptide revealed a new band of M(r) 6000-6500, indicating that the extracellular bromelain cleavage site is located near the C terminus of the molecule. The band of M(r) 6000-6500 was not obtained in erythrocyte membranes derived from bromelain treated D-negative erythrocytes. Erythrocytes of the rare -D- phenotype appear to either totally lack, or have gross alterations in, the Cc/Ee polypeptide(s), since the bromelain treatment of these cells resulted in the total loss of staining in the M(r) 35,000-37,000 region and the concomitant appearance of the new band of M(r) 6000-6500.
authors.affiliation: International Blood Group Reference Laboratory, Southmead, Bristol, United Kingdom.
authors.forename: N D
authors.initials: ND
authors.lastname: Avent
authors.forename: S K
authors.initials: SK
authors.lastname: Butcher
authors.forename: W
authors.initials: W
authors.lastname: Liu
authors.forename: W J
authors.initials: WJ
authors.lastname: Mawby
authors.forename: G
authors.initials: G
authors.lastname: Mallinson
authors.forename: S F
authors.initials: SF
authors.lastname: Parsons
authors.forename: D J
authors.initials: DJ
authors.lastname: Anstee
authors.forename: M J
authors.initials: MJ
authors.lastname: Tanner
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D007106:Immune Sera; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*; D011233:Precipitin Tests
chemical_list: D007106:Immune Sera; D010455:Peptides; D002268:Carboxypeptidases; D043422:Carboxypeptidases A