Isolation of cDNA clones for a 50 kDa glycoprotein of the human erythrocyte membrane associated with Rh (rhesus) blood-group antigen expression

Abstract

The Rh blood-group antigens are associated with human erythrocyte membrane proteins of approx. 30 kDa (the Rh30 polypeptides). Heterogeneously glycosylated membrane proteins of 50 and 45 kDa (the Rh50 glycoproteins) are coprecipitated with the Rh30 polypeptides on immunoprecipitation with anti-Rh-specific mono- and poly-clonal antibodies. We have isolated cDNA clones representing a member of the Rh50 glycoprotein family (the Rh50A glycoprotein). We used PCR with degenerate primers based on the N-terminal amino acid sequence of the Rh50 glycoproteins and human genomic DNA as a template and cloned and sequenced three types of PCR product of the expected size. Two of these products, Rh50A and Rh50B, gave the same translated amino acid sequence which corresponded to the expected Rh50 glycoprotein sequence but had only 75% DNA sequence similarity. The third product (Rh50C) contained a single base deletion, and the translated amino acid sequence contained an in-frame stop codon. We have isolated cDNA clones containing the full coding sequence of the Rh50A glycoprotein. This sequence predicts that it is a 409-amino acid N-glycosylated membrane protein with up to 12 transmembrane domains. The Rh50A glycoprotein shows clear similarity to the Rh30A protein in both amino acid sequence and predicted topology. Our results are consistent with the Rh30 and Rh50 groups of proteins being different subunits of an oligomeric complex which is likely to have a transport or channel function in the erythrocyte membrane. We mapped the Rh50A gene to human chromosome 6p21-qter, showing that genetic differences in the Rh30 rather than the Rh50 genes specify the major polymorphic forms of the Rh antigens.

The RHD gene is highly detectable in RhD-negative Japanese donors.

“…In these Rhnull cases, the Rh50 glycoproteins have shown genomic alterations of frameshift, nucleotide mutations, and failure of amplification. The Rh50 glycoproteins that were immunoprecipitated by Moore and Green (36) are heterogeneously glycosylated membrane proteins of 50 and 45 kD, coprecipitated with the Rh30 protein on immunoprecipitation with antiRh-specific antibodies (37). It is thought that the Rh30 polypeptide, which is a nonglycosylated 30-kD erythrocyte membrane protein, is associated with Rh antigen (38).…”

Immunochemical Analysis of the Human Erythrocyte Rh Polypeptides

“…The Rh proteins span the membrane bilayer 12 times based on hydropathy analysis and immunochemical studies (8,13,14). The Rh proteins are tightly associated with an ancestrally related glycoprotein component, the Rh glycoprotein (15)(16)(17). Although the involvement of the Rh glycoprotein in Rh antigen expression is not known, it is critical for assembly of the Rh complex, as defects in its structural gene result in the Rh null phenotype (18).…”

“…4) corresponded to residue 40 of the Rh glycoprotein sequence (17). This peptide was shown to contain the C terminus of the Rh glycoprotein by immunoblot analysis of this preparative immunoprecipitate with anti-Rh glycoprotein Cter serum (not shown).…”

International Society of Blood Transfusion Committee on Terminology for Red Blood Cell Surface Antigens: Macao report

“…Antigens of this system appear to be located on the Rh-associated glycoprotein (CD241) encoded by the RHAG gene [6]. RHAG1 (Duclos) was previously the high-incidence antigen 901013.…”