title: Immunochemical analysis of the human erythrocyte Rh polypeptides.
abstract: We have used rabbit polyclonal antisera raised against synthetic peptides complementary to different domains of the Rh polypeptides and Rh glycoprotein to examine the topography and organization of these proteins in the human erythrocyte membrane. Previously unrecognized exofacial protease sites have been identified on Rh CcEe, D proteins, and Rh glycoprotein. The Rh D protein has two specific bromelain cleavage sites located within the first and sixth predicted external domains, with the site of cleavage localized in the sixth domain to lie between residues 353 and 354. All Rh polypeptide species were found to be susceptible to cleavage with trypsin and subtilisin within the first external domain of these proteins. The Rh glycoprotein has two bromelain cleavage sites within the first external domain. These flank the single N-glycosylation site (Asn37), with the cleavage site toward the C-terminal side of this residue being between residues 39 and 40. Bromelain treatment was found to deglycosylate the Rh glycoprotein. Immunoprecipitation experiments have revealed that anti-C, -c,E, -e, and -D immune complexes are reactive with antisera raised against the fourth predicted external loop of the Rh proteins and the C-terminal domain. These data indicate that the hypothesis that suggests Rh C/c antigens are expressed on truncated Rh polypeptides by a mechanism of alternate splicing is incorrect and support the hypothesis that Rh Cc and Ee antigens are expressed on a single polypeptide chain.
authors.affiliation: International Blood Group Reference Laboratory, Southmead Rd., Southmead, Bristol BS10 5ND, United Kingdom.
authors.forename: N D
authors.initials: ND
authors.lastname: Avent
authors.forename: W
authors.initials: W
authors.lastname: Liu
authors.forename: K M
authors.initials: KM
authors.lastname: Warner
authors.forename: W J
authors.initials: WJ
authors.lastname: Mawby
authors.forename: J W
authors.initials: JW
authors.lastname: Jones
authors.forename: K
authors.initials: K
authors.lastname: Ridgwell
authors.forename: M J
authors.initials: MJ
authors.lastname: Tanner
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001963:Bromelains; D004910:Erythrocyte Membrane / Q000276:immunology / Q000648:ultrastructure; D006801:Humans; D007106:Immune Sera; D015151:Immunoblotting; D008961:Models, Structural; D008969:Molecular Sequence Data; D010206:Papain; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D010641:Phenotype; D017433:Protein Structure, Secondary; D011817:Rabbits; D012204:Rh-Hr Blood-Group System / Q000737:chemistry* / Q000276:immunology; D013381:Subtilisins; D014357:Trypsin
chemical_list: D007106:Immune Sera; D010446:Peptide Fragments; D012204:Rh-Hr Blood-Group System; D001963:Bromelains; D013381:Subtilisins; D014357:Trypsin; D010206:Papain